JP6446875B2 - Biological component measuring method and measuring composition - Google Patents

Description

The present invention relates to a measurement method for measuring a biological component using a redox reaction in clinical diagnosis and a measurement composition used therefor, and more particularly, to avoid the influence of interfering substances coexisting in the biological component. To the above methods and compositions.

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. This method has been known to be susceptible to the effects of interfering substances coexisting in serum, such as bioreductive substances such as ascorbic acid and bilirubin, but various countermeasures have been studied depending on the respective interfering substances. Have been overcome.

Recently, dobutamine has been found as a new interfering substance (Non-patent Document 1). Dobutamine is a pharmaceutical agent used as a cardiotonic agent, and is administered to a patient by intravenous injection. Therefore, it may be mixed with body fluids such as serum and affect the measured value. According to Non-Patent Document 1, it is considered that the addition of sodium periodate to a sample was effective in avoiding the influence of dobutamine. However, this method is very complicated because it requires pre-treatment of individual samples, and sodium periodate is a substance having a very strong oxidizing power classified as a first class hazardous material. Care was also needed.

Medical Examination, Vol. 56, No. 9, 1216-1220 (2007)

The object of the present invention is to create a simpler, safer and more effective method to avoid the effects of dobutamine.

The inventors have 2,2,6,6-tetramethylpiperidine 1-oxyl free radical (also known as TEMPO free radical or 2,2,6,6-tetramethylpiperidine 1-oxyl free radical) (CAS number) to the reaction system. : 2564-83-2) (hereinafter also referred to as TEMPO), it was found that the influence of dobutamine can be avoided. Furthermore, by adding TEMPO to the measurement composition in advance, it was found that measurement without being affected by dobutamine can be performed without pretreatment of the sample, and the present invention was completed.
That is, the present invention has the following configuration.

[Claim 1]
Biological component measuring reagent (1) Peroxidase (2) Redox coloring reagent that reacts with hydrogen peroxide in the presence of peroxidase (1) to (3) 3) 2,2,6,6-Tetramethylpiperidine 1-Oxyl Free Radical (2,2,6,6-tetramethylpiperidine 1-oxyl free radical)
[Section 2]
A biological component measuring method comprising coexisting the following components (1) to (3) in a process of measuring a biological component using an oxidation-reduction reaction.
(1) Peroxidase (2) Redox coloring reagent that reacts with hydrogen peroxide in the presence of peroxidase to develop color (3) 2,2,6,6-Tetramethylpiperidine 1-Oxyl Free Radical (2,2,6, 6-tetramethylpiperidine 1-oxyl free radical)

According to the present invention, in the measurement of biological components by an enzyme method using an oxidase-peroxidase-color former system, measurement that is not affected by dobutamine can be performed without pretreatment of the sample.

(Biological component measurement method)
The biological component measurement method to which the present invention is applied is a biological component measurement method based on an enzyme method, particularly a method using an oxidase-peroxidase-coloring agent system, that is, an enzyme reaction of a measurement target substance in a specimen to produce hydrogen peroxide. Is generated and reacted with a color former in the presence of peroxidase to perform colorimetric determination.
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 biological components in various samples, and the mode is not particularly limited.
For example, a method for measuring biological components such as uric acid (UA), creatinine (CRE), triglyceride (TG), cholesterol (CHO) and the like can be exemplified.

For example, when measuring UA, hydrogen peroxide generated by the reaction of uricase (oxidase) using UA as a substrate can be quantified by a peroxidase-color former system.
On the other hand, when measuring CRE, hydrogen peroxide is not directly generated in the reaction of creatinine amidinohydrolase using CRE as a substrate. Therefore, it reacts with creatine amide hydrolase in which creatine generated in the reaction of creatinine amidinohydrolase is added to the reagent in advance. By designing a so-called coupling reaction in which sarcosine is generated and hydrogen peroxide is generated using sarcosine oxidase (oxidase) in which sarcosine has been added to the reagent in advance, the CRE concentration of the peroxidase-chromogenic system can be increased. Quantification becomes possible.
In the case of measuring TG, a peroxidase-coloring agent system is produced by generating hydrogen peroxide using lipoprotein lipase using TG as a substrate and glycerol kinase and glycerol triphosphate oxidase (oxidase) as conjugate enzymes. Quantification of TG concentration is possible.
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.

