JP2019195300A - Biogenic substance measurement kit and method in which measurement sensitivity is improved - Google Patents

Abstract

To provide a measurement kit and measurement method of a biogenic substance capable of achieving stable measurement accuracy without reducing sensitivity, in measurement of a biogenic substance which is performed using color reaction generated by oxidation condensation of 4-amino antipyrine and hydrogen donator in coexistence of hydrogen peroxide generated by making an oxidation enzyme act to a biogenic substance and peroxidase.SOLUTION: There are provided a measurement kit and a measurement method of biogenic substance, in the measurement kit of biogenic substance, hydrogen peroxide generated by making an oxidation enzyme act to a biogenic substance performs oxidation condensation of 4-amino antipyrine and hydrogen donator in coexistence of peroxidase, therefore color reaction of a reaction liquid is generated, the color reaction is subjected to colorimetric determination. In the measurement kit and measurement method, a density of 4-hydroxy antipyrine in the reaction liquid is equal to or smaller than 1.5 μg/ml.SELECTED DRAWING: None

Description

  The present invention relates to a composition for measuring a biological component and a method for analyzing the biological component using the composition in clinical diagnosis. More specifically, the coloration of the reaction solution produced by oxidative condensation of 4-aminoantipyrine and hydrogen donor in the coexistence of peroxidase with hydrogen peroxide generated by the action of oxidase on biological components is measured by colorimetric determination. The present invention relates to a composition to be used and a kit used in a method for analyzing a biological component using the composition.

  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 color former) system, that is, a measurement target in a specimen. There has been widely used a method for 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. (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.

  In addition, a method for analyzing and quantifying 4-aminoantipyrine, 4-hydroxyantipyrine, and the like in the blood and urine as metabolites in vivo of a drug by a HPLC method using a reverse phase column has been reported. (Non-Patent Documents 2 and 3)

BUNSEKI KAGAKU Vol. 45, no. 2, pp. 111-124 (1996) JOURNAL OF CHROMATOGRAPHY Vol. 375, pp. 91-99 (1986) ANALYST, JANUARY Vol. 107, pp. 61-70 (1982)

  The purpose of the present invention is to confirm the presence and amount of contaminants that reduce the measurement sensitivity of biological components in advance with respect to the measurement of biological components. The second is to prevent the production of a biological component measurement kit, and the second is to prevent measurement errors and measurement errors due to a decrease in measurement sensitivity without recognition when measuring biological components.

  In recent years, high-sensitivity measurement is required for some biological components, and three measurement kits and measurement methods that can provide stable measurement results even when the content of biological components is extremely small are provided. It is for the purpose.

  The present inventor has prepared an enzyme-peroxidase-coloring agent system biocomponent measurement reagent using 4-aminoantipyrine and a hydrogen donor, and experienced a decrease in measurement sensitivity of unknown cause when used for biocomponent measurement. did.

  Since the degree of decrease in measurement sensitivity varied depending on the lot of reagents prepared for measurement, the present inventor 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. When 4-hydroxyantipyrine is present in the reagent, structurally, 4-hydroxyantipyrine does not react with the hydrogen donor, but in the presence of hydrogen peroxide, 4-hydroxyantipyrine and peroxidase react to consume hydrogen peroxide. As a result, hydrogen peroxide generated by reacting the enzyme with the measurement target substance in the sample is consumed by 4-hydroxyantipyrine, and the original 4-aminoantipyrine-hydrogen donor It was thought that the amount of color developed by the reaction decreased and the sensitivity decreased. Then, by making the 4-hydroxyantipyrine concentration in the reagent below a certain level, it was found that a good measurement result of a biological component could be obtained without lowering from the designed measurement sensitivity level, and the present invention was completed. It was.

  That is, the present invention has the following configuration.

(Claim 1)
Biological component measurement kit for colorimetric determination of the color of the reaction solution produced by the oxidative condensation of 4-aminoantipyrine and hydrogen donor in the presence of peroxidase with hydrogen peroxide generated by the action of oxidase on the biological component A biological component measurement kit, wherein the concentration of 4-hydroxyantipyrine in the reaction solution is 1.5 μg / ml or less.

(Section 2)
The biological component measurement kit according to claim 1, wherein the concentration of 4-hydroxyantipyrine in the reaction solution is 0.9 μg / ml or less.

