WO2019216405A1

WO2019216405A1 - Method for suppressing reduction in sensitivity of biological-component-measuring reagent kit

Info

Publication number: WO2019216405A1
Application number: PCT/JP2019/018704
Authority: WO
Inventors: 研吾西村; 木全　伸介
Original assignee: 東洋紡株式会社
Priority date: 2018-05-10
Filing date: 2019-05-10
Publication date: 2019-11-14
Also published as: JP7314931B2; JPWO2019216405A1

Abstract

The present invention provides an agent for suppressing a reduction in sensitivity of measurement, the agent being applied to a biological component measurement method in which an aminoantipyrine compound is used as a coupler of a redox coloring reagent, wherein the agent for suppressing a reduction in sensitivity of measurement of a biological component is characterized by containing a copper-containing substance. Preferably, the biological component measurement method involves the use of a reagent or a reagent set that satisfies requirements (a)-(d) below, the method being characterized in that the copper-containing substance is used by being added to a reagent that satisfies requirement (d): (a) contains an oxidase capable of generating hydrogen peroxide. (b) contains peroxidase. (c) contains a redox coloring reagent that colors upon reaction with hydrogen peroxide in the presence of peroxidase. (d) contains an aminoantipyrine compound as a coupler of the redox coloring reagent.

Description

Method for inhibiting sensitivity reduction of biological component measurement reagent kit

The present invention relates to a method for suppressing sensitivity reduction 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 a Trinder reagent combining an aminoantipyrine compound as a coupler and a hydrogen donor), The present invention relates to a method for suppressing a decrease in sensitivity in a biological component measurement reagent kit that performs colorimetric determination of coloration generated by oxidative condensation in the presence of peroxidase.

Conventionally, in clinical diagnosis, biological components are measured by an enzyme method, and in particular, a method using an oxidase-peroxidase-oxidation-reduction color reagent (hereinafter also referred to as a color former), that is, a measurement target in a specimen. A method of performing colorimetric determination by reacting a substance with an enzyme to generate hydrogen peroxide and reacting it with a color former in the presence of peroxidase is widely performed (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 a Trinder method in which a hydrogen donor and a coupler are oxidized and condensed with hydrogen peroxide in the presence of peroxidase to form a dye. For example, 4-aminoantipyrine (hereinafter also referred to as 4AA) is known as a coupler used in this method.

The present inventors have prepared an enzyme-peroxidase-coloring agent system biocomponent measurement reagent using an aminoantipyrine compound and a hydrogen donor, and when using it for biocomponent measurement, the measurement sensitivity of unknown cause is reduced. Experienced.

Since the degree of decrease in measurement sensitivity varied depending on the lot of reagent composition prepared for measurement, the present inventors conducted various studies on the reagent composition of the measurement kit. As a result, it was surprisingly found that a very small amount of 4-hydroxyantipyrine (hereinafter also referred to as 4HA) exists in 4-aminoantipyrine. Although not wishing to be bound by theory, when 4-hydroxyantipyrine is present in the reagent, 4-hydroxyantipyrine is structurally not coupled to the hydrogen donor, but 4-hydroxyantipyrine in the presence of hydrogen peroxide. As a result, the hydrogen peroxide generated by reacting the enzyme with the analyte in the sample is consumed by 4-hydroxyantipyrine. It was considered that the amount of color developed due to 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 a decrease in sensitivity due to 4-hydroxyantipyrine in order to solve the above-mentioned problems that have not been known so far.

As a result of intensive investigations to achieve the above object, the present inventors unexpectedly used a copper-containing substance such as a component that generates copper ions in preparing a biological component measurement reagent kit. Thus, the inventors have found that the decrease in sensitivity due to 4-hydroxyantipyrine can be suppressed, and have completed the present invention. That is, the present invention has the following configuration.

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

[Claim 2] The biological component measurement method uses a reagent or a reagent set that satisfies the following requirements (a) to (d), and is added to the reagent that satisfies the requirement (d): Item 2. The measurement sensitivity lowering inhibitor of a biological component according to Item 1, which is used.

(A) An oxidase capable of generating hydrogen peroxide is included.

(B) Contains peroxidase.

(C) A redox coloring reagent that reacts with hydrogen peroxide in the presence of peroxidase to develop color.

(D) An aminoantipyrine compound is included as a coupler of the redox coloring reagent.

CLAIM | ITEM 3 The measurement sensitivity fall inhibitor of the biological component of claim | item 1 or 2 characterized by suppressing the fall of the measurement sensitivity of the biological component resulting from 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 for a redox coloring reagent, wherein the measurement sensitivity of a biological component is characterized by using a copper-containing substance. How to suppress the decrease.

[Item 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 copper is contained 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) An oxidase capable of generating hydrogen peroxide is included.

(B) Contains peroxidase.

(C) A redox coloring reagent that reacts with hydrogen peroxide in the presence of peroxidase to develop color.

(D) An aminoantipyrine compound is included as a coupler of the redox coloring reagent.

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

CLAIM | ITEM 7 The measurement sensitivity fall of the biological component of claim | item 5 or 6 characterized by the density | concentration of the copper containing substance coexisted in the reagent satisfy | filling the requirements of said (d) being 0.00001-1 mM. Suppression method.

CLAIM | ITEM 8 The said biological component is either creatinine or glycated hemoglobin, The suppression method of the measurement sensitivity fall of biological component in any one of claim | item 4 thru | or 7 characterized by the above-mentioned.

[Item 9] A biological component measurement reagent kit using an aminoantipyrine compound as a redox coloring reagent coupler, comprising the measurement sensitivity decrease inhibitor according to Item 1.

[Item 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 reagent that satisfies the two requirements at the same time: A biological component measuring reagent kit.

(A) An oxidase capable of generating hydrogen peroxide is included.

(B) Contains peroxidase.

(C) A redox coloring reagent that reacts with hydrogen peroxide in the presence of peroxidase to develop color.

(D) An aminoantipyrine compound is included as a coupler of the redox coloring reagent.

(E) Contains a copper-containing material.

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

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

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

(A) An oxidase capable of generating hydrogen peroxide is included.

(B) Contains peroxidase.

(C) A redox coloring reagent that reacts with hydrogen peroxide in the presence of peroxidase to develop color.