Means for carrying out the above method include a method using a liquid reagent (or kit) configured to be applicable to a general-purpose automatic analyzer (for example, Hitachi 7170 type automatic analyzer), a means such as lyophilization. Various methods such as a method using a reagent (or kit) composed of a combination of the produced dry preparation and a solution, a method using a kit or a sensor called a so-called dry system in which an enzyme is supported on an appropriate carrier, etc. A form can be illustrated.
Preferably, the analysis is performed by an automatic analyzer using a liquid reagent in which the reagent is divided into two (hereinafter also referred to as a two-reagent liquid reagent). In this method, the first type of reagent (hereinafter also referred to as the first reagent or R1) is first added to the sample and allowed to react for a certain period of time, and then the second type of reagent (hereinafter also referred to as the first reagent or R1). The target component can be quantified by adding and reacting, and measuring the change in absorbance during this period.

(Biological component measurement reagent)
The biological component measurement reagent of the present invention is not particularly limited as long as it contains the following components (1) to (3). As described above, various forms can be adopted, and the composition of the reagent composition and the like can be appropriately set by those skilled in the art based on the description of the present specification.
(1) Peroxidase (2) Redox coloring reagent that reacts with hydrogen peroxide in the presence of peroxidase to develop color (3) 2,2,6,6-Tetramethylpiperidine 1-Oxyl Free Radical (2,2,6, 6-tetramethylpiperidine 1-oxyl free radical)

The sample containing the biological component to be measured is not particularly limited, and examples thereof include blood, beverages, and foods.

In particular, when a body fluid of a patient administered with dobutamine (for example, a sample derived from blood such as serum or plasma) is used as a measurement target, it is preferable in that an effect of reducing the influence of dobutamine can be obtained. That is, the present invention also has one aspect as a method for reducing the influence of dobutamine.

(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 (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 solution, 1 mL of 0.147M hydrogen peroxide solution and 2 mL of 100 mM phosphate buffer (pH 6.0) were sequentially mixed, and then pre-controlled 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 solution, 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 0.147 M 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.

U / mL
= {ΔOD (OD _test -OD _blank ) × dilution ratio} / {0.117 × 1 (mL))
= ΔOD x 8.547 x dilution factor

U / mg = {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 present invention, any kind of dye may be used as long as it reacts with hydrogen peroxide and develops color, for example, hydrogen donor and coupler, leuco body, tetrazolium salt, etc. 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.
Known hydrogen donors used in the Trinder method include phenol, phenol derivatives, aniline derivatives, naphthol, naphthol derivatives, naphthylamine, naphthylamine derivatives, and the like.
For example, N-ethyl-N-sulfopropyl-3-methoxyaniline, N-ethyl-N-sulfopropylaniline, N-ethyl-N-sulfopropyl-3,5-dimethoxyaniline, N-sulfopropyl-3,5 -Dimethoxyaniline, N-ethyl-N-sulfopropyl-3,5-dimethylaniline, N-ethyl-N-sulfopropyl-3-methylaniline, N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methoxyaniline, N-ethyl-N- (2-hydroxy-3-sulfopropyl) aniline, N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline, N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline, N-ethyl-N- (2-hydroxy-3-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. These hydrogen donors can be used in combination with a coupler.
As couplers, 4-aminoantipyrine (4AA), aminoantipyrine derivatives, vanillindiamine sulfonic acid, methylbenzthiazolinone hydrazone (MBTH), sulfonated methylbenzthiazolinone hydrazone (SMBTH) and the like are known.

Examples of leuco bodies include triphenylmethane derivatives, phenothiazine derivatives, diphenylamine derivatives, and the like. Specifically, 4,4′-benzylidenebis (N, N-dimethylaniline), 4,4′-bis [N-ethyl-N- (3-sulfopropylamino) -2,6-dimethylphenyl] methane 1- (ethylaminothiocarbonyl) -2- (3,5-dimethoxy-4-hydroxyphenyl) -4,5-bis (4-diethylaminophenyl) imidazole, 4,4′-bis (dimethylamino) diphenylamine, N- (carboxymethylaminocarbonyl) -4,4′-bis (dimethylamino) diphenylamine salt (DA64), 10- (carboxymethylaminocarbonyl) -3,7-bis (dimethylamino) phenothiazine salt (DA67), etc. Can be mentioned.

