JPWO2019216405A1 - Method for suppressing decrease in sensitivity of biological component measurement reagent kit - Google Patents

Abstract

The present invention is an agent for suppressing a decrease in measurement sensitivity applied to a method for measuring a biological component using an aminoantipyrine compound as a coupler of a redox color-developing reagent, which comprises a copper-containing substance for measuring a biological component. An agent for suppressing a decrease in sensitivity is provided. Preferably, the biological component measuring method is a biological component measuring method using a reagent or a reagent set satisfying the following requirements (a) to (d), and the copper-containing substance is contained in a reagent satisfying the requirement (d). It is characterized by being added and used: (a) contains an oxidase capable of generating hydrogen peroxide. (B) Contains peroxidase. (C) Contains a redox color-developing reagent that reacts with hydrogen peroxide to develop color in the presence of peroxidase. (D) An aminoantipyrine compound is contained as a coupler of the redox color-developing reagent.

Description

The present invention relates to a method for suppressing a decrease in sensitivity of a biological component measurement reagent kit used in clinical diagnosis. More specifically, hydrogen peroxide generated from a biological component using a redox reaction and a redox color-developing reagent (such as a Trinder reagent in which an aminoantipyrine compound as a coupler and a hydrogen donor are combined) are used. The present invention relates to a method for suppressing a decrease in sensitivity in a biological component measurement reagent kit for quantifying and measuring coloration generated by oxidative condensation in the presence of peroxidase.

Conventionally, in clinical diagnosis, biological components are measured by an enzyme method, and in particular, a method using an oxidase-peroxidase-oxidation-reduction color-developing reagent (hereinafter, also referred to as a color-developing agent) system, that is, a measurement target in a sample. A method of enzymatically reacting a substance to generate hydrogen peroxide and reacting this with a color former in the presence of peroxidase to perform colorimetric quantification is widely used (Non-Patent Document 1).

Examples of the redox color-developing reagent system include a method using a hydrogen donor and a coupler. A typical example is the Trinder method in which a hydrogen donor and a coupler are oxidatively condensed with hydrogen peroxide in the presence of peroxidase to form a dye. As a coupler used in this method, for example, 4-aminoantipyrine (hereinafter, also referred to as 4AA) is known.

BUNSEKI KAGAKU Vol. 45, No. 2, pp. 111-124 (1996)

The present inventors prepared an enzyme-peroxidase-color former biocomponent measurement reagent using an aminoantipyrine compound and a hydrogen donor, and used it for biocomponent measurement to reduce the measurement sensitivity of unknown cause. Experienced.

Since the degree of decrease in measurement sensitivity varied depending on the lot of reagent composition prepared for measurement, the present inventors examined various reagent compositions of the measurement kit. As a result, it was surprisingly found that a very small amount of a substance called 4-hydroxyantipyrine (hereinafter, also referred to as 4HA) is present in 4-aminoantipyrine. Although not bound by theory, when 4-hydroxyantipyrine is present in the reagent, 4-hydroxyantipyrine structurally does not couple with the hydrogen donor, but in the presence of hydrogen peroxide, 4-hydroxyantipyrine. And peroxidase react with each other, and hydrogen peroxide is considered to be consumed. As a result, hydrogen peroxide generated by reacting the enzyme with the substance to be measured in the sample is consumed by 4-hydroxyantipyrine. It was considered that the amount of color developed by the reaction of the original 4-aminoantipyrine-hydrogen peroxide donor was reduced, and the sensitivity was lowered.

Therefore, an object of the present invention is to provide a means for suppressing a decrease in sensitivity due to 4-hydroxyantipyrine in order to solve the above-mentioned problems that have not been known so far.

As a result of diligent studies to achieve the above object, the present inventors unexpectedly used a copper-containing substance such as a component that generates copper ions in preparing a reagent kit for measuring biological components. , 4-Hydroxyantipyrine has been found to be able to suppress the decrease in sensitivity, and the present invention has been completed. That is, the present invention has the following configuration.

[Item 1] Measurement of biological components, which is a measurement sensitivity reduction inhibitor applied to a biological component measurement method using an aminoantipyrine compound as a coupler of a redox color-developing reagent, and is characterized by containing a copper-containing substance. Sensitivity reduction inhibitor.

[Item 2] The biological component measuring method is a biological component measuring method using a reagent or a reagent set satisfying the following requirements (a) to (d), and is added to a reagent satisfying the requirement (d). Item 2. The agent for suppressing a decrease in measurement sensitivity of a biological component according to Item 1.

(A) Contains an oxidase capable of generating hydrogen peroxide.

(B) Contains peroxidase.

(C) Contains a redox color-developing reagent that reacts with hydrogen peroxide to develop color in the presence of peroxidase.

(D) An aminoantipyrine compound is contained as a coupler of the redox color-developing reagent.

[Item 3] The agent for suppressing a decrease in measurement sensitivity of a biological component according to Item 1 or 2, which suppresses a decrease in measurement sensitivity of a biological component caused by 4-hydroxyantipyrine.

[Item 4] A method for suppressing a decrease in measurement sensitivity applied to a method for measuring a biological component using an aminoantipyrine compound as a coupler of a redox color-developing reagent, which comprises using a copper-containing substance for measuring the sensitivity of the biological component. How to control the decline.

[Item 5] A method for suppressing a decrease in measurement sensitivity in a biological component measurement method using a reagent or a reagent set that satisfies the following requirements (a) to (d), and copper is included in the reagent that satisfies the requirement (d). A method for suppressing a decrease in measurement sensitivity of a biological component, which comprises adding a contained substance.

(A) Contains an oxidase capable of generating hydrogen peroxide.

(B) Contains peroxidase.

(C) Contains a redox color-developing reagent that reacts with hydrogen peroxide to develop color in the presence of peroxidase.

(D) An aminoantipyrine compound is contained as a coupler of the redox color-developing reagent.

[Item 6] The method for suppressing a decrease in measurement sensitivity of a biological component according to Item 4 or 5, wherein the decrease in measurement sensitivity of a biological component due to 4-hydroxyantipyrine is suppressed.

[Item 7] Decrease in measurement sensitivity of the biological component according to Item 5 or 6, wherein the concentration of the copper-containing substance coexisting in the reagent satisfying the requirement (d) is 0.00001 to 1 mM. How to suppress.

[Item 8] The method for suppressing a decrease in measurement sensitivity of a biological component according to any one of Items 4 to 7, wherein the biological component is either creatinine or glycated hemoglobin.

[Item 9] A biological component measurement reagent kit using an aminoantipyrine compound as a coupler of a redox color-developing reagent, which comprises the measurement sensitivity reduction inhibitor according to item 1.

[Item 10] A reagent kit for measuring biological components that satisfies the following requirements (a) to (e), and the reagents that satisfy the requirements (d) and (e) are one reagent that simultaneously satisfies the two requirements. A reagent kit for measuring biological components.

(A) Contains an oxidase capable of generating hydrogen peroxide.

(B) Contains peroxidase.

(C) Contains a redox color-developing reagent that reacts with hydrogen peroxide to develop color in the presence of peroxidase.

(D) An aminoantipyrine compound is contained as a coupler of the redox color-developing reagent.

(E) Contains copper-containing substances.

Item 11. The biological component measuring reagent according to Item 10, wherein one reagent that simultaneously satisfies the two requirements (d) and (e) has passed at least one month after production. kit.

[Item 12] A method for measuring a biological component, which comprises using the biological component measuring kit according to any one of Items 9 to 11.

[Item 13] A method for producing a biological component measurement reagent kit that satisfies the following requirements (a) to (e) and suppresses a decrease in sensitivity due to 4-hydroxyantipyrine, wherein (d) and (e) are used. A method for producing a reagent kit for measuring biological components, which comprises producing a reagent that satisfies the above requirements as one reagent that simultaneously satisfies the two requirements.

(A) Contains an oxidase capable of generating hydrogen peroxide.

(B) Contains peroxidase.

(C) Contains a redox color-developing reagent that reacts with hydrogen peroxide to develop color in the presence of peroxidase.

(D) An aminoantipyrine compound is contained as a coupler of the redox color-developing reagent.

(E) Contains copper-containing substances.

[Item 14] Regarding the reagents satisfying the requirements (d) and (e), one reagent that simultaneously satisfies the two requirements is an intermediate reagent in the manufacturing process of the biological component measurement reagent kit. 13. The method for producing a biological component measurement reagent kit according to 13.

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to suppress a decrease in sensitivity due to 4-hydroxyantipyrine in the measurement of biological components by an enzymatic method using an oxidase-peroxidase-color former. Therefore, according to the present invention, it is possible to measure good biological components. In particular, stable measurement is possible in the measurement of biological components requiring high sensitivity such as the content of biological components being extremely small.

It is a figure which shows the HPLC fraction as a result of analysis of 4-aminoantipyrine drug substance. It is a figure which shows the structural formula of 4-hydroxyantipyrine. It is a figure which shows the relationship between the concentration in the 2nd reagent of 4-hydroxyantipyrine and the sample measurement sensitivity.

The embodiments of the present invention will be described in detail as follows, but the present invention is not limited thereto. It should be understood that the terms used herein are used in the meaning commonly used in the art unless otherwise specified.