(Section 3)
Item 3. The biological component measurement kit according to Item 1 or 2, wherein the concentration of 4-hydroxyantipyrine in the reaction solution is 0.6 μg / ml or less.

(Claim 4)
The biological component measurement kit according to any one of Items 1 to 3, wherein the concentration of 4-hydroxyantipyrine in the reaction solution is 0.3 μg / ml or less.

(Section 5)
The biological component measuring kit according to any one of Items 1 to 4, wherein the biological component is either creatinine or glycated hemoglobin.

(Claim 6)
Item 6. The biological component measurement kit according to any one of Items 1 to 5, wherein the redox coloring reagent is a Trinder reagent.

(Claim 7)
Biological component measurement method for colorimetric determination of the color of a reaction solution produced by oxidative condensation of 4-aminoantipyrine and hydrogen donor in the presence of peroxidase with hydrogen peroxide generated by the action of an oxidase on a biological component And the concentration of 4-hydroxyantipyrine in the reaction solution is 1.5 μg / ml or less.

(Section 8)
The biological component measurement method according to item 7, wherein the concentration of 4-hydroxyantipyrine in the reaction solution is 0.9 μg / ml or less.

(Claim 9)
Item 9. The biological component measurement method according to Item 7 or 8, wherein the concentration of 4-hydroxyantipyrine in the reaction solution is 0.6 μg / ml or less.

(Section 10)
Item 10. The biological component measurement method according to any one of Items 7 to 9, wherein the concentration of 4-hydroxyantipyrine in the reaction solution is 0.3 µg / ml or less.

(Item 11)
The biological component measuring method according to any one of Items 7 to 10, wherein the biological component is either creatinine or glycated hemoglobin.

(Clause 12)
Item 12. The biological component measurement method according to any one of Items 7 to 11, wherein the redox coloring reagent is a Trinder reagent.

According to the present invention, it is possible to obtain a biological component measurement reagent without reducing the sensitivity from the designed level for each reagent production lot in the biological component measurement by an enzyme method using an oxidase-peroxidase-color former system. Good measurement was possible using the reagent.
In addition, stable measurement is possible even in the measurement of biological components that require high sensitivity, for example, the content of biological components is extremely small.

It is a figure which shows the chemical structural formula of 4-aminoantipyrine and 4-hydroxyantipyrine. It is a figure which shows the HPLC fraction at the time of analyzing 4-aminoantipyrine. It is a figure which shows the detection sensitivity of the determination method by the HPLC method of 4-hydroxyantipyrine. It is a figure which shows the fixed_quantity | quantitative_assay of the determination method by the HPLC method of 4-hydroxyantipyrine. It is a figure which shows the relationship between 4-hydroxyantipyrine density | concentration in a reaction liquid, and sample measurement sensitivity (mABS).

(Biological components)
Examples of biological components used in the measurement of biological components of the present invention include AST (GOT), ALT (GPT), LDH (lactate dehydrogenase) and isozymes, ALP (alkaline phosphatase) and isozymes, and CK (creatine kinase). 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 (UA), bilirubin (Bil), ammonia, cholesterol, HDL cholesterol (HDL C, also called high density lipoprotein cholesterol), LDL cholesterol (LDL-C, also called low density lipoprotein cholesterol), neutral fat (triglyceride) (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. It is done.
Among these biological components, creatinine (CRE) and glycated hemoglobin (HbA1c) are particularly preferably used because they require highly sensitive measurement.

(Sample)
Examples of the specimen containing a biological component used for the biological component measurement of the present invention include human body fluids such as blood (particularly serum and plasma), urine, ascites, and cerebrospinal fluid, and people such as beverages and foods. Ingestion and so on.

(Biological component measurement method)
The biological component measurement method of the present invention is not particularly limited as long as it 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 step in which the hydrogen peroxide generated in step (1) causes the reaction liquid to color by oxidative condensation of 4-aminoantipyrine and hydrogen donor by the action of peroxidase in the presence of peroxidase. ,
(3) A step of colorimetrically determining the reaction product colored in step (2).

The biological component measuring method of the present invention is a biological component measuring method by an enzyme method, particularly a method using an oxidase-peroxidase-color former system, that is, the amount of the biological component by reacting the biological component in the specimen with an enzyme. 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.

