JPH0622794A - Indicator for measuring peroxide-decomposable substance and reagent kid - Google Patents

Abstract

PURPOSE:To enable the long period preservation of a peroxide-decomposable substance-measuring indicator containing a peroxide and a benzidine coloring agent. CONSTITUTION:The benzidine coloring agent-containing indicator for measuring peroxide-decomposable substances contains a buffer solution as a medium, a peroxide, the benzidine coloring agent and further a solubilizing agent.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention is used in the fields of enzyme immunoassay, biochemical assay, etc., and is used as an indicator containing a benzidine color-developing agent for the purpose of measuring a peroxide decomposable substance such as peroxidase. The present invention relates to a reagent kit containing this indicator.

[0002]

2. Description of the Related Art Conventionally, in the field of enzyme immunoassay and the like, an indicator in which a peroxide such as hydrogen peroxide and a benzidine color former are dissolved in an aqueous medium is known. This indicator uses a peroxide decomposable substance or a substance labeled with this substance as a measurement target, and the measurement of this peroxide decomposable substance is performed in the reaction between this substance and peroxide. This is performed by color development of the accompanying benzidine-based color developing agent.

Unlike the color formers such as ortho-phenylenediamine, which have been used for a long time, the benzidine-based color formers used as the indicator have low toxicity and have the advantage of high sensitivity, but they have the advantage of being highly sensitive to water. Dissolves only slightly and has a problem in storage stability at low temperatures. Therefore, conventionally, a benzidine-based color-developing agent was dissolved at a high concentration in an aqueous solution containing an organic solvent such as methanol or 1-methyl-2-pyrrolidone, and it was used after being diluted to an appropriate concentration with a buffer solution or the like before use ( For example, JP-A 63-199270 and JP-A 6-1994
2-500882, JP-A-56-76048, JP-A-4
9-133092).

[0004]

However, conventional benzidine-based indicators containing a large amount of organic solvent are relatively stable when the concentration of the organic solvent before preparation of the indicator is high, but dilute to the use concentration. After being used as an indicator, it is unstable, and a color former precipitates, color develops despite the absence of peroxide-decomposable substances such as peroxidase (blank value increases), and the degree of color development decreases. Since it cannot be stored for a long time at the use concentration, it was necessary to prepare it just before use. These things severely limit their usefulness as analysis means such as enzyme immunoassay. An object of the present invention is to provide an aqueous medium having excellent sensitivity and good storage stability when using a benzidine-based color former as an indicator for measurement.

[0005]

Means for Solving the Problems The inventors of the present invention have conducted extensive studies to achieve the above object, and as a result, by using a specific compound as a solubilizing agent for a benzidine-based color former, it can be stored for a long time as an indicator, and The inventors have found that an indicator capable of accurately measuring a low-concentration peroxide-decomposing substance can be obtained, and have reached the present invention. That is, the present invention comprises the following <1> and <2>. <1> A peroxide (A) and a benzidine-based color former (B) are dissolved in an aqueous medium, and the peroxide decomposable substance (C) is measured by the reaction between (A) and (C). The indicator which can be carried out by the color development of (B), further comprising at least one solubilizer (D) selected from compounds represented by the following general formulas (1) to (4): Indicator for measuring peroxide-decomposable substances. (In the formula, R ₁ is each independently an alkylene group having 2 to 4 carbon atoms, R ₂ is an alkyl group having 1 to 4 carbon atoms, R ₃ is hydrogen or an alkyl group having 1 to ₄ carbon atoms, and R ₄ is a carbon number. 1-4 alkylene groups, R ₅ is independently hydrogen or C 1
~ 4 alkyl group, m is an integer of 2-20, n is 1-20
And an integer of X + Y + Z is an integer of 1 to 20) <2> A reagent kit containing a peroxide-decomposable substance (C) or a compound labeled with (C) as a component,
A reagent kit comprising at least the indicator according to the above <1> or each component of the indicator together as a component.

In the present invention, examples of the peroxide (A) include hydrogen peroxide, inorganic peroxides (sodium perborate, potassium perborate, etc.), organic peroxides (cumene peroxide, t-). Butyl peroxide, diisopropylbenzene peroxide, 2,5-dimethylhexane-
2,5-peroxide and the like). Preferred among these is hydrogen peroxide.

