JP7276327B2 - METHOD FOR SUPPRESSING SENSITIVITY REDUCTION OF BIOLOGICAL COMPONENT MEASUREMENT REAGENT KIT - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for suppressing sensitivity deterioration of a biological component measurement reagent kit used in clinical diagnosis. More specifically, hydrogen peroxide generated from a biological component using an oxidation-reduction reaction and an oxidation-reduction coloring reagent (such as Trinder's reagent, which is a combination of an aminoantipyrine-based compound and a hydrogen donor as a coupler), The present invention relates to a method for suppressing a decrease in sensitivity in a reagent kit for measuring biological components, which is used for colorimetric measurement of color produced by oxidative condensation in the presence of peroxidase, and the like.

Conventionally, in clinical diagnosis, measurement of biological components by an enzymatic method has been performed, and in particular, a method using an oxidase-peroxidase-redox coloring reagent (hereinafter also referred to as a coloring agent) system, that is, a measurement target in a specimen. A widely used method is to cause an enzymatic reaction of a substance to generate hydrogen peroxide, which is then reacted with a coloring agent in the presence of peroxidase to carry out colorimetric determination (Non-Patent Document 1).

Examples of the redox coloring 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 have prepared an enzyme-peroxidase-chromogenic agent-based biological component measurement reagent using an aminoantipyrine compound and a hydrogen donor, and when using it for biological component measurement, the measurement sensitivity has decreased for unknown reasons. Experienced.

Since the degree of decrease in measurement sensitivity varied depending on the lot of the reagent composition prepared for measurement, the present inventors conducted various studies on the reagent composition of the measurement kit. As a result, it was unexpectedly found that 4-aminoantipyrine contains a very small amount of a substance called 4-hydroxyantipyrine (hereinafter also referred to as 4HA). Without wishing to be bound by theory, when 4-hydroxyantipyrine is present in the reagent, structurally 4-hydroxyantipyrine does not undergo coupling reactions with hydrogen donors, but in the presence of hydrogen peroxide, 4-hydroxyantipyrine It is thought that peroxidase reacts with and consumes hydrogen peroxide, and as a result, hydrogen peroxide generated by reacting the enzyme with the substance to be measured in the specimen is consumed by 4-hydroxyantipyrine, It was thought that the amount of color developed by the reaction of the original 4-aminoantipyrine-hydrogen donor was reduced and the sensitivity was lowered.

Accordingly, an object of the present invention is to provide a means for suppressing the decrease in sensitivity caused by 4-hydroxyantipyrine, in order to solve the hitherto unknown problems described above.

As a result of intensive studies to achieve the above object, the present inventors have unexpectedly discovered that an iron-containing substance such as a component that generates iron ions can be used in the preparation of a biological component measurement reagent kit. , 4-hydroxyantipyrine, can be suppressed, and have completed the present invention. That is, the present invention consists of the following configurations.

[Item 1] Measurement of biocomponents, which is applied to a biocomponent measurement method using an aminoantipyrine-based compound as a coupler of a redox coloring reagent, and which is characterized by containing an iron-containing substance. Sensitivity reduction inhibitor.

[Claim 2] The biocomponent measurement method uses a reagent or reagent set that satisfies the following requirements (a) to (d), and is added to the reagent that meets the requirements (d): Item 1. The agent for suppressing decrease in measurement sensitivity of biological components according to Item 1, which is used for the following purposes.

(a) contains an oxidase capable of generating hydrogen peroxide;

(b) contains peroxidase;

(c) contains a redox coloring reagent that reacts with hydrogen peroxide in the presence of peroxidase to develop a color;

(d) contains an aminoantipyrine-based compound as a coupler of the redox coloring reagent.

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

[Item 4] A method for suppressing a decrease in measurement sensitivity applied to a biological component measurement method using an aminoantipyrine compound as a coupler of a redox coloring reagent, wherein the biological component measurement sensitivity is characterized by using an iron-containing substance. Degradation suppression method.

[Claim 5] A method for suppressing a decrease in measurement sensitivity in a biological component measurement method using a reagent or reagent set that satisfies the following requirements (a) to (d), wherein iron in the reagent that satisfies the requirement (d) A method for suppressing a decrease in measurement sensitivity of a biological component, comprising adding a contained substance.

(a) contains an oxidase capable of generating hydrogen peroxide;

(b) contains peroxidase;

(c) contains a redox coloring reagent that reacts with hydrogen peroxide in the presence of peroxidase to develop a color;

(d) contains an aminoantipyrine-based compound as a coupler of the redox coloring reagent.

[Item 6] A method for suppressing a decrease in sensitivity for measuring a biological component according to Item 4 or 5, characterized by suppressing a decrease in sensitivity for measuring a biological component caused by 4-hydroxyantipyrine.

[Item 7] Decrease in measurement sensitivity of a biological component according to item 5 or 6, characterized in that the concentration of the iron-containing substance coexisting in the reagent that satisfies the requirement (d) is 0.001 to 1 mM. suppression method.

[Item 8] The method for suppressing 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 the oxidation-reduction coloring reagent, comprising the agent for suppressing decrease in measurement sensitivity according to Item 1.

[Claim 10] A biological component measurement reagent kit that satisfies the following requirements (a) to (e), and the reagent that satisfies the requirements (d) and (e) is a single reagent that satisfies the two requirements at the same time. A biological component measurement reagent kit characterized by:

(a) contains an oxidase capable of generating hydrogen peroxide;

(b) contains peroxidase;

(c) contains a redox coloring reagent that reacts with hydrogen peroxide in the presence of peroxidase to develop a color;

(d) contains an aminoantipyrine-based compound as a coupler of the redox coloring reagent.

(e) contains iron-containing substances;

[Item 11] The reagent for measuring biological components according to item 10, wherein at least one month has passed since the manufacturing of the one reagent that simultaneously satisfies the two requirements (d) and (e). kit.

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

[Claim 13] A method for manufacturing a biological component measurement reagent kit that satisfies the following requirements (a) to (e) and suppresses a decrease in sensitivity caused by 4-hydroxyantipyrine, comprising (d) and (e) A method for manufacturing a reagent kit for measuring biological components, characterized in that the reagents satisfying the requirements of (1) are manufactured 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 coloring reagent that reacts with hydrogen peroxide in the presence of peroxidase to develop a color;

(d) contains an aminoantipyrine-based compound as a coupler of the redox coloring reagent.

(e) contains iron-containing substances;

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

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to suppress the decrease in sensitivity caused by 4-hydroxyantipyrine in biocomponent measurement by an enzymatic method using an oxidase-peroxidase-coloring agent system. Therefore, the present invention enables good measurement of biological components. In particular, stable measurement is possible in the measurement of biological components that require high sensitivity, such as when the content of biological components is extremely small.

