JPH06165696A - Indicator for measuring peroxide decomposing substance and reagent kit - Google Patents

Abstract

PURPOSE:To improve performances of an indicator for measuring a substance decomposable with a peroxide, comprising a peroxide and a benzidine-based color coupler. CONSTITUTION:A buffer solution as a medium is mixed with a peroxide, a benzidine-based color coupler and gamma-cyclodextrin and the benzidine-based color coupler is dissolved to give an indicator for measuring a substance decomposable with a peroxide.

Description

Detailed Description of the Invention

[0001]

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

[0002]

2. Description of the Related Art In the fields of enzyme immunoassays, biochemical assays and the like, indicators in which a peroxide such as hydrogen peroxide and a benzidine type color former are dissolved in an aqueous medium are known. This indicator uses a peroxide decomposable substance or a compound 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 for this indicator have many advantages of low toxicity and high sensitivity, but they are not suitable for water. Only slightly soluble. Therefore, methanol, an aqueous solution containing an organic solvent such as 1-methyl-2-pyrrolidone, a benzidine-based color developing agent is dissolved at a high concentration, and diluted with a buffer solution or the like to an appropriate concentration before use (for example, JP-A-63-19927
0, JP-A-62-500882, JP-A-59-1668
65, JP-A-49-133092).

However, while these indicators are relatively stable when the organic solvent before dilution is at a high concentration, when they are diluted to the use concentration, they are not sufficiently stable in storage and cannot be stored with the passage of time. Accurate measurement is not possible because the color developing agent is deposited, the sensitivity is reduced, and the oxidation reaction of peroxide-decomposable substances such as peroxidase is inhibited, so that the absorbance proportional to the amount of peroxidase cannot be obtained. There were many problems such as difficulty in doing so.

On the other hand, for use in a system containing no organic solvent, a chelating agent or β-cyclodextrin (β-
Attempts have been made to improve the storage stability by CD (for example, JP-A-59-193354 and JP-A-62-5021).
00, JP-A-63-61953).

[0006]

[Problems to be Solved by the Invention] However, these
In this system, precipitation of the color former was still observed over time.
As a result, a decrease in sensitivity was observed, and peroxide-decomposable substances
Accurate measurement of quality, especially in direct proportion to peroxidase
Was still difficult. These things are important for accuracy
In analysis means such as enzyme immunoassay that is considered as an element,
It significantly limits its usefulness. The invention is Ben
Prepared when using a dizine-based coloring agent as an indicator for measurement
The sensitivity is high and no decrease in sensitivity is observed with the passage of time.
The correct absorbance is proportional to the amount of peroxide-decomposable substance
The purpose is to provide an indicating agent.

[0007]

Means for Solving the Problems As a result of intensive studies for improving the above-mentioned problems, the present inventors have found that the use of a specific compound as a stabilizer for a benzidine-based color former provides high sensitivity during preparation, Further, they have found that the sensitivity does not decrease for a long period of time, and that an accurate value proportional to the amount of the peroxide decomposable substance can be measured, and the present invention has been accomplished. That is, the present invention includes 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 a reaction between (A) and (C). In the indicator which can be performed by the color development of (B),
An indicator for measuring a peroxide-decomposable substance, characterized by containing CD. <2> A reagent kit comprising a peroxide decomposable substance (C) or a compound labeled with (C) as a component,
A reagent kit comprising at least the indicator described in 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, a benzidine-based coloring agent
Examples of (B) include benzidine and nuclear-substituted benzidi.
And N, N'-substituted benzidines, their hydrochlorides, etc.
Conventionally known various benzidine-based color formers can be mentioned.
Specific examples of (B) include benzidine and benzidine.2.
HCl, O-Tolidine, O-Tolidine.2HCl, O-
Dianisidine, 3,3 ', 5,5'-tetra (lower alkane
Kill) benzidine, 3,3 ', 5,5'-tetra (lower)
Alkyl) benzidine ・ 2HCl ・ 2H2 O, 2, 7-
Examples thereof include diaminofluorene. For use here
The term "lower alkyl" refers to an alkyl group having 1 to 6 carbon atoms (meth
Ru, ethyl, n-propyl and isopropyl, and seeds
Butyl, pentyl and hexyl isomers)
It Two or more of the materials exemplified above as (B) are used in combination.
Good. Of these, preferred are 3,3 ', 5,5'
-Tetra (lower alkyl) benzidine, particularly preferred
Is 3,3 ', 5,5'-tetramethylbenzidine
is there. The preparation of these benzidine-based color-developing agents (B) is carried out using salt
It can be added to the aqueous medium as it is.
If it is not in salt form, it is dissolved in a small amount of organic solvent and is aqueous.
Add it to the medium and remove the solvent to the extent that it does not affect the performance.
You may leave.