In the present invention, examples of the benzidine-based color former (B) include various conventionally known benzidine-based color formers such as benzidine, nucleus-substituted benzidine, N-, N'-substituted benzidine and their hydrochlorides. To be
Specific examples of (B) include benzidine and benzidine.2.
HCl, O-Tolidine, O-Tolidine.2HCl, O-
Dianisidine, 3,3 ', 5,5'-tetra (lower alkyl) benzidine, 3,3', 5,5'-tetra (lower alkyl) benzidine · 2HCl · 2H ₂ O, 2,7-
Diaminofluorene and mixtures thereof may be mentioned.
As used herein, the term "lower alkyl" is methyl, ethyl,
n-propyl and isopropyl, and various butyls,
1 to 6 carbon atoms including pentyl and hexyl isomers
Means an alkyl group. Of these, preferred is 3,3 ', 5,5'-tetra (lower alkyl) benzidine, and particularly preferred is 3,3', 5,5'-tetramethylbenzidine. In the preparation of these benzidine-based color formers (B), the salt form can be added as it is to an aqueous medium, and the non-salt form can be dissolved in a small amount of an organic solvent and added to the aqueous medium. Good.

In the present invention, as the peroxide decomposing substance (C), for example, enzymes such as peroxidase (peroxidase extracted from horseradish, milk, leukocytes) or catalase, and the same action as the enzyme is exhibited. Hemoglobin, cytochrome-C and their derivatives,
UV, organic and inorganic metal salts are mentioned. Among these, preferred is peroxidase, and particularly preferred is peroxidase extracted from horseradish.

In the present invention, the solubilizer (D) is represented by the following general formula (In the formula, R ₁ is each independently an alkylene group having 2 to 4 carbon atoms, R ₂ is an alkyl group having 1 to 4 carbon atoms, R ₃ is hydrogen or an alkyl group having 1 to ₄ carbon atoms, and R ₄ is a carbon number. 1-4 alkylene groups, R ₅ is independently hydrogen or C 1
~ 4 alkyl group, m is an integer of 2-20, n is 1-20
And an integer of X + Y + Z is an integer of 1 to 20).

In the present invention, the compound represented by the general formula (1) in the solubilizer (D) includes diethylene glycol, triethylene glycol, tetraethylene glycol, polyethylene glycol 200 to 800 (weight average molecular weight 200 to 800). Oxyethylene addition polymer), dipropylene glycol, tripropylene glycol, polypropylene glycol 200 to 800 (oxypropylene addition polymer having a weight average molecular weight of 200 to 800), polyoxyethylene / polyoxypropylene copolymer, and the like. . Of these, preferred are triethylene glycol, tetraethylene glycol, and polyethylene glycols 200 to 800.

The compound represented by the general formula (2) in (D) is a mono- or dialkyl of the above ethylene glycols or propylene glycols (eg,
Examples thereof include methyl, ethyl, propyl, butyl) ether, ethylene oxide and propylene oxide copolymers of n-butanol, and the like. Of these, the preferred one is
A mono- or dimethyl ether of ethylene glycol or diethylene glycol or triethylene glycol.

Examples of the compound represented by the general formula (3) in (D) include ethylene oxide addition polymer of glycerin, propylene oxide addition polymer of glycerin, and ethylene oxide and propylene oxide addition copolymer of glycerin. Of these, preferred are ethylene oxide addition polymers of glycerin and ethylene oxide and propylene oxide addition copolymers of glycerin.

Further, among the compounds represented by the general formula (4) in (D), 2-methyl-2,4-pentanediol, 2,4-dimethyl-2,4-pentanediol,
2,5-dimethyl-2,5-hexanediol may be mentioned. Among these, preferred is 2-methyl-2,
It is 4-pentanediol.

The content of each component constituting the indicator of the present invention will be described. The respective concentrations of the peroxide (A), the benzidine color-developing agent (B) and the solubilizing agent (D) in the indicator of the present invention are not optimal depending on the measuring method and the conditions for promoting the color-developing reaction. must not.
Further, it is preferable to consider the balance of the mutual concentrations of these (A), (B) and (D). Although there are these premise, the content of each component in the indicator of the present invention, the peroxide (A) is usually 0.005-5.0 mg / ml, preferably 0.05-0.2 mg / ml. ml. The color former (B)
Is usually 0.01 to 10.0 mg / ml, preferably 0.03 to 2.0 mg / ml. The solubilizer (D)
Is usually 5.0 to 800 mg / ml, preferably 50.0
~ 500 mg / ml.