(D) An aminoantipyrine compound is included as a coupler of the redox coloring reagent.

(E) Contains a copper-containing material.

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

According to the present invention, it is possible to suppress a decrease in sensitivity due to 4-hydroxyantipyrine in biological component measurement by an enzyme method using an oxidase-peroxidase-color former system. Therefore, according to the present invention, it is possible to measure a favorable biological component. In particular, stable measurement is possible in the measurement of biological components that require high sensitivity, for example, the content of the biological components is extremely small.

It is a figure which shows the HPLC fraction of the result of having analyzed 4-aminoantipyrine API. It is a figure which shows the structural formula of 4-hydroxyantipyrine. It is a figure which shows the relationship between the density | concentration in a 2nd reagent of 4-hydroxyantipyrine, and sample measurement sensitivity.

An embodiment of the present invention will be described in detail as follows, but the present invention is not limited to this. It should be understood that the terms used in this specification are used in the meaning normally used in the art unless otherwise specified.

In addition, all of the non-patent documents and patent documents described in this specification are incorporated herein by reference. In the present specification, “to” means “above and below”. For example, if “X to Y” is described in the specification, it means “from X to Y”. In the present specification, “and / or” means either one or both. Also, in this specification, it should be understood that the expression of the singular includes the concept of the plural unless specifically stated otherwise.

(Inhibitor for decrease in measurement sensitivity of biological components)

The biological component measurement sensitivity decrease inhibitor of the present invention is a measurement sensitivity decrease inhibitor applied to a biological component measurement method using an aminoantipyrine compound as a coupler of a redox coloring reagent, and is a copper-containing substance (for example, copper A component that generates ions).

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

(A) An oxidase capable of generating hydrogen peroxide is included.

(B) Contains peroxidase.

(C) A redox coloring reagent that reacts with hydrogen peroxide in the presence of peroxidase to develop color.

(D) An aminoantipyrine compound is included as a coupler of the redox coloring reagent.

In the biological component measurement sensitivity lowering inhibitor of the present invention, it is preferable to add and use a copper-containing substance (for example, a component that generates copper ions) in a reagent that satisfies the requirement (d).

The aminoantipyrine compound contained in the reagent satisfying the requirement (d) may contain a trace amount of 4-hydroxyantipyrine as a by-product during the production thereof. It has been found that 4-hydroxyantipyrine causes a decrease in measurement sensitivity of biological components. Furthermore, the present inventors have found that such a decrease in sensitivity can be suppressed by allowing a copper-containing substance to coexist with 4-hydroxyantipyrine, thereby completing the present invention.

That is, the biological component measurement sensitivity lowering inhibitor of the present invention is preferably used by adding a copper-containing substance to a reagent satisfying the requirement (d), and the biological component measurement sensitivity caused by 4-hydroxyantipyrine. The purpose of this is to suppress the decrease.

Here, the reagent or reagent set may be prepared as one reagent that satisfies the requirements of (a) to (d), or is composed of 2 to 3 or more by packaging the reagents. It may be a reagent set. The reagent or reagent set may be in a form such as a kit packaged in a single packaging container, or may be a form in which each reagent is prepared separately and used in a set.

(Suppression method for measurement sensitivity reduction of biological components)

In one embodiment, the method for suppressing a decrease in measurement sensitivity of a biological component of the present invention is a method for suppressing a decrease in measurement sensitivity applied to a biological component measurement method using an aminoantipyrine compound as a redox coloring reagent coupler, A copper-containing material is used.

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

(A) An oxidase capable of generating hydrogen peroxide is included.

(B) Contains peroxidase.

(C) A redox coloring reagent that reacts with hydrogen peroxide in the presence of peroxidase to develop color.

(D) An aminoantipyrine compound is included as a coupler of the redox coloring reagent.

The present inventors speculate that the decrease in measurement sensitivity occurs as a result of consumption of hydrogen peroxide by the reaction of 4-hydroxyantipyrine with peroxidase. In the present invention, the reaction of 4-hydroxyantipyrine The present invention was completed by finding that the coexistence can be suppressed by the coexistence of a copper-containing substance (preferably, copper ions that can be generated from the copper-containing substance).

That is, the method for reducing the measurement sensitivity of the biological component of the present invention adds the copper-containing substance (for example, a component that generates copper ions) to the reagent that satisfies the requirement (d). A biological component caused by 4-hydroxyantipyrine by coexisting 4-hydroxyantipyrine and a copper-containing substance mixed in a reagent satisfying the above requirements and reacting both to suppress the reactivity of 4-hydroxyantipyrine It is characterized by suppressing a decrease in measurement sensitivity.

In the reagent containing the aminoantipyrine compound mixed with 4-hydroxyantipyrine, the time for reacting the copper-containing substance (copper ions that can be generated in a specific embodiment) is not particularly limited as long as the effect of the present invention is exhibited. . For example, from the time when the copper-containing substance is added to the reagent satisfying the requirement (d), at least at 1 ° C. to 10 ° C. in the state where the copper-containing substance coexists in the reagent satisfying the requirement (d). 2 weeks, at least 1 week at 11 ° C to 25 ° C, at least 2 days at 26 ° C to 40 ° C, at least 5 hours at 41 ° C to 60 ° C, at least 1 hour at 61 ° C to 80 ° C Can be obtained. Therefore, even if the temperature is not strictly controlled, the effect of the present invention can be obtained if the copper-containing substance is reacted after being stored for about one month or longer. In the present invention, it is preferable to use a reagent that satisfies the above-mentioned requirement (d) after the 4-hydroxyantipyrine and the copper-containing substance are allowed to coexist for a period as described above. In addition, said reaction may be performed during a stock storage and a distribution | circulation stage after manufacture.

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

As a specific embodiment of the present invention, for example, when the reagent satisfying the requirement (d) is a product reagent (for example, a reagent containing a protein such as an enzyme), a copper-containing substance is added to the product reagent. After that, the reaction proceeds by refrigeration or storage near room temperature in the coexistence of a copper-containing substance, and storage before use, so that the reagent that satisfies the requirement (d) before use What is necessary is just to use it, after at least 2 weeks have passed since the date of manufacture of the biological component measurement reagent kit manufactured by adding a copper-containing substance.