In addition, all of the non-patent documents and patent documents described in the present specification are incorporated herein by reference. "-" In the present specification means "greater than or equal to, less than or equal to". For example, if "X to Y" is described in the present specification, it means "more than or equal to X, less than or equal to Y". In addition, "and / or" in the present specification means either one or both. It should also be understood herein that the singular representation also includes the concept of the plural, unless otherwise stated.

(Inhibitor of measurement sensitivity decrease of biological components)

The measurement sensitivity reduction inhibitor of the present invention is a measurement sensitivity reduction inhibitor applied to a biological component measurement method using an aminoantipyrine compound as a coupler of a redox color-developing reagent, and is a copper-containing substance (for example, copper). It is characterized by containing a component that generates ions, etc.).

The biological component measurement method to which the biocomponent measurement sensitivity reduction inhibitor of the present invention is applied is preferably a biological component measurement method using a reagent or a reagent set satisfying the following requirements (a) to (d).

(A) Contains an oxidase capable of generating hydrogen peroxide.

(B) Contains peroxidase.

(C) Contains a redox color-developing reagent that reacts with hydrogen peroxide to develop color in the presence of peroxidase.

(D) An aminoantipyrine compound is contained as a coupler of the redox color-developing reagent.

In the agent for suppressing the decrease in measurement sensitivity of biological components of the present invention, it is preferable to add a copper-containing substance (for example, a component that generates copper ions) to a reagent that satisfies the above requirement (d).

The aminoantipyrine compound contained in the reagent satisfying the requirement (d) may be contaminated with a very small amount of 4-hydroxyantipyrine as a by-product during its production. It was found that 4-hydroxyantipyrine causes a decrease in the measurement sensitivity of biological components. Furthermore, the present inventors have found that it is possible to suppress such a decrease in sensitivity by coexisting a copper-containing substance with 4-hydroxyantipyrine, and completed the present invention.

That is, the agent for suppressing the decrease in measurement sensitivity of biological components of the present invention is preferably used by adding a copper-containing substance to a reagent satisfying the requirement (d), and the measurement sensitivity of biological components caused by 4-hydroxyantipyrine. The purpose is to suppress the decrease in the amount of copper.

Here, the reagent or the reagent set may be prepared as one reagent satisfying the requirements of (a) to (d), or the reagent is packaged and composed of 2 to 3 or more. It may be a reagent set. Further, the reagent or the reagent set may be in the form of a kit or the like packaged in one packaging container, or may be in the form in which each reagent is prepared separately and used as a set at the time of use.

(Method of suppressing decrease in measurement sensitivity of biological components)

In one embodiment, the method for suppressing a decrease in measurement sensitivity of a biological component of the present invention is a method for suppressing a decrease in measurement sensitivity applied to a method for measuring a biological component using an aminoantipyrine compound as a coupler of a redox color-developing reagent. It is characterized by using a copper-containing substance.

In a further embodiment, the method for suppressing a decrease in measurement sensitivity of a biological component of the present invention is a method for suppressing a decrease in measurement sensitivity in a method for measuring a biological component using a reagent or a reagent set satisfying the following requirements (a) to (d). It is characterized in that a copper-containing substance is added to a reagent that satisfies the requirement (d).

(A) Contains an oxidase capable of generating hydrogen peroxide.

(B) Contains peroxidase.

(C) Contains a redox color-developing reagent that reacts with hydrogen peroxide to develop color in the presence of peroxidase.

(D) An aminoantipyrine compound is contained as a coupler of the redox color-developing reagent.

The present inventors presume that the decrease in measurement sensitivity occurs as a result of the consumption of hydrogen peroxide due to the reaction of 4-hydroxyantipyrine with peroxidase. The present invention has been completed by finding that the property can be suppressed by coexisting with a copper-containing substance (preferably, copper ions that can be generated from the copper-containing substance).

That is, in the method for suppressing the measurement sensitivity of a biological component of the present invention, a copper-containing substance (for example, a component that generates copper ions) is added to a reagent that satisfies the requirement (d). 4-Hydroxyantipyrine mixed in a reagent that meets the requirements of 4-hydroxyantipyrine and a copper-containing substance coexist and react with each other to suppress the reactivity of 4-hydroxyantipyrine, thereby causing a biological component derived from 4-hydroxyantipyrine. It is characterized by suppressing a decrease in measurement sensitivity of.

In the reagent containing an aminoantipyrine compound mixed with 4-hydroxyantipyrine, the time for reacting the copper-containing substance (in a specific embodiment, the copper ion that can be generated from the copper ion) is not particularly limited as long as the effect of the present invention is exhibited. .. For example, from the time when the copper-containing substance is added to the reagent satisfying the requirement (d), at least at 1 ° C. to 10 ° C. in a state where the copper-containing substance coexists in the reagent satisfying the requirement (d). Sufficient effect is obtained by reacting for 2 weeks, at least 1 week at 11 ° C to 25 ° C, at least 2 days at 26 ° C to 40 ° C, at least 5 hours at 41 ° C to 60 ° C, and at least 1 hour at 61 ° C to 80 ° C. Can be obtained. Therefore, even when strict temperature control is not performed, the effect of the present invention can be obtained by storing the copper-containing substance for about one month or longer and reacting the copper-containing substance. In the present invention, it is preferable to use a reagent that satisfies the above requirement (d) after the 4-hydroxyantipyrine and the copper-containing substance are allowed to coexist and react for the above period. The above reaction may be carried out during the inventory storage or distribution stage after production.

As for the storage temperature, the lower limit temperature is preferably a temperature at which the reagent does not freeze, and the upper limit temperature is preferably a temperature at which various components in the reagent do not cause quality deterioration such as alteration or denaturation.

As a specific embodiment of the present invention, for example, when the reagent satisfying the requirement (d) is a product reagent (for example, a reagent containing a protein such as an enzyme), a copper-containing substance is added to the product reagent. After that, the reaction proceeds by refrigerating or storing in stock, distributing, and storing before use in a state where the copper-containing substance coexists. Therefore, a reagent that satisfies the above requirement (d) before use. The biological component measurement reagent kit produced by adding a copper-containing substance to the product may be used after at least two weeks have passed from the production date.

As another specific embodiment of the present invention, the reagent satisfying the requirement (d) is an intermediate reagent (for example, an aminoantipyrine compound having a concentration several times to several tens of times higher than that of the product reagent as a buffer solution component or the like. In the case of the adjusted reagent), after adding the copper-containing substance to the intermediate reagent, it is stored for several days under temperature conditions of 30 ° C. or higher, for several hours under temperature conditions of 45 ° C. or higher, and diluted to a predetermined concentration after storage. Then, the product reagent may be prepared.

When a copper-containing substance is added to the intermediate reagent, a copper-containing substance (in a specific embodiment, copper ions that can be generated from the copper ion) coexisting in the reagent satisfying the requirement (d) is added at the stage of preparing the product reagent. Except for this, it may be used as a product reagent.

The present inventors have found that such a decrease in measurement sensitivity due to 4-hydroxyantipyrine can be suppressed by coexisting a copper-containing substance with 4-hydroxyantipyrine in advance. Although the mechanism by which the copper-containing substance suppresses the decrease in sensitivity due to 4-hydroxyantipyrine is not clear, the present inventors have caused some structural changes in 4-hydroxyantipyrine due to the copper-containing substance (for example, copper ions that may be generated from this). It is presumed that it does not react with peroxidase and does not consume hydrogen peroxide.

Regarding this point, the structural change of 4-hydroxyantipyrine is presumed to be an irreversible change, and the decrease in measurement sensitivity of biological components due to 4-hydroxyantipyrine suppressed by the copper-containing substance is subsequently caused by the coexisting reagent. The present inventors speculate that the reaction between 4-hydroxyantipyrine and peroxidase will not be restored even if the copper-containing substance (in a specific embodiment, the copper ion that can be generated from the copper ion) is removed from the inside.

In the method for suppressing a decrease in measurement sensitivity of a biological component of the present invention, the concentration of the copper-containing substance coexisting in the reagent satisfying the requirement (d) is preferably 0.00001 to 1 mM, preferably 0.00003 to 0. 3 mM is more preferable, 0.0001 to 0.1 mM is further preferable, and 0.0001 to 0.01 mM is particularly preferable.

When the concentration of the copper-containing substance is less than 0.00001 mM, the inhibitory effect is small, and when the concentration of the copper-containing substance is more than 1 mM, the color development caused by the copper-containing substance becomes large in the biological component measurement reaction, and a measurement error occurs.

The preferable concentration range of the copper-containing substance coexisting in the reagent satisfying the requirement (d) depends on the concentration of 4-hydroxyantipyrine mixed in the reagent satisfying the requirement (d). -If the concentration of hydroxyantipyrine is low, the decrease in measurement sensitivity of biological components can be suppressed even if the concentration of copper-containing substance is low, and if the concentration of 4-hydroxyantipyrine is high, it is effective to use a higher concentration of copper-containing substance. Therefore, in the method for suppressing a decrease in sensitivity for measuring biological components of the present invention, it is preferable to appropriately set the concentration of the copper-containing substance according to the concentration of 4-hydroxyantipyrine mixed therein.

As a method for measuring the copper ion concentration that can be generated from a copper-containing substance, ICP emission spectrometric analysis (ICP-AES), ICP mass spectrometry (ICP-MS), or the like can be preferably adopted. Specifically, in the present invention, the concentration of copper ions can be measured by ICP-AES (using SPECTROBLUE manufactured by AMETEK, Inc.). When the detection limit is exceeded by ICP-AES, it can be measured by ICP-MS (using Agilent 7700s ICP-MS manufactured by Agilent Technologies).

