JP2023063609A - Method for suppressing decrease in sensitivity of reagent kit for measuring biological component - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide means for suppressing a decrease in sensitivity due to 4-hydroxy antipyrine in a biological component measurement reagent kit.

SOLUTION: The present invention provides an agent for suppressing a decrease in measurement sensitivity, applied to a biological component measurement method which uses an aminoantipyrine-based compound as a coupler for an oxidation-reduction color reagent, wherein the agent contains at least one buffer selected from the group consisting of piperazine-1,4-bis(2-ethanesulfonate), tris (hydroxymethyl) aminomethane, and salts thereof.

SELECTED DRAWING: None

COPYRIGHT: (C)2023,JPO&INPIT

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-coloring 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. 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 unexpectedly found that piperazine-1,4-bis(2-ethanesulfonic acid) or 4-Hydroxyantipyrine by using a buffer such as its salt (hereinafter also referred to as "PIPES"), or tris(hydroxymethyl)aminomethane or a salt thereof (hereinafter also referred to as "Tris") The inventors have found that it is possible to suppress the resulting decrease in sensitivity, and have completed the present invention. That is, the present invention consists of the following configurations.

[Item 1] A measurement sensitivity reduction inhibitor applied to a biological component measurement method using an aminoantipyrine compound as a coupler of a redox coloring reagent, comprising piperazine-1,4-bis(2-ethanesulfonic acid), tris 1. An agent for suppressing decrease in measurement sensitivity of biological components, comprising at least one buffer selected from the group consisting of (hydroxymethyl)aminomethane and salts thereof.
[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 satisfies the requirement (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 deterioration in measurement sensitivity applied to a biological component measurement method using an aminoantipyrine compound as a coupler of a redox coloring reagent, comprising piperazine-1,4-bis(2-ethanesulfonic acid), tris A method for suppressing a decrease in the measurement sensitivity of a biological component, which comprises using at least one buffer selected from the group consisting of (hydroxymethyl)aminomethane and salts thereof.
[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 the reagent that satisfies the requirement (d) includes: characterized by adding at least one buffer selected from the group consisting of piperazine-1,4-bis(2-ethanesulfonic acid), tris(hydroxymethyl)aminomethane, and salts thereof. A method for suppressing a decrease in component measurement sensitivity.
(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] Selected from the group consisting of piperazine-1,4-bis(2-ethanesulfonic acid), tris(hydroxymethyl)aminomethane, and salts thereof coexisting in a reagent that satisfies the requirements of (d) above Item 7. The method for suppressing a decrease in the measurement sensitivity of biological components according to item 5 or 6, wherein the concentration of the at least one buffering agent used is 20 to 2000 mM.
[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) at least one buffer selected from the group consisting of piperazine-1,4-bis(2-ethanesulfonic acid), tris(hydroxymethyl)aminomethane, and salts thereof.
[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) at least one buffer selected from the group consisting of piperazine-1,4-bis(2-ethanesulfonic acid), tris(hydroxymethyl)aminomethane, and salts thereof.
[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 biocomponent 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 comprises piperazine-1,4-bis (2-ethanesulfonic acid), tris(hydroxymethyl)aminomethane, and at least one buffer selected from the group consisting of salts thereof (hereinafter collectively referred to as "PIPES and/or Tris" It is characterized by containing

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 add PIPES and/or Tris 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 found that such a decrease in sensitivity can be suppressed by coexisting PIPES and/or Tris with 4-hydroxyantipyrine, and completed the present invention.
That is, the agent for suppressing decrease in measurement sensitivity of biological components of the present invention is preferably used by adding PIPES and/or Tris to a reagent that satisfies the above requirement (d). It aims at suppressing the deterioration of the measurement sensitivity of the component.

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 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 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 at least one buffer selected from the group consisting of piperazine-1,4-bis(2-ethanesulfonic acid), tris(hydroxymethyl)aminomethane, and salts thereof.
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): At least one selected from the group consisting of piperazine-1,4-bis(2-ethanesulfonic acid), tris(hydroxymethyl)aminomethane, and salts thereof in the reagent that satisfies the requirements of (d) It is characterized by the addition of a seed buffer.
(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 inventors have found that the coexistence of PIPES and/or Tris can suppress the sexuality, and completed the present invention.
That is, the method for suppressing low measurement sensitivity of a biological component of the present invention is performed by adding PIPES and/or Tris to a reagent that satisfies the requirement (d). Contaminating 4-hydroxyantipyrine PIPES and/or Tris are allowed to coexist and react with each other to suppress the reactivity of 4-hydroxyantipyrine, thereby improving the measurement sensitivity of biological components caused by 4-hydroxyantipyrine. It is characterized by suppressing the decrease.