Examples of the tetrazolium salt include 2,3,5-triphenyltetrazolium salt, 2,5-diphenyl-3- (1-naphthyl) -2H-tetrazolium salt, 3,3 ′-[3,3′-dimethoxy- (1 , 1′-biphenyl) -4,4′-diyl] -bis [2- (4-nitrophenyl) -5-phenyl-2H-tetrazolium] salt, 3,3 ′-[3,3′-dimethoxy- ( 1,1′-biphenyl) -4,4′-diyl] -bis (2,5-diphenyl-2H-tetrazolium) salt, 2- (4-iodophenyl) -3- (4-nitrophenyl) -5 (2,4-disulfophenyl) -2H-tetrazolium salt, 3,3 ′-(1,1′-biphenyl-4,4′-diyl) -bis (2,5-diphenyl-2H-tetrazolium) salt, 3- (4,5-dimethyl-2 Thiazolyl) -2,5-diphenyl -2H- tetrazolium salts.

(TEMPO)
TEMPO used in the present invention is available from, for example, Tokyo Chemical Industry.

The content of TEMPO is not particularly limited, but the preferred lower limit is 0.01 mmol / L, the more preferred lower limit is 0.02 mmol / L, the more preferred lower limit is 0.05 mmol / L, the more preferred lower limit is 0.1 mmol / L, and further A preferred lower limit is 0.2 mmol / L, and a more preferred lower limit is 0.3 mmol / L. On the other hand, the preferred upper limit is 10 mmol / L, the more preferred upper limit is 5.0 mmol / L, the more preferred upper limit is 2.0 mmol / L, the more preferred upper limit is 1.0 mmol / L, the more preferred upper limit is 0.8 mmol / L, and further A preferable upper limit is 0.7 mmol / L, a more preferable upper limit is 0.6 mmol / L, a further preferable upper limit is 0.5 mmol / L, and a further preferable upper limit is 0.4 mmol / L.

The present invention preferably contains a buffer component. Examples of the buffer include Tris buffer, phosphate buffer, borate buffer, carbonate buffer, and GOOD buffer. 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 present invention, ascorbate oxidase, preservatives, salts, enzyme stabilizers, chromogen stabilizers and the like may be added within a range that does not affect the reaction. There are no particular restrictions on the amount used or the form of addition. All of these can be obtained as commercial products.
Examples of antiseptics include azides, chelating agents, antibiotics, and antibacterial agents.
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.

(Enzyme stabilization method)
Moreover, although it was unpredictable at the beginning, it turned out that TEMPO used for the biological component measuring method of this invention has an enzyme stabilization effect as described in the below-mentioned Example. That is, the present invention also has one aspect as a method for stabilizing an enzyme.

  Here, the enzyme to be stabilized is not particularly limited. For example, each enzyme used for the measurement of UA, CRE, TG, CHO, etc., peroxidase, etc. shown in the above description of the biological component measurement method.

By the way, in these methods, there are known problems that are easily affected by interfering substances coexisting in samples such as serum, for example, bioreductive substances such as ascorbic acid and bilirubin. Various countermeasures such as a so-called erasing system have been studied and overcome. For example, ascorbic acid can be eliminated by allowing ascorbate oxidase to act on the sample. Further, bilirubin can be eliminated by allowing bilirubin oxidase to act on the sample. Reagents employing such a method include ascorbate oxidase and / or bilirubin oxidase. These enzymes can also be subject to stabilization.
Furthermore, when several stages of conjugation reactions are designed, a positive error occurs due to the inclusion of the reaction intermediate in the sample. A typical method is to cause an enzyme other than the enzyme that directly acts on the measurement target substance to act on the sample to generate hydrogen peroxide, erase the reaction intermediate with catalase, and then react the enzyme that acts directly on the measurement target substance. In this method, hydrogen peroxide is generated in addition to the system, and this is reacted with a color former in the presence of peroxidase, and at the same time, the action of catalase is virtually stopped to perform colorimetric determination. For example, in the case of CRE measurement, an enzyme other than creatinine amidinohydrolase (creatine amide hydrolase and sarcosine oxidase) is allowed to act on the sample and hydrogen peroxide generated due to the reaction intermediate (creatine, etc.) is eliminated by catalase. , Creatinine amidinohydrolase was added to the reaction system, and hydrogen peroxide generated due to the CRE to be measured was reacted with a color former in the presence of peroxidase for colorimetric determination. The action is substantially stopped.) Reagents employing such a method include catalase. This enzyme can also be subject to stabilization.

  Specific examples include ascorbate oxidase, sarcosine oxidase, creatine amidinohydrolase, and catalase. Of these, ascorbate oxidase and catalase are preferable.

In the case of a two-reagent system, TEMPO may be added to either reagent from the viewpoint of enzyme stabilization. If it is desired to obtain the effect of avoiding the influence of dobutamine, it is preferably added to the first reagent.

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
TEMPO was prepared as the first reagent of the following creatinine measurement reagent so that the final concentration in the reagent would be 0.01 mmol / L to 10 mmol / L, and used as a measurement reagent.
As a sample, dobutamine hydrochloride was added to liquid nescor N (Alfresa Pharma) so that the final concentration in the sample would be 4 μg / mL or 20 μg / mL. did.
As a comparative example, the same study was performed using a creatinine measuring reagent not added with TEMPO.

(Preparation of reagents)
A creatinine measuring reagent having the following composition was prepared.
First reagent PIPES-NaOH 50 mM pH 7.4
Ascorbate oxidase (ASO-311 manufactured by Toyobo Co., Ltd.) 3 U / mL
Sarcosine oxidase (SAO-351, Toyobo Co., Ltd.) 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. Absorbance at 546 nm was measured by a two-point end method in which the absorbances of the first reaction and the second reaction were corrected for the liquid volume and the difference between the absorbances was taken.
The creatinine concentration of the sample with unknown creatinine concentration was calculated from the measured absorbance of purified water and a 5 mg / dL creatinine aqueous solution.

The results are shown in Table 1. In all the examples (the amount of TEMPO added was 0.01 mmol / L or more and 10 mmol / L or less), the effect of dobutamine was reduced as compared with the comparative example. In particular, when the concentration of dobutamine was 4 μg / mL, the effect of dobutamine was within 5% when the addition amount of TEMPO was 0.01 to 10 mmol / L compared to the comparative example. .

(Example 2)
As the first reagent of the creatinine measuring reagent described in Example 1, TEMPO was prepared to a final concentration of 0.3 mmol / L in the reagent to prepare a measuring reagent. Immediately after the preparation, after storage at 35 ° C. for 7 days and 35 The reagent after storage at 14 ° C. for 14 days was measured under the following measurement conditions.
As samples, purified water and creatinine 5 mg / dL aqueous solution were measured, and a reagent blank (purified water measurement absorbance) and sensitivity (absorbance obtained by subtracting purified water measurement absorbance from creatinine 5 mg / dL aqueous solution absorbance) were determined.
Further, as in Example 1, as a sample, liquid nescoll N (Alfresa Pharma) was added with dobutamine hydrochloride so that the final concentration in the sample was 20 μg / mL, and liquid nescol N without addition of dobutamine hydrochloride was used as a control. The degree of influence was confirmed.
As a comparative example, the same study was performed using a creatinine measuring reagent not added with TEMPO.

(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. Absorbance at 546 nm was measured by a two-point end method in which the absorbances of the first reaction and the second reaction were corrected for the liquid volume and the difference between the absorbances was taken.
The creatinine concentration of the sample with unknown creatinine concentration was calculated from the measured absorbance of purified water and a 5 mg / dL creatinine aqueous solution.

The results are shown in Table 2. When TEMPO was added, the effect of dobutamine could be avoided even after storage at 35 ° C. for 7 days and 14 days.
The blank values and sensitivity of the examples were good after storage at 35 ° C. for 7 days and 14 days.

Example 3
For each reagent used in Example 2, the stability of each enzyme in the first reagent was confirmed. Each enzyme was measured by the following measurement method.
The component activity or remaining amount in the reagent after storage for 14 days at 35 ° C. for 7 days was confirmed with the measured value of the reagent immediately after preparation as 100%.

The results are shown in Table 3. In the example in which TEMPO was added, the enzyme activity in the 35 ° C. storage reagent was higher for each enzyme than in the comparative example without addition. In particular, ascorbate oxidase and catalase were significantly improved in storage stability when TEMPO was added.