In the method for suppressing a decrease in measurement sensitivity of a biological component of the present invention, a copper-containing substance (for example, a component that generates copper ions) is added so that the concentration of 4-hydroxyantipyrine present in the reagent satisfying the requirement (d) is high. The effect is particularly likely to be obtained when the concentration before the above is about 0.1 to 50 μg / ml.

When the concentration of 4-hydroxyantipyrine present in the reagent satisfying the requirement (d) before adding the copper-containing substance is less than 0.1 μg / ml, the measurement sensitivity of biological components is lowered by 4-hydroxyantipyrine. Is 1% or less, and there is little need to suppress a decrease in measurement sensitivity by coexisting with a copper-containing substance.

When the concentration of 4-hydroxyantipyrine present in the reagent satisfying the requirement (d) before adding the copper-containing substance is more than 50 μg / ml, the decrease in measurement sensitivity of biological components due to 4-hydroxyantipyrine is suppressed. Therefore, a high-concentration copper-containing substance is required, and therefore, a side reaction caused by the copper-containing substance tends to increase the color development and cause a measurement error.

According to the studies by the present inventors, it was found that a general aminoantipyrine compound (4-aminoantipyrine) drug substance contains about 0.005 to 0.30 w / w% of 4-hydroxyantipyrine. doing. Therefore, in the present invention, as an example, a reagent containing an aminoantipyrine compound (4-aminoantipyrine) of about 0.01 to 100 g / l, preferably an aminoantipyrine compound (4-amino) of about 0.01 to 10 g / l. It is effective to use it as a reagent containing (antipyrine), more preferably about 0.01 to 5 g / l of an aminoantipyrine compound (4-aminoantipyrine).

When the amount of 4-hydroxyantipyrine mixed is large, it is preferable to remove 4-hydroxyantipyrine from the aminoantipyrine compound in advance by a method for removing 4-hydroxyantipyrine, which will be described later, and then use the biocomponent measurement. ..

(Biological component)

The biological component to be measured by the biological component measuring reagent kit of the present invention is not particularly limited, and can be used for measuring various biological components. For example, the biological components used for the biological component measurement of the present invention are uric acid (UA), creatinine (CRE), triglyceride (TG), cholesterol (CHO), AST (GOT), ALT (GPT), LDH (lactic acid dehydrogenation). Enzymes) and isozymes, ALP (alkaline phosphatase) and isozymes, CK (creatin kinase) and isozymes, amylase (Amy) and isozymes, lipase, γ-GTP (γ-glutamyl transpeptidase), cholinesterase (ChE), sodium (Na) , Potassium (K), Cholesterol (Cl), Calcium (Ca), Phosphorus (P) [Inorganic phosphorus (IP)], Iron (Fe), Magnesium (Mg), Total protein (TP), Serum protein fraction (PF) ), Urea nitrogen (BUN), creatinine (CRE), uric acid (UA), birylbin (Bil), ammonia, cholesterol, HDL cholesterol (HDL-C, high density lipoprotein cholesterol), LDL cholesterol (LDL-C, low density) Lipoprotein cholesterol), neutral fat (triglyceride) (TG), cholesterol (CHO), BTR (BTR, total branched amino acid / tyrosine ratio), tyrosine measurement reagent (TYR), blood glucose (BS, GLU), 1,5 -Anhydro-D-glucitol (1,5-AG), saccharified albumin (GA), saccharified hemoglobin (HbA1c) and the like can be mentioned, but are not limited thereto.

Hydrogen peroxide can be generated by allowing any oxidase and, if necessary, other enzymes (for example, hydrolases) to act on these biological components. For example, with respect to uric acid (UA), creatinine (CRE), triglyceride (TG), and glycated hemoglobin (HbA1c), specific aspects of biological component measurement will be described below.

When measuring uric acid (UA), hydrogen peroxide produced by the reaction of uricase (oxidase) using uric acid (UA) as a substrate can be quantified by a peroxidase-color former system.

When measuring creatinine (CRE), hydrogen peroxide is not directly generated in the reaction of creatinine amidinohydrolase using creatinine (CRE) as a substrate. Therefore, creatine produced by the reaction of creatinine amidinohydrolase is added to the reagent in advance. Peroxidase-color former system by designing a so-called conjugated reaction in which creatinine is produced by reacting with amide hydrolase, and hydrogen peroxide is produced using sarcosine oxidase (oxidizing enzyme) in which creatine is added to a reagent in advance. Allows quantification of creatinine (CRE) concentration.

When measuring triglyceride (TG), lipoprotein lipase using triglyceride (TG) as a substrate, and glycerol kinase and glycerol triphosphate oxidase (oxidase) as conjugate enzymes are used to generate hydrogen peroxide. , Peroxidase-color former system enables quantification of triglyceride (TG) concentration.

When glycated hemoglobin (HbA1c) is measured, hydrogen peroxide produced by the reaction of glycated hemoglobin oxidase (for example, fructosyl amino acid oxidase) using glycated hemoglobin as a substrate can be quantified in a peroxidase-color former system.

In this way, a reaction that can change the measurement target to a substrate of an oxidase that can generate hydrogen oxide without a suitable enzyme that catalyzes the reaction that directly oxidizes the measurement target to generate hydrogen peroxide. It is also possible to measure the concentration or amount of biological components other than the above by appropriately designing a conjugated reaction in which the catalytic enzyme (the enzyme reaction of several steps may be linked) and the oxidizing enzyme. Is. Even when measuring other biological components, hydrogen peroxide can be generated by a method well known in the art in the same manner as described above.

Among the biological components listed above, creatinine (CRE) and glycated hemoglobin (HbA1c) have extremely low contents of biological components, and therefore, particularly highly sensitive measurement is required. According to the present invention, the influence of 4-hydroxyantipyrine that inhibits the reaction of the oxidase-peroxidase-color former system can be suppressed, and the decrease in sensitivity of biological component measurement can be suppressed. Therefore, such high-sensitivity measurement is required. It is useful for measuring biological components. Therefore, the present invention is suitable for the measurement of creatinine and glycated hemoglobin, and above all, it is preferably used for the measurement of creatinine.

(Biological component measurement kit)

In one embodiment, the biological component measurement kit of the present invention comprises a measurement sensitivity reduction inhibitor containing a copper-containing substance as described above.

In a further embodiment, the biological component measurement kit of the present invention satisfies the following requirements (a) to (e), and simultaneously satisfies two requirements for a reagent satisfying the following requirements (d) and (e). It is characterized by being a single reagent. (A) Contains an oxidase capable of generating hydrogen peroxide. (B) Contains peroxidase. (C) Contains a redox color-developing reagent that reacts with hydrogen peroxide to develop color in the presence of peroxidase. (D) An aminoantipyrine compound is contained as a coupler of a redox color-developing reagent. (E) Contains copper-containing substances.

Regarding the reagents satisfying the requirements (d) and (e), one reagent satisfying the two requirements at the same time generates a copper-containing substance (for example, copper ions) in the reagent satisfying the requirement (d) as described above. It can be produced by adding a component, etc.).

In the biological component measurement kit of the present invention, it is preferable that one reagent satisfying the two requirements (d) and (e) has passed at least one month after production.

The present inventors speculate that 4-hydroxyantipyrine reacts with peroxidase, resulting in the consumption of hydrogen peroxide. The reactivity of 4-hydroxyantipyrine is determined by adding a copper-containing substance to 4-hydroxyantipyrine. It has been found in the present invention that it can be suppressed by adding it.

The reaction rate of this inhibitory reaction may be affected by the reaction temperature and the reaction time, but the inhibitory effect is produced when the storage period is several weeks to one month or more under the normal storage conditions of the biological component measurement kit. It is preferable that at least one month has passed since the production of the biological component measurement kit.

Normally, it is considered that it takes one to several months for such a measurement kit to be used after being manufactured through an inventory / distribution process, during which the inhibitory reaction proceeds and the effect of the present invention is exhibited. It will be.

(Biological component measurement method)

The biological component measuring method of the present invention is characterized in that the biological component is measured using the above-mentioned biological component measuring kit.

(Manufacturing method of biological component measurement kit)

The method for producing a biological component measurement kit of the present invention is a method for producing a biological component measurement reagent kit that satisfies the following requirements (a) to (e) and suppresses a decrease in sensitivity due to 4-hydroxyantipyrine. , (D) and (e) are produced as one reagent that satisfies the two requirements at the same time.

(A) Contains an oxidase capable of generating hydrogen peroxide.

(B) Contains peroxidase.

(C) Contains a redox color-developing reagent that reacts with hydrogen peroxide to develop color in the presence of peroxidase.

(D) An aminoantipyrine compound is contained as a coupler of the redox color-developing reagent.

(E) Contains copper-containing substances.

Here, it is preferable that the reagent satisfying the above requirements (d) and (e) is produced as one reagent satisfying the two requirements at the same time, and the measurement of the biological component caused by the 4-hydroxyantipyrine. The reason why it is preferable that a sufficient period (for example, about one month or more) has passed to suppress the decrease in sensitivity is as described above.