The reaction time of PIPES and/or Tris in a reagent containing an aminoantipyrine compound contaminated with 4-hydroxyantipyrine is not particularly limited as long as the effects of the present invention are achieved. For example, from the time when the PIPES and/or Tris are added to the reagent satisfying the requirement (d), the PIPES and/or the Tris are allowed to coexist in the reagent satisfying the requirement (d). , 1°C to 10°C for at least 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. A sufficient effect can be obtained by reacting over a range of Therefore, even if the temperature is not strictly controlled, the effect of the present invention can be obtained by storing the PIPES and/or Tris for about one month or more to react. In the present invention, it is preferable to react 4-hydroxyantipyrine with PIPES and/or Tris 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), the product reagent contains PIPES and / or After the addition of the Tris, the reaction proceeds by storing, distributing, and storing before use in a state in which the PIPES and/or Tris are coexisting in the presence of the PIPES and/or Tris. The biocomponent measurement reagent kit manufactured by adding PIPES and/or Tris to reagents satisfying the requirements (d) 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 adjusted reagents), after adding PIPES and/or Tris to the intermediate reagent, for example, it is stored for several days under temperature conditions of 30 ° C. or higher and for several hours under temperature conditions of 45 ° C. or higher. A product reagent may be prepared by diluting to a predetermined concentration.
When PIPES and/or Tris are added to the intermediate reagent, at the stage of preparing the product reagent, PIPES and/or Tris coexisting in the reagent satisfying the requirements of (d) are excluded from the product reagent. good too.

The present inventors have found that such decrease in measurement sensitivity caused by 4-hydroxyantipyrine can be suppressed by allowing PIPES and/or Tris to coexist with 4-hydroxyantipyrine in advance. Although the mechanism by which PIPES and/or Tris suppress sensitivity reduction due to 4-hydroxyantipyrine is not clear, the present inventors discovered that PIPES and/or Tris cause some structural change in 4-hydroxyantipyrine and peroxidase. It is assumed that it does not react and consumes 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 PIPES and / or Tris We speculate that the reaction between 4-hydroxyantipyrine and peroxidase will not be restored even if PIPES and/or Tris are removed from the coexisting reagents.

In the method for suppressing decrease in measurement sensitivity of biological components of the present invention, the concentration of PIPES and/or Tris coexisting in the reagent satisfying the requirement (d) is preferably 20 to 2000 mM, preferably 50 to 1000 mM. More preferably, it is 100 to 1000 mM, and particularly preferably 200 to 800 mM. As shown in the results of the test examples described later, the higher the concentration of PIPES and/or Tris coexisting in the reagent that satisfies the requirement (d), the more effectively lowering the measurement sensitivity of biological components. can be suppressed. Therefore, by using PIPES and/or Tris within the range described above, it is possible to obtain a sufficiently high suppressing effect while suppressing costs and the risk of measurement errors.
If the concentration of PIPES and/or Tris is less than 20 mM, the inhibitory effect is small, and if the concentration of PIPES and/or Tris is more than 2000 mM, measurement errors may occur in the biological component measurement reaction.
The preferred concentration range of the PIPES and/or Tris coexisting in the reagent that satisfies the requirement (d) above is the concentration of 4-hydroxyantipyrine mixed in the reagent that satisfies the requirement (d). Depending on the concentration, if the concentration of 4-hydroxyantipyrine is low, the decrease in the measurement sensitivity of biological components can be suppressed even if the concentration of PIPES and/or Tris is low, and if the concentration of 4-hydroxyantipyrine is high, PIPES at a higher concentration can be suppressed. Since it is effective to use Tris and/or Tris, the concentration of PIPES and/or Tris depends on the concentration of 4-hydroxyantipyrine contaminating in the method for suppressing deterioration in biocomponent measurement sensitivity of the present invention. It is preferable to set it as appropriate.