(Enzyme activity measurement method)
Ascorbate oxidase (ASO)
The amount of enzyme that oxidizes 1 micromole of ascorbic acid per minute under the following conditions is defined as 1 unit (U).
Reagent (A) 1.0 mM ascorbic acid solution [10 mM L-ascorbic acid solution is diluted 10-fold with 0.2 M KH ₂ PO ₄ solution containing 1.0 mM EDTA] (prepared when used)
A 10 mM L-ascorbic acid solution is prepared by accurately weighing 176 mg of L-ascorbic acid (reagent grade, MW = 176.13) and dissolving it in 100 mL of 1.0 mM HCl solution containing 1.0 mM EDTA.
(B) 10 mM Na ₂ HPO ₄ solution (C) 0.2 N HCl solution Test enzyme solution: A preparation containing the enzyme was dissolved in distilled water cooled in advance with ice (60 U / mL or more), and 0.05 immediately before the analysis. Dilute to 0.15-0.25 U / mL with 10 mM Na ₂ HPO ₄ solution (ice cooling) containing% BSA.
Procedure (1) Prepare the following reaction mixture in a test tube and preheat at 30 ° C. for about 5 minutes.
0.5 mL substrate solution (A)
0.5 mL Na ₂ HPO ₄ solution (B)
(The pH of the reaction mixture is 5.6)
(2) Add 0.1 mL of the test enzyme solution and start the reaction.
(3) After reacting at 30 ° C. for exactly 5 minutes, 3.0 mL of HCl solution (C) is added to stop the reaction. The absorbance at 245 nm is measured for this solution (OD _test ).
(4) The blind test is prepared by allowing the reaction mixture (1) to stand at 30 ° C. for 5 minutes, adding 3.0 mL of HCl solution (C) and mixing, and then adding 0.1 mL of enzyme solution. Similarly, the absorbance is measured (OD _blank ).
Formula U / mL = (ΔOD (OD _blank −OD _test ) × 4.1 (mL) × dilution rate) / (10.0 × 1.0 × 5 (min) × 0.1 (mL)) = ΔOD × 0.82 × dilution factor 10.0: millimolar molecular extinction coefficient (F / micromole) of ascorbic acid under the above measurement conditions (pH 1.0)
1.0: Optical path length (cm)

Sarcosine oxidase (SAO)
The amount of enzyme that produces 1 micromole of H ₂ O ₂ per minute under the following conditions is defined as 1 unit (U).
Reagent (A) 0.2 M sarcosine solution [1.78 g of sarcosine (MW = 89.09) dissolved in 80 mL of 0.125 M Tris-HCl buffer (pH 8.0) containing 0.125% Triton X-100 Then, adjust the pH to 8.0 (25 ° C) with 1.0 N NaOH or HCl, and make it 100 mL with distilled water.]
(B) 0.1% 4-AA aqueous solution (100 mg of 4-aminoantipyrine is dissolved in 100 mL of distilled water)
(C) 0.1% phenol aqueous solution (100 mg of phenol is dissolved in 100 mL of distilled water)
(D) Peroxidase aqueous solution [25 mg of peroxidase (POD) (110 purprogalin units / mg) is dissolved in 100 mL of distilled water]
(E) 0.25% SDS aqueous solution [1.25 g of sodium dodecylsulfate (SDS) is dissolved in 500 mL of distilled water]
Test enzyme solution: A preparation containing the enzyme was dissolved in a 20 mM Tris-HCl buffer solution (pH 8.0) containing 2.0 mM EDTA previously cooled in ice, and 0.07 to 0.17 U in the same buffer solution immediately before the analysis. Dilute to / mL.
Procedure (1) The following reaction mixture is prepared (ice-cooled in a brown bottle).
50 mL sarcosine solution (A)
10 mL 4-AA aqueous solution (B)
20mL phenol aqueous solution (C)
20 mL peroxidase aqueous solution (D)
(2) Take 1.0 mL of the reaction mixture in a test tube and preheat at 37 ° C. for about 5 minutes.
(3) Add 0.05 mL of the test enzyme solution and start the reaction.
(4) After reacting accurately at 37 ° C. for 10 minutes, 2.0 mL of an aqueous SDS solution (E) is added to stop the reaction. The absorbance at 500 nm is measured for this solution (OD _test ).
(5) In the blind test, an enzyme dilution solution (20 mM Tris-HCl buffer solution (pH 8.0) containing 2.0 mM EDTA) is used instead of the enzyme solution, and the operation is performed in the same manner as described above to measure the absorbance (OD _blank). ).
Formula U / mL = (ΔOD (OD _test −OD _blank ) × 3.05 (mL) × dilution rate) / (13.3 × (1/2) × 1.0 × 10 (min) × 0.05 (ML)) = ΔOD × 0.917 × dilution ratio 13.3: Quinoneimine dye molar absorption coefficient (F / micromole) under the above measurement conditions
1/2: Quinoneimine dye formed from one molecule of H ₂ O ₂ produced by the enzymatic reaction is a factor of 1/2 molecule 1.0: Optical path length (cm)