In the method for producing a biological component measurement kit of the present invention, for the reagents satisfying the above requirements (d) and (e), one reagent that simultaneously satisfies the two requirements is an intermediate reagent in the manufacturing process of the biological component measurement reagent kit. Therefore, a product reagent may be prepared using the intermediate reagent.

As described above, in the biological component measurement kit of the present invention, it is necessary to add a copper-containing substance to 4-hydroxyantipyrine to suppress the reactivity of 4-hydroxyantipyrine, and this suppression reaction is used as a product reagent as described above (d). ) May be carried out by coexisting a copper-containing substance with 4-hydroxyantipyrine mixed in a reagent that satisfies the requirements, but this inhibitory reaction does not necessarily have to be carried out in the product reagent, for example, an aminoantipyrine compound. In the process of preparing the reagent to be contained, a copper-containing substance may be added in the state of an intermediate reagent or the like, and then an inhibitory reaction may be carried out, and then a product reagent may be produced using the intermediate reagent and provided as a biological component measurement kit.

The above-mentioned reagent intermediate containing an aminoantipyrine compound is, for example, the concentration of an aminoantipyrine compound obtained by diluting the original of the aminoantipyrine compound with a buffer solution or the like in the step of producing a reagent containing the aminoantipyrine compound. It means an intermediate reagent adjusted to a concentration of several times to several tens of times that of the product reagent.

In the biological component measurement kit of the present invention, when a copper-containing substance is added to an intermediate reagent containing an aminoantipyrine compound to carry out an inhibitory reaction, the copper-containing substance is added to the intermediate reagent until the product manufacturing date. It is preferable that the total of the period and the period from the product manufacturing date to the product usage date is at least one month.

The copper-containing substance of the present invention (for example, a component that generates copper ions) is allowed to coexist with 4-hydroxyantipyrine mixed as an impurity of an aminoantipyrine compound to measure the sensitivity of biological components caused by 4-hydroxyantipyrine. It is presumed that the decrease will be suppressed.

Although it is not clear why the decrease in sensitivity of biological component measurement is suppressed by coexisting the copper-containing substance of the present invention with 4-hydroxyantipyrine, the copper-containing substance (for example, copper ions that can be generated from this) is 4 Direct or indirect action on −hydroxyantipyrine causes some changes in 4-hydroxyantipyrine, resulting in reduced consumption of hydrogen peroxide due to the reaction of 4-hydroxyantipyrine with peroxidase. It is considered that the decrease in sensitivity is suppressed. Here, the change in 4-hydroxyantipyrine is expected to be irreversible, and once denatured and detoxified, 4-hydroxyantipyrine may cause a decrease in sensitivity under normal storage conditions of a biological component measurement kit. It is presumed that there is no such thing.

Therefore, when a copper-containing substance is added to the intermediate reagent, the product reagent is produced by removing the copper-containing substance (in a specific embodiment, copper ions that can be generated from the copper ion) in the subsequent stage of producing the product reagent. Although it is considered possible, a reagent kit for measuring biological components produced using such a product reagent and a method for producing the same are also within the scope of the present invention.

The copper-containing substance used in the present invention is preferably a component that generates copper ions, and is not particularly limited, and examples thereof include a copper salt containing copper ions, a copper protein containing copper ions as a complementary factor, and a pigment containing copper ions. Can be done. For example, as components that generate copper ions, CuBr, CuBr ₂ , CuC ₂ , Cu ₂ C ₂ , CuCl, CuCl ₂ , CuF, CuF ₂ , CuH, CuI, CuI ₂ , CuN ₃ , Cu (N ₃ ) ₂ , CuO, CuO ₂ , Cu ₂ O, Cu ₃ P, CuP ₂ , CuS, Cu ₂ S, Cu ₃ (AsO ₄ ) ₂ , Cu (BF ₄ ) ₂ , Cu (ClO ₃ ) ₂ , Cu (ClO ₄ ) ₂ , CuCN, Cu (CN) ₂ , CuCrO ₄ , Cu (IO ₃ ) ₂ , Cu (IO ₄ ) ₂ , Cu (NO ₃ ) ₂ , CuOH, Cu (OH) ₂ , Cu ₃ (PO ₄ ) ₂ , CuSO ₄ , Cu ₂ SO ₄ , Cu (CH ₃ COO), Cu (CH ₃ COO) ₂ , CuCO _{3 ·} Cu (OH) ₂ , CuSCN, Cu (SCN) _{2 and} other copper salts; Proclin 200 (5-chloro-) Mixtures containing copper salts such as 2-methyl-4-isothiazolin-3-one, 2-methyl-4-isothiazolin-3-one, magnesium salts, copper salts, preservatives containing water); ascorbic acid oxidase (ascorbin) Acid oxidase), azurin, stellacyanin, plastocyanin, quercetin-2,3-dioxygenase, rusticianin, pseudoazurin, lacquerze, copper-containing nitrite reductase, superoxide dismutase, galactose oxidase, dopamine-β- Hydroxylase, dopamine-β-monooxygenase, peptidylglycine α-amidating monooxygenase, membrane-bound methanemonooxygenase, copper amine oxidase, galactose oxidase, tyrosinase, catechol oxidase, celluloplasmin, cytochrome c oxidase, hemocyanin, zinc nitric reductase, copper proteins _{etc; CuCO 3 · Cu (OH)} 2 ( _{patina), CuSO 4 · 3Cu (OH} ) 2 (Brochantite), Cu 2 (OH) 3 Cl (Atacamite) _{, Cu 2 (OH) 3 Cl} (Paratacamite), Cu (C 2 H 3 O 2) 2 · 3Cu (AsO 2) 2 ( Paris _{Green), CuCO 3 · Cu (OH} ) 2 (Malachite), (Cu, Zn ) ₂ (CO ₃ ) (OH) ₂ (Rosasite), (Zn, Cu) ₂ (CO3) (O H) ₂ (Zincrosite), (Zn, Cu) ₂ (AsO ₄ ) (OH) ₂ (Adamite), (Cu, Zn) ₆ (AsO ₄ , PO ₄ ) ₂ (OH) ₆ · H ₂ O (Philipsburgite) Pigments containing copper ions and the like, but are not limited thereto.

The components that generate these copper ions can be appropriately selected, for example, depending on the biological component to be measured, the biological sample, and the like. Copper (I) chloride, copper (II) chloride, copper acetate, copper sulfate, copper nitrate, proclinic 200, ascorbic acid oxidation from the viewpoint of excellent handling convenience and expectation of even higher effects. It is preferable to use an enzyme.

The amount of the copper-containing substance used in the present invention is not particularly limited as long as the effects of the present invention are exhibited.

As described above, in the present invention, the copper-containing substance is preferably used as an intermediate reagent of an aminoantipyrine compound or a product reagent blended together with an aminoantipyrine compound, and as an example, copper in the intermediate reagent or the product reagent. It may be prepared and used so that the concentration of the contained substance is 0.00001 to 1 mM, preferably the concentration of the copper-containing substance in the reagent intermediate or the reagent is 0.00003 to 0.3 mM, more preferably 0. The compounding amount may be adjusted and used so as to be 0001 to 0.1 mM, more preferably 0.0001 to 0.01 mM. By using the copper-containing substance in such an amount, it is possible to effectively suppress the decrease in sensitivity of the biological component measurement reagent kit due to 4-hydroxyantipyrine.

The copper-containing substance used in the present invention has an effect of suppressing a decrease in sensitivity of biological component measurement due to 4-hydroxyantipyrine contained as an impurity of the aminoantipyrine compound after being blended with the aminoantipyrine compound. Is preferably allowed to elapse for a certain period of time after blending.

In order for the copper-containing substance used in the present invention to exhibit the effect of suppressing the decrease in sensitivity of biological component measurement due to 4-hydroxyantipyrine, the storage temperature is 1 ° C. to 10 ° C. for 2 weeks or more, 11 ° C. -25 ° C for 1 week or longer, 26 ° C-40 ° C for 2 days or longer, 41 ° C-60 ° C for 5 hours or longer, 61 ° C-80 ° C for 1 hour or longer Preferably, depending on the storage temperature, the higher the temperature, the shorter the time, and the lower the temperature, the longer the time. The upper limit of the reaction time is not particularly limited, but can be, for example, 10 years or less.

The copper-containing substance used in the present invention suppresses the decrease in sensitivity of biological component measurement due to 4-hydroxyantipyrine as long as the quality of the reagent used in the biological component measurement kit is not deteriorated even if the upper limit of the elapsed time is exceeded. The effect is maintained.

In the above, in the product life cycle of a general biological component measurement kit, it takes about several weeks to one month or more to be mixed with a reagent in the production process, and to be quality tested, shipped, distributed, stored, and used. The measurement kit containing the reagent in which the copper-containing substance of the present invention coexists with the aminoantipyrine compound is in a state in which the effect of suppressing the decrease in sensitivity by the copper-containing substance used in the present invention is exhibited and maintained at the stage of use. However, when the product life cycle is shortened, it can be prepared by manufacturing process control or product control at the time of shipment.

(Oxidase)

The oxidase used in the present invention can be used without limitation as long as it can generate hydrogen peroxide from the substrate, depending on the target measurement target. Specific examples include, but are not limited to, uricase, sarcosine oxidase, glycerol triphosphate oxidase, fructosyl amino acid oxidase, and the like. As commercially available products, UAO-211 (manufactured by Toyobo), SAO-351 (manufactured by Toyobo), G3O-311 (manufactured by Toyobo) and the like are preferably used. The amount used and the form of addition are not particularly limited.