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 satisfying the requirement (d) is 0 at the concentration before adding PIPES and / or Tris. The effect is particularly likely to be obtained when it is about 1 to 50 µg/ml.
If the concentration of 4-hydroxyantipyrine present in the reagent that satisfies the requirements of (d) before adding the PIPES and/or Tris is less than 0.1 μg/ml, the biocomponent due to 4-hydroxyantipyrine is 1% or less, and there is little need to suppress the decrease in measurement sensitivity by allowing PIPES and/or Tris to coexist.
If the concentration of 4-hydroxyantipyrine present in the reagent that satisfies the requirements of (d) before adding the ions is more than 50 μg/ml, in order to suppress the decrease in the measurement sensitivity of biological components due to 4-hydroxyantipyrine. requires high concentrations of PIPES and/or Tris and is therefore prone to measurement errors due to side reactions caused by PIPES and/or Tris.
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, 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 biological component 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 is selected from the group consisting of piperazine-1,4-bis(2-ethanesulfonic acid), tris(hydroxymethyl)aminomethane, and salts thereof as described above. A measurement desensitization inhibitor containing at least one selected buffering agent is included.
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) at least one buffer selected from the group consisting of piperazine-1,4-bis(2-ethanesulfonic acid), tris(hydroxymethyl)aminomethane, and salts thereof.

Regarding the reagents satisfying the requirements (d) and (e) above, one reagent that satisfies the two requirements at the same time is to add PIPES and/or Tris to the reagent that satisfies the requirements of (d) as described above. It can be manufactured by

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. Or, in the present invention, it was found that it can be suppressed by adding Tris.
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 the 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) at least one buffer selected from the group consisting of piperazine-1,4-bis(2-ethanesulfonic acid), tris(hydroxymethyl)aminomethane, and salts thereof.
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 PIPES and/or Tris to 4-hydroxyantipyrine to suppress the reactivity of 4-hydroxyantipyrine. may be carried out by allowing PIPES and/or Tris to coexist with 4-hydroxyantipyrine mixed in the reagent that satisfies the requirements of (d) above, but this inhibitory reaction does not necessarily have to be carried out in the product reagent. Instead, for example, in the process of preparing a reagent containing an aminoantipyrine compound, PIPES and/or Tris are added in the state of an intermediate reagent, etc., and after performing an inhibitory reaction, the intermediate reagent is used to produce a product reagent. may be 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 PIPES and/or Tris are added to an intermediate reagent containing an aminoantipyrine compound to perform an inhibitory reaction, PIPES and/or Tris are added to the intermediate reagent. The total period from the date of manufacture of the product after addition to the date of use of the product is preferably at least one month.

It is presumed that the PIPES and/or Tris of the present invention suppresses the decrease in the measurement sensitivity of biological components caused by 4-hydroxyantipyrine by coexisting with 4-hydroxyantipyrine that is mixed as an impurity in aminoantipyrine compounds. do.
It is not clear why the coexistence of the PIPES and/or Tris of the present invention with 4-hydroxyantipyrine suppresses the decrease in the sensitivity of biocomponent measurement, but the PIPES and/or Tris are combined with 4-hydroxyantipyrine By acting directly or indirectly on 4-hydroxyantipyrine, some changes occur in 4-hydroxyantipyrine, and as a result, the consumption of hydrogen peroxide due to the reaction of 4-hydroxyantipyrine with peroxidase is reduced, resulting in decreased sensitivity. It is considered to be 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.
Therefore, when PIPES and/or Tris are added to the intermediate reagent, it is possible to manufacture the product reagent by removing PIPES and/or Tris in the subsequent stage 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.

Piperazine-1,4-bis(2-ethanesulfonic acid) and its salts used in the present invention are one of so-called good buffers, also called PIPES. The salt form of PIPES is not limited as long as the effect of the present invention is exhibited, and examples thereof include sodium salt, potassium salt, calcium salt and the like. The salt form of PIPES is preferably a sodium salt (eg piperazine-1,4-bis(2-ethanesulfonic acid) disodium).

Moreover, tris(hydroxymethyl)aminomethane and its salts used in the present invention are one of well-known buffers, also called Tris. The form of Tris salt is not limited as long as the effect of the present invention is exhibited, and examples thereof include hydrochloride and acetate. The salt form of Tris is preferably hydrochloride (tris(hydroxymethyl)aminomethane hydrochloride, also referred to as “Tris-HCl”). Tris-HCl can be used by adding HCl dropwise to an aqueous solution of Tris (about pH 10.5) to adjust the desired pH.

The amount of PIPES and/or Tris 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, PIPES and/or Tris are preferably used as intermediate reagents for aminoantipyrine compounds or product reagents formulated together with aminoantipyrine compounds. The concentration of PIPES and/or Tris in the reagent may be adjusted to 20 to 2000 mM, preferably the PIPES and/or Tris concentration in the reagent intermediate or reagent is 50 to 1000 mM. It may be used by adjusting the blending amount so that it is preferably 100 to 1000 mM, more preferably 200 to 800 mM. As shown in the results of test examples described later, it has been confirmed that the use of higher concentrations of PIPES and/or Tris enhances the lowering of measurement sensitivity for biological components. Therefore, by using PIPES and/or Tris in such amounts, it is possible to effectively suppress the decrease in sensitivity of the reagent kit for measuring biological components caused by 4-hydroxyantipyrine.