  Hereinafter, specific aspects of the biological component measuring method of the present invention will be described by taking uric acid (UA), creatinine (CRE), triglyceride (TG), and glycated hemoglobin (HbA1c) as examples.

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

  In the case of measuring creatinine (CRE), hydrogen peroxide is not directly generated in the reaction of creatinine amidinohydrolase using creatinine (CRE) as a substrate. Peroxidase-coloring agent system by designing a so-called conjugation reaction in which sarcosine is generated by reaction with amide hydrolase, and hydrogen peroxide is generated using sarcosine oxidase (oxidase) to which sarcosine has been added in advance. Quantitative determination of creatinine (CRE) concentration

  When measuring triglyceride (TG), 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 by a peroxidase-color former system.

  When glycated hemoglobin (HbA1c) is measured, hydrogen peroxide produced by the reaction of glycated hemoglobin oxidase (eg, fructosyl amino acid oxidase) using glycated hemoglobin as a substrate can be quantified in a peroxidase-color former 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, the reaction can change the measurement target to a substrate of oxidase that can generate hydrogen peroxide. It is also possible to measure the concentration or amount of biological components other than those described above by appropriately designing a conjugation reaction in which an enzyme that catalyzes the reaction (may be linked to several stages of enzyme reaction) and the oxidase. Is possible.

(Biological component measurement kit)
The biological component measurement kit of the present invention is not particularly limited as long as it satisfies the following requirements (a) to (d).
(A) It contains an oxidase capable of generating hydrogen peroxide by acting on a biological component.
(B) Contains peroxidase.
(C) It contains a redox coloring reagent that reacts with hydrogen peroxide in the presence of peroxidase to develop color.
(D) Contains 4-aminoantipyrine as a coupler for the redox coloring reagent.

  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.

  Examples of the constitution of the biological component measurement kit of the present invention include a kit composed of one reagent and a kit composed of 2 to 3 or more by packaging the reagent.

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

  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.

  The biological component measurement reagent of the present invention preferably contains a buffer component. In the biological component measurement reagent of the present invention, ascorbic acid oxidase, preservatives, salts, enzyme stabilizers, chromogen stabilizers and the like may be added within a range that does not affect the reaction.

  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.

(Oxidase)
As long as the oxidase used in the biological component measurement method of 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 uricase, sarcosine oxidase, glycerol triphosphate oxidase and the like as described above. As a commercial item, UAO-211 (made by Toyobo), SAO-351 (made by Toyobo), G3O-311 (made by Toyobo), etc. are used suitably. There are no particular restrictions on the amount used or the form of addition.

(Peroxidase)
The peroxidase used in the biological component measurement method of the present invention may be any kind of enzyme as long as it catalyzes the reaction between hydrogen peroxide and a redox coloring reagent, for example, plant-derived or bacterial-derived. And peroxidases derived from basidiomycetes. Among these, horseradish, rice, soybean-derived peroxidase is preferable, and horseradish-derived peroxidase is more preferable because of purity, availability, and price. PEO-131 (made by Toyobo), PEO-301 (made by Toyobo), PEO-302 (made by Toyobo), etc. are used suitably as a commercial item. 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 ratio} / {0.117 × 1 (mL))
= ΔOD × 8.547 × dilution ratio peroxidase activity (U / mg) = peroxidase activity (U / mL) × 1 / C
0.117: Absorbance at 420 nm of a 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 biological component measurement method 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 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)
The hydrogen donor is not particularly limited as long as it has the effect of the present invention as long as it has the property of donating hydrogen. 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. Such a hydrogen donor is also called an oxidative coloring reagent.

  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) Examples include -2,5-dimethylaniline, N-ethyl-N- (3-methylphenyl) -N'-succinylethylenediamine, N-ethyl-N- (3-methylphenyl) -N'-acetylethylenediamine.

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

  Examples of couplers used in the measurement of biological components of the present invention include 4-aminoantipyrine (4AA), aminoantipyrine derivatives, vanillin diamine sulfonic acid, methylbenzthiazolinone hydrazone (MBTH), sulfonated methylbenzthiazolinone hydrazone (SMBTH). ) Etc. are used.

(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.

  The chemical structural formulas of 4-aminoantipyrine and 4-hydroxyantipyrine are shown in FIG.