(Peroxidase)

As the peroxidase used in the present invention, any kind of enzyme may be used as long as it is an enzyme that catalyzes the reaction between hydrogen peroxide and a redox color-developing reagent. Can be mentioned. Among these, horseradish, rice, and soybean-derived peroxidase are preferable, and horseradish-derived peroxidase is more preferable, for reasons of purity, availability, price, and the like. As commercially available products, PEO-131 (manufactured by Toyobo), PEO-301 (manufactured by Toyobo), PEO-302 (manufactured by Toyobo) and the like are preferably used. The amount used and the form of addition are not particularly limited.

Peroxidase activity is defined in the following way.

After sequentially mixing 14 mL of distilled water, 2 mL of a 5% (W / V) pyrogallol aqueous solution, 1 mL of 0.147 M hydrogen peroxide solution, and 2 mL of 100 mM phosphate buffer (pH 6.0), preliminary temperature adjustment at 20 ° C. for 5 minutes. Then, add 1 mL of the sample solution and start the enzymatic reaction.

After carrying out the reaction for 20 seconds, the reaction is stopped by adding 1 mL of a 2N sulfuric acid aqueous solution, and the produced purpurogallin is extracted 5 times with 15 mL of ether.

After combining the extracts, the total volume is 100 mL, and the absorbance at a wavelength of 420 nm is measured (ΔOD _test ).

On the other hand, in blinding, 14 mL of distilled water, 2 mL of 5% pyrogallol aqueous solution, 1 mL of 0.147 M hydrogen peroxide solution and 2 mL of 100 mM phosphate buffer (pH 6.0) were sequentially mixed, and then 1 mL of 2N sulfuric acid aqueous solution was added and mixed. Then, 1 mL of the sample solution is added to prepare.

The absorbance of this solution is measured by performing ether extraction in the same manner as described above (ΔOD _blank ).

The amount of purpurogallin produced is calculated from the difference in absorbance between the _{ΔOD test} and the ΔOD _{blank, and the peroxidase activity is calculated.}

The amount of enzyme that produces 1.0 mg of purpurogallin in 20 seconds under the above conditions is defined as 1 purpurogallin unit (U). The calculation formula is as shown below.

Peroxidase activity (U / mL) = {ΔOD (OD _{test- OD blank} ) x dilution ratio} / {0.117 x 1 (mL)) = ΔOD x 8.547 x dilution ratio

Peroxidase activity (U / mg) = Peroxidase activity (U / mL) x 1 / C

0.117: Absorbance of 1 mg% purprogalin ether solution at 420 nm

C: Enzyme concentration at dissolution (c mg / mL)

(1 proprugalin unit corresponds to 13.5 international units (using o-dianisidine as a substrate and reaction conditions at 25 ° C.).)

In the above measurement, the sample solution was dissolved in a 0.1 M phosphate buffer solution pH 6.0 that had been ice-cooled in advance, and diluted with the same buffer solution to 3.0 to 6.0 pullprogalin units (U) / mL. It is preferable to use it for measurement.

(Redox color development reagent)

As the redox color-developing reagent used for the measurement of biological components of the present invention, any kind of dye may be used as long as it reacts with hydrogen peroxide to develop color. For example, a combination of a hydrogen donor and a coupler may be used. Can be mentioned. The amount used and the form of addition are not particularly limited. For all of these, commercially available products and the like can be obtained.

A typical example using a hydrogen donor and a coupler is the Trinder method in which a hydrogen donor and a coupler are oxidatively condensed with hydrogen peroxide in the presence of peroxidase to form a dye.

(Hydrogen donor)

In the biological component measurement method of the present invention, phenol, phenol derivative, aniline derivative, naphthol, naphthol derivative, naphthylamine, naphthylamine derivative and the like are used as the hydrogen donor used in the Trinder method and the like.

For example, N-ethyl-N-sulfopropyl-3-methoxyaniline, N-ethyl-N-sulfopropylaniline, N-ethyl-N-sulfopropyl-3,5-dimethoxyaniline, N-sulfopropyl-3,5 -Dimethoxyaniline, N-ethyl-N-sulfopropyl-3,5-dimethylaniline, N-ethyl-N-sulfopropyl-3-methylaniline, N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methoxyaniline, N-ethyl-N- (2-hydroxy-3-sulfopropyl) aniline, N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline, N- (2-Hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline, N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3,5-dimethylaniline, N-ethyl-N- (2) -Hydroxy-3-sulfopropyl) -3-methoxyaniline, N-sulfopropylaniline, N- (2-hydroxy-3-sulfopropyl) -2,5-dimethylaniline, N-ethyl-N- (3-methyl) Examples thereof include phenyl) -N'-succinylethylenediamine and N-ethyl-N- (3-methylphenyl) -N'-acetylethylenediamine.

(Coupler)

These hydrogen donors can be used in combination with the coupler.

As the coupler, aminoantipyrine compounds such as 4-aminoantipyrine (4AA) and aminoantipyrine derivatives; vanillindiaminesulfonic acid compounds such as vanillindiaminesulfonic acid; methylbenzthiazolinone hydrazone (MBTH), sulfonated methylbenzthia. Examples thereof include methylbenzthiazolinone hydrazone compounds such as zolinone hydrazone (SMBTH).

Since the present invention can suppress the decrease in sensitivity of the biological component measurement reagent kit caused by a very small amount of 4-hydroxyantipyrine contained in the aminoantipyrine compound, it is effective when the aminoantipyrine compound is used as a coupler. is there. In particular, the present invention is useful when 4-aminoantipyrine is used as a coupler.

The coupler used in the present invention may be two or more kinds of aminoantipyrine compounds, or may be used in combination with other couplers in addition to the aminoantipyrine compounds, but one kind of aminoantipyrine compounds is preferable. , And more preferably 4-aminoantipyrine.

The amount of the aminoantipyrine compound used in the present invention is not particularly limited as long as the effects of the present invention are exhibited. The aminoantipyrine compound may contain a very small amount of 4-hydroxyantipyrine that causes a decrease in sensitivity, and it is desired to reduce the amount of 4-hydroxyantipyrine. However, according to the present invention, a copper-containing substance. By coexisting with, the decrease in sensitivity can be suppressed even if a small amount of 4-hydroxyantipyrine is contained. From this point of view, as an example, the concentration of 4-hydroxyantipyrine mixed in the reagent containing the aminoantipyrine compound before coexistence with the copper-containing substance is preferably 0.1 to 50 μg / ml, more preferably 0.1 to 50 μg / ml. It is effective to prepare and use the amount of the aminoantipyrine compound used for the measurement of biological components so as to be 0.3 to 20 μg / ml, particularly preferably 1 to 10 μg / ml.

When the concentration of 4-hydroxyantipyrine is lower than the above range, the influence of the decrease in measurement sensitivity of biological components by 4-hydroxyantipyrine is small, and it is less necessary to suppress the decrease in measurement sensitivity due to the coexistence of copper-containing substances.

If the concentration of 4-hydroxyantipyrine exceeds the above range, a high concentration of copper-containing substance is required to suppress the decrease in measurement sensitivity of biological components due to 4-hydroxyantipyrine, and therefore a color development side reaction due to the copper-containing substance occurs. As a result, the measurement accuracy of biological components tends to decrease.

If the amount of 4-hydroxyantipyrine mixed is large, the 4-hydroxyantipyrine mixed in the aminoantipyrine compound should be removed in advance by a method for removing 4-hydroxyantipyrine, which will be described later, and then subjected to biological component measurement. Is preferable.

Considering the amount of 4-hydroxyantipyrine mixed in a general aminoantipyrine compound (4-aminoantipyrine), the present invention provides, for example, an aminoantipyrine compound (4-aminoantipyrine) of about 0.01 to 100 g / l. Reagent containing, preferably a reagent containing an aminoantipyrine compound (4-aminoantipyrine) of about 0.01 to 10 g / l, more preferably an aminoantipyrine compound (4-aminoantipyrine) of about 0.01 to 5 g / l. It is effective to use it in a reagent containing. By using the aminoantipyrine compound in such a range, the effect of the present invention can be exhibited even more effectively.

(Method for quantifying 4-hydroxyantipyrine)

The content of 4-hydroxyantipyrine in the aminoantipyrine compound is, for example, high performance liquid chromatography (hereinafter, also referred to as HPLC method), gas chromatography (hereinafter, also referred to as GC method), mass spectrometry (hereinafter, MS). It can be measured by performing a quantitative method such as a nuclear magnetic resonance method (hereinafter, also referred to as an NMR method) alone or in an arbitrary combination. Although not particularly limited, the HPLC method is preferably used from the viewpoint of ease of operation and economic efficiency of systems and equipment. As the column of the HPLC method, a reverse phase column is preferable, and it is more preferable that the reverse phase column is a silica-based porous column.

Hereinafter, specific examples of the HPLC method will be described. In this specification, 4-hydroxyantipyrine was quantified under the following HPLC method conditions.