FIG. 4 shows HPLC fractions from analysis of 4-aminoantipyrine drug substance. Figure 1 shows the structural formula of 4-hydroxyantipyrine; FIG. 4 is a diagram showing the relationship between the concentration of 4-hydroxyantipyrine in the second reagent and sample measurement sensitivity.

Detailed descriptions of the embodiments of the present invention are as follows, but the present invention is not limited thereto. It should be understood that the terms used in this specification have the meanings commonly used in the relevant field unless otherwise specified.

Also, all non-patent documents and patent documents mentioned in this specification are incorporated herein by reference. In the present specification, "~" means "more than or equal to or less than". For example, "X to Y" in the specification means "X or more and Y or less". Also, "and/or" in this specification means either or both. Also, in this specification, it should be understood that the singular terms also include the plural terms unless stated otherwise.

(Suppressant for decrease in measurement sensitivity of biocomponents)

The biological component 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 coloring reagent, and is an iron-containing substance (e.g., iron component that generates ions, etc.).

The biological component measurement method to which the agent for suppressing decrease in sensitivity of biological component measurement of the present invention is applied is preferably a biological component measurement method using a reagent or reagent set that satisfies the following requirements (a) to (d).

(a) contains an oxidase capable of generating hydrogen peroxide;

(b) contains peroxidase;

(c) contains a redox coloring reagent that reacts with hydrogen peroxide in the presence of peroxidase to develop a color;

(d) contains an aminoantipyrine-based compound as a coupler of the redox coloring reagent.

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

An aminoantipyrine compound contained in a reagent that satisfies the above 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 reduction in the measurement sensitivity of biocomponents. Furthermore, the present inventors have completed the present invention by discovering that such a decrease in sensitivity can be suppressed by allowing an iron-containing substance to coexist with 4-hydroxyantipyrine.

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

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

(Method for Suppressing Decrease in Measurement Sensitivity of Biological Component)

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 that is applied to a method for measuring a biological component using an aminoantipyrine compound as a coupler of a redox coloring reagent, It is characterized by using an iron-containing substance.

In a further embodiment, the method for suppressing decrease in measurement sensitivity of a biological component of the present invention is a method for suppressing decrease in measurement sensitivity in a method for measuring a biological component using a reagent or reagent set that satisfies the following requirements (a) to (d): and is characterized by adding an iron-containing substance to the reagent that satisfies the requirement (d).

(a) contains an oxidase capable of generating hydrogen peroxide;

(b) contains peroxidase;

(c) contains a redox coloring reagent that reacts with hydrogen peroxide in the presence of peroxidase to develop a color;

(d) contains an aminoantipyrine-based compound as a coupler of the redox coloring reagent.

The present inventors presume that the decrease in measurement sensitivity is caused by the consumption of hydrogen peroxide due to the reaction of 4-hydroxyantipyrine with peroxidase. The present inventors have found that the coexistence of an iron-containing substance (preferably, iron ions that can be generated from the iron-containing substance) can suppress the resistance to heat, thereby completing the present invention.

That is, the method for suppressing low measurement sensitivity of a biological component of the present invention is achieved by adding an iron-containing substance (e.g., a component that generates iron ions, etc.) to a reagent that satisfies the requirements of (d). 4-Hydroxyantipyrine and an iron-containing substance mixed in a reagent that satisfies the requirements of (1) are allowed to coexist and react with each other, thereby suppressing the reactivity of 4-hydroxyantipyrine, thereby reducing the reactivity of 4-hydroxyantipyrine to biological components caused by 4-hydroxyantipyrine. It is characterized by suppressing a decrease in the measurement sensitivity of.

In a reagent containing an aminoantipyrine compound contaminated with 4-hydroxyantipyrine, the reaction time of the iron-containing substance (in a specific embodiment, iron ions that can be generated from this) is not particularly limited as long as the effects of the present invention are exhibited. . For example, from the time the iron-containing substance is added to the reagent that satisfies the requirement (d), the iron-containing substance is allowed to coexist in the reagent that satisfies the requirement (d), and at least at 1 ° C. to 10 ° C. 2 weeks, 11°C to 25°C for at least 1 week, 26°C to 40°C for at least 2 days, 41°C to 60°C for at least 5 hours, 61°C to 80°C for at least 1 hour are sufficient effects. can be obtained. Therefore, even if the temperature is not strictly controlled, the effects of the present invention can be obtained by storing the iron-containing substance for about one month or more to react. In the present invention, it is preferable to react 4-hydroxyantipyrine and an iron-containing substance in the coexistence for the period described above, and then use a reagent that satisfies the above requirement (d). It should be noted that the above reactions may be carried out during inventory storage and distribution stages after manufacture.

Regarding 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 quality deterioration such as alteration or denaturation of various components in the reagent does not occur.

As a specific embodiment of the present invention, for example, when the reagent satisfying the requirement (d) is a product reagent (e.g., a reagent containing a protein such as an enzyme), an iron-containing substance is added to the product reagent After that, the reaction progresses by storing, distributing, and storing before use in a refrigerator or near room temperature in the presence of an iron-containing substance. The biological component measurement reagent kit manufactured by adding an iron-containing substance to the kit may be used after at least two weeks have passed from the date of manufacture.

In another specific embodiment of the present invention, the reagent that satisfies the requirement (d) is an intermediate reagent (for example, an aminoantipyrine compound with a concentration several times to several tens of times higher than that of the product reagent as a buffer component). In the case of the adjusted reagent), after adding the iron-containing substance to the intermediate reagent, for example, it is stored for several days at a temperature of 30 ° C. or higher and for several hours at a temperature of 45 ° C. or higher, and diluted to a predetermined concentration after storage. to prepare the product reagent.

When adding an iron-containing substance to the intermediate reagent, the iron-containing substance coexisting in the reagent that satisfies the requirements of (d) (in a specific embodiment, iron ions that can be generated from this) is added at the stage of preparing the product reagent. It may be removed and used as a product reagent.

The present inventors have found that such decrease in measurement sensitivity caused by 4-hydroxyantipyrine can be suppressed by allowing an iron-containing substance to coexist with 4-hydroxyantipyrine in advance. Although the mechanism by which an iron-containing substance suppresses the decrease in sensitivity caused by 4-hydroxyantipyrine is not clear, the present inventors have found that 4-hydroxyantipyrine undergoes some kind of structural change due to an iron-containing substance (for example, iron ions that can be generated from it). 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 the measurement sensitivity of biological components caused by 4-hydroxyantipyrine suppressed by an iron-containing substance is caused by the coexisting reagent We speculate that removing the iron-containing material (and in certain embodiments, the iron ions that may be generated therefrom) does not restore the reaction between 4-hydroxyantipyrine and peroxidase.

In the method for suppressing reduction in measurement sensitivity of biological components of the present invention, the concentration of the iron-containing substance coexisting in the reagent satisfying the requirement (d) is preferably 0.001 to 1 mM, and is preferably 0.005 to 1 mM. It is more preferable if it is present, and more preferable if it is 0.01 to 0.5 mM.