  However, the present invention shows that 4-hydroxyantipyrine consumes hydrogen peroxide and exerts a great influence on the color reaction in the coloring reaction in the biological component measurement method of the present invention even if the mixing amount is very small. Found.

(Sample preparation used for quantification of 4-hydroxyantipyrine)
Examples of the sample used for quantification of 4-hydroxyantipyrine according to the present invention include a 4-aminoantipine drug substance or a reagent containing 4-aminoantipyrine used in a biological component measurement kit. In this specification, the reagent includes a state of a reagent composition in which two or more kinds of reagents are mixed.
The 4-aminoantipyrine drug substance used in the present invention is a 4-aminoantipyrine stock solution / raw material used for producing a reagent containing 4-aminoantipyrine among the reagents constituting the biological component measurement kit of the present invention. Say the end.

As described above, the biological component measurement kit used in the present invention includes 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, and an appropriate carrier. Various forms such as a kit called a so-called dry system in which an enzyme or the like is supported and a kit using a sensor are exemplified, but the 4-aminoantipin active substance or 4-aminoantipyrine used in the present invention is exemplified. The reagent containing is in a solution state prescribed in a state normally used when measuring biological components according to the prescription of the kit, and has a good affinity with an aqueous solution and is excellent in solubility of 4-hydroxyantipyrine. Dilute for 4-hydroxyantipyrine quantification.
The dilution ratio is preferably about 4/3 to 5 times, more preferably about 2 times so as not to affect the stability of the prepared measurement sample and the measurement sensitivity of HPLC.

  The dilution solvent for sample preparation used for quantification of 4-aminoantipyrine used in the present invention is not particularly limited, and low molecular weight alcohols and mobile phase solvents used in HPLC methods are used. Affinity with aqueous solution and solubility of 4-hydroxyantipyrine are well used.

  When quantifying 4-hydroxyantipyrine, a deproteinization operation may be appropriately performed prior to measurement. The method for deproteinization is not limited, and examples thereof include a method for deproteinization by ultrafiltration. Specifically, it is possible to deproteinize by centrifuging using an Amicon Ultra 30K filter (MILLIPORE) at a centrifugal acceleration of 2900 g for about 20 minutes and ultrafiltration.

(4-hydroxyantipyrine detection limit)
The detection sensitivity of 4-hydroxyantipyrine in the 4-hydroxyantipyrine quantification method of the present invention is preferably 0.1 μg / ml or less, more preferably 0.05 μg / ml or less, and further preferably 0.03 μg / ml or less.

  As will be described later, the allowable concentration of 4-hydroxyantipyrine in the reaction solution in the biological component measurement of the present invention is preferably 1.5 μg / ml or less, more preferably 0.9 μg / ml or less, and still more preferably 0. .6 μg / ml or less, particularly preferably 0.3 μg / ml or less, and taking into account the dilution by several times at the time of quantification by the HPLC method, in order to detect and quantify 4-hydroxyantipyrine, -The detection sensitivity of hydroxyantipyrine is preferably 0.1 μg / ml or less, and more highly desirable biological component measurement is more preferably 0.05 μg / ml or less, and 0.03 μg / ml. The following is more preferable.

(Quantitative method of 4-hydroxyantipyrine)
As a method for quantifying 4-hydroxyantipyrine of the present invention, a high performance liquid chromatographic method (hereinafter also referred to as HPLC method) is preferably used.

  When high performance liquid chromatography is used, 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.

  When using high performance liquid chromatography, general reverse phase column chromatography is preferably used. The carrier for reverse phase column chromatography is not particularly limited, but for example, a carrier such as silica gel or polymer is preferably used, and among these, porous silica gel is more preferably used. When silica gel is used as a carrier, the presence or absence of end cap treatment is not particularly limited.

  The apparatus for high performance liquid chromatography can be used by appropriately adjusting the conditions according to the purpose in any system of low pressure, medium pressure and high pressure.

  The type of ligand that binds to the high performance liquid 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 mobile phase of high performance liquid chromatography should be water and a water-soluble solvent such as methanol or acetonitrile. To avoid ionic interaction with the silanol groups of silica gel, the pH of the mobile phase should be acidic according to conventional methods. The ion pair reagent may be added in a small amount.

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

(Acceptable concentration of 4-hydroxyantipyrine in the reaction solution)
The permissible concentration of 4-hydroxyantipyrine in the reaction mixture in the biological component measurement of the present invention is preferably 1.5 μg / ml or less, more preferably 0.9 μg / ml or less, and further preferably 0.6 μg / ml or less. Particularly preferably, it is 0.3 μg / ml or less.

  Hereinafter, the influence of 4-hydroxyantipyrine on the reduction in the sensitivity of biological component measurement and the allowable concentration of 4-hydroxyantipyrine in the reaction solution will be described using an example of a biological component to be measured as creatinine.

  In recent years, eGFR 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.

On the other hand, in the case of Example 5 or the like, 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 the in vitro diagnostic agent. % Normal distribution) is calculated to be about 0.117 to 0.296 mABS.
Therefore, it is possible to detect as a signal if there is an absorbance equal to or greater than the maximum value of 2.6σ of 0.296 mABS, that is, about 0.3 mABS.

  In other words, about 0.3 mABS is required as the minimum detection sensitivity when the creatinine concentration is 0.03 mg / dL, which corresponds to about 50 mABS when replaced when the creatinine concentration is 5 mg / dL. .

  In Example 4, a measurement reagent to which various concentrations of 4-hydroxyantipyrine were added was prepared using a 5 mg / dL creatinine aqueous solution, the measurement sensitivity of creatinine was measured, and the results are shown in FIG.

  From FIG. 5, it can be read that the concentration of 4-hydroxyantipyrine when the detection sensitivity is 50 mABS is about 1.5 μg / ml.

From the above, it can be seen that the allowable concentration of 4-hydroxyantipyrine in the reaction solution in the measurement of the biological component of the present invention is preferably 1.5 μg / ml or less.
In addition, when a more sensitive measurement is required, the allowable concentration of 4-hydroxyantipyrine in the reaction solution in the biological component measurement of the present invention is more preferably 0.9 μg / ml or less, and still more preferably 0. 0.6 μg / ml or less, particularly preferably 0.3 μg / ml or less.

  In the method for producing a biological component measurement kit of the present invention, the method for quantifying 4-hydroxyantipyrine used in the step of quantifying 4-hydroxyantipyrine precursor or 4-hydroxyantipyrine contained in the reagent is as described above.

  In the method for producing a biological component measurement kit of the present invention, a reagent containing 4-aminoantipyrine contained in the biological component measurement kit so that the concentration of 4-hydroxyantipyrine in the reaction solution is 1.5 μg / ml or less. It is preferable to adjust the concentration of 4-hydroxyantipyrine therein.

  In the method for producing a biological component measurement kit of the present invention, as a method for adjusting the concentration of 4-hydroxyantipyrine in a reagent containing 4-aminoantipyrine, a 4-aminoantipyrine raw material containing a high concentration of 4-hydroxyantipyrine is used. The concentration of 4-hydroxyantipyrine in the reaction solution is adjusted to be below the allowable concentration by avoiding the use of the body or by mixing with a 4-aminoantipyrine raw material containing a low concentration of 4-hydroxyantipyrine. The method etc. are mentioned.

  In addition, in consideration of the fact that 4-hydroxyantipyrine is easily decomposed in an aqueous solution at a low temperature (30 to 40 ° C.), a reagent containing 4-aminoantipyrine and an intermediate thereof when producing a measurement kit A method of reducing the concentration of 4-hydroxyantipyrine over time by placing the solution in an aqueous solution at a low temperature for a certain period of time, oxidizing the 4-hydroxyantipyrine, and a composition capable of decomposing 4-hydroxyantipyrine. The method of adjusting the density | concentration of 4-hydroxyantipyrine by adding, etc. are mentioned.

  Hereinafter, the present invention will be specifically described by way of examples, but the present invention is not limited thereto.

(Example 1) Confirmation of impurities of 4-aminoantipyrine drug substance The degree of decrease in reagent sensitivity (color intensity of reaction solution: mABS) at the time of measurement of biological components is due to the lot difference of the 4-aminoantipyrine drug substance. As a result, it was assumed that the content of impurities contained in a very small amount in the 4-aminoantipyrine drug substance was different for each lot, and the impurities in the 4-aminoantipyrine drug substance were detected by HPLC.