(1) Column Imtakat Cadenza CD-C18 2.0 × 150 mm

(2) Mobile phase A: 0.1% formic acid, B: methanol

(3) Gradient condition

0min (A95%, B5%)-(Linear gradient during this period) -15min (A2%, B98%) -25min (A2%, B98%)

(4) Flow velocity 0.2 mL / min

(5) Column temperature 40 ° C

(6) Sample injection amount 5 μL

(7) Detection wavelength UV250nm

A 4HA (Sigma) product with a known concentration was used as a standard product, and the coincidence of the detection position of the HPLC fraction peak with the measurement sample was confirmed. In addition, the area of the fraction peak was compared with that of the measurement sample for quantification. Sigma's 4HA (4-Hydroxyantipyline) includes Cas. NO. A product of 1672-63-5, product number 109428-5G, and 99% purity was used.

In the present invention, it is preferable to use the above method as a method for quantifying 4-hydroxyantipyrine.

(4-Hydroxyantipyrine)

4-Hydroxyantipyrine has a structure in which the amino group at the 4-position of 4-aminoantipyrine is converted into a hydroxyl group, and is considered to be a by-product produced and mixed in the production process of 4-aminoantipyrine.

It has been clarified by the studies by the present inventors that 4-hydroxyantipyrine has a great influence on the color reaction in the color reaction in the biological component measurement method even if the amount of the mixture is very small. This phenomenon is caused by the fact that the reaction rate of 4-hydroxyantipyrine is faster than that of aminoantipyrine compounds (for example, 4-aminoantipyrine) originally intended to react as a coupler, and hydrogen peroxide is consumed. It is inferred that.

In the biocomponent measurement reagent kit of the present invention, the drug substance of an aminoantipyrine compound that can contain 4-hydroxyantipyrine may be used as it is, or the drug substance of the aminoantipyrine compound having a low 4-hydroxyantipyrine content. May be used, or the drug substance of the aminoantipyrine compound whose 4-hydroxyantipyrine content is reduced by removing 4-hydroxyantipyrine may be selected and used. ..

There is no particular limitation on the method for reducing the 4-hydroxyantipyrine content from the aminoantipyrine compound. For example, any method well known in the art such as a method using chromatography such as HPLC from a biological component measurement reagent, a method of adsorbing 4-hydroxyantipyrine on an adsorbent such as a resin after dissolving it in water or a solvent, and removing it. Separation / removal may be performed by means.

When chromatography is used as a means for removing 4-hydroxyantipyrine from an aminoantipyrine compound, the physicochemical principle of its separation is not particularly limited. For example, various principles such as partitioning (forward / reverse phase), adsorption, molecular exclusion, and ion exchange can be mentioned. Separation may be performed by a method of adsorbing to a resin to which a ligand is bound or an adsorbent.

As the 4-hydroxyantipyrine removing means, general reverse phase chromatography can be used. The carrier for reverse phase chromatography is not particularly limited. For example, silica gel is preferable, but a polymer-based carrier may also be used. When silica gel is used as a carrier, one with end cap treatment and one without end cap treatment can be selected. Further, the chromatography apparatus can be used regardless of the low pressure, medium pressure, or high pressure chromatography system by appropriately preparing the conditions according to the purpose.

The type of ligand that binds to the chromatography carrier is also not particularly limited. As the ligand, in addition to the widely used octadecyl group (ODS), a phenyl group and an octyl group can also be selected by optimizing the conditions. The binding of the ligand may be monomeric or polypeptide. Any filler can be used by optimizing the separation conditions.

The mobile phase of the chromatography may be water and a water-soluble solvent such as methanol or acetonitrile, and the pH of the mobile phase of the chromatography is acidic according to a conventional method in order to avoid ionic interaction with the silanol group of silica gel. It may be prepared on the side or a small amount of ion pair reagent may be added.

The flow velocity of the mobile phase may be optimized according to the capacity of the system used. Further, it may be eluted and separated by stepwise instead of linear gradient.

(Other ingredients, etc.)

The biological component measurement reagent kit of the present invention preferably contains a reagent containing a buffer solution component. In addition, ascorbic acid oxidase, preservatives, salts, enzyme stabilizers, chromogen stabilizers, etc. are added to the reagents included in the biological component measurement reagent kit of the present invention as long as they do not affect the reaction. May be good.

Examples of the buffer solution component that can be contained in the biological component measuring reagent of the present invention include Tris buffer solution, phosphate buffer solution, boric acid buffer solution, carbonic acid buffer solution, GOOD buffer solution and the like. The amount used, the set pH, the form of addition, etc. are not particularly limited. For all of these, commercially available products and the like can be obtained.

GOOD buffers include N- (2-acetamido) -2-aminoethanesulfonic acid (ACES), N, N-bis (2-hydroxyethyl) -2-aminoethanesulfonic acid (BES), N-cyclohexyl- 2-Aminoethanesulfonic acid (CHES), 2- [4- (2-hydroxyethyl) -1-piperazinyl] ethanesulfonic acid (HEPES), 2-morpholinoetansulfonic acid (MES), piperazin-1,4-bis (2-Ethan Sulfonic Acid) (PIPES), N-Tris (Hydroxymethyl) Methyl-2-aminomethane Sulfonic Acid (TES), N-Cyclohexyl-3-aminoPropane Sulfonic Acid (CAPS), N-Cyclohexyl-2- Hydroxy-3-aminopropanesulfonic acid (CAPSO), 3- [N, N-bis (2-hydroxyethyl) amino] -2-hydroxypropanesulfonic acid (DIPSO), 3- [4- (2-hydroxyethyl)) -1-piperazinyl] propanesulfonic acid (EPPS), 2-hydroxy-3- [4- (2-hydroxyethyl) -1-piperazinyl] propanesulfonic acid (HEPPSO), 3-morpholinopropanesulfonic acid (MOPS), 2 -Hydroxy-3-morpholinopropanesulfonic acid (MOPSO), piperazin-1,4-bis (2-hydroxy-3-propanesulfonic acid) (POPSO), N-tris (hydroxymethyl) methyl-3-aminopropanesulfonic acid (TAPSO), N- (2-acetamido) iminonic acid (ADA), N, N-bis (2-hydroxyethyl) glycine (Bicine), N- [tris (hydroxymethyl) methyl] glycine (Tricine), etc. Illustrated.

In the biological component measuring reagent of the present invention, the amount of ascorbic acid oxidase, preservative, salts, enzyme stabilizer, chromogen stabilizer, etc. used and the form of addition are not particularly limited. For all of these, commercially available products and the like can be obtained.

Examples of preservatives include proclinic 150, proclinic 200, proclinic 300, proclinic 950, azides, chelating agents, antibiotics, antibacterial agents and the like.

Examples of the chelating agent include ethylenediaminetetraacetic acid and salts thereof.

Examples of the antibiotic include gentamicin, kanamycin, chloramphenicol and the like.

Examples of the antibacterial agent include methylisothiazolinone, imidazolidinylurea and the like.

Examples of salts include sodium chloride, potassium chloride, aluminum chloride and the like.

Examples of the enzyme stabilizer include sucrose, trehalose, cyclodextrin, gluconate, amino acids and the like.

Examples of the chromogen stabilizer include chelating agents such as ethylenediaminetetraacetic acid and salts thereof, cyclodextrin and the like.

The reagent included in the biological component measurement reagent kit of the present invention may be a liquid reagent dissolved in an arbitrary solvent (for example, purified water, organic solvent, etc.), or may be dissolved in the same solvent as described above before use. It may be a dry powder reagent (for example, freeze-dried powder) or the like used in the above.

(Measurement method using biological component measurement reagent kit)

When measuring a biological component using the biological component measuring kit of the present invention, a general-purpose automatic analyzer (for example, Hitachi 7180 type automatic analyzer) can be used. The biological component measurement kit of the present invention may be configured to be applicable to such an automatic analyzer. The embodiment is not particularly limited, and for example, a kit composed of a liquid reagent, a kit composed of a combination of a drying reagent and a solution produced by means such as freeze-drying, an enzyme or the like is supported on an appropriate carrier. Various forms such as a kit called a so-called dry system in a form and a kit in a form using a sensor can be exemplified.

As the constitution of the biological component measurement kit of the present invention, a kit composed of one reagent or a kit composed of two or three or more reagents packaged is used. When the reagents are packaged into a kit composed of two or more reagents, for example, (a) an oxidase capable of generating hydrogen peroxide and (c) a hydrogen donor among the redox coloring reagents are used. The kit may be configured by packaging the reagent containing the reagent and a reagent containing 4-aminoantipyrine as a coupler among (b) peroxidase and (d) redox coloring dye.

Hereinafter, an example of a form of a kit composed of a liquid reagent in which a reagent is packaged in two (hereinafter, also referred to as a two-reagent system liquid reagent) will be described.

In the method of analyzing with an automatic analyzer using this form of reagent, the first type of reagent (hereinafter, also referred to as the first reagent or R1) is first added to the sample and reacted for a certain period of time, and then the second type of reagent is used. The target component can be quantified by further adding the reagent (hereinafter, also referred to as the second reagent or R2) and reacting, and measuring the change in absorbance during this period.

When the biological component measurement reagent of the present invention is supplied in two or more packages in consideration of application to an automatic analyzer as described above, the concentration of the copper-containing substance in each packaged reagent and the concentration of the copper-containing substance are determined. It is determined whether or not the concentration of the other component is within the range of the preferable concentration of each of the above components.

(Biological component measurement method)

The biological component measuring method targeted by the present invention includes the following steps (1) to (3).