In the present invention, as the peroxide decomposing substance (C), an active enzyme such as peroxidase (peroxidase extracted from horseradish, milk, leukocytes, etc.) or catalase, and hemoglobin exhibiting the same action as this enzyme. , Cytochrome C and their derivatives,
Ultraviolet rays, organic and inorganic metal salts and the like can be mentioned. Of these, preferred are peroxidases, especially peroxidase extracted from horseradish.

In the present invention, the cationic surfactant (D) includes amine salt type, quaternary ammonium salt type and the like, and quaternary ammonium salt type is preferable. Examples of the quaternary ammonium salt type include benzalkonium chloride, benzethonium chloride, stearyldimethylbenzylammonium chloride, myristyldimethylbenzylammonium chloride, methyllauryldimethylbenzylammonium chloride and didecyldimethylammonium chloride. Particularly preferred among these is benzalkonium chloride.

The content of each component constituting the indicator of the present invention will be described. The concentrations of the peroxide (A), the benzidine-based color-developing agent (B), γ-CD and the cationic surfactant (D) in the indicator of the present invention are optimal depending on the measuring method and the conditions for promoting the color-developing reaction. The concentration must be set. Moreover, these (A), (B), and γ-
The balance of the mutual concentrations of CD and (D) may also be considered. Although these are premised, the content of (A) is usually 0.005 to 5 in the content of each component in the indicator of the present invention.
5 mg / ml, preferably 0.05-0.2 mg / ml
Is. For (B), usually 0.01 to 5.0 mg
/ Ml, preferably 0.03 to 1.0 mg / ml. γ-CD is usually 0.1 to 50 mg / ml,
It is preferably 0.5 to 30 mg / ml. Further, the cationic surfactant (D) is not particularly limited, but may be a concentration usually used. Specifically 0.01 to 50 mg
/ Ml, preferably 0.5-10 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).
It is preferable to carry out at pH 3.0 to 5.
When measured at 5, stable and good sensitivity can be 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 /
Examples thereof include sodium tetraborate buffer, potassium hydrogen citrate / sodium tetraborate buffer, disodium hydrogen phosphate / citrate buffer, sodium acetate / hydrochloric acid buffer, acetic acid / sodium acetate buffer. At this time, the preferred buffer concentration is 0.1 to 100 mM, particularly 1 to 50 mM.
Is.

[0014] The method for preparing the indicator of the present invention includes
The dizine-based color-developing agent (B) may be added directly.
Add (B) dissolved in a high concentration to a solvent, etc.
After dissolution, the solvent may be removed if necessary. Above these
Add peroxide (A) and peroxide decomposable substance (C)
To obtain the indicator of the present invention, but is particularly limited to these
Not a thing.

[0015] The temperature for the color reaction is peroxide decomposable
The most suitable temperature for the reaction of substance (C) (peroxidase
In the case of enzymes such as, it is usually carried out at 20-50 ° C)
Is preferred.

Even if the peroxide decomposing substance (C) to be measured is in a free state, the binding substance (eg, antigen, antibody, hapten, protein A, avidin, biotin, etc.) such as an immunoreactive substance can be obtained by (C). (Nucleic acid) may be in a labeled state, or the labeled substance may be in a complex state after binding with another substance.

The reagent kit of the present invention comprises a peroxide-decomposable substance (C) or a compound labeled with (C),
At least the indicator of the present invention or each component of the indicator of the present invention is a component. As the compound referred to herein, a binding substance such as an immunoactive substance as exemplified above can be used.

Since the indicator of the present invention has high sensitivity, can be stored for a long period of time, and can be accurately measured in proportion to the concentration of a peroxide decomposing substance such as peroxidase, it is thus a component of the reagent kit of the present invention. Therefore, there is an advantage that each component of the indicator does not have to be compounded each time as in the conventional case. 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 are advantages such as improvement in sensitivity and accuracy.