The measurement of the peroxide decomposable substance (C) using the indicator of the present invention was carried out on the acidic side (pH 2.5 to 6.5).
Is preferable, and particularly when measured at pH 3.0 to 5.5, stable and good color development is obtained. As the buffer solution used as the place for the color development reaction, any kind of buffer solution can be used as long as it satisfies the above pH, but potassium hydrogen phthalate / sodium hydroxide buffer solution, citrate diacid solution can be used. Sodium / hydrochloric acid buffer, potassium dihydrogen citrate / sodium hydroxide buffer, succinic acid / sodium tetraborate buffer, potassium hydrogen citrate / sodium tetraborate buffer, sodium hydrogen phosphate / citrate buffer, Examples thereof include sodium acetate / hydrochloric acid buffer solution, acetic acid / sodium acetate buffer solution and the like. At this time, the preferable buffer solution concentration is 0.1 to 100 mM, particularly 1 to 50 mM.

The method for preparing the indicator of the present invention is as follows:
(B) may be added directly to (D), or one obtained by previously dissolving (B) in a high concentration in another solvent or the like may be added.
The above-mentioned buffer solution, peroxide (A) and peroxide decomposable substance (C) are added to these to obtain the indicator of the present invention, but it is not particularly limited thereto.

The color development reaction is preferably carried out at a temperature most suitable for the reaction (C) (in the case of an enzyme such as peroxidase, it is usually 20 to 50 ° C.).

Even if the peroxide-decomposing substance (C) to be measured is in a free state, the binding substance (eg, antigen, antibody, single-stranded nucleic acid, hapten, protein A) such as an immunoreactive substance can be obtained depending on (C). , Avidin and biotin) may be labeled, or the labeled substance may be in a complex state after binding with another substance.

The reagent kit of the present invention comprises, as components, at least the indicator of the present invention or each component of the indicator of the present invention, together with the peroxide decomposable substance (C) or a substance labeled with (C). Is. As the substance to be labeled here, a binding substance such as the immunoactive substance as exemplified above can be used. Since the indicator of the present invention has good storage stability, it can be used as a component of the reagent kit of the present invention in this way, so that each component of the indicator does not need to be blended each time as in the conventional case. There are good advantages. Further, the components of the indicator of the present invention can be used as the components of the kit of the present invention, and it is necessary to mix them at the time of use, but there is still an advantage that the stability after mixing increases. .

[0020] As an example used in the enzyme immunoassay, an example of a kit containing the "enzyme-labeled antibody" and the "indicator of the present invention" as components will be given below. The antibody is bound in advance to a solid support such as a test tube, glass beads, plastic beads, microparticles, nylon membrane, filter paper. A sample is added to this, and the first reaction is performed with the antigen that is the substance to be measured to form a "carrier-bound antibody-antigen" complex. Unreacted material is removed, then "enzyme-labeled antibody" that has an affinity for the complex is added, and a second reaction is performed.
A complex "carrier-bound antibody-antigen-enzyme labeled antibody" is formed. The unreacted material is removed again, and the enzyme bound to the carrier is finally reacted with the indicator of the present invention to measure the absorbance.
Since this absorbance is proportional to the amount of the substance to be measured in the sample, the amount of the substance to be measured in the sample can be obtained from the calibration curve prepared by measuring the standard substance having a known concentration.

The reagent kit of the present invention utilizes, for example, an enzyme immunoreaction, α-fetoprotein (AFP), carcinoembryonic protein (CEA), tumor markers such as ferritin, thyroid stimulating hormone (TSH), luteinizing hormone. (L
H), follicle stimulating hormone (FSH), hormones such as insulin, cortisol, triiodothyronine (T
3), hapten hormones such as thyroxine (T4), drugs such as digoxin and theophylline, infectious substances such as bacteria and viruses, hepatitis antibodies and the like, but not limited thereto.

As a test reagent other than the enzyme immunoassay, an example of a hemoglobin quantitative reagent kit containing the "indicator of the present invention" as a component is given. "Indicator of the invention",
Mix strontium peroxide. Then, a sample containing hemoglobin is added and reacted for a certain period of time. Absorbance is measured using the blank as a control. The concentration of hemoglobin in the sample is obtained from the calibration curve created by measuring hemoglobin of known concentration.

[0023]

The present invention will be further described with reference to the following examples.
The present invention is not limited to this. Further, the indicator of the present invention, if necessary, various additives (eg, stabilizers such as chelating agents, bactericides and antiseptics to prevent decay,
It is also possible to add a surfactant, a colorant, an antifoaming agent, etc.).