When the concentration of the iron-containing substance is less than 0.001 mM, the inhibitory effect is small, and when the concentration of the iron-containing substance is more than 1 mM, the measurement sensitivity is lowered due to the iron-containing substance in the biological component measurement reaction.

The preferred concentration range of the iron-containing substance coexisting in the reagent that satisfies the requirements of (d) depends on the concentration of 4-hydroxyantipyrine mixed in the reagent that satisfies the requirements of (d). - If the concentration of hydroxyantipyrine is low, it is possible to suppress the deterioration of the measurement sensitivity of biological components even if the concentration of the iron-containing substance is low, and if the concentration of 4-hydroxyantipyrine is high, it is effective to use a higher concentration of iron-containing substance. For this reason, in the method for suppressing a decrease in biological component measurement sensitivity of the present invention, it is preferable to appropriately set the concentration of the iron-containing substance according to the concentration of 4-hydroxyantipyrine mixed therein.

ICP emission spectrometry (ICP-AES), ICP mass spectrometry (ICP-MS), and the like can be suitably used as methods for measuring the concentration of iron ions that can be generated by iron-containing substances. Specifically, in the present invention, the concentration of iron ions can be measured by ICP-AES (using SPECTROBLUE manufactured by Ametech). If the ICP-AES is below the detection limit, it can be measured by ICP-MS (using Agilent 7700sICP-MS from Agilent Technologies).

In the method for suppressing decrease in measurement sensitivity of biological components of the present invention, the concentration of 4-hydroxyantipyrine present in the reagent that satisfies the above requirement (d) is such that iron-containing substances (for example, components that generate iron ions, etc.) are added. The effect is particularly likely to be obtained when the concentration before treatment is about 0.1 to 50 μg/ml.

When the concentration of 4-hydroxyantipyrine present in the reagent that satisfies the requirements of (d) before adding the iron-containing substance is 0.1 μg/ml or less, 4-hydroxyantipyrine reduces the measurement sensitivity of biological components. is less than 1%, and there is little need to suppress the decrease in measurement sensitivity by coexisting an iron-containing substance.

If the concentration of 4-hydroxyantipyrine present in the reagent that satisfies the requirements of (d) above before adding the iron-containing substance is greater than 50 μg/ml, the decrease in the measurement sensitivity of biological components due to 4-hydroxyantipyrine is suppressed. In order to do so, a high-concentration iron-containing substance is required, and as a result, side reactions caused by the iron-containing substance tend to increase color development and easily cause measurement errors.

A study by the present inventors revealed that a general aminoantipyrine compound (4-aminoantipyrine) drug substance contains about 0.005 to 0.30 w/w% of 4-hydroxyantipyrine. are doing. Therefore, the present invention provides, for example, a reagent containing about 0.01 to 100 g/l of an aminoantipyrine compound (4-aminoantipyrine), preferably about 0.01 to 10 g/l of an aminoantipyrine compound (4-aminoantipyrine). antipyrine), more preferably about 0.01 to 5 g/l of an aminoantipyrine compound (4-aminoantipyrine).

In addition, when the amount of 4-hydroxyantipyrine mixed in is large, it is preferable to remove 4-hydroxyantipyrine from the aminoantipyrine-based compound in advance by the method for removing 4-hydroxyantipyrine described later, etc., before subjecting it to measurement of biological components. .

(biological component)

The biological component to be measured by the biological component measurement reagent kit of the present invention is not particularly limited, and can be used to measure various biological components. For example, the biocomponents used in the biocomponent measurement of the present invention include uric acid (UA), creatinine (CRE), triglyceride (TG), cholesterol (CHO), AST (GOT), ALT (GPT), LDH (lactate dehydrogenation enzyme) and isozyme, ALP (alkaline phosphatase) and isozyme, CK (creatine kinase) and isozyme, amylase (Amy) and isozyme, lipase, γ-GTP (γ-glutamyltranspeptidase), cholinesterase (ChE), sodium (Na) , potassium (K), chloride (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), bilirubin (Bil), ammonia, cholesterol, HDL cholesterol (HDL-C, high density lipoprotein cholesterol), LDL cholesterol (LDL-C, low density Lipoprotein Cholesterol), Triglycerides (TG), Cholesterol (CHO), BTR (BTR, Total Branched Chain Amino Acids/Tyrosine Ratio), Tyrosine Measuring Reagent (TYR), Blood Glucose (BS, GLU), 1,5 - anhydro-D-glucitol (1,5-AG), glycated albumin (GA), glycated hemoglobin (HbA1c), etc., but not limited thereto.

Any oxidase and, if necessary, other enzymes (for example, hydrolase) can act on these biological components to generate hydrogen peroxide. For example, uric acid (UA), creatinine (CRE), triglyceride (TG), and glycosylated hemoglobin (HbA1c) will be described below in specific aspects of biocomponent measurement.

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

When measuring creatinine (CRE), hydrogen peroxide is not directly produced in the reaction of creatinine amidinohydrolase with creatinine (CRE) as a substrate, so creatine produced by the reaction of creatinine amidinohydrolase was previously added to the reagent. By designing a so-called coupled reaction in which sarcosine is produced by reacting with an amidohydrolase and then hydrogen peroxide is produced using sarcosine oxidase (oxidase) to which sarcosine is added in advance to the reagent, a peroxidase-chromogenic agent system allows quantification of creatinine (CRE) concentration by

When measuring triglyceride (TG), lipoprotein lipase using triglyceride (TG) as a substrate and glycerol kinase and glycerol 3-phosphate oxidase (oxidase) as coupled enzymes are used to generate hydrogen peroxide. , allowing the quantification of triglyceride (TG) concentrations by a peroxidase-chromogen system.

When measuring glycated hemoglobin (HbA1c), 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-coloring agent system.

In this way, even if there is no suitable enzyme that catalyzes the reaction that directly oxidizes the measurement target to generate hydrogen peroxide, a reaction that can change the measurement target into a substrate of an oxidase that can generate hydrogen oxide can be performed. It is also possible to measure the concentration or amount of biological components other than the above by appropriately designing a coupled reaction that combines the catalyzing enzyme (which may be linked to several steps of enzymatic reactions) and the oxidase. 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 biocomponents listed above, creatinine (CRE) and glycosylated hemoglobin (HbA1c) are extremely low in biocomponent content, and therefore highly sensitive measurement is required. According to the present invention, the influence of 4-hydroxyantipyrine, which inhibits the reaction of the oxidase-peroxidase-chromogenic agent system, can be suppressed, and the decrease in sensitivity of biocomponent measurement can be suppressed. Therefore, such highly sensitive measurement is desired. It is useful for measuring biological components. Therefore, the present invention is suitable for measuring creatinine and glycosylated hemoglobin, and is particularly suitable for measuring creatinine.