(1) A step of causing an oxidase to act on a biological component to generate hydrogen peroxide.

(2) The hydrogen peroxide generated in the step (1) causes the reaction solution to be colored by oxidatively condensing 4-aminoantipyrine and the redox coloring reagent by the action of peroxidase in the coexistence of peroxidase. Process,

(3) A step of colorimetrically quantifying the reaction product colored in the step (2).

The method for measuring biological components of the present invention is a method for measuring biological components by an enzymatic method, particularly a method using an oxidase-peroxidase-color former system, that is, an amount of biological components by enzymatically reacting the biological components in a sample. The measurement principle is to generate hydrogen peroxide according to the above and react it with a color former in the presence of peroxidase to quantify the color development generated.

A method for measuring biological components using this principle has already been established in the art. Therefore, the knowledge can be applied to the present invention to measure the amount or concentration of biological components in various samples, and the embodiment is not particularly limited.

The present inventors have found that 4-hydroxyantipyrine contained in a trace amount in the drug substance of an aminoantipyrine compound (particularly 4-aminoantipyrine) inhibits the reaction of this oxidase-peroxidase-color former system and is sensitive. For the first time, I found that it caused a decline. The mechanism by which 4-hydroxyantipyrine inhibits the above reaction is not always clear, but due to its structure, the redox coloring reagent and 4-hydroxyantipyrine cause a condensation reaction in the presence of hydrogen peroxide due to the action of peroxidase, resulting in hydrogen peroxide. It is inferred that it will consume. Then, the present inventors have found that such reaction inhibition caused by 4-hydroxyantipyrine can be suppressed by reacting 4-hydroxyantipyrine with a copper-containing substance in advance, and a decrease in sensitivity can be suppressed. .. The mechanism by which the copper-containing substance suppresses the decrease in sensitivity due to 4-hydroxyantipyrine is not clear, but the copper-containing substance (for example, copper ions that may be generated from this) causes some structural change in 4-hydroxyantipyrine and reacts with peroxidase. It is presumed that it will become difficult and will not consume hydrogen peroxide.

(Sample)

Examples of the sample containing the biological component used for the biological component measurement of the present invention include biological body fluids such as blood (particularly serum and plasma), urine, ascites, and cerebrospinal fluid, and humans such as beverages and foods. Examples include those to be ingested. Of these, it is preferable to use human body fluids (samples derived from blood such as serum and plasma, samples derived from urine, etc.) as samples to be measured.

(Detection sensitivity of biological component measurement reagent kit)

By using a copper-containing substance, the present invention has a detection sensitivity due to 4-hydroxyantipyrine in the measurement of biological components by an enzymatic method using an oxidase-peroxidase-color former system as compared with the case where a copper-containing substance is not used. Can be suppressed.

Hereinafter, the detection sensitivity of the biological component measurement reagent kit will be described using creatinine as an example of the biological component.

In recent years, the measurement accuracy of creatinine up to the last two digits of the decimal point has been required for the calculation of eGFR (also referred to as estimated glomerular filtration rate), and the minimum detection sensitivity is required to be about 0.03 mg / dL in creatinine concentration. There is.

On the other hand, as the measurement accuracy of the automatic analyzer, the fluctuation of the blank of the creatinine reagent is about σ = 0.045 to 0.114 mABS, which is generally 2.6σ (99.5), which is the minimum detection sensitivity of the in vitro diagnostic agent. % Normal distribution) is about 0.117 to 0.296 mABS.

Therefore, if there is an absorbance of 0.296 mABS, which is the maximum value of 2.6σ, that is, about 0.3 mABS or more, it can be detected as a signal, and it is considered that the existence or nonexistence of creatinine and the quantification of creatinine can be determined.

According to the present invention, by using a copper-containing substance, it is possible to suppress a decrease in sensitivity of the biological component measurement reagent kit due to 4-hydroxyantipyrine and increase the detection sensitivity to the above level.

The present invention is not limited to the configurations described above, and various modifications can be made within the scope of the claims, and the technical means disclosed in different embodiments and examples. The embodiments and examples obtained by appropriately combining, modifying or replacing the above are also included in the technical scope of the present invention.

Hereinafter, the present invention will be specifically described with reference to Examples, but the present invention is not limited thereto.

(Example 1) Separation of 4HA by HPLC

Since it was found that the degree of decrease in reagent sensitivity is due to the lot difference of 4AA, it is estimated that the content of impurities contained in a very small amount in 4AA varies from lot to lot, and the impurities are determined by the above-mentioned 4HA quantification method (HPLC method). Was detected. The purity of 4AA used for examining the lot difference was 98.0% or more according to JIS-K8048.

The HPLC fraction when 4AA is analyzed is shown in FIG. In FIG. 1, the large peak observed in the elution time on the horizontal axis of the graph around 7 to 8 minutes is the peak of 4-aminoantipyrine. In this HPLC fraction, as shown in FIG. 1, three kinds of impurities other than 4-aminoantipyrine were confirmed.

(Example 2) Identification of 4HA

When the content of each lot was examined for the three types of impurities (a, b, c) seen in FIG. 1 of Example 1, it was found that the content of only the impurity b was significantly different for each lot. Therefore, the molecular weight and structure of these three types of substances were analyzed by mass spectrometry (MS spectral method).

As a result, the impurity b was identified as 4-hydroxyantipyrine (4HA). FIG. 2 shows the structural formula of 4HA.

When 4HA (Sigma) having a known concentration was used as a standard product and HPLC was carried out under the same conditions as in Example 1, the fraction peaks of 4HA and the impurity b were in agreement.

(Example 3) Dependence of 4HA contamination concentration on creatinine measurement sensitivity

Using creatinine as a biological component, the dependence on the amount of 4HA mixed in was evaluated with respect to the decrease in measurement sensitivity caused by 4HA mixed in the reagent.

4HA was added to the second reagent of the following creatinine measuring reagent so that the final concentration in the reagent was 0.13 to 8.75 μg / ml, and each measuring reagent was prepared. A 5 mg / dL creatinine aqueous solution was used as a biological component sample.

[Preparation of reagents]

Creatinine measuring reagents having the following compositions were prepared. Here, as 4-aminoantipyrine, a commercially available 4-aminoantipyrine drug substance was purified to produce 4-aminoantipyrine containing no 4-hydroxyantipyrine and used.

First reagent

PIPES-NaOH 50 mM pH 7.4

Ascorbic acid oxidase (ASO-311 manufactured by Toyobo) 3U / mL

Zarcosin oxidase (SAO-351 manufactured by Toyobo) 10U / mL

Creatine amidinohydrolase (CRH-229 manufactured by Toyobo) 40U / mL

Catalase (CAO-509 manufactured by Toyobo) 130U / mL

N-Ethyl-N- (3-sulfopropyl) -3-methoxyaniline 0.14 g / L

Second reagent

PIPES-NaOH 50 mM pH 7.4

Creatinine Amide Hydrolase (CNH-311 manufactured by Toyobo) 400U / mL

Peroxidase (PEO-302 manufactured by Toyobo) 10U / mL

4-Aminoantipyrine 0.6 g / L

[Measurement method]

A Hitachi 7180 automatic analyzer was used. 120 μL of the first reagent was added to 2.7 μL of the sample and incubated at 37 ° C. for 5 minutes to prepare the first reaction. Then, 40 μL of the second reagent was added and incubated for 5 minutes to prepare a second reaction. The absorbance at 546 nm (main wavelength) and the absorbance at 800 nm (secondary wavelength) were measured by a two-point end method in which the absorbances of the first reaction and the second reaction were corrected by the amount of liquid and the difference between the absorbances was taken. The absorbance obtained by subtracting the sub-wavelength from the main wavelength was calculated and obtained.

The concentration of 4HA during the reaction under these measurement conditions is 0.03 to 2.14 μg / ml.

The results are shown in Table 1 and FIG. It was confirmed that the sample measurement sensitivity decreased as the 4HA concentration in the second reagent increased.

(Example 4) Effect of suppressing decrease in sensitivity due to copper salt

Regarding the copper salt, the effect of suppressing the decrease in sensitivity due to 4HA was confirmed.

As for the experimental conditions, for the reagents other than the control (a), 4HA was added to the second reagent of the creatinine measuring reagent so that the final concentration in the reagent was 10 μg / ml, and a 5 mg / dL creatinine aqueous solution was used as the biological component sample. The procedure was carried out under the same conditions as in Example 3 except that the test was carried out.

After adding various copper salts to the final concentration in the second reagent of the predetermined creatinine measurement reagent, the second reagent was stored for 3 days under the temperature conditions of each refrigeration condition (6 ° C.) and acceleration test (35 ° C.). The measurement sensitivity (mABS) was examined using. The blank value of each reagent was also measured at the same time, and the value obtained by subtracting the blank value from the measurement sensitivity was calculated as the STD sensitivity. Then, the ratio [vs control (%)] of the measurement sensitivity (mABS) of each reagent to the measurement sensitivity (mABS) of the control (a) to which 4HA was not added was calculated.

The results are shown in Table 2.

From this result, it was confirmed that the coexistence of various copper salts can effectively suppress the decrease in sensitivity caused by 4HA.