[0019] Below, as an example used for enzyme immunoassay
"Enzyme-labeled antibody" and "Indicator of the present invention"
Examples of kits including Test tube, glass bee
, Plastic beads, micro particles, ny
Antibodies are previously bound to a solid phase carrier such as a Ron membrane or filter paper.
Ku. A sample is added to this, and the first
Reaction to form the "antibody-binding carrier-antigen" complex
To do. Unreacted material is removed and then the affinity of the complex
Add a certain "enzyme-labeled antibody" and perform the second reaction,
Form a complex "antibody-binding carrier-antigen-enzyme-labeled antibody"
To achieve. Unreacted material is removed again, and finally bound to the carrier.
The enzyme is reacted with the indicator of the present invention and the absorbance is measured.
This absorbance is proportional to the amount of target substance in the sample
From a calibration curve created by measuring a standard substance of known concentration
The amount of the substance to be measured in the sample can be calculated.

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),
It can be used for measuring drugs such as digoxin and theophylline, infectious substances such as bacteria and viruses, hepatitis antibodies, antibodies such as IgE, but is not necessarily limited to these items.

[0021] In addition, as a test reagent other than enzyme immunoassay
And hemoglobin containing the "indicator of the present invention" as a component
An example of a quantitative reagent kit is given below. "Indicator of the invention",
Mix strontium peroxide. Then hemoglobin
And a sample containing the same is reacted for a certain period of time. Absorbance
Rank is measured as a control. Known concentration of hemoglobin
From the calibration curve created by measuring
Calculate the concentration.

[0022]

EXAMPLES The present invention will be further described below with reference to examples.
However, the present invention is not limited to this. Also,
The light indicator may have various additives (e.g.
Rate agents, bactericides and antiseptics to prevent spoilage, surfactants,
It is also possible to further add a colorant, an antifoaming agent, etc.).

[0023]Example 1 ¹⁾ Preparation of indicators [1] to [3]  Add 10 ml of 20 mM citrate buffer (pH 4.0) to
Add hydrogen oxide so that the amount of hydrogen oxide becomes 0.05 mg / ml. This
Γ-CD to a final concentration of 1.0, 4.0 and 1
It was added to 6.0 mg / ml, and
3 ', 5,5'-tetramethylbenzidine.2HCl
Add 5.0 mg (hereinafter abbreviated as TMB salt) and stir well
By doing so, the indicators of the present invention [1] to
Obtained [3].

[0024]²⁾ Preparation of indicators [ratio 1] and [ratio 2]  Add 10 ml of 20 mM citrate buffer (pH 4.0) to
Add hydrogen oxide so that the amount of hydrogen oxide becomes 0.05 mg / ml. This
Solution of N- (2-hydroxyethyl) ethylenedia
Mineral triacetate iron chelate (^Fe-HEDTA) * 1 And β-CD
Add 5.0 mg / ml each, and add T
By adding 5.0 mg of MB salt and stirring well,
The indicators [ratio 1] and [ratio 2] were obtained.  * 1 Commercially available_HEDTAas well as_FeCl3・ 6H₂Equimolar amount of O in aqueous solution
Mixed with iron: chelate 1: 1 (mol: mol) Fe-HEDT
Prepare solution A

3) Peroxidase solution (with POD solution
Abbreviated) 1% bovine serum albumin, 0.85% NaCl, pH 7.
2, 0.02M phosphate buffer (hereinafter abbreviated as PBS) 1
0.5 ml peroxidase (hereinafter abbreviated as POD)
Dissolve mg and further dilute it 500 times with PBS,
D solution <1> (1 μg / ml) was obtained. In addition, the POD solution <1> is diluted 2, 4, or 8 times with PBS to obtain P
OD concentrations of 0.5, 0.25 and 0.125 μg / ml were prepared and used as POD solutions <2>, <3> and <4>, respectively.

4) Sensitivity test of indicators [1] to [3], [ratio 1] and [ratio 2] 300 each
Add 50 μl of POD solution <1> to μl,
It was reacted for 15 minutes. Then, using a spectrophotometer,
The absorbance at 0 nm was measured using a solution prepared by reacting with water instead of the POD solution as a control. 4 ° C, 2
With respect to the same indicator stored at 5 ° C. for 1, 3 and 6 months, the POD solution <1> was reacted and the absorbance was measured in the same manner. The results are shown in Table 1 below. As is clear from the results shown in Table 1, the indicators [1] to [3] of the present invention show significantly less decrease in sensitivity at the time of preparation and over time than the indicators [ratio 1] and [ratio 2]. .

