JPS63246665A - Stabilization of reagent for measuring biological component - Google Patents

Abstract

PURPOSE:To stabilize a reagent, by adding peroxidase or catalase to a reagent containing no peroxidase in a two-reagent system reagent for determining hydrogen peroxide formed by an oxidation enzyme. CONSTITUTION:A solution is prepared containing an oxidation enzyme such as acylcoenzyme A oxidase, coenzyme A, ATP, 4-aminoantipyrine and magnesium chloride and peroxidase or catalase as stabilizer is added to the solution to make a first reagent. A second reagent is prepared as solution containing acylcoenzyme A oxidase, FAD, N-ethylmaleimide, peroxidase and N-ethyl-N-(2- hydroxy-3-sulfopropyl)-m-toluisine. Thus, secular rise of a reagent blank can be prevented.

Description

[Detailed Description of the Invention] [Industrial Application Field 1] The present invention aims to reduce and stabilize the blind value (blank) of a two-reagent system reagent for measuring biological components using the reaction of oxidase. It is mainly used to improve the quality of clinical diagnostic reagents.

[Prior Art] Until now, reagents for measuring biological components, such as free fatty acids in serum, triglycerides, cholesterol, uric acid,
Various reagents for measuring glucose and the like have been developed.

Particularly in recent years, #identification has been developed, making it possible to measure these substances more accurately in a shorter time. Enzymatic methods mainly act on the biological components themselves to be measured.
Alternatively, an oxidizing enzyme that acts on a substance secondarily produced by an enzyme from the biological component is used. That is, hydrogen peroxide produced by the production of oxidase is converted into peroxidase using a suitable phenol-based or aniline-based hydrogen donor and a suitable oxidizing dye coupler such as 4-aminoantipyrine or methylbenzthiazolone hydrazone. The biological component of interest is measured by developing color in the presence of the dye (oxidative dye coupling reaction) and measuring its absorbance.

These #identifications are performed using a one-step method or a two-step method; the former uses a single reagent system in which all the reagent components for measurement are combined, and the latter uses a two-reagent system in which the reagent components are divided into two. use. When comparing both methods, the two-step method is generally more advantageous in terms of stability of the reagent and less influence by reducing substances contained in the sample to be measured.

[Problems to be Solved by the Invention] However, even with biological component measuring reagents based on the two-step method, reagent blanks rise during storage, although the degree of reagents differs to a certain extent from the one-step method, which poses a major problem. In the one-step method, color-forming reagents, i.e., peroxidase, oxidizing dye couplers such as 4-7 minoantibiline or methylbenzthiazolone hydrazone, and hydrogen donors such as phenol or aniline, coexist in one reagent. Therefore, during storage of the reagent, these dyes are easily oxidized and develop color, which tends to increase the reagent blank.
Since the two-step method uses a two-reagent system, the color-forming reagent can be separated into two, the oxidizing dye coupler and the aqueous donor, so in principle there should be no rise in reagent blanks.

However, in reality, the reagent blank gradually rises, although it is milder than in the one-step method.

As a result of detailed study by the present inventors regarding the problem of increase in reagent blank in the two-reagent system, we found that the reagent containing no peroxidase (first reagent or second reagent)
It was determined that the cause was the reagent.

An object of the present invention is to provide a stable method for improving the composition of the above-mentioned peroxidase-free reagent and suppressing blank rise in the measurement reagent.

[Means for Solving the Problems] The present invention measures a target biological component by colorimetrically quantifying hydrogen peroxide produced by the action of an oxidase in the presence of peroxidase through an oxidative dye coupling reaction. In a two-reagent system measurement reagent, the reagent that does not contain peroxidase is especially
Alternatively, it is a method for stabilizing a reagent for measuring biological components, which is characterized by adding catalase.

To carry out the present invention, the hydrogen donor and oxidative dye coupler of the coloring reagent are separately formulated into the first reagent and the second reagent among the reagents for two-reagent system measurement, and peroxidase is contained. To the other reagent, in particular peroxidase or catalase is added as a stabilizer. In the case of peroxidase, it ends up being prescribed to both the first reagent and the @2 reagent. In the case of catalase, it is added only to the one that does not contain peroxidase, or it is added to both the first reagent and the second reagent.

The peroxidase used for this purpose may be of any origin, and a clear effect is seen when it is added to the reagent at a concentration of 0.001 U/yr1 or more. Regarding catalase, it is preferable that the concentration of catalase added is at least 1 Ulz1, regardless of its origin.

In the present invention, the oxidase reagent that generates hydrogen peroxide corresponds to the target biological component measurement, and includes acylphenzyme A oxygase, glycerin-3-phosphate oxygase, glucose oxidase, cholesterol oxygase, uricase, etc. Show.

As the hydrogen donor of the color-forming reagent, a known 7-ol compound or aniline compound can be used, and as the oxidizing dye coupler, 4-amino/antipyrine or methylbenzthiazolone hydrazone can be used. can.

The present invention is also effective in reagents for measuring biological components containing substantially any buffering agent. Generally, the pH is in the range of 4 to 9, and phosphoric acid, Tris, PES, HEPES
, ) Known buffering agents such as lysine and glycine are used.

[Effect] In a two-reagent system measuring reagent for measuring various biological components, by adding peroxidase or catalase to the reagent that does not contain peroxidase,
It is now possible to prevent the reagent blank from increasing over time.

Hereinafter, the present invention will be illustrated in detail with reference to Examples.

Example 1 Stabilization method of reagent for measuring free fatty acids (1) Preparation of reagent ■Reagent 1 50mM potassium hydroxide t&W
B fL (pH 7,0) with 7silcoenzyme A synthetase 0.4 U/xi, coenzyme A 0.3 mM
.

ATP 1.5mM, 4-aminoantipyrine 2
a+M.

Prepare a solution containing 1 mM of magnesium chloride, and add peroxidase as a stabilizer at IU/x (l) or catalase at 100 U/M. Use this as the first reagent in a light-tight container for 2 to 30 minutes. Store at 8°C for 7 days.

■Reagent 2 7silcoenzyme A oxyguse 5 in 10mM potassium dihydrogen phosphate-potassium hydroxide buffer (pH 7.0)
U/xl, FAD 3μM, N-ethylmaleimide 1
mM, peroxidase 4U/xi, N-ethyl-N-
A solution containing 3mM of (2-hydroxy-3-sulfopropyl)m-)ruinone is prepared. This is stored as a second reagent in a light-shielded container at 2 to 8°C for 7 days.

(2) Measurement procedure Add reagent 1 to 0.05+N purified water. Oif plus 37
℃ for 5 minutes, then add reagent 2 to 2. OR1 plus 3
After reaction for 5 minutes at 7°C, absorbance is measured at 555 nm. (Water as a control) Comparative Example 1 Reagent 1.2 was prepared which was completely the same as in Reagent 1 of Example 1 except for peroxidase or catalase, and
The operating method is also the same as in Example 1.

(3) Results As shown in Table 1, by adding peroxidase or catalase to the first reagent not containing peroxidase, the increase in the reagent blank was clearly suppressed.

Table 1 and a half CAT: Catalase, POD: Peroxyguze Xinhao 1st and 2nd reagent storage period Example 2 Stabilization method of triglyceride measurement reagent (1) Preparation of reagent ■Reagent 1 20mM PIPES-sodium hydroxide buffer ( pH7
, 0) with 6 U of glycerol-3-phosphate oxidase/
xi, glycerokinase 0.5 U/x1, ATP
1.5mM, magnesilla chloride A 20mM, peroxidase 3U/all SN-(2-carboxyethyl)-N-ethyl-ra-)luidine 2mM
Prepare a complete solution. This is used as the first reagent and stored in a light-shielded container at 2 to 8°C for 30 days.

■Reagent 2 Lipoprotein lipase 500U/20mM PIPES-sodium hydroxide buffer (pH 7.0)
zf.

A solution containing 1.5mM of 4-7 minoantipyrine was prepared, and 0.0% of peroxidase was added as a stabilizer to this solution.
Add IU/z (1) as the second reagent and store it in a light-tight container at 2-8°C for 30 days.

(2) Measurement procedure Add 1.5x1 of reagent 1 to 0.02xI of purified water, 37
The mixture was allowed to stand for 5 minutes at °C, then 1.5xl of Reagent 2 was added, and after reacting at 37 °C for 5 minutes, the absorbance was measured at 550 nm. (Water as a control) Comparative Example 2 Reagent 1.2, which is completely the same except for peroxidase in Reagent 2 of Example 2, is prepared, and the procedure is the same as in Example 2.

(3) Results As shown in Table 2, the stability of the reagent blank was significantly improved by adding peroxidase to the second reagent that did not contain peroxidase. .

Table 2

Claims (1)

[Claims] Contains peroxidase in a two-reagent measuring reagent that measures target biological components by colorimetrically quantifying hydrogen peroxide produced by the action of oxidase in the presence of peroxidase using an oxidative dye coupling reaction. 1. A method for stabilizing a reagent for measuring biological components, which comprises adding peroxidase and/or catalase to the other reagent.