That is, the measurement sensitivity of the control (b) to which 4HA was added so as to be 10 μg / ml was 32% at 3 days of refrigeration and 45% at 35 ° C. for 3 days, whereas the results using various copper salts showed. The measurement sensitivity was 75% to 100% in 3 days of refrigeration and 71% to 100% in 3 days at 35 ° C. From this result, it can be seen that, as an overall tendency, it is easier to obtain a stable and high sensitivity reduction suppressing effect when stored at 35 ° C. than when stored under refrigerated conditions. Since the evaluation at 35 ° C is equivalent to an accelerated test under refrigerated storage conditions, there is a tendency that the decrease in sensitivity due to 4-hydroxyantipyrine can be more effectively suppressed by reacting various copper salts for a longer period of time. It was presumed that there was.

In the example in which 0.1 mM of copper (II) chloride was added in Experiment No. 3, color development presumed to be dependent on copper (II) chloride was observed, and when a high concentration of copper salt (II) was used, it was measured. It was thought that the attention was required.

(Example 5) Effect of suppressing decrease in sensitivity due to a mixture containing copper protein / copper salt

Using ASO-311 (ascorbic acid osicidase) as a copper protein and Proclin 200 as a mixture containing a copper salt, the effect of suppressing the decrease in sensitivity due to 4HA was confirmed.

As for the experimental conditions, for the reagents other than the control (a), 4HA was added to the second reagent of the creatinine measuring reagent so that the final concentration in the reagent was 10 μg / ml, and a 5 mg / dL creatinine aqueous solution was used as the biological component sample. The procedure was carried out under the same conditions as in Example 3 except that the test was carried out.

ASO-311 and Procline 200 were added to the second reagent of the creatinine measurement reagent so as to have the final concentration in the predetermined second reagent, and then stored for 3 days under the temperature conditions of the accelerated test (35 ° C.), respectively. The measurement sensitivity (mABS) was examined using. The blank value of each reagent was also measured at the same time, and the value obtained by subtracting the blank value from the measurement sensitivity was calculated as the STD sensitivity. Then, the ratio [vs control (%)] of the measurement sensitivity (mABS) of each reagent to the measurement sensitivity (mABS) of the control (a) to which 4HA was not added was calculated.

The results are shown in Table 3.

From this result, it was confirmed that the decrease in sensitivity due to 4HA can be suppressed even when these components are used. Here, ASO-311 (ascorbic acid oxidase) is known to be a copper protein containing copper ions. Procrine 200 may contain a copper salt as a stabilizer in addition to the main components 5-chloro-2-methyl-4-isothiazolin-3-one and 2-methyl-4-isothiazolin-3-one. Are known. Therefore, it was confirmed that the effect of the present invention is exhibited even when such a mixture containing a copper protein or a copper salt is used.

That is, the measurement sensitivity of the control (b) to which 4HA was added so as to be 10 μg / ml was 41% at 35 ° C. for 3 days, whereas the measurement sensitivity using ASO-311 was 35 ° C. 3 It was 90% to 100% in days, and the measurement sensitivity was 96% to 98% in 3 days at 35 ° C. as a result of using Proclin 200.

(Example 6) Concentration dependence of copper salt / 4HA on the effect of suppressing sensitivity decrease

Regarding the effect of suppressing the decrease in sensitivity caused by 4HA using a copper salt, the concentration dependence of the copper salt and 4HA was confirmed.

The experimental conditions were 600 μg / ml, 300 μg / ml, and 100 μg / ml as the final concentration of 4AA in the second reagent of the creatinine measuring reagent for the reagents other than the control (a), and 4HA in the second reagent. The reagent was added so as to have a final concentration of 10 μg / ml, 5 μg / ml, and 1.6 μg / ml, and the same conditions as in Example 3 were used except that a 5 mg / dL creatinine aqueous solution was used as the biological component sample.

After adding various copper salts to the second reagent of the creatinine measuring reagent so as to have the final concentration in the predetermined second reagent, the second reagent was stored for 3 days under the temperature conditions of the accelerated test (35 ° C.). , The measurement sensitivity (mABS) was examined. The blank value of each reagent was also measured at the same time, and the value obtained by subtracting the blank value from the measurement sensitivity was calculated as the STD sensitivity. Then, the ratio [vs control (%)] of the measurement sensitivity (mABS) of each reagent to the measurement sensitivity (mABS) of the control (a) of each 4AA concentration to which 4HA was not added was calculated.

The results are shown in Tables 4-6.

From this result, the effect of suppressing the decrease in sensitivity due to 4HA due to the coexistence of the copper salt depends on the copper ion concentration and the 4HA concentration, but in a wide copper ion concentration range, the effect on the decrease in sensitivity due to 4HA It was confirmed that it can be effectively suppressed.

That is, both copper (I) and (II) chloride were found to have an effect of suppressing the decrease in sensitivity due to 4HA in the concentration range of 0.00001 mM to 0.01 mM. However, when the 4HA concentration was high, it was found that the effect of suppressing the decrease in sensitivity due to 4HA was slightly weak.

Copper (I) chloride and copper (II) chloride showed the same tendency in the effect of suppressing the decrease in sensitivity due to 4HA, and no significant difference was observed depending on the valence of copper ions.

It can be applied to a measuring method for measuring a biological component using a redox reaction, and a reagent or composition used in the method.

Claims (14)

An inhibitor for reducing the measurement sensitivity of a biological component, which is applied to a method for measuring a biological component using an aminoantipyrine compound as a coupler of a redox color-developing reagent, and is characterized by containing a copper-containing substance. ..

The biological component measuring method is a biological component measuring method using a reagent or a reagent set that satisfies the following requirements (a) to (d), and is used by being added to a reagent that meets the requirements of (d). The characteristic agent for suppressing a decrease in measurement sensitivity of a biological component according to claim 1.

(A) Contains an oxidase capable of generating hydrogen peroxide.

(B) Contains peroxidase.

(C) Contains a redox color-developing reagent that reacts with hydrogen peroxide to develop color in the presence of peroxidase.

(D) An aminoantipyrine compound is contained as a coupler of the redox color-developing reagent.

The agent for suppressing a decrease in the measurement sensitivity of a biological component according to claim 1 or 2, wherein the decrease in the measurement sensitivity of the biological component due to 4-hydroxyantipyrine is suppressed.

A method for suppressing a decrease in measurement sensitivity, which is applied to a method for measuring a biological component using an aminoantipyrine compound as a coupler of a redox color-developing reagent, which comprises using a copper-containing substance. ..

A method for suppressing a decrease in measurement sensitivity in a biological component measurement method using a reagent or a reagent set that satisfies the following requirements (a) to (d), and a copper-containing substance is added to the reagent that satisfies the requirement (d). A method for suppressing a decrease in measurement sensitivity of a biological component, which is characterized by the above.

(A) Contains an oxidase capable of generating hydrogen peroxide.

(B) Contains peroxidase.

(C) Contains a redox color-developing reagent that reacts with hydrogen peroxide to develop color in the presence of peroxidase.

(D) An aminoantipyrine compound is contained as a coupler of the redox color-developing reagent.

The method for suppressing a decrease in the measurement sensitivity of a biological component according to claim 4 or 5, wherein the decrease in the measurement sensitivity of the biological component due to 4-hydroxyantipyrine is suppressed.

The method for suppressing a decrease in measurement sensitivity of a biological component according to claim 5 or 6, wherein the concentration of the copper-containing substance coexisting in the reagent satisfying the requirement (d) is 0.00001 to 1 mM. ..

The method for suppressing a decrease in measurement sensitivity of a biological component according to any one of claims 4 to 7, wherein the biological component is either creatinine or glycated hemoglobin.

A biological component measurement reagent kit using an aminoantipyrine compound as a coupler of a redox color-developing reagent, which comprises the measurement sensitivity reduction inhibitor according to claim 1.

It is a biological component measurement reagent kit that meets the following requirements (a) to (e), and the reagents that meet the requirements (d) and (e) are characterized by being one reagent that simultaneously meets the two requirements. Reagent kit for measuring biological components.

(A) Contains an oxidase capable of generating hydrogen peroxide.

(B) Contains peroxidase.

(C) Contains a redox color-developing reagent that reacts with hydrogen peroxide to develop color in the presence of peroxidase.

(D) An aminoantipyrine compound is contained as a coupler of the redox color-developing reagent.

(E) Contains copper-containing substances.

The biological component measurement reagent kit according to claim 10, wherein one reagent that simultaneously satisfies the two requirements (d) and (e) has passed at least one month after production.

A method for measuring a biological component, which comprises using the biological component measuring kit according to any one of claims 9 to 11.

A method for producing a biological component measurement reagent kit in which a decrease in sensitivity due to 4-hydroxyantipyrine is suppressed, which satisfies the following requirements (a) to (e), and satisfies the requirements (d) and (e). A method for producing a reagent kit for measuring biological components, which comprises producing a reagent as one reagent that simultaneously satisfies two requirements.

(A) Contains an oxidase capable of generating hydrogen peroxide.

(B) Contains peroxidase.

(C) Contains a redox color-developing reagent that reacts with hydrogen peroxide to develop color in the presence of peroxidase.

(D) An aminoantipyrine compound is contained as a coupler of the redox color-developing reagent.

(E) Contains copper-containing substances.

13. The reagent according to claim 13, wherein one reagent that simultaneously satisfies the two requirements for the reagents that satisfy the requirements (d) and (e) is an intermediate reagent in the manufacturing process of the biological component measurement reagent kit. How to manufacture a biological component measurement reagent kit.

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