Creatine amidinohydrolase (CRH)
The amount of enzyme that produces 1 micromole of yellow pigment per minute under the following conditions is defined as 1 unit (U).
Reagent (A) 0.1 M creatine solution [1.49 g creatine (manufactured by Nacalai Tesque) is dissolved in 50 mM phosphate buffer pH 7.5 to make 100 mL]
(B) DAB solution (2.0 g of p-dimethylaminobenzaldehyde is dissolved in 100 P of dimethyl sulfoxide, and then 15 mL of concentrated hydrochloric acid is added)
Test enzyme solution: A preparation containing the enzyme is dissolved in a 50 mM phosphate buffer (pH 7.5) previously cooled in ice, and diluted to 2.0 to 3.0 U / mL with the same buffer immediately before the analysis.
Procedure (1) Take 1.0 mL of the substrate solution (A) in a test tube and preheat at 37 ° C. for about 5 minutes.
(2) Add 0.1 mL of the test enzyme solution and start the reaction.
(3) After reacting accurately at 37 ° C. for 10 minutes, 2.0 mL of DAB solution (B) is added to stop the reaction.
(4) After standing at 25 ° C. for 20 minutes, the absorbance at 435 nm is measured (OD _test ).
(5) In the blind test, 1.0 mL of the substrate solution (A) is allowed to stand at 37 ° C. for 10 minutes, then 2.0 mL of DAB solution (B) is added and mixed, and then 0.1 mL of the enzyme solution is added. Similarly, the absorbance is measured after standing at 25 ° C. for 20 minutes (OD _blank ).
Formula U / mL = (ΔOD (OD _test −OD _blank ) × 3.1 (mL) × dilution rate) / (0.321 × 1.0 × 10 (min) × 0.1 (mL)) = ΔOD X 9.65 x dilution factor 0.321: millimolar molecular extinction coefficient of yellow pigment (F / micromole)
1.0: Optical path length (cm)

Catalase (CAO)
The amount of enzyme that hydrolyzes 1 micromole of H ₂ O ₂ per minute under the following conditions is defined as 1 unit (U).
Reagent (A) 10 mM phosphate buffer pH 7.0 (at 25 ° C.)
(B) H ₂ O ₂ solution 16 mM (0.182 ml of 30% (W / V) is dissolved in 100 mL of (A).))
(C) Titanium reagent 1.0 g of TiO ₂ and 10 g of K ₂ SO ₄ are mixed together with 150 mL of concentrated sulfuric acid at 180-220 ° C. for 2-3 hours on a mantle heater. Cool the mixture and dilute the solution portion to 1.5 L with water.
(D) Enzyme diluent (A) is used.
Procedure (1) 0.25 mL of the substrate solution (B) is put in a test tube and pre-warmed at 25 ° C. for about 5 minutes.
(2) Add and mix 0.25 mL of the test enzyme solution.
(3) After reacting accurately at 25 ° C. for 5 minutes, 2.5 mL of titanium reagent (C) is added to stop the reaction.
(4) The absorbance at 410 nm is measured (OD _test ).
(5) The blind test is prepared by adding 0.25 mL of the substrate solution (B) at 25 ° C. for 5 minutes, adding 2.5 mL of the titanium reagent (C), mixing, and then adding 0.25 mL of the enzyme solution. (OD _blank ).
Formula U / mL = (ΔOD (OD _test −OD _blank ) × 3.0 (mL) × dilution rate) / (F × 1.0 × 5 (min) × 0.25 (mL)) = ΔOD × 2 4 × (1 / F) × dilution factor F: extinction coefficient of Titaniu color product produced by the presence of 1.0 mM H ₂ O ₂ (usually about 7.0, but with known concentrations of H ₂ O ₂ Use concentration to determine for each lot.)
1.0: Optical path length (cm)

(Example 4)
For each first reagent of the following uric acid measurement reagent (UA), neutral fat measurement reagent (TG), total cholesterol measurement reagent (TC), HDL-C measurement reagent (HDL), and LDL-C measurement reagent (LDL) TEMPO was prepared to a final concentration of 0.3 mmol / L in the reagent, and used as a measurement reagent.
As a sample, dobutamine hydrochloride was added to liquid nescor N (Alfresa Pharma Co., Ltd.) so that the final concentration in the sample was 20 μg / mL, and the degree of influence was confirmed using liquid nescor N without addition of dobutamine hydrochloride as a control.
As a comparative example, the same examination was performed with each measurement reagent to which TEMPO was not added.