(Biological component measurement kit)

In one embodiment, the biological component measurement kit of the present invention contains an agent for suppressing decrease in measurement sensitivity containing an iron-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 the two requirements for reagents that satisfy the following requirements (d) and (e): It is characterized by becoming one reagent. (a) contains an oxidase capable of generating hydrogen peroxide; (b) contains peroxidase; (c) contains a redox coloring reagent that reacts with hydrogen peroxide in the presence of peroxidase to develop a color; (d) contains an aminoantipyrine-based compound as a coupler of the redox coloring reagent; (e) contains iron-containing materials;

Regarding the reagents satisfying the requirements (d) and (e) above, one reagent that simultaneously satisfies the two requirements is, as described above, an iron-containing substance (e.g., iron ion generated in the reagent that satisfies the requirements of (d) components, etc.).

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

The present inventors speculate that 4-hydroxyantipyrine reacts with peroxidase, resulting in the consumption of hydrogen peroxide. In the present invention, it was found that the addition can be suppressed.

The reaction speed of this inhibitory reaction may be affected by the reaction temperature and reaction time, but under normal storage conditions for the biological component measurement kit, the inhibitory effect occurs after a storage period of several weeks to one month or longer. It is preferable that at least one month has passed since the manufacturing of the biological component measurement kit.

In general, it is considered that it takes one to several months after manufacturing such a measurement kit to go through the process of inventory and distribution until it is used, and the inhibitory reaction progresses during this time, and the effects of the present invention are exhibited. It will happen.

(Biological component measurement method)

The biological component measuring method of the present invention is characterized by measuring the biological component using the biological component measuring kit described above.

(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 caused by 4-hydroxyantipyrine, , (d) and (e) are produced as a single 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 coloring reagent that reacts with hydrogen peroxide in the presence of peroxidase to develop a color;

(d) contains an aminoantipyrine-based compound as a coupler of the redox coloring reagent.

(e) contains iron-containing substances;

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

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

As described above, the biological component measurement kit of the present invention requires the addition of an iron-containing substance to 4-hydroxyantipyrine to suppress the reactivity of 4-hydroxyantipyrine. ) may be carried out by allowing an iron-containing substance to coexist with 4-hydroxyantipyrine mixed in a reagent that satisfies the requirements of ), but this inhibitory reaction does not necessarily have to be carried out in the product reagent. In the process of preparing the containing reagent, the iron-containing substance may be added in the form of an intermediate reagent or the like, and after the inhibitory reaction is performed, a product reagent may be produced using the intermediate reagent and provided as a biological component measurement kit.

The reagent intermediate containing the aminoantipyrine compound described above is, for example, the process of manufacturing the reagent containing the aminoantipyrine compound, by diluting the active ingredient of the aminoantipyrine compound with a buffer solution, etc., so that the concentration of the aminoantipyrine compound is means an intermediate reagent or the like adjusted to a concentration several times to several tens of times that of the product reagent.

In the biological component measurement kit of the present invention, when an inhibitory reaction is performed by adding a component that generates an iron-containing substance to an intermediate reagent containing an aminoantipyrine compound, the product is obtained after adding the iron-containing substance to the intermediate reagent. It is preferable that the sum of the period until the date of manufacture and the period from the date of manufacture of the product to the date of use of the product is at least one month.

The iron-containing substance of the present invention (for example, a component that generates iron ions, etc.) is allowed to coexist with 4-hydroxyantipyrine that is mixed as an impurity in aminoantipyrine compounds, thereby improving the measurement sensitivity of biological components caused by 4-hydroxyantipyrine. It is presumed that it suppresses the decline.

Although it is not clear why the coexistence of the iron-containing substance of the present invention with 4-hydroxyantipyrine suppresses the decrease in the sensitivity of biocomponent measurement, the iron-containing substance (for example, iron ions that can be generated from this) is 4-hydroxyantipyrine. - by acting directly or indirectly on hydroxyantipyrine, causing some change in 4-hydroxyantipyrine, resulting in a decrease in the 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 4-hydroxyantipyrine once denatured and detoxified does not cause a decrease in sensitivity under normal storage conditions for biological component measurement kits. presumed not.

For this reason, when an iron-containing substance is added to the intermediate reagent, the product reagent is manufactured by removing the iron-containing substance (in a specific embodiment, iron ions that can be generated from this) in the subsequent step of manufacturing the product reagent. However, a biological component measurement reagent kit manufactured using such product reagents and a manufacturing method thereof are also within the scope of the present invention.

The iron-containing substance used in the present invention is preferably a component that generates iron ions, and is not particularly limited. Examples thereof include iron-containing compounds containing iron ions, iron proteins, iron oxide pigments, and the like. In the present specification, iron ion refers to an ion containing iron in its composition, and is a concept including, for example, ferricyanide ion and/or ferrocyanide ion. For example, FeBr ₂ , FeBr ₃ , Fe ₃ C, FeCl ₂ , FeCl ₃ , FeF ₂ , FeF 3 , FeH ₂ , FeH ₃ , FeI ₂ , FeI _{3 ,} FeN, Fe ₃ N, _{Fe3N2 , Fe} ( _{N3 ) 2} , FeO, _{Fe2O3 ,} Fe3O4 _, FeS, _FeS2 , _{Fe2S3 , Fe3S4} , FeSe, _{Fe2Se3 , FeSi2} , Fe ( _C5H5 ) ₂ , Fe _{( ClO3} ) ₃ , Fe(ClO4) ₂ , Fe( _{ClO4 ) 3} , Fe(CN) ₂ , Fe(CN) ₃ , FeCO3 _, Fe(CO) ₅ , _FeC2O4 , Fe2 _{( CO3} ) _{3 , Fe2} (CO) ₉ , Fe2( _{C2O4 ) 3} , _Fe3 (CO) ₁₂ , _Fe2 ( _CrO4 ) _{3 , Fe2} ( _{Cr2 O7} ) ₃ , _Fe5 ( _IO6 ) ₂ , _FeMnO4 , FeMoO4, Fe( _NO3 ) ₂ , Fe( _NO3 ) ₃ , Fe(OH) ₂ , Fe(OH) _{3 ,} FeO(OH), _FePO4 , _Fe3 ( _PO4 ) ₂ , FeSeO4, _FeSO3 , _FeSO4 , _Fe2 ( _SO4 ) ₃ , _H2FeO4 , _{BaFeO4 , K2FeO4 , Fe} ( _IO3 ) ₂ , Fe( _IO3 ) ₃ , _FeWO4 , [Fe( _C5H5 ) ₂ ] _BF4 , Fe(CH3COO) ₂ , Fe( _CH3COO )3, Fe( _HCOO ) _{2 ,} Fe( _OCN ) ₂ , Fe iron-containing compounds such as (SCN) ₂ , Fe(SCN) ₃ , H ₃ [Fe(CN) ₆ ], H ₄ [Fe(CN) ₆ ], (NH ₄ ) ₂ Fe(SO ₄ ) ₂ ; Iron proteins such _{as hemoglobin} , myoglobin, cytochrome ( _cytochrome ), hemopexin _, transferrin, hemosiderin, and ferritin; FeOFe ₂ O ₃ )), yellow brown pigments (ZnOFe ₂ O ₃ , MgOFe ₂ O ₃ ), transparent iron oxides (α-Fe ₂ O ₃ , α-FeOOH) and other iron oxide pigments. , but not limited to.

These components that generate iron ions can be appropriately selected according to, for example, the biological component or biological sample to be measured. FeCl ₂ , FeCl ₃ iron chloride (I) (FeCl ₂ ), iron chloride (II) (FeCl ₃ ), ferrocyanine It is preferable to use a compound (H ₃ [Fe(CN) ₆ ]), a ferricyanide (H ₄ [Fe(CN) ₆ ]), a peroxidase, or the like.

The amount of the iron-containing substance used in the present invention is not particularly limited as long as the effect of the present invention is exhibited.

As described above, in the present invention, the iron-containing substance is preferably used as an intermediate reagent for an aminoantipyrine compound or a product reagent blended with an aminoantipyrine compound. The concentration of the contained substance may be adjusted to 0.001 to 1 mM, preferably the iron-containing substance concentration in the reagent intermediate or reagent is 0.005 to 1 mM, more preferably 0.01 to 1 mM. It may be used by adjusting the blending amount so as to be 0.5 mM. By using the iron-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 iron-containing substance used in the present invention exhibits the effect of suppressing the decrease in sensitivity of biological component measurement caused by 4-hydroxyantipyrine contained as an impurity of the aminoantipyrine compound after being blended with the aminoantipyrine compound. is preferably allowed to pass for a certain period of time after blending.

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

Even if the elapsed time exceeds the upper limit, the iron-containing substance used in the present invention suppresses the decrease in sensitivity of biocomponent measurement caused by 4-hydroxyantipyrine as long as the quality of the reagent used in the biocomponent measurement kit does not deteriorate. Effect is maintained.

In the above, in the product life cycle of a general biological component measurement kit, it takes about several weeks to a month or more from reagent formulation in the production process to quality testing, shipment, distribution, storage, and use. When the measurement kit containing the reagent in which the iron-containing substance of the present invention is coexistent with the aminoantipyrine compound is used, the effect of suppressing the decrease in sensitivity due to the iron-containing substance used in the present invention is expressed and maintained. However, when the product life cycle is shortened, it is possible to prepare by controlling the manufacturing process and product control at the time of shipment.

(oxidase)

The oxidase used in the present invention can be used without limitation according to the target measurement target, as long as it can generate hydrogen peroxide from a substrate. Specific examples include, but are not limited to, uricase, sarcosine oxidase, glycerol-3-phosphate 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. There are no particular restrictions on the amount used or the form of addition.

(peroxidase)

As the peroxidase used in the present invention, any kind of enzyme may be used as long as it catalyzes the reaction between hydrogen peroxide and a redox coloring reagent. is mentioned. Among these, horseradish, rice, and soybean-derived peroxidases are preferred, and horseradish-derived peroxidases are more preferred, in terms 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. There are no particular restrictions on the amount used or the form of addition.

Peroxidase activity is defined in the following manner.

14 mL of distilled water, 2 mL of 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) were mixed in order, and then preliminarily adjusted at 20 ° C. for 5 minutes. and add 1 mL of the sample solution to initiate the enzymatic reaction.

After reacting for 20 seconds, 1 mL of 2N aqueous sulfuric acid solution is added to stop the reaction, and the produced purpurogallin is extracted 5 times with 15 mL of ether.

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

On the other hand, in the blind test, 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, then 1 mL of 2N sulfuric acid aqueous solution was added and mixed. , then add 1 mL of sample solution to prepare.

This liquid is subjected to ether extraction in the same manner as above, and the absorbance is measured (ΔOD _blank ).

The amount of purpurogallin produced is calculated from the difference in absorbance between ΔOD _test and 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 ) × dilution ratio} / {0.117 × 1 (mL)) = ΔOD × 8.547 × dilution ratio

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

0.117: absorbance at 420 nm of 1 mg% purpurogallin ether solution

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

(One propurgarine unit corresponds to 13.5 international units (using o-dianisidine as a substrate under reaction conditions of 25°C).)

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

(Redox coloring reagent)

Any kind of dye may be used as the redox color-developing reagent used in the biological component measurement of the present invention as long as it reacts with hydrogen peroxide to develop a color. For example, a combination of a hydrogen donor and a coupler can be used. mentioned. There are no particular restrictions on the amount used or the form of addition. All of these can be obtained as commercially available products.

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

(hydrogen donor)

In the biological component measuring method of the present invention, phenol, phenol derivatives, aniline derivatives, naphthols, naphthol derivatives, naphthylamine, naphthylamine derivatives and the like are used as hydrogen donors 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 phenyl)-N'-succinylethylenediamine, N-ethyl-N-(3-methylphenyl)-N'-acetylethylenediamine and the like.

(Coupler)

These hydrogen donors can be used in combination with couplers.

Examples of couplers include aminoantipyrine compounds such as 4-aminoantipyrine (4AA) and aminoantipyrine derivatives; vanillindiaminesulfonic acid compounds such as vanillindiaminesulfonic acid; methylbenzthiazolinone hydrazone (MBTH) and sulfonated methylbenzthia. Examples include methylbenzthiazolinone hydrazone compounds such as zolinone hydrazone (SMBTH).

INDUSTRIAL APPLICABILITY The present invention can suppress the decrease in sensitivity of a biological component measurement reagent kit caused by a very small amount of 4-hydroxyantipyrine contained in an aminoantipyrine compound, so it is effective when an aminoantipyrine compound is used as a coupler. be. In particular, the present invention is beneficial when 4-aminoantipyrine is used as a coupler.

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

The amount of the aminoantipyrine compound used in the present invention is not particularly limited as long as the effect of the present invention is exhibited. Aminoantipyrine compounds may contain a very small amount of 4-hydroxyantipyrine, which causes a decrease in sensitivity, and it is desirable to reduce the amount of 4-hydroxyantipyrine. However, according to the present invention, iron-containing substances coexistence of 4-hydroxyantipyrine, even if it contains a little 4-hydroxyantipyrine, it is possible to suppress the decrease in sensitivity. From such a point of view, as an example, the concentration of 4-hydroxyantipyrine mixed in the reagent containing the aminoantipyrine compound before coexistence with the iron-containing substance is preferably 0.1 to 50 μg / ml, more preferably It is effective to adjust the amount of the aminoantipyrine compound to be used for biological component measurement so that the concentration is 0.3 to 20 μg/ml, particularly preferably 1 to 10 μg/ml.

When the concentration of 4-hydroxyantipyrine is below the above range, the influence of 4-hydroxyantipyrine on lowering the measurement sensitivity of biological components is small, and there is little need to suppress the reduction in measurement sensitivity due to coexistence of an iron-containing substance.

When the concentration of 4-hydroxyantipyrine exceeds the above range, a high concentration of an iron-containing substance is required in order to suppress the deterioration of the measurement sensitivity of biological components due to 4-hydroxyantipyrine, and therefore the iron-containing substance inhibits the measurement sensitivity. This tends to reduce the measurement accuracy of biological components.

If the amount of 4-hydroxyantipyrine mixed in is large, the 4-hydroxyantipyrine mixed in the aminoantipyrine compound should be removed in advance by the method for removing 4-hydroxyantipyrine described later, and then subjected to biocomponent measurement. is preferred.

Considering the amount of 4-hydroxyantipyrine mixed in a general aminoantipyrine compound (4-aminoantipyrine), the present invention provides, for example, about 0.01 to 100 g / l aminoantipyrine compound (4-aminoantipyrine). Reagent containing, preferably about 0.01 to 10 g / l aminoantipyrine compound (4-aminoantipyrine), more preferably about 0.01 to 5 g / l aminoantipyrine compound (4-aminoantipyrine) It is effective to use a reagent containing

By using the aminoantipyrine compound in such a range, the effects of the present invention can be exhibited more effectively.

(Method for quantifying 4-hydroxyantipyrine)

The content of 4-hydroxyantipyrine in aminoantipyrine compounds can be determined, for example, by high-performance liquid chromatography (hereinafter also referred to as HPLC method), gas chromatography (hereinafter also referred to as GC method), mass spectrometry (hereinafter, MS method), nuclear magnetic resonance method (hereinafter also referred to as NMR method), and the like alone or in any combination. Although not particularly limited, the HPLC method is preferably used from the viewpoint of simplicity of operation and economy of systems and equipment. As the column for the HPLC method, a reversed-phase column is preferred, and a silica-based porous column is more preferred as the reversed-phase column.

A specific embodiment of the HPLC method will be described below. In this specification, 4-hydroxyantipyrine was quantified by the following HPLC method conditions.

(1) Column Imtakt Cadenza CD-C18 2.0 x 150 mm

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

(3) Gradient conditions

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

(4) Flow rate 0.2 mL/min

(5) Column temperature 40°C

(6) Sample injection volume 5 μL

(7) Detection wavelength UV250nm

4HA (manufactured by Sigma) with a known concentration was used as a standard, and the coincidence of the detection position of the HPLC fraction peak with the measurement sample was confirmed. In addition, quantification was performed by comparing the area of the fraction peak with that of the measurement sample. Cas. NO. 1672-63-5, product number 109428-5G, 99% pure product 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 position 4 of 4-aminoantipyrine is converted to a hydroxyl group, and is considered to be a by-product produced and mixed in the manufacturing process of 4-aminoantipyrine.

It has been clarified by the studies of the present inventors that 4-hydroxyantipyrine, even if the amount of contamination is extremely small, has a great effect on the color development reaction in the biological component measurement method. This phenomenon is due to the fact that the reaction rate of 4-hydroxyantipyrine is faster than that of aminoantipyrine compounds (e.g., 4-aminoantipyrine) that were originally intended to react as couplers, and hydrogen peroxide is consumed. It is speculated that

In the biological component measurement reagent kit of the present invention, the drug substance of an aminoantipyrine compound that may contain 4-hydroxyantipyrine may be used as it is, or the drug substance of an aminoantipyrine compound having a low 4-hydroxyantipyrine content may be used. may be used after quantifying and screening, or those with reduced 4-hydroxyantipyrine content, such as by removing 4-hydroxyantipyrine from the drug substance of aminoantipyrine compounds, may be selected and used. .

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

When chromatography is used as a means of removing 4-hydroxyantipyrine from aminoantipyrine compounds, the physicochemical principle of the separation is not particularly limited. Examples include various principles such as partition (normal phase/reverse phase), adsorption, molecular exclusion, and ion exchange. Separation may be performed by a method of adsorbing to a ligand-bound resin or an adsorbent.

Common reversed-phase chromatography can be used as the means for removing 4-hydroxyantipyrine. The carrier for reversed-phase chromatography is not particularly limited. For example, silica gel is suitable, but a polymer-based carrier may also be used. When silica gel is used as a carrier, one with or without end capping can be selected. In addition, any chromatography system of low pressure, medium pressure, or high pressure can be used by properly adjusting the conditions according to the purpose.

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

The mobile phase for chromatography may be water and a water-soluble solvent such as methanol or acetonitrile. In order to avoid ionic interactions with the silanol groups of silica gel, the pH of the mobile phase for chromatography is adjusted to acidic according to the usual method. Alternatively, a small amount of ion pair reagent may be added.

The mobile phase flow rate may be optimized according to the capabilities of the system used. Alternatively, separation may be performed by stepwise elution instead of linear gradient.

(Other ingredients, etc.)

The biological component measurement reagent kit of the present invention preferably contains reagents containing buffer components. In addition, ascorbic acid oxidase, preservatives, salts, enzyme stabilizers, chromogen stabilizers, and the like are added to the reagents contained in the reagent kit for measuring biological components of the present invention within a range that does not affect the reaction. good too.

Examples of buffer components that can be contained in the biological component measurement reagent of the present invention include Tris buffer, phosphate buffer, borate buffer, carbonate buffer, GOOD buffer and the like. The amount used, the set pH, the mode of addition, etc. are not particularly limited. All of these can be obtained as commercially available products.

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-morpholinoethanesulfonic acid (MES), piperazine-1,4-bis (2-ethanesulfonic acid) (PIPES), N-tris(hydroxymethyl)methyl-2-aminomethanesulfonic acid (TES), N-cyclohexyl-3-aminopropanesulfonic 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), piperazine-1,4-bis(2-hydroxy-3-propanesulfonic acid) (POPSO), N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPSO), N-(2-acetamido)iminodiacetic acid (ADA), N,N-bis(2-hydroxyethyl)glycine (Bicine), N-[tris(hydroxymethyl)methyl]glycine (Tricine), etc. exemplified.

In the reagent for measuring a biological component of the present invention, the amount of ascorbate oxidase, preservatives, salts, enzyme stabilizers, chromogen stabilizers, etc. used and the form of addition thereof are not particularly limited. All of these can be obtained as commercially available products.

Antiseptics include Proclin 150, Proclin 200, Proclin 300, Proclin 950, azides, chelating agents, antibiotics, antibacterial agents, and the like.

Chelating agents include ethylenediaminetetraacetic acid and salts thereof.

Antibiotics include gentamicin, kanamycin, chloramphenicol and the like.

Antibacterial agents include methylisothiazolinone, imidazolidinyl urea, and the like.

Salts include sodium chloride, potassium chloride, aluminum chloride and the like.

Enzyme stabilizers include sucrose, trehalose, cyclodextrin, gluconate, amino acids and the like.

Chromogenic stabilizers include chelating agents such as ethylenediaminetetraacetic acid and salts thereof, cyclodextrin, and the like.

The reagents contained in the biological component measurement reagent kit of the present invention may be liquid reagents dissolved in any solvent (e.g., purified water, organic solvent, etc.), or may be dissolved in the same solvent as above before use. It may also be a dry powdery reagent (eg, freeze-dried powder) used in other applications.

(Measurement method using biological component measurement reagent kit)

When measuring a biological component using the biological component measurement kit of the present invention, a general-purpose automatic analyzer (for example, Hitachi 7180 automatic analyzer) can be used. The biological component measurement kit of the present invention may be constructed so as 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 dry reagent and a solution prepared by means such as freeze drying, and an appropriate carrier carrying an enzyme or the like Various forms can be exemplified, such as a kit in the form of a so-called dry system and a kit in the form of using a sensor.

As the configuration of the biological component measurement kit of the present invention, a kit composed of one reagent, or a kit composed of two or more reagents divided into packages is used. When reagents are packaged to form a kit composed of two or more reagents, for example, (a) an oxidase capable of generating hydrogen peroxide and (c) a hydrogen donor among redox coloring reagents and a reagent containing 4-aminoantipyrine as a coupler of (b) peroxidase and (d) a redox coloring dye.

An example of a form of a kit composed of liquid reagents (hereinafter also referred to as two-reagent liquid reagents) in which the reagent is divided into two will be described below.

In the method of analyzing with an automatic analyzer using this form of reagent, first, the first type of reagent (hereinafter also referred to as the first reagent or R1) is added to the sample and reacted for a certain period of time, and then the second type (hereinafter also referred to as the second reagent or R2) is further added and reacted, and the change in absorbance during this reaction is measured to quantify the target component.

In addition, when the biological component measurement reagent of the present invention is supplied in two or more divided packages in consideration of application to an automatic analyzer as described above, the concentration of the iron-containing substance in each packaged reagent, It is determined whether the concentrations of the other components are within the preferred concentration range for each of the 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 step (1) oxidizes and condenses 4-aminoantipyrine and a redox coloring reagent through the action of peroxidase in the coexistence of peroxidase, thereby coloring the reaction solution. process,

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

The biological component measuring method of the present invention is a biological component measuring method based on an enzymatic method, particularly a method using an oxidase-peroxidase-coloring agent system. The principle of the measurement is to generate hydrogen peroxide according to the reaction rate, react it with a coloring agent in the presence of peroxidase, and colorimetrically determine the resulting color.

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

The present inventors have found that 4-hydroxyantipyrine, which is contained in an extremely small amount in the drug substance of aminoantipyrine compounds (particularly 4-aminoantipyrine), inhibits the reaction of this oxidase-peroxidase-chromogen system, resulting in increased sensitivity. For the first time, I found that it causes a decline. Although the mechanism by which 4-hydroxyantipyrine inhibits the above reaction is not necessarily clear, from its structure, the redox coloring reagent and 4-hydroxyantipyrine undergo a condensation reaction in the presence of hydrogen peroxide due to the action of peroxidase, resulting in hydrogen peroxide. is consumed. The present inventors have found that such reaction inhibition caused by 4-hydroxyantipyrine can be suppressed by allowing an iron-containing substance to coexist with 4-hydroxyantipyrine in advance to react, thereby suppressing sensitivity reduction. . Although the mechanism by which an iron-containing substance suppresses the decrease in sensitivity caused by 4-hydroxyantipyrine is also unclear, 4-hydroxyantipyrine undergoes some kind of structural change due to an iron-containing substance (for example, iron ions that can be generated from it), and reacts with peroxidase. It is assumed that it becomes difficult and does not consume hydrogen peroxide.

(specimen)

Specimens containing biological components used in the biological component measurement of the present invention include, for example, blood (especially serum, plasma, etc.), urine, ascitic fluid, cerebrospinal fluid, and other biological fluids, beverages, foods, and the like. Things to ingest, etc. Among them, it is preferable to use human body fluids (samples derived from blood such as serum and plasma, samples derived from urine, etc.) as specimens to be measured.

(Detection sensitivity of biological component measurement reagent kit)

By using an iron-containing substance, the present invention can improve the detection sensitivity attributed to 4-hydroxyantipyrine in biological component measurement by an enzymatic method using an oxidase-peroxidase-coloring agent system, compared to the case where an iron-containing substance is not used. can be suppressed.

The detection sensitivity of the biological component measurement reagent kit will be described below using creatinine as an example of a biological component.

In recent years, the calculation of eGFR (also called estimated glomerular filtration rate) requires creatinine measurement accuracy to two decimal places, and the minimum detection sensitivity is required to be about 0.03 mg/dL in terms of creatinine concentration. there is

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

Therefore, if there is an absorbance of 0.296mABS, which is the maximum value of 2.6σ, that is, about 0.3mABS or more, it can be detected as a signal, and it is considered possible to determine the presence or absence of creatinine and to quantify creatinine.

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

It should be noted that the present invention is not limited to the configurations described above, and can be modified in various ways within the scope of the claims. Also included in the technical scope of the present invention are embodiments and examples obtained by appropriately combining, modifying, or replacing the above.

EXAMPLES The present invention will be specifically described below with reference to Examples, but the present invention is not limited to these.

(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 was speculated that the content of the impurity contained in 4AA in a very small amount differs from lot to lot, and the impurity was detected by the above-mentioned 4HA quantitative method (HPLC method). was detected. The purity of 4AA used for examining lot differences was 98.0% or higher according to JIS-K8048.

FIG. 1 shows the HPLC fractions for the analysis of 4AA. In FIG. 1, the large peak observed around 7 to 8 minutes of elution time on the horizontal axis of the graph is the peak of 4-aminoantipyrine. In this HPLC fraction, as shown in FIG. 1, 3 kinds of impurities were confirmed in addition to 4-aminoantipyrine.

(Example 2) Identification of 4HA

When the contents of the three types of impurities (a, b, c) seen in FIG. 1 of Example 1 were investigated in each lot, it was found that only the impurity b content varied greatly from lot to lot. Therefore, the molecular weights and structures of these three substances were analyzed by mass spectrometry (MS spectrum method).

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

When HPLC was performed under the same conditions as in Example 1 using 4HA (Sigma) with a known concentration as a standard, the fraction peaks of 4HA and impurity b coincided.

(Example 3) Contamination concentration dependence of 4HA on creatinine measurement sensitivity

Using creatinine as a biological component, the dependency of the amount of 4HA contaminating the reagent on the decrease in measurement sensitivity caused by 4HA contaminating the reagent was evaluated.