The PIPES and/or Tris used in the present invention have the effect of suppressing the decrease in the sensitivity of biological component measurement due to 4-hydroxyantipyrine contained as an impurity of the aminoantipyrine compound after being blended with the aminoantipyrine compound. In order to develop the effect, it is preferable to allow a certain period of time to pass after blending.
In order for the PIPES and/or Tris used in the present invention to exhibit the effect of suppressing the sensitivity reduction in biological component measurement caused by 4-hydroxyantipyrine, it takes 2 weeks or more at a storage temperature of 1°C to 10°C. 1 week or more at 11°C to 25°C, 2 days or more at 26°C to 40°C, 5 hours or more at 41°C to 60°C, 1 hour or more at 61°C to 80°C. is preferably longer than that. Depending on the storage temperature, the higher the temperature, the shorter the required time, and the lower the temperature, the longer the required time. 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, as long as the quality of the reagent used in the biological component measurement kit does not deteriorate, the PIPES and/or Tris used in the present invention are 4-hydroxyantipyrine. The effect of suppressing the decline 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. At the stage of using the measurement kit containing the reagent in which the PIPES and/or Tris of the present invention are coexistent with an aminoantipyrine compound, the PIPES and/or Tris used in the present invention have the effect of suppressing the decrease in sensitivity. It is expressed and maintained, but if the product life cycle is shortened, it can be adjusted by manufacturing process control, product control at the time of shipment, or the like.

Regarding reagents satisfying the requirements (d) and (e), the pH of one reagent that satisfies the two requirements at the same time is not limited as long as the effects of the present invention are exhibited, and can be, for example, pH 6 to 12. In certain embodiments, the pH of the reagent may be less than 8, eg, pH 6-8, preferably pH 7-8.

(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, for the 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. 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 sequentially mixed, and then pre-heated 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, and 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 a dye is formed by oxidizing and condensing a hydrogen donor and a coupler with hydrogen peroxide in the presence of peroxidase.

(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 that causes a decrease in sensitivity, and it is desired to reduce the amount of 4-hydroxyantipyrine. However, according to the present invention, PIPES and / Or the coexistence of Tris can suppress the decrease in sensitivity 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 a reagent containing an aminoantipyrine compound before coexistence with PIPES and/or Tris is preferably 0.1 to 50 μg/ml. , more preferably 0.3 to 20 μg/ml, and 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 it is necessary to suppress the reduction in measurement sensitivity due to the coexistence of PIPES and/or Tris. Less is.
When the concentration of 4-hydroxyantipyrine exceeds the above range, high concentrations of PIPES and/or Tris are required in order to suppress the decrease in the measurement sensitivity of biological components due to 4-hydroxyantipyrine. Alternatively, a tris-based color-developing side reaction tends to occur, which tends to lower 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 × 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 freeze-drying or other means, 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 reagent for measuring biological components of the present invention is supplied in two or more packages in consideration of application to an automatic analyzer as described above, PIPES and/or Tris in each package reagent It is determined whether or not the similar concentration and the concentration of other components are within the preferred concentration range of each component.

(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 PIPES and/or Tris to coexist with 4-hydroxyantipyrine in advance, thereby suppressing the decrease in sensitivity. I found what I can do. Although the mechanism by which PIPES and/or Tris suppress sensitivity reduction due to 4-hydroxyantipyrine is also unclear, PIPES and/or Tris cause some structural change in 4-hydroxyantipyrine, making it less reactive with peroxidase. It is presumed that hydrogen peroxide is no longer consumed.

(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)
According to the present invention, by using PIPES and/or Tris, compared to the case where PIPES and/or Tris are not used, biocomponent measurement by an enzymatic method using an oxidase-peroxidase-coloring agent system can achieve 4 - It is possible to suppress the decrease in detection sensitivity caused by hydroxyantipyrine.
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 PIPES and/or Tris, 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. becomes.