Example 1 1) Preparation of indicators [1] to [15] The following compositions were prepared as shown in Table 1 below to obtain indicators [1] to [15], respectively. 30 mM citrate buffer (pH 5.1) solubilizer 5% hydrogen peroxide solution 3,3 ′, 5,5′-tetramethylbenzidine solution (3,3 ′, 5,5′-tetramethylbenzidine (Dojindo Kagaku) (Manufactured) was dissolved in acetone to prepare 200 mg / ml)

[0025]

[Table 1]

2) Comparative indicators [ratio 1] and [ratio 2]
Comparative indicators [ratio 1] and [ratio 2] were obtained in the same manner as above except that methanol or 1-methyl-2-pyrrolidone was used as the prepared solubilizer. It is shown in Table 2 below.

[0027]

[Table 2]

3) 3,3 ', 5,5'-tetramethylbe
Effect of solubilizers on benzidine In order to compare the stability (solubility) of the indicators after preparation, indicators [1] to [15] and [ratio 1] and [ratio 2] were used for 1 week and 1 month immediately after preparation. Table 3 below shows the results of examining the presence or absence of crystal precipitation after storage at 4 ° C for 1 year.

[0029]

[Table 3]

Example 2 1) Preparation of peroxidase solution (abbreviated as POD solution) 1% bovine serum albumin, 0.85% NaCl, pH 7.
2, 0.02M phosphate buffer (hereinafter abbreviated as PBS) 1m
0.5 mg of peroxidase (hereinafter abbreviated as POD; manufactured by Toyobo) was dissolved in 1 and further diluted 500 times with PBS to obtain a POD solution (1 μg / ml).

2) Blank test of indicator Example 1 The spectrophotometer (manufactured by Shimadzu Corp.) was used to measure the absorbance of the prepared indicators [3], [6], [14] and [ratio 1], [ratio 2] at 650 nm. UV-160A) was used to measure water as a control. In addition, a similar indicator at 4 ° C and 25
With respect to the samples stored at 1, 3, 6 months respectively, blanks were similarly measured using water as a control.
The results are shown in Table 4 below.

[0032]

[Table 4]

Example 3 1) Color development test of indicator Each of the indicators [3], [6], [14] and [ratio 1], [ratio 2] 300 μl prepared in Example 1 was treated with 5 parts of POD solution.
0 μl was added, and the mixture was reacted at 37 ° C. for 15 minutes. afterwards,
Using a spectrophotometer, the degree of color development at 650 nm was measured.
Furthermore, at 4 ° C and 25 ° C, the POD solution was also reacted with the same indicator stored for 1, 3 and 6 months respectively, and the absorbance was measured using the solution reacted by adding water instead of the POD solution as a control. did. The results are also shown in Table 5 below.

[0034]

[Table 5]

[0035]

The indicator of the present invention has the following remarkable effects. (1) Since the indicator of the present invention has good storage stability, it can be used as a component of a reagent kit. Conventional indicators are
It is unstable after preparation, and the color former precipitates, and color develops (blank value increases) even in the absence of peroxide-decomposable substances such as peroxidase, and the degree of coloration decreases, so it is prepared immediately before use. It solves the problem of having to do. (2) The surface tension is reduced, and the accuracy measured by the device is increased.

Claims (6)

[Claims] 1. A method in which a peroxide-decomposable substance (C) is prepared by dissolving a peroxide (A) and a benzidine-based color former (B) in an aqueous medium. In the indicator which can be performed by the color development of (B) accompanying the reaction,
An indicator for measuring a peroxide decomposable substance, which further comprises at least one solubilizer (D) selected from compounds represented by the following general formulas (1) to (4). (In the formula, R ₁ is each independently an alkylene group having 2 to 4 carbon atoms, R ₂ is an alkyl group having 1 to 4 carbon atoms, R ₃ is hydrogen or an alkyl group having 1 to ₄ carbon atoms, and R ₄ is a carbon number. 1-4 alkylene groups, R ₅ is independently hydrogen or C 1
~ 4 alkyl group, m is an integer of 2-20, n is 1-20
, X + Y + Z is an integer of 1 to 20) 2. The content of (D) is 5 to 800 mg / ml.
The indicator according to claim 1, which is 3. The indicator according to claim 1, wherein (B) is 3,3 ′, 5,5′-tetramethylbenzidine. 4. The content of (B) is 0.01 to 10.0 mg.
The indicator according to any one of claims 1 to 3, which is / ml. 5. The indicator according to claim 1, wherein (C) is peroxidase. 6. A reagent kit comprising a peroxide decomposable substance (C) or a substance labeled with (C) as a constituent, at least the indicator according to any one of claims 1 to 5 or each component of the indicator. A reagent kit, characterized in that both are components.