JPH08205892A - Method for stabilizing reagent for measuring biological component - Google Patents

Abstract

PURPOSE: To reduce and stabilize a blank test value (blank) of a two-reagent- based reagent for measuring a biological component utilizing reaction of an oxidase. CONSTITUTION: This method for stabilizing a reagent for measuring a biological component is to especially add a catalase to a reagent without containing a peroxidase and prescribe the reagent so as to separately include a hydrogen donor of a color developing reagent and an oxidizing coloring matter coupler in the first and the second reagents in the two-reagent-based reagent for measuring the objective biological component by colorimetrically determining hydrogen peroxide produced under the actions of an oxidase in the presence of the peroxidase according to oxidizing coloring matter coupling reaction.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention is intended to reduce or stabilize the blind value (blank) of a two-reagent system reagent for measuring biological components utilizing the reaction of oxidase, and is mainly used for clinical diagnostic agents. It is useful for improving quality.

[0002]

2. Description of the Related Art Reagents for measuring biological components, for example, various reagents for measuring free fatty acid, triglyceride, cholesterol, uric acid, glucose and the like in serum have been developed to date. Particularly in recent years, an enzymatic method has been developed, and it has become possible to measure these substances accurately in a shorter time. In the enzymatic method, an oxidase that mainly acts on a biological component itself to be measured or that acts on a substance secondarily generated by an enzyme from the biological component is used.
That is, hydrogen peroxide produced by the action of an oxidase is peroxidized with a suitable hydrogen donor of a phenol compound or an aniline compound and a suitable oxidizing dye coupler such as 4-aminoantipyrine or methylbenzthiazolone hydrazone. Color is developed in the presence of (oxidative dye coupling reaction), and the absorbance is measured to measure the target biological component.

[0003] Such an enzymatic method is carried out by a one-step method or a two-step method. The former is a one-reagent system in which all reagent components for measurement are combined, and the latter is a two-reagent system in which reagent components are divided into two. Use a reagent system. Comparing the two methods, the two-step method is generally more advantageous in that the stability of the reagent or the effect of reducing substances contained in the sample to be measured is less likely.

[0004]

However, even in the biological component measuring reagent by the two-step method, there is a degree of difference from the one-step method, and there is a rise in the reagent blank during storage, which is a serious problem. In the one-step method, a chromogenic reagent, that is, an oxidizing dye coupler such as peroxidase, 4-aminoantipyrine or methylbenzthiazolone hydrazone, and a hydrogen donor such as a phenol compound or an aniline compound are coexistent in one reagent. Therefore, there is a strong tendency that these dyes are easily oxidized and colored during storage of the reagent and the reagent blank rises. On the other hand, since the two-step method is a two-reagent system, the coloring reagent can be separated into the oxidizing dye coupler and the hydrogen donor, and in principle, there should be no rise in the reagent blank. However, in reality, the reagent blank gradually rises although it is lighter than the one-step method.

[0005] The inventors of the present invention have conducted detailed studies on the problem of an increase in the reagent blank in such a two-reagent system, and as a result, have found that the reagent containing no peroxidase (No.
(Reagent or the second reagent).

An object of the present invention is to provide a stable method for improving the composition of the reagent which does not contain peroxidase and suppressing the blank increase of the measuring reagent.

[0007]

According to the present invention, hydrogen peroxide produced by the action of an oxidase is colorimetrically determined by an oxidative dye coupling reaction in the presence of peroxidase to measure a target biological component. In the two-reagent type measuring reagent, catalase is added to the reagent not containing peroxidase, and the hydrogen donor and the oxidative dye coupler of the coloring system reagent are separately provided for the first reagent and the second reagent. The method for stabilizing a reagent for measuring a biological component is characterized by prescribing as follows.

In order to carry out the present invention, a hydrogen donor and an oxidative dye coupler of a chromogenic reagent out of two reagent reagents are prescribed separately in a first reagent and a second reagent, and a peroxidase is prepared. In particular, catalase is added as a stabilizer to the reagent containing no. Catalase is added only to the one that does not contain peroxidase, or is added and formulated to both the first and second reagents.

[0009] Further, peroxidase may be added to the reagent not containing peroxidase together with catalase.

Regarding the catalase used for this purpose, the addition concentration is preferably 1 U / ml or more regardless of the origin.

In the present invention, the oxidase reagent for producing hydrogen peroxide corresponds to the target biological component measurement, and refers to acyl coenzyme A oxidase, glycerin-3-phosphate oxidase, glucose oxidase, cholesterol oxidase, uricase and the like. Indicate.

Known hydrogen donors such as phenol compounds or aniline compounds can be used as the hydrogen donor of the color forming reagent, and 4-aminoantipyrine or methylbenzthiazolone hydrazone can be used as the oxidizing dye coupler.

The present invention is effective for a reagent for measuring a biological component containing substantially any buffer. In general, p
H4-9, phosphoric acid, Tris, PIPES,
Known buffering agents such as HEPES, tricine and glycine are used.

[0014]

EXAMPLES The present invention will be described in detail below with reference to examples.

Example Method for stabilizing reagent for measuring free fatty acid

(1) Preparation of Reagent 1 Reagent 1 50 mM potassium dihydrogen phosphate-potassium hydroxide buffer (pH 7.0) was added with acyl coenzyme A synthetase 0.4 U / ml, coenzyme A 0.3 mM, ATP1.
A solution containing 5 mM, 4-aminoantipyrine 2 mM and magnesium chloride 1 mM was prepared, and catalase was added thereto as a stabilizer at 0.1 U / ml, 1 U / ml, 10 U / m 2.
l, 100 U / ml, or 1000 U / ml. Using this as the first reagent, in a light-shielding container, 2-8
Store at 7 ° C for 7 days.

Reagent 2 Acyl coenzyme A oxidase 5 was added to 10 mM potassium dihydrogen phosphate-potassium hydroxide buffer (pH 7.0).
U / ml, FAD 3 μM, N-ethylmaleimide 1 mM,
Peroxidase 4 U / ml, N-ethyl-N- (2-hydroxy-3-sulfopropyl) -m-toluidine 3 mM
A solution containing is prepared. This is stored as a second reagent in a light-tight container at 2 to 8 ° C. for 7 days.

(2) Measurement procedure 1.0 ml of reagent 1 was added to 0.05 ml of purified water and left at 37 ° C. for 5 minutes, then 2.0 ml of reagent 2 was added and reacted for 5 minutes at 37 ° C. Measure at 555 nm. (Control water)

Comparative Example Reagents 1 and 2 which were exactly the same except for the catalase in the reagent 1 of the example were prepared, and the procedure was the same as that of the example.

(3) Results As shown in Table 1, by adding catalase to the first reagent containing no peroxidase, the rise of the reagent blank was significantly suppressed.

[0021]

[Table 1]

[0022]

INDUSTRIAL APPLICABILITY In a two-reagent system measuring reagent for measuring various biological components, particularly, catalase is added to a reagent not containing peroxidase, and a hydrogen donor and an oxidative dye coupler of a color-forming reagent are added. It was possible to prevent the reagent blank from increasing with time by prescribing so that the first reagent and the second reagent were separated.

Claims (1)

[Claims] 1. A two-reagent measuring reagent for measuring a target biological component by colorimetrically quantifying hydrogen peroxide produced by the action of an oxidase in the presence of peroxidase by an oxidative dye coupling reaction. In particular, catalase is added to the reagent that does not contain peroxidase, and the hydrogen donor of the chromogenic reagent and the oxidative dye coupler are formulated separately for the first reagent and the second reagent. And a method for stabilizing a reagent for measuring a biological component.