As another specific embodiment of the present invention, a reagent that satisfies the requirement (d) is an intermediate reagent (for example, an aminoantipyrine compound having a concentration several to several tens of times that of a product reagent as a buffer component). In the case of an adjusted reagent), after adding a copper-containing substance to the intermediate reagent, for example, store it for several days at a temperature condition of 30 ° C. or more, and for several hours at a temperature condition of 45 ° C. or more. Thus, a product reagent may be prepared.

In addition, when adding a copper-containing substance to the intermediate reagent, at the stage of preparing the product reagent, a copper-containing substance (copper ions that can be generated in the specific embodiment) coexisting in the reagent that satisfies the requirement of (d) is added. Except for the product reagent.

The present inventors have found that such a decrease in measurement sensitivity caused by 4-hydroxyantipyrine can be suppressed in advance by coexisting a copper-containing substance with 4-hydroxyantipyrine. Although the mechanism by which the copper-containing substance suppresses the sensitivity decrease due to 4-hydroxyantipyrine is not clear, the present inventors have caused some structural change of 4-hydroxyantipyrine due to the copper-containing substance (for example, copper ions that can be generated in the future). It is assumed that hydrogen peroxide is not consumed without reacting with peroxidase.

In this regard, 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 the copper-containing substance is The present inventors presume that the reaction between 4-hydroxyantipyrine and peroxidase will not be restored even if the copper-containing material (copper ions that can be generated in the specific embodiment) is removed from the medium.

In the method for suppressing a decrease in measurement sensitivity of a biological component of the present invention, the concentration of the copper-containing substance coexisting in the reagent satisfying the requirement (d) is preferably 0.00001 to 1 mM, and 0.00003 to 0.00. 3 mM is more preferable, 0.0001 to 0.1 mM is more preferable, and 0.0001 to 0.01 mM is particularly preferable.

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

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

As a method for measuring the concentration of copper ions that can be generated from a copper-containing substance, ICP emission spectroscopy (ICP-AES), ICP mass spectrometry (ICP-MS), and the like can be suitably employed. Specifically, in the present invention, the concentration of copper ions can be measured by ICP-AES (using SPECTROBLUE manufactured by Ametech). When the detection limit is below the ICP-AES, it can be measured by ICP-MS (using Agilent 7700s ICP-MS manufactured by Agilent Technologies).

In the method for suppressing a decrease in measurement sensitivity of a biological component of the present invention, the concentration of 4-hydroxyantipyrine present in the reagent that satisfies the requirement (d) is added with a copper-containing substance (for example, a component that generates copper ions). When the concentration is about 0.1 to 50 μg / ml, the effect is particularly easily obtained.

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

When the concentration of 4-hydroxyantipyrine present in the reagent that satisfies the requirement (d) before adding the copper-containing substance is more than 50 μg / ml, the decrease in measurement sensitivity of biological components due to 4-hydroxyantipyrine is suppressed. In order to achieve this, a high concentration of copper-containing material is required, and therefore, color development tends to increase due to side reactions caused by the copper-containing material, which tends to cause measurement errors.

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

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

(Biological components)

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 for measurement of various biological components. For example, the biological components used in the biological component measurement of the present invention are uric acid (UA), creatinine (CRE), triglyceride (TG), cholesterol (CHO), AST (GOT), ALT (GPT), LDH (lactic acid dehydrogenation). Enzymes) and isozymes, ALP (alkaline phosphatase) and isozymes, CK (creatine kinase) and isozymes, amylases (Amy) and isozymes, lipases, γ-GTP (γ-glutamyl transpeptidase), cholinesterase (ChE), sodium (Na) , Potassium (K), chlor (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 (U ), Bilirubin (Bil), ammonia, cholesterol, HDL cholesterol (HDL-C, high density lipoprotein cholesterol), LDL cholesterol (LDL-C, low density lipoprotein cholesterol), neutral fat (triglyceride) (TG), cholesterol (CHO), BTR (BTR, total branched chain amino acid / tyrosine ratio), tyrosine measuring reagent (TYR), blood glucose (BS, GLU), 1,5-anhydro-D-glucitol (1,5-AG), glycated albumin (GA), glycated hemoglobin (HbA1c) and the like can be mentioned, but are not limited thereto.

These biological components can be reacted with any oxidase and, if necessary, other enzymes (for example, hydrolases) to generate hydrogen peroxide. For example, specific aspects of biological component measurement will be described below for uric acid (UA), creatinine (CRE), triglyceride (TG), and glycated hemoglobin (HbA1c).

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

When measuring creatinine (CRE), hydrogen peroxide is not directly generated in the reaction of creatinine amidinohydrolase using creatinine (CRE) as a substrate. Therefore, creatine obtained by adding creatine generated in the reaction of creatinine amidinohydrolase to the reagent in advance is used. Peroxidase-coloring agent system by designing a so-called conjugation reaction in which sarcosine is generated by reacting with amide hydrolase, and hydrogen peroxide is generated using sarcosine oxidase (oxidase) with sarcosine added to the reagent in advance. Quantification of creatinine (CRE) concentration by means of

When triglyceride (TG) is measured, hydrogen peroxide is generated by using lipoprotein lipase using triglyceride (TG) as a substrate and glycerol kinase and glycerol triphosphate oxidase (oxidase) as conjugate enzymes. The triglyceride (TG) concentration can be quantified using a peroxidase-color former system.

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

Thus, even if there is no suitable enzyme that catalyzes the reaction that directly oxidizes the measurement target to generate hydrogen peroxide, the reaction that can change the measurement target to the substrate of oxidase that can generate hydrogen oxide. It is also possible to measure the concentration or amount of biological components other than the above by appropriately designing a conjugation reaction that combines an enzyme to be catalyzed (which may connect several stages of enzyme reaction) and the oxidase. It 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 above-described biological components, creatinine (CRE) and glycated hemoglobin (HbA1c) have extremely low biological component content, and therefore, particularly sensitive measurement is required. According to the present invention, the influence of 4-hydroxyantipyrine, which inhibits the reaction of the oxidase-peroxidase-color former system, can be suppressed and the decrease in sensitivity of biological component measurement can be suppressed, and thus high sensitivity measurement is required. Useful for measuring biological components. Therefore, the present invention is suitable for measurement of creatinine and glycated hemoglobin, and in particular, it is suitably used for measurement of creatinine.

(Biological component measurement kit)

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

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

One reagent that satisfies the two requirements simultaneously for the reagent that satisfies the requirements (d) and (e) generates a copper-containing substance (for example, copper ions) in the reagent that satisfies the requirement (d) as described above. And the like.

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

The present inventors presume that 4-hydroxyantipyrine reacts with peroxidase, and as a result, hydrogen peroxide is consumed. The reactivity of 4-hydroxyantipyrine is determined by adding a copper-containing substance to 4-hydroxyantipyrine. It discovered in this invention that it can suppress by adding.

The reaction rate of the suppression reaction may affect the reaction temperature and reaction time. However, under the normal storage conditions of the biological component measurement kit, the suppression effect is produced when the storage period is several weeks to one month or more. It is preferable that at least one month has passed since the biological component measurement kit of the present invention.

In general, such a measurement kit is considered to take one month to several months after being manufactured and used through an inventory / distribution process, during which the suppression reaction proceeds and the effect of the present invention is exhibited. It will be.

(Biological component measurement method)

The biological component measuring method of the present invention is characterized by measuring biological components using the aforementioned biological component measuring kit.

(Method for producing biological component measurement kit)

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

(A) An oxidase capable of generating hydrogen peroxide is included.

(B) Contains peroxidase.

(C) A redox coloring reagent that reacts with hydrogen peroxide in the presence of peroxidase to develop color.

(D) An aminoantipyrine compound is included as a coupler of the redox coloring reagent.

(E) Contains a copper-containing material.

Here, it is preferable that the reagent that satisfies the requirements (d) and (e) is preferably manufactured as one reagent that simultaneously satisfies the two requirements, and that the biological component derived from 4-hydroxyantipyrine is measured. The reason why it is preferable that a sufficient period (for example, about one month or more) has passed to suppress the decrease in sensitivity is as described above.

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

As described above, the biological component measurement kit of the present invention needs to suppress the reactivity of 4-hydroxyantipyrine by adding a copper-containing substance to 4-hydroxyantipyrine. ) May be carried out by allowing a copper-containing substance to coexist with 4-hydroxyantipyrine mixed in a reagent that satisfies the requirements of (1), but this suppression reaction is not necessarily carried out in the product reagent. For example, an aminoantipyrine compound is added. In the process of preparing the reagent to be contained, after adding a copper-containing substance in a state such as an intermediate reagent and then performing an inhibition reaction, a product reagent may be produced using the intermediate reagent and provided as a biological component measurement kit.

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

The biological component measurement kit of the present invention, when adding a copper-containing substance to an intermediate reagent containing an aminoantipyrine compound and performing an inhibitory reaction, adds the copper-containing substance to the intermediate reagent and then ends the product production date. The sum of the period and the period from product manufacture date to product use date is preferably at least one month.

The copper-containing substance of the present invention (for example, a component that generates copper ions) is allowed to coexist with 4-hydroxyantipyrine mixed as an impurity of an aminoantipyrine compound, thereby measuring the biological component attributed to 4-hydroxyantipyrine. Presumed to suppress the decrease.

Although it is not clear why the coexistence of the copper-containing substance of the present invention with 4-hydroxyantipyrine suppresses a decrease in the sensitivity of biological component measurement, there are 4 copper-containing substances (for example, copper ions that can be generated from this). -Directly or indirectly acting on hydroxyantipyrine causes some change in 4-hydroxyantipyrine, resulting in reduced consumption of hydrogen peroxide by reacting 4-hydroxyantipyrine with peroxidase, It is considered that the decrease in sensitivity is suppressed. Here, the change of 4-hydroxyantipyrine is expected to be irreversible, and once denatured and rendered harmless, 4-hydroxyantipyrine does not cause a decrease in sensitivity under normal biological component measurement kit storage conditions. I guess it is not.

For this reason, when a copper-containing substance is added to the intermediate reagent, the product reagent is produced by removing the copper-containing substance (in a specific embodiment, copper ions that can be generated in the future) in the stage of producing the product reagent thereafter. Although it is considered possible, a biological component measurement reagent kit manufactured using such a product reagent and its manufacturing method are also within the scope of the present invention.

The copper-containing substance used in the present invention is preferably a component that generates copper ions, and is not particularly limited. Examples thereof include copper salts containing copper ions, copper proteins containing copper ions as prosthetic factors, and pigments containing copper ions. Can do. For example, as a component that generates copper ions, CuBr, CuBr ₂ , CuC ₂ , Cu ₂ C ₂ , CuCl, CuCl ₂ , CuF, CuF ₂ , CuH, CuI, CuI ₂ , CuN ₃ , Cu (N ₃ ) ₂ , _{_{_{_{CuO, CuO 2, Cu 2 O}}}} , Cu 3 P, CuP 2, CuS, Cu 2 S, Cu 3 (AsO 4) 2, Cu (BF 4) 2, Cu (ClO 3) 2, Cu (ClO 4) 2 , CuCN, Cu (CN) ₂ , CuCrO ₄ , Cu (IO ₃ ) ₂ , Cu (IO ₄ ) ₂ , Cu (NO ₃ ) ₂ , CuOH, Cu (OH) ₂ , Cu ₃ (PO ₄ ) ₂ , CuSO ₄ , Cu ₂ SO ₄ , Cu (CH ₃ COO), Cu (CH ₃ COO) ₂ , CuCO _{3 ·} Cu (OH) ₂ , CuSCN, Cu (SCN) _{2 and} other copper salts; 200 containing a copper salt such as 200 (5-chloro-2-methyl-4-isothiazolin-3-one, 2-methyl-4-isothiazolin-3-one, magnesium salt, copper salt, preservative containing water); Ascorbate oxidase (also called ascorbate oxidase), azurin, stellacyanin, plastocyanin, quercetin-2,3-dioxygenase, rustycyanin, pseudoazurin, laccase, copper-containing nitrite reductase, superoxide dismutase, galactose oxidation Enzyme, dopamine-β-hydroxylase, dopamine-β-monooxygenase, peptidylglycine α-amidating monooxygenase, membrane-bound methane monooxygenase, copper amine oxidase, galactose oxidase, tyrosinase, catechol oxidase , Ceruloplasmin, cytochrome c oxidase, hemocyanin, zinc nitric reductase, copper and the like _{_{proteins; CuCO 3 · Cu (OH)}} 2 ( _{_{patina), CuSO 4 · 3Cu (OH}} ) 2 (Brochantite), Cu 2 (OH ) ₃ Cl (Atacamite), Cu ₂ (OH) ₃ Cl (Paratacamite), Cu (C ₂ H ₃ O ₂ ) ₂ · 3Cu (AsO ₂ ) ₂ (flower patina), CuCO ₃ · Cu (OH) ₂ (Malachite) _{_{_{_{), (Cu, Zn) 2}}}} (CO 3) (OH) 2 (Rosasite), (Zn, Cu) 2 (CO3) (OH) 2 (Zincrosasite), (Zn, Cu) 2 (AsO 4) (OH) _{_{_{2 (Adamite), (Cu,}}} Zn) 6 (AsO 4, PO 4) 2 (OH) 6 · H 2 O (P And pigment or the like containing copper ions Ilipsburgite), etc., but not limited thereto.