Each measurement reagent was prepared by adding 4HA to the second reagent of the following creatinine measurement reagents so that the final concentration in the reagent was 0.13 to 8.75 μg/ml. A 5 mg/dL creatinine aqueous solution was used as a biological component sample.

[Preparation of reagent]

A creatinine measuring reagent having the following composition was prepared. Here, 4-aminoantipyrine was used by purifying commercially available 4-aminoantipyrine raw material to produce 4-aminoantipyrine containing no 4-hydroxyantipyrine.

First reagent

PIPES-NaOH 50 mM pH 7.4

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

Sarcosine oxidase (Toyobo SAO-351) 10 U/mL

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

Catalase (Toyobo CAO-509) 130U/mL

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

Second reagent

PIPES-NaOH 50 mM pH 7.4

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

Peroxidase (Toyobo PEO-302) 10 U/mL

4-aminoantipyrine 0.6g/L

[Measurement method]

Hitachi 7180 type 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 obtain the first reaction. After that, 40 μL of the second reagent was added and incubated for 5 minutes to obtain the second reaction. Absorbance at 546 nm (main wavelength) and absorbance at 800 nm (sub-wavelength) were measured by a two-point end method in which the absorbances of the first reaction and the second reaction were corrected for liquid volume and the difference was taken. It was obtained by calculating the absorbance obtained by subtracting the sub-wavelength from the main wavelength.

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

Results are shown in Table 1 and FIG. It was confirmed that the higher the 4HA concentration in the second reagent, the lower the sample measurement sensitivity.

(Example 4) Sensitivity reduction suppressing effect by iron-containing compound

Using ferric chloride as the iron-containing compound, the effect of suppressing sensitivity reduction caused by 4HA was confirmed.

For the reagents other than control (a), 4HA was added to the second reagent of the creatinine measurement 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. It was carried out under the same conditions as in Example 3, except that

After adding ferric chloride to the final concentration in the second reagent of the predetermined creatinine measurement reagent, the measurement sensitivity ( mABS) were examined. A blank value of each reagent was also measured at the same time, and a value obtained by subtracting the blank value from the measurement sensitivity was calculated as the STD sensitivity. Then, the ratio of the measurement sensitivity (mABS) of each reagent to the measurement sensitivity (mABS) of the control (a) at each 4AA concentration to which 4HA was not added [vs. control (%)] was calculated.

The results are shown in Tables 2-4.

From this result, the effect of suppressing sensitivity reduction caused by 4HA by coexistence of an iron salt depends on both the iron ion concentration and the 4HA concentration. It was confirmed that the reduction in sensitivity caused by 4HA can be suppressed particularly effectively when adjusted to 0.05 mM. Also, even when the concentration of ferric chloride in the second reagent was adjusted to 0.001 mM, a certain effect of suppressing the decrease in sensitivity was observed when compared with the blank. Here, the evaluation at 35° C. corresponds to an accelerated test under refrigerated storage conditions. Therefore, it is speculated that even under refrigeration conditions, the decrease in sensitivity caused by 4-hydroxyantipyrine can be suppressed by reacting various iron salts over a long period of time.

(Example 5) Effect of a mixture containing iron protein/iron salt to suppress sensitivity deterioration

Potassium ferricyanide and potassium ferrocyanide were used as iron-containing compounds, and PEO-302 (peroxidase) was used as an iron protein to confirm the effect of suppressing sensitivity reduction caused by 4HA.

For the reagents other than control (a), 4HA was added to the second reagent of the creatinine measurement 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. It was carried out under the same conditions as in Example 3, except that

Potassium ferricyanide, potassium ferrocyanide, and PEO-302 were added to the second reagent of the creatinine measurement reagent so as to have a predetermined final concentration in the second reagent, and then each was stored for 3 days under the temperature conditions of the accelerated test (35 ° C.). Measurement sensitivity (mABS) was investigated using the second reagent. A blank value of each reagent was also measured at the same time, and a value obtained by subtracting the blank value from the measurement sensitivity was calculated as the STD sensitivity. Then, the ratio of the measurement sensitivity (mABS) of each reagent to the measurement sensitivity (mABS) of the control (a) to which 4HA was not added [vs. control (%)] was calculated.

Table 5 shows the results.

From this result, it was confirmed that both potassium ferricyanide and potassium ferrocyanide can suppress the decrease in sensitivity caused by 4HA. As for PEO-302, since the concentration in the second reagent was low, the effect of suppressing sensitivity deterioration was not large, but it was observed that the suppressing effect increased as the concentration increased.

The present invention can be applied to measurement methods for measuring biological components using redox reactions, and reagents and compositions used in the methods.

Claims (8)

A measurement sensitivity reduction inhibitor applied to a biological component measurement method using an aminoantipyrine compound containing 4-hydroxyantipyrine as a coupler of a redox coloring reagent, characterized by containing an iron-containing substance, Agent for suppressing decrease in measurement sensitivity of biocomponents. The biocomponent measurement method is a biocomponent measurement method using a reagent or reagent set that satisfies the following requirements (a) to (d), and is added to a reagent that satisfies the requirement (d). The agent for suppressing decrease in measurement sensitivity of biological components according to claim 1.
(a) contains an oxidase capable of generating hydrogen peroxide;
(b) contains peroxidase;
(c) contains a redox coloring reagent that reacts with hydrogen peroxide in the presence of peroxidase to develop a color;
(d) includes an aminoantipyrine compound containing 4-hydroxyantipyrine as a coupler for the redox coloring reagent. 3. The agent for suppressing decrease in sensitivity for measurement of biological components according to claim 1 or 2, which suppresses decrease in sensitivity for measurement of biological components caused by 4-hydroxyantipyrine. A method for suppressing decrease in measurement sensitivity applied to a biological component measurement method using an aminoantipyrine compound containing 4-hydroxyantipyrine as a coupler of a redox coloring reagent, characterized in that an iron-containing substance is used. A method for suppressing a decrease in component measurement sensitivity. A method for suppressing reduction in measurement sensitivity in a biological component measurement method using a reagent or reagent set that satisfies the following requirements (a) to (d), wherein an iron-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, characterized by:
(a) contains an oxidase capable of generating hydrogen peroxide;
(b) contains peroxidase;
(c) contains a redox coloring reagent that reacts with hydrogen peroxide in the presence of peroxidase to develop a color;
(d) includes an aminoantipyrine compound containing 4-hydroxyantipyrine as a coupler for the redox coloring reagent. 6. The method for suppressing a decrease in sensitivity for measuring a biological component according to claim 4 or 5, wherein the decrease in sensitivity for measuring a biological component caused by 4-hydroxyantipyrine is suppressed. The concentration of the iron-containing substance coexisting in the reagent that satisfies the requirements of (d) is 0.001 to 1 mM
7. The method for suppressing a decrease in the measurement sensitivity of biological components according to claim 5 or 6, characterized in that: 8. The method for suppressing reduction 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.

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