The 4-aminoantipyrine drug substance of each lot was diluted with methanol and adjusted so that the 4-aminoantipyrine concentration was 0.6 g / L.
Using these as test solutions, the 4-hydroxyantipyrine impurities were detected under the following HPLC conditions.

(HPLC analysis conditions)
(1) Column Imtakt Cadenza CD-C18 2.0 × 150 mm
(2) Mobile phase A: 0.1% formic acid, B: methanol (3) Gradient condition 0 min (A 95%, B 5%)-(During this time linear gradient) -15 min (A 2%, B 98%) -25 min (A 2%, B98%)
(4) Flow rate 0.2mL / min
(5) Column temperature 40 ° C
(6) Sample injection volume 5μL
(7) Detection wavelength UV250nm

  The purity of 4-aminoantipyrine in the 4-aminoantipyrine base used for examination of the lot difference was 98.0% or more according to JIS-K8048.

  The HPLC fraction when one lot of 4-aminoantipyrine drug substance is analyzed is shown in FIG. The peak of elution time 7-8 minutes on the horizontal axis of the graph is the peak of 4-aminoantipyrine. Three types of impurities other than the 4-aminoantipyrine peak were confirmed.

(Example 2) Identification of 4-hydroxyantipyrine in 4-aminoantipyrine drug substance The content of each lot was examined for the three types of impurities (a, b, c) seen in FIG. It was found that the content of only the impurity b is greatly different from lot to lot.
Therefore, the molecular weight and structure of these three types of impurities were analyzed by mass spectrometry (MS spectrum method).

  As a result, impurity b was identified as 4-hydroxyantipyrine.

  4-hydroxyantipyrine (Sigma) was used as a standard product of 4-hydroxyantipyrine. When HPLC was performed under the same conditions as in Example 1, the elution time values of the fraction peaks of 4-hydroxyantipyrine (Sigma) and impurity b coincided.

(4-hydroxyantipyrine standard)
4-Hydroxyantipyrine Sigma Cas. NO. 1672-63-5,
Product number 109428-5G, purity 99%

(Example 3) Detection sensitivity and quantification accuracy of 4-hydroxyantipyrine quantification method The detection sensitivity of 4-hydroxyantipyrine quantification method was measured.

4-Hydroxyantipyrine (Sigma) was diluted with methanol to adjust the 4-hydroxyantipyrine concentration to 0.1 μg / ml, 0.05 μg / ml, and 0.02 μg / ml.
These were analyzed as test solutions under the following HPLC analysis conditions.

(HPLC analysis conditions)
(1) Column Waters BEH-C18 2.1 x 150mm
(2) Mobile phase A: 0.1% formic acid, B: acetonitrile (3) Gradient conditions 0 min (A 98%, B 2%)-(linear gradient during this time)-25 min (A 82%, B 18%)-26 min (A 2%, B98%)-30 min (A2%, B98%)
(4) Flow rate 0.2mL / min
(5) Column temperature 40 ° C
(6) Sample injection volume 5μL
(7) Detection wavelength UV250nm

  A chart in which the concentration of 4-hydroxyantipyrine is 0.02 μg / ml is shown in FIG.

The peak height when the concentration of 4-hydroxyantipyrine is 0.02 μg / ml is 18 mm, and the maximum and minimum amplitudes of signals other than the peak are measured as 12 mm. The peak half-width method (of the baseline before and after the peak) Dionex Technical Review TR015YS-0083 Nippon Dionex Co., Ltd.) Obtains the maximum and minimum amplitude fluctuations of signals other than the peak in the time width 10 to 20 times the peak half-value width, and uses 1/2 of that as noise. Thus, the noise height was measured to be 6 mm.
From the above results, when S / N was 3, 6 mm × 3 = 18 mm, that is, 0.02-μg / ml as 4-hydroxyantipyrine concentration was determined as the detection sensitivity.
Moreover, the same tendency could be confirmed even in charts having 4-hydroxyantipyrine concentrations of 0.1 μg / ml and 0.05 μg / ml.

  Using a diluted solution of 4-hydroxyantipyrine at each concentration as a measurement sample, the area of the fraction peak was measured, and the correlation between the concentration of 4-hydroxyantipyrine and the area of the fraction peak was evaluated.