These components that generate copper ions can be appropriately selected according to, for example, a biological component or a biological sample to be measured. From the viewpoint of excellent handling convenience and the expectation that even higher effects can be exhibited, copper chloride (I), copper chloride (II), copper acetate, copper sulfate, copper nitrate, proclin 200, ascorbic acid oxidation It is preferable to use an enzyme.

The usage-amount of the copper containing material used for this invention is not specifically limited as long as there exists an effect of this invention.

As described above, in the present invention, the copper-containing substance is preferably used as a product reagent mixed with an aminoantipyrine compound intermediate reagent or an aminoantipyrine compound, and as an example, the copper reagent in the intermediate reagent or product reagent The concentration of the contained substance may be adjusted so as to be 0.00001 to 1 mM, and the concentration of the copper-containing substance in the reagent intermediate or reagent is preferably 0.00003 to 0.3 mM, more preferably 0.8. The blending amount may be adjusted to 0001 to 0.1 mM, more preferably 0.0001 to 0.01 mM. By using the copper-containing substance in such an amount, it is possible to effectively suppress a decrease in sensitivity of the biological component measurement reagent kit caused by 4-hydroxyantipyrine.

The copper-containing substance used in the present invention has an effect of suppressing the decrease in the 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 elapse for a certain time after blending.

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

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

In the above, in the product life cycle of a general biological component measurement kit, it takes about several weeks or more than one month until it is blended with reagents in the production process and used for quality testing, shipping, distribution, storage and use. The measurement kit including the reagent in which the copper-containing substance of the present invention is coexistent with the aminoantipyrine compound is in a state where the sensitivity reduction suppressing effect by the copper-containing substance used in the present invention is expressed and maintained at the stage of use. However, when the product life cycle is shortened, it can be prepared by manufacturing process management, product management at the time of shipment, or the like.

(Oxidase)

As long as the oxidase used in the present invention can generate hydrogen peroxide from a substrate, it can be used without limitation depending on the target measurement target. Specific examples include, but are not limited to, uricase, sarcosine oxidase, glycerol triphosphate oxidase, fructosyl amino acid oxidase and the like. As commercially available products, UAO-211 (manufactured by Toyobo), SAO-351 (manufactured by Toyobo), G3O-311 (manufactured by Toyobo) and the like are preferably used. 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. For example, peroxidases derived from plants, bacteria, and basidiomycetes Is mentioned. Among these, horseradish, rice, soybean-derived peroxidase is preferable, and horseradish-derived peroxidase is more preferable because of purity, availability, and price. PEO-131 (Toyobo), PEO-301 (Toyobo), PEO-302 (Toyobo) and the like are preferably used as commercially available products. There are no particular restrictions on the amount used or the form of addition.

Peroxidase activity is defined by the following method.

14 mL of distilled water, 2 mL of 5% (W / V) pyrogallol aqueous solution, 1 mL of 0.147M hydrogen peroxide solution and 2 mL of 100 mM phosphate buffer solution (pH 6.0) were sequentially mixed, and then pre-conditioned at 20 ° C. for 5 minutes. Then, 1 mL of the sample solution is added to start the enzyme reaction.

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

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

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

About this liquid, ether extraction is performed in the same manner as described 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 follows.

Peroxidase activity (U / mL) = {ΔOD (OD _test −OD _blank ) × dilution factor} / {0.117 × 1 (mL)) = ΔOD × 8.547 × dilution factor

Peroxidase activity (U / mg) = peroxidase activity (U / mL) × 1 / C

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

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

(One propargalin unit corresponds to 13.5 international units (with o-dianisidine as a substrate and reaction conditions at 25 ° C.).)

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

(Redox coloring reagent)

As the redox coloring reagent used in the measurement of the biological component of the present invention, any kind of dye may be used as long as it reacts with hydrogen peroxide and develops color. For example, a combination of a hydrogen donor and a coupler is used. Can be mentioned. There are no particular restrictions on the amount used or the form of addition. All of these can be obtained as commercial products.

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

(Hydrogen donor)

In the biological component measurement method of the present invention, phenol, a phenol derivative, an aniline derivative, naphthol, a naphthol derivative, a naphthylamine, a naphthylamine derivative, or the like is used as a hydrogen donor used in the Trinder method or 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-sulfop Pill) -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-methylphenyl) -N'-succinylethylenediamine, N-ethyl-N- (3-methylphenyl) -N'-acetylethylenediamine, and the like.

(Coupler)

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

Examples of couplers include 4-aminoantipyrine (4AA), aminoantipyrine compounds such as aminoantipyrine derivatives; vanillin diamine sulfonic acid compounds such as vanillin diamine sulfonic acid; methylbenzthiazolinone hydrazone (MBTH), sulfonated methylbenzthia. Examples thereof include methylbenzthiazolinone hydrazone compounds such as zolinone hydrazone (SMBTH).

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

The coupler used in the present invention may be two or more aminoantipyrine compounds, or may be used in combination with another coupler in addition to the aminoantipyrine compound, but preferably one aminoantipyrine compound. It is preferable to use 4-aminoantipyrine.