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. The technical scope of the present invention also includes 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 lot differences in 4AA, it was speculated that the content of trace impurities contained in 4AA differs from lot to lot. Impurities were detected by a 4HA quantitative method (HPLC method). 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 As a result of examining the content of each lot for the three types of impurities (a, b, c) seen in Fig. 1 of Example 1, the content of only impurity b was large for each lot. turned out to be different. 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 Dependence of 4HA on Creatinine Measurement Sensitivity Using creatinine as a biological component, the dependence of the amount of 4HA contaminant 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) Effect of TrisHCl/PIPES in suppressing sensitivity reduction The effect of TrisHCl and PIPES in 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 TrisHCl or PIPES instead of PIPES-NaOH so that the final concentration in the second reagent of the predetermined reagent for creatinine measurement is reached, the second reagent was stored for 3 days under the temperature conditions of the accelerated test (35 ° C.). The sensitivity of the measurement (mABS) was investigated. 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. Also, the pH of each second reagent used in the test was actually measured.
Table 2 shows the results.

From this result, it was confirmed that the coexistence of TrisHCl or PIPES can effectively suppress the decrease in sensitivity caused by 4HA.
In other words, the control (b) to which 4HA was added at 10 μg/ml had a measurement sensitivity of 47% after 3 days at 35°C, whereas the results using TrisHCl or PIPES showed a measurement sensitivity of 35°C at 35°C. It ranged from 56% to 99% during the day. Here, the evaluation at 35° C. corresponds to an accelerated test under refrigerated storage conditions. Therefore, it is speculated that even under refrigerated conditions, the sensitivity reduction caused by 4-hydroxyantipyrine can be suppressed by reacting TrisHCl or PIPES over a long period of time. Moreover, as shown in the above test results, it has been clarified that the higher the concentration of TrisHCl and PIPES used, the more effectively the decrease in sensitivity can be suppressed.

(Example 5) Effect of PIPES to Suppress Decrease in Sensitivity Using PIPES, the effect of suppressing decrease in sensitivity 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 PIPES to the second reagent of the creatinine measurement reagent so as to have a predetermined final concentration in the second reagent, the second reagent was stored for 3 days under the temperature conditions of the accelerated test (35 ° C.). Sensitivity (mABS) was investigated. 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. Also, the pH of each second reagent used in the test was actually measured.
Table 3 shows the results.

From this result, it was confirmed that sensitivity reduction due to 4HA can be suppressed even when these components are used.
In other words, the control (b) to which 4HA was added at 10 μg/ml had a measurement sensitivity of 45% after 3 days at 35°C, whereas the results using PIPES showed a sensitivity of measurement of 45% after 3 days at 35°C. 54% to 90%. It was also confirmed that, as in Example 4 above, the effect of suppressing the decrease in sensitivity is more effectively enhanced as the concentration of PIPES increases.

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 (7)

The biological component measurement reagent kit that satisfies the following requirements (a) to (e), and the reagent that satisfies the requirements (d) and (e) is one reagent that satisfies the two requirements at the same time. A biological component measurement reagent kit, wherein the reagent is used after being stored for 3 days or more.
(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) at least one buffer selected from the group consisting of piperazine-1,4-bis(2-ethanesulfonic acid), tris(hydroxymethyl)aminomethane, and salts thereof. 2. The biological component measurement reagent kit according to claim 1, wherein one reagent that simultaneously satisfies the two requirements (d) and (e) has been manufactured for at least one month. 3. The biological component measurement reagent kit according to claim 1 or 2, wherein one reagent that simultaneously satisfies the two requirements (d) and (e) contains the buffering agent (e) at a concentration of 20 to 2000 mM. A biological component measuring method, which comprises using the biological component measuring kit according to any one of claims 1 to 3. A method for producing a biological component measurement reagent kit that suppresses sensitivity reduction due to 4-hydroxyantipyrine, satisfying the following requirements (a) to (e), and satisfying the requirements (d) and (e): A method for manufacturing a biological component measurement reagent kit, characterized in that one reagent satisfying two requirements at the same time is manufactured, and the one reagent is stored for three days or longer.
(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) at least one buffer selected from the group consisting of piperazine-1,4-bis(2-ethanesulfonic acid), tris(hydroxymethyl)aminomethane, and salts thereof. 6. The method for producing a biological component measurement reagent kit according to claim 5, wherein one reagent that simultaneously satisfies the two requirements (d) and (e) contains the buffering agent (e) at a concentration of 20 to 2000 mM. Regarding the reagents satisfying the requirements (d) and (e), one reagent that satisfies the two requirements at the same time is an intermediate reagent in the manufacturing process of the reagent kit for measuring biological components. 3. The method for producing the biological component measurement reagent kit according to 1.

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