(Preparation of reagents)
Uric acid measurement reagent (UA)
First reagent PIPES-NaOH 50 mM pH 7.0
Peroxidase (Toyobo PEO-301) 2U / mL
Second reagent PIPES-NaOH 50 mM pH 7.0
N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methylaniline (manufactured by Dojin Chemical) 0.4 g / L
Uricase (UAO-211 manufactured by Toyobo Co., Ltd.) 0.8 U / mL
Neutral fat measurement reagent (TG)
First reagent PIPES-NaOH 100 mM pH 6.6
MgCl ₂ 1 mmol / L
Adenosine triphosphate 2Na salt 1mmol / L
4-aminoantipyrine 0.4 mmol / L
Flavin adenine dinucleotide 2Na salt 8 μmol / L
Glycerol kinase (Toyobo GYK-311) 3 U / mL
Catalase (Toyobo) 200U / mL
Glycerophosphate oxidase (G3O-321 manufactured by Toyobo Co., Ltd.) 3 U / mL
ASO (Toyobo ASO-311) 1U / mL
Second reagent PIPES-NaOH 50 mM pH 7.4
Creatinine amide hydrolase (Toyobo CNH-311) 400 U / mL
Peroxidase (Toyobo PEO-302) 10 / mL
4-Aminoantipyrine 0.6g / L

Total cholesterol measuring reagent (TC)
First reagent PIPES-NaOH 50 mM pH 7.0
Ascorbate oxidase (ASO-311 manufactured by Toyobo Co., Ltd.) 3 U / mL
Cholesterol esterase (Toyobo COE-311) 2 U / mL
Sodium cholate 5g / L
N-ethyl-N- (3-sulfopropyl) -3-methoxyaniline 0.14 g / L
Second reagent PIPES-NaOH 50 mM pH 7.0
Cholesterol oxidase (Toyobo COO-321) 2 U / mL
Peroxidase (Toyobo PEO-302) 10 / mL
4-Aminoantipyrine 0.6g / L

HDL-C measurement reagent (HDL)
First reagent PIPES-NaOH 50 mM pH 6.5
Ethylenediaminetetraacetic acid disodium salt 0.2mmol / L
Magnesium chloride 5mmol / L
Calcium chloride 0.1mmol / L
N, N-bis (3-D-gluconamidopropyl) deoxycollamide (manufactured by Dojindo) 0.1%
Digitonin (Dojin Chemical Co., Ltd.) 0.01%
Cholesterol esterase (Toyobo COE-301) 2 U / mL
Cholesterol oxidase (Toyobo COO-321) 3 U / mL
Catalase (Toyobo CAO-509) 100 U / mL
N-ethyl-N-sulfopropyl-3-methoxyaniline (manufactured by Dojin Chemical) 0.1 g / L
Second reagent MOPS-NaOH 100 mM pH 7.5
Ethylenediaminetetraacetic acid disodium salt 0.2 mmol / L
Calcium chloride 0.1mmol / L
Emulgen 120 (Kao Corporation) 0.3%
Chemically modified cholesterol esterase (Toyobo COE-313) 1 U / mL
Peroxidase (Toyobo PEO-301) 8U / mL
4-aminoantipyrine 0.4 g / L

LDL-C measurement reagent (LDL)
First reagent PIPES-NaOH 50 mM pH 7.5
Ethylenediaminetetraacetic acid disodium salt 0.2 mmol / L
Magnesium chloride 10mmol / L
Calcium chloride 0.1mmol / L
N, N-bis (3-D-gluconamidopropyl) deoxycholamide (manufactured by Dojindo) 0.2%
Chemically modified cholesterol esterase (Toyobo COE-313) 0.5 U / mL
Unmodified cholesterol oxidase (Toyobo COO-321) 3 U / mL
Catalase (Toyobo CAO-509) 100 U / mL
4-Aminoantipyrine 0.1g / L
Second reagent PIPES-NaOH 50 mM pH 7.5
Ethylenediaminetetraacetic acid disodium salt 0.2 mmol / L
Magnesium chloride 10mmol / L
Calcium chloride 0.1mmol / L
Polyoxypropylene (2) polyoxyethylene decyl ether (Emalex DAPE0207: manufactured by Nippon Emulsion Co., Ltd.) 0.3%
Peroxidase (Toyobo PEO-301) 8U / mL
N-ethyl-N-sulfopropyl-3-methoxyaniline (manufactured by Dojin Chemical) 0.4 g / L