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

When the concentration of 4-hydroxyantipyrine is lower than the above range, there is little influence of a decrease in measurement sensitivity of biological components due to 4-hydroxyantipyrine, and there is little need to suppress a decrease in measurement sensitivity due to the coexistence of a copper-containing substance.

When the concentration of 4-hydroxyantipyrine exceeds the above range, a high concentration of copper-containing material is required to suppress a decrease in measurement sensitivity of biological components due to 4-hydroxyantipyrine. This tends to reduce the measurement accuracy of biological components.

If the amount of 4-hydroxyantipyrine mixed in is large, remove 4-hydroxyantipyrine mixed in the aminoantipyrine compound in advance by a method for removing 4-hydroxyantipyrine, which will be described later. Is preferred.

In consideration of the amount of 4-hydroxyantipyrine mixed in a general aminoantipyrine compound (4-aminoantipyrine), the present invention, as an example, has an aminoantipyrine compound (4-aminoantipyrine) of about 0.01 to 100 g / l. A reagent containing, preferably about 0.01 to 10 g / l of an aminoantipyrine compound (4-aminoantipyrine), more preferably about 0.01 to 5 g / l of an aminoantipyrine compound (4-aminoantipyrine) It is effective to use for a reagent containing. By using an aminoantipyrine compound in such a range, the effects of the present invention can be more effectively exhibited.

(Quantitative method for 4-hydroxyantipyrine)

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

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

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

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

(3) Gradient conditions

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

(4) Flow rate 0.2mL / min

(5) Column temperature 40 ° C

(6) Sample injection volume 5μL

(7) Detection wavelength UV250nm

4HA (Sigma) having a known concentration was used as a standard product, and the coincidence of the detection position of the HPLC fraction peak with the measurement sample was confirmed. Moreover, it quantified by comparing the area of the fraction peak with a measurement sample. As Sigma's 4HA (4-Hydroxyantipyrine), Cas. NO. 1672-63-5, product number 109428-5G, 99% purity product was used.

In the present invention, the above method is preferably used as a method for quantifying 4-hydroxyantipyrine.

(4-hydroxyantipyrine)

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

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

In the biological component measurement reagent kit of the present invention, an aminoantipyrine compound drug substance that can contain 4-hydroxyantipyrine may be used as it is, or an aminoantipyrine compound drug substance that has a low 4-hydroxyantipyrine content. May be used by selecting the amount of 4-hydroxyantipyrine content reduced by removing 4-hydroxyantipyrine from the aminoantipyrine compound drug substance. .

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

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

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

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

The chromatographic mobile phase may be water and a water-soluble solvent such as methanol or acetonitrile. In order to avoid ionic interaction with the silanol groups of silica gel, the pH of the chromatographic mobile phase should be acidic. A small amount of preparation or ion pair reagent may be added to the side.

The mobile phase flow rate may be optimized according to the capacity of the system used. Further, elution may be performed stepwise instead of using a linear gradient.

(Other ingredients)

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

Examples of the buffer solution component that can be contained in the biological component measurement reagent of the present invention include Tris buffer solution, phosphate buffer solution, borate buffer solution, carbonate buffer solution, and GOOD buffer solution. There are no particular limitations on the amount used, the set pH, the form of addition, and the like. All of these can be obtained as commercial 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-hydro Cyethyl) 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) iminoniacetic acid (ADA), N, N-bis (2 -Hydroxyethyl) glycine (Bicine), N- [Tris (hydro Shimechiru) methyl] glycine (Tricine), etc. are exemplified.

In the biological component measurement reagent of the present invention, there are no particular limitations on the amount used or the form of addition of ascorbic acid oxidase, preservatives, salts, enzyme stabilizers, chromogen stabilizers, and the like. All of these can be obtained as commercial products.

Examples of preservatives include Procrine 150, Procrine 200, Procrine 300, Procrine 950, azide, chelating agent, antibiotic, antibacterial agent and the like.

Examples of the chelating agent include ethylenediaminetetraacetic acid and its salt.

Antibiotics include gentamicin, kanamycin, chloramphenicol and the like.

Examples of antibacterial agents include methyl isothiazolinone and imidazolidinyl urea.

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

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

Examples of chromogen stabilizers include chelating agents such as ethylenediaminetetraacetic acid and its salts, and cyclodextrins.

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

(Measurement method using biological component measurement reagent kit)

When measuring biological components 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 configured to be applicable to such an automatic analyzer. The mode is not particularly limited. For example, a kit composed of a liquid reagent, a kit composed of a combination of a dry reagent and a lysate produced by means such as lyophilization, an enzyme or the like supported on an appropriate carrier. Various forms such as a kit called a so-called dry system and a kit using a sensor can be exemplified.

As a configuration of the biological component measurement kit of the present invention, a kit composed of one reagent or a kit composed of 2 to 3 or more by packaging the reagent is used. When a reagent is 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 of the redox coloring reagent. The kit may be configured by being packaged into a reagent containing 4-aminoantipyrine as a coupler among (b) peroxidase and (d) redox coloring dye.

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

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 allowed to react for a certain period of time, and then the second type. The target component can be quantified by further adding and reacting the reagent (hereinafter also referred to as the second reagent or R2) and measuring the change in absorbance during this period.

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

(Biological component measurement method)

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

(1) a step of generating hydrogen peroxide by causing an oxidase to act on a biological component;

(2) The hydrogen peroxide generated in step (1) reacts with peroxidase in the presence of peroxidase, whereby the reaction solution is colored by oxidative condensation of 4-aminoantipyrine and redox coloring reagent. Process,

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

The biological component measurement method of the present invention is a biological component measurement method by an enzyme method, particularly a method using an oxidase-peroxidase-color former system, that is, the amount of biological component by enzymatic reaction of the biological component in the specimen. The measurement principle is to perform colorimetric determination of the color produced by generating hydrogen peroxide according to the above and reacting it with a color former in the presence of peroxidase.

Biological component measurement methods using this principle have already been established in the art. Therefore, the knowledge can be applied to the present invention to measure the amount or concentration of a biological component in various samples, and the mode is not particularly limited.

The present inventors have found that 4-hydroxyantipyrine contained in a trace amount in an active ingredient of an aminoantipyrine compound (especially 4-aminoantipyrine) inhibits the reaction of this oxidase-peroxidase-color-developer system. We found for the first time that it caused a decline. The mechanism by which 4-hydroxyantipyrine inhibits the above reaction is not necessarily clear, but its structure causes a redox coloring reagent and 4-hydroxyantipyrine to undergo a condensation reaction in the presence of hydrogen peroxide by the action of peroxidase. It is guessed that it consumes. Then, the present inventors have found that such reaction inhibition caused by 4-hydroxyantipyrine can be suppressed in advance by reacting 4-hydroxyantipyrine in the presence of a copper-containing substance, and sensitivity reduction can be suppressed. . The mechanism by which the copper-containing substance suppresses the decrease in sensitivity due to 4-hydroxyantipyrine is not clear, but 4-hydroxyantipyrine undergoes some structural change due to the copper-containing substance (for example, copper ions that can be generated in the future) and reacts with peroxidase. It is assumed that it becomes difficult and does not consume hydrogen peroxide.

(Sample)

Examples of the specimen containing a biological component used for the biological component measurement of the present invention include biological fluids such as blood (particularly serum and plasma), urine, ascites, and cerebrospinal fluid, and people such as beverages and foods. Ingestion and so on. In particular, it is preferable to use human body fluid (a sample derived from blood such as serum or plasma, a sample derived from urine, or the like) as a specimen to be measured.

(Detection sensitivity of biological component measurement reagent kit)

The present invention uses a copper-containing substance, and in comparison with the case where no copper-containing substance is used, the detection sensitivity due to 4-hydroxyantipyrine in the measurement of biological components by an enzyme method using an oxidase-peroxidase-color former system. Can be suppressed.

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

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

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

Therefore, if there is an absorbance of 0.296 mABS, which is the maximum value of 2.6σ, that is, an absorbance of about 0.3 mABS 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 a copper-containing substance, it is possible to suppress a decrease in sensitivity of the biological component measurement reagent kit caused by 4-hydroxyantipyrine and increase detection sensitivity to the above level.

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

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

Example 1 Separation of 4HA by HPLC

Since it was found that the degree of decrease in reagent sensitivity was due to the difference in lots of 4AA, it was assumed that the content of impurities contained in trace amounts in 4AA differed from lot to lot, and impurities were determined by the aforementioned 4HA quantitative method (HPLC method). Was detected. The purity of 4AA used for examination of lot difference was 98.0% or more according to JIS-K8048.

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

(Example 2) Identification of 4HA

Examining the contents of each lot for the three types of impurities (a, b, c) seen in FIG. 1 of Example 1, it was found that the contents of only the impurities b differ greatly from lot to lot. Therefore, the molecular weight and structure of these three kinds of substances were analyzed by mass spectrometry (MS spectrum method).

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

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

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

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

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

[Preparation of reagents]

A creatinine measuring reagent having the following composition was prepared. Here, as 4-aminoantipyrine, commercially available 4-aminoantipyrine raw material was purified to produce 4-aminoantipyrine free of 4-hydroxyantipyrine and used.

First reagent

PIPES-NaOH 50 mM pH 7.4

Ascorbate oxidase (Toyobo ASO-311) 3 U / mL

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

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

Catalase (Toyobo CAO-509) 130 U / mL

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

Second reagent

PIPES-NaOH 50 mM pH 7.4

Creatinine amide hydrolase (Toyobo CNH-311) 400 U / mL

Peroxidase (Toyobo PEO-302) 10 U / mL

4-Aminoantipyrine 0.6g / L

[Measurement method]

A Hitachi 7180 automatic analyzer was used. 120 μL of the first reagent was added to 2.7 μL of the sample and incubated at 37 ° C. for 5 minutes to prepare the first reaction. Thereafter, 40 μL of the second reagent was added and incubated for 5 minutes to form a second reaction. The absorbance at 546 nm (major wavelength) and the absorbance at 800 nm (subwavelength) were measured by a two-point end method that takes the difference between the absorbances obtained by correcting the absorbances of the first reaction and the second reaction. The absorbance was calculated by subtracting the sub wavelength from the main wavelength.

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

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

(Example 4) Sensitivity reduction suppression effect by copper salt

About the copper salt, the inhibitory effect of the sensitivity fall resulting from 4HA was confirmed.

The experimental conditions were as follows. For the reagents other than the 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. The process was performed under the same conditions as in Example 3 except that.

Second reagent stored for 3 days under the respective refrigeration conditions (6 ° C) and accelerated test (35 ° C) temperature conditions after adding various copper salts to the final concentration in the second reagent of the specified creatinine measurement reagent Was used to examine measurement sensitivity (mABS). The 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 STD sensitivity. 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.

The results are shown in Table 2.

From this result, it was confirmed that the decrease in sensitivity due to 4HA can be effectively suppressed by coexisting various copper salts.

That is, the measurement sensitivity of the control (b) to which 4HA was added to 10 μg / ml was 32% for 3 days of refrigeration and 45% for 3 days at 35 ° C. The measurement sensitivity was 75% to 100% after 3 days of refrigeration, and 71% to 100% after 3 days at 35 ° C. From this result, it can be seen that, as a general tendency, storage at 35 ° C. is more stable and a higher sensitivity reduction suppressing effect can be obtained more easily than storage under refrigerated conditions. Since the evaluation at 35 ° C. corresponds to an accelerated test under refrigerated storage conditions, the sensitivity decrease due to 4-hydroxyantipyrine tends to be more effectively suppressed by reacting various copper salts for a longer period of time. It was estimated that there was.

In addition, in the example in which 0.1 mM of copper chloride (II) in Experiment No. 3 was added, color development presumed to depend on copper (II) chloride was observed, and in the case of using a high concentration of copper salt (II), It was thought that the attention of was needed.

(Example 5) Inhibition of sensitivity reduction by a mixture containing copper protein / copper salt

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

The experimental conditions were as follows. For the reagents other than the 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. The process was performed under the same conditions as in Example 3 except that.

Second reagent stored for 3 days under the temperature conditions of the accelerated test (35 ° C) after adding ASO-311 and Procrine 200 to the second reagent of the creatinine measurement reagent so as to have a final concentration in the predetermined second reagent Was used to examine measurement sensitivity (mABS). The 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 STD sensitivity. 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.

The results are shown in Table 3.

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

That is, the measurement sensitivity of the control (b) to which 4HA was added to 10 μg / ml was 41% at 35 ° C. for 3 days, whereas the measurement sensitivity was 35 ° C. 3 in the result using ASO-311. The measurement sensitivity was 96% to 98% at 35 ° C. for 3 days.

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

The copper salt and the concentration dependency of 4HA were confirmed with respect to the effect of suppressing the decrease in sensitivity due to 4HA.

The experimental conditions were as follows. For reagents other than the control (a), 4AA was used as the second reagent in the creatinine measurement reagent as the final concentration in the second reagent, and 600 μg / ml, 300 μg / ml, 100 μg / ml, and 4HA in the second reagent. The final conditions were 10 μg / ml, 5 μg / ml, and 1.6 μg / ml, and a 5 mg / dL creatinine aqueous solution was used as a biological component sample.

After adding various copper salts to the second reagent of the creatinine measurement reagent so as to have a final concentration in the predetermined second reagent, each was stored for 3 days under the temperature condition of the accelerated test (35 ° C.). The measurement sensitivity (mABS) was examined. The blank value of each reagent was also measured at the same time, and a value obtained by subtracting the blank value from the measurement sensitivity was calculated as STD sensitivity. Then, the ratio [vs control (%)] 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 was calculated.

The results are shown in Tables 4-6.

From this result, although the suppression effect of the sensitivity fall resulting from 4HA by coexisting a copper salt has a copper ion concentration and 4HA density | concentration dependence, in a wide copper ion concentration range, with respect to the sensitivity fall resulting from 4HA. It was confirmed that it can be effectively suppressed.

That is, for both copper chlorides (I) and (II), the effect of suppressing the decrease in sensitivity due to 4HA was observed in the concentration range of 0.00001 mM to 0.01 mM. However, when the 4HA concentration was high, it was recognized that the effect of suppressing the decrease in sensitivity due to 4HA was somewhat weak.

In addition, copper chloride (I) and copper chloride (II) showed the same tendency in the inhibitory effect of the sensitivity fall resulting from 4HA, and the big difference by the valence of a copper ion was not recognized.

The present invention can be applied to a measurement method for measuring a biological component using an oxidation-reduction reaction, and a reagent or composition used in the method.

Claims

A measurement sensitivity reduction inhibitor applied to a biological component measurement method using an aminoantipyrine compound as a coupler for a redox coloring reagent, characterized by containing a copper-containing substance, .
The biological component measurement method is a biological component measurement method using a reagent or a reagent set that satisfies the following requirements (a) to (d), and is used by being added to a reagent that satisfies the requirement (d): The biological component measurement sensitivity decrease inhibitor according to claim 1, wherein

(A) An oxidase capable of generating hydrogen peroxide is included.

(B) Contains peroxidase.

(C) A redox coloring reagent that reacts with hydrogen peroxide in the presence of peroxidase to develop color.

(D) An aminoantipyrine compound is included as a coupler of the redox coloring reagent.
The biological component measurement sensitivity decrease inhibitor according to claim 1 or 2, which suppresses a decrease in measurement sensitivity of the biological component caused by 4-hydroxyantipyrine.
A method for suppressing a decrease in measurement sensitivity of a biological component, characterized by using a copper-containing substance, and a method for suppressing a decrease in measurement sensitivity applied to a biological component measurement method using an aminoantipyrine compound as a coupler for a redox coloring reagent. .
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 a copper-containing substance is added to the reagent that satisfies the requirement (d) A method for suppressing a decrease in measurement sensitivity of a biological component, characterized in that:

(A) An oxidase capable of generating hydrogen peroxide is included.

(B) Contains peroxidase.

(C) A redox coloring reagent that reacts with hydrogen peroxide in the presence of peroxidase to develop color.

(D) An aminoantipyrine compound is included as a coupler of the redox coloring reagent.
6. The method for suppressing a decrease in measurement sensitivity of a biological component according to claim 4 or 5, wherein a decrease in the measurement sensitivity of the biological component due to 4-hydroxyantipyrine is suppressed.
The method for suppressing a decrease in measurement sensitivity of a biological component according to claim 5 or 6, wherein the concentration of the copper-containing substance coexisting in the reagent satisfying the requirement (d) is 0.00001 to 1 mM. .
The method for suppressing a decrease in measurement sensitivity of a biological component according to any one of claims 4 to 7, wherein the biological component is either creatinine or glycated hemoglobin.
A biological component measurement reagent kit using an aminoantipyrine compound as a coupler for a redox coloring reagent, comprising the measurement sensitivity reduction inhibitor according to claim 1.
A biological component measurement reagent kit that satisfies the following requirements (a) to (e), wherein the reagent that satisfies the requirements (d) and (e) is a single reagent that simultaneously satisfies the two requirements: A biological component measurement reagent kit.

(A) An oxidase capable of generating hydrogen peroxide is included.

(B) Contains peroxidase.

(C) A redox coloring reagent that reacts with hydrogen peroxide in the presence of peroxidase to develop color.

(D) An aminoantipyrine compound is included as a coupler of the redox coloring reagent.

(E) Contains a copper-containing material.
11. The biological component measurement reagent kit according to claim 10, wherein one reagent that satisfies the two requirements (d) and (e) has passed at least one month after the production.
A biological component measurement method using the biological component measurement kit according to claim 9.
A method for producing a biological component measurement reagent kit that satisfies the following requirements (a) to (e) and suppresses a decrease in sensitivity due to 4-hydroxyantipyrine, which satisfies the requirements (d) and (e): A method for producing a reagent kit for measuring a biological component, wherein the reagent is produced as one reagent that simultaneously satisfies two requirements.

(A) An oxidase capable of generating hydrogen peroxide is included.

(B) Contains peroxidase.

(C) A redox coloring reagent that reacts with hydrogen peroxide in the presence of peroxidase to develop color.

(D) An aminoantipyrine compound is included as a coupler of the redox coloring reagent.

(E) Contains a copper-containing material.
The reagent that satisfies the two requirements simultaneously for the reagent that satisfies the requirements of (d) and (e) is an intermediate reagent in the manufacturing process of the biological component measurement reagent kit. A method for producing a biological component measurement reagent kit.