  The results are shown in FIG. As shown in FIG. 4, a high correlation tendency was confirmed.

(Example 4) Relationship between 4-hydroxyantipyrine concentration (concentration in reaction solution) and creatinine measurement sensitivity The second reagent of the following creatinine measurement reagent has 4HA as a final concentration in the reagent of 0.13 to 8.75 μg / ml. Thus, each measurement reagent was prepared.

  As a comparative control, a commercially available 4-aminoantipyrine drug substance was purified, and 4-aminoantipyrine free from 4-hydroxyantipyrine was produced and used.

  A 5 mg / dL creatinine aqueous solution was used as a sample.

[Preparation of reagents]
A creatinine measuring reagent having the following composition was prepared.
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 (Toyobo CRH-229) 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.

  The concentration of 4-hydroxyantipyrine in the reaction solution under the 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 concentration of 4-hydroxyantipyrine in the reaction solution increased.
The 4-hydroxyantipyrine concentration in the reaction solution is preferably 1.5 μg / ml or less, more preferably 0.9 μg / ml or less, even more preferably 0.6 μg / ml or less, and A particularly preferable result was 3 μg / ml or less.

(Example 5) Difference between lots Using different lots of 4-aminoantipyrine drug substance, 10 types of creatinine measurement reagent kits (S1 to S10) were produced. Hydroxyantipyrine concentration (μg / ml) and reagent measurement sensitivity (mABS) were measured.
The measurement of 4-hydroxyantipyrine concentration (μg / ml) in the reaction solution was carried out under substantially the same conditions as in Example 3, and the protein was removed using an Amicon Ultra 30K filter (MILLIPORE) before quantification of 4-hydroxyantipyrine. Processed. The reagent measurement sensitivity (mABS) was measured under the same conditions as in Example 4.

  As a control product, a creatinine measurement reagent kit was produced using 4-aminoantipyrine containing no 4-hydroxyantipyrine purified from commercially available 4-aminoantipyrine, and measured in the same manner as described above.

  The results are shown in Table 2.

  The present invention can be applied to a method for measuring a biological component in clinical diagnosis, a measurement kit used in the measurement method, and a reagent or a composition constituting the kit.

Claims (12)

  Biological component measurement kit for colorimetric determination of the color of the reaction solution produced by the oxidative condensation of 4-aminoantipyrine and hydrogen donor in the presence of peroxidase with hydrogen peroxide generated by the action of oxidase on the biological component A biological component measurement kit, wherein the concentration of 4-hydroxyantipyrine in the reaction solution is 1.5 μg / ml or less.   The biological component measurement kit according to claim 1, wherein the concentration of 4-hydroxyantipyrine in the reaction solution is 0.9 µg / ml or less.   The biological component measurement kit according to claim 1 or 2, wherein the concentration of 4-hydroxyantipyrine in the reaction solution is 0.6 µg / ml or less.   The biological component measurement kit according to any one of claims 1 to 3, wherein the concentration of 4-hydroxyantipyrine in the reaction solution is 0.3 µg / ml or less.   The biological component measurement kit according to any one of claims 1 to 4, wherein the biological component is either creatinine or glycated hemoglobin.   The biological component measurement kit according to any one of claims 1 to 5, wherein the redox reagent is a Trinder reagent.   Biological component measurement method for colorimetric determination of the color of a reaction solution produced by oxidative condensation of 4-aminoantipyrine and hydrogen donor in the presence of peroxidase with hydrogen peroxide generated by the action of an oxidase on a biological component And the concentration of 4-hydroxyantipyrine in the reaction solution is 1.5 μg / ml or less.   The biological component measurement method according to claim 7, wherein the concentration of 4-hydroxyantipyrine in the reaction solution is 0.9 μg / ml or less.   The biological component measurement method according to claim 7 or 8, wherein the concentration of 4-hydroxyantipyrine in the reaction solution is 0.6 µg / ml or less.   The biological component measurement method according to claim 7, wherein the concentration of 4-hydroxyantipyrine in the reaction solution is 0.3 μg / ml or less.   11. The biological component measuring method according to claim 7, wherein the biological component is either creatinine or glycated hemoglobin.   The biological component measuring method according to claim 7, wherein the redox reagent is a Trinder reagent.