(Measurement method)
A Hitachi 7180 automatic analyzer was used. The first reagent was added to the sample and incubated at 37 ° C. for 5 minutes to form the first reaction. Then, the second reagent was added and incubated for 5 minutes to form a second reaction. Absorbance at a predetermined wavelength was measured by a two-point end method in which the absorbances of the first reaction and the second reaction were corrected for liquid amounts and the difference between the absorbances was taken.
The following samples (S), reagent amounts (R1, R2), and measurement wavelengths were used. (S / R1, R2 / measured wavelength)
UA 2.7 μL / 120 μL, 60 μL / 600 nm
TC 1.5μL / 120μL, 60μL / 600nm
TG 1.5μL / 120μL, 60μL / 600nm
HDL 1.6μL / 150μL, 50μL / 600nm
LDL 1.6μL / 150μL, 50μL / 600nm
The concentration was calculated from the measured absorbance of purified water and standard solutions for each item.
The following standard solution concentrations were used.
UA 6.9mg / dL
TC 221mg / dL
TG 124mg / dL
HDL 89mg / dL
LDL 91mg / dL

Results are shown in Table 4. As compared with the comparative example, in each of the examples in which TEMPO was added to all the reagents, the effect of dobutamine could be reduced.

(Example 5)
The same experiment as in Example 3 was carried out by changing the concentration of TEMPO in the range of 0.01 to 10 mmol / L as described in Table 5 and setting the measured value of the reagent immediately after preparation as 100% at 35 ° C. for 14 days after storage. The component activity or remaining amount in the reagent was confirmed.

The results are shown in Table 5. In all the examples (the amount of TEMPO added was 0.1 mmol / L or more and 1 mmol / L or less), each enzyme had a higher enzyme activity in the 35 ° C. storage reagent than the comparative example without TEMPO addition. In particular, ascorbate oxidase and catalase were significantly improved in storage stability when TEMPO was added.

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 (10)

The following (1) to (3), characterized in that it comprises a component of a biological component measurement reagents for measuring a biological sample that may contain a dobutamine.
(1) Peroxidase (2) Redox coloring reagent that reacts with hydrogen peroxide in the presence of peroxidase to develop color (3) 2,2,6,6-Tetramethylpiperidine 1-Oxyl Free Radical (2,2,6, 6-tetramethylpiperidine 1-oxyl free radical) (3) The biological component measurement reagent according to claim 1, wherein the final concentration of 2,2,6,6-tetramethylpiperidine 1-oxyl free radical in the reagent is 0.01 mmol / L to 10 mmol / L. The biological component measuring reagent according to claim 1 or 2, wherein the biological component measuring reagent is a two-reagent system, and the first reagent contains (3) 2,2,6,6-tetramethylpiperidine 1-oxyl free radical. . The biological component measurement reagent according to any one of claims 1 to 3, which is a creatinine measurement reagent, a uric acid measurement reagent, a neutral fat measurement reagent, a total cholesterol measurement reagent, an HDL-C measurement reagent, or an LDL-C measurement reagent. The biological component measurement reagent according to any one of claims 1 to 4, further comprising at least one enzyme selected from the group consisting of ascorbate oxidase, sarcosine oxidase, creatine amidinohydrase, and catalase. A method for reducing the influence of dobutamine in a biological component measurement method , wherein the following components (1) to (3) are allowed to coexist in the process of measuring a biological component using an oxidation-reduction reaction.
(1) Peroxidase (2) Redox coloring reagent that reacts with hydrogen peroxide in the presence of peroxidase to develop color (3) 2,2,6,6-Tetramethylpiperidine 1-Oxyl Free Radical (2,2,6, 6-tetramethylpiperidine 1-oxyl free radical) (3) The method according to claim 6, wherein the final concentration of 2,2,6,6-tetramethylpiperidine 1-oxyl free radical in the reagent is 0.01 mmol / L to 10 mmol / L. The biological component measuring method is a two-reagent system, and (3) 2,2,6,6-tetramethylpiperidine 1-oxyl free radical is contained in the first reagent and used together, The method described. The method according to any one of claims 6 to 8, which is carried out for creatinine measurement, uric acid measurement, neutral fat measurement, total cholesterol measurement, HDL-C measurement, or LDL-C measurement. (3) 2,2,6,6-tetramethylpiperidine 1-oxyl free radical further coexists with at least one enzyme selected from the group consisting of ascorbate oxidase, sarcosine oxidase, creatine amidinohydrase, and catalase The method according to any one of claims 6 to 9.