[0027]

[Table 1]

5) Dilution linearity test indicator [2], [ratio 1] and [ratio 2] 300 μl each,
POD solutions <1>, <2>, <3> and <4> 50 each
μl was added, and the mixture was reacted at 37 ° C. for 15 minutes, 4) of Example 1.
The absorbance of each POD solution was measured according to the sensitivity test of 1.
The results are shown in Table 2 below and FIG. In Figure 1, the horizontal axis is P
6 is a graph showing the relationship between POD concentration and absorbance by taking the OD concentration and the absorbance at 650 nm on the vertical axis. Compared to the indicators [ratio 1] and [ratio 2], the indicator [2] is the POD in the entire area
There is linearity in the concentration, indicating that accurate measurement is possible.

[0029]

[Table 2]

Example 2 1) Preparation of indicator [4] Hydrogen peroxide was added to 10 ml of 20 mM citrate buffer (pH 4.0) so that the concentration would be 0.05 mg / ml. To this solution, benzalkonium chloride and γ-CD are added to final concentrations of 5.0 mg / ml and 4.0 mg / ml, respectively. Further, 5.0 mg of TMB salt was added and stirred well to obtain the indicator [4] of the present invention.

2) Preparation of indicators [ratio 3] and [ratio 4] The indicator [ratio 3] was prepared in the same manner as the indicator [4] except that Fe-HEDTA and β-CD were added instead of γ-CD. ]
And [ratio 4] were obtained.

3) Sensitivity test of indicator Example except that indicator [4], [ratio 3] and [ratio 4] are used instead of indicator [1] to [3], [ratio 1] and [ratio 2]. The absorbance was measured by the same method as in 1) 4). The results are shown in Table 3 below. As is clear from the results of Table 3, the indicator [4] of the present invention shows a markedly smaller decrease in sensitivity at the time of preparation and over time, as compared with the indicators [ratio 3] and [ratio 4].

[0033]

[Table 3]

4) Dilution linearity test Example 1 except that the indicators [4], [ratio 3] and [ratio 4] were used instead of the indicators [1] to [3] and [ratio 1].
The absorbance was measured by the same method as 5) above. The results are shown in Table 4.
And shown in FIG. In FIG. 2, the horizontal axis represents POD concentration and the vertical axis represents 6
It is the graph which took the light absorbency of 50 nm, and showed the relationship between POD concentration and sensitivity. Compared to the indicators [ratio 3] and [ratio 4], the indicator [4] has linearity even in the entire region, which indicates that accurate measurement is possible.

[0035]

[Table 4]

[0036]

【The invention's effect】

(1) Improvement of sensitivity The indicator of the present invention has high sensitivity during preparation in the concentration range of peroxidase used. Therefore, the significant difference can be clearly discriminated even in the measurement in the region where the content of peroxidase is extremely low. This is very suitable for a test reagent that requires high sensitivity. (2) Improvement of storage stability Since the indicator of the present invention has good storage stability, it can be used as a component of a reagent kit. The conventional indicator solves the problem that it has to be prepared immediately before use because its sensitivity decreases with time. Further, with respect to the deposition of the color-developing agent over time, the deposition of crystals does not occur, and it is possible to stably supply the reagent for a long period of time. (3) Improvement of accuracy Even when the indicator of the present invention is in a high concentration range or a low concentration range of a peroxide-decomposable substance such as peroxidase, it is extremely proportional to the amount of the peroxide-decomposable substance. It has the advantage that it can be measured accurately. That is, the sensitivity is not lowered in the low value region due to the inhibition of the color development reaction as seen with the conventional indicators, and as a result, the measurement error does not occur.
This is very preferable for test reagents that require strict performance. (4) Prevention of carryover The indicator of the present invention is a carryover (in particular, when a highly sensitive sample is measured with a measuring device such as a flow cell, the measured value of the next sample is actually added by adding a cationic surfactant. Higher) can be prevented. This is very advantageous when automatically measuring a large number of samples. (5) Antiseptic effect The indicator of the present invention containing a cationic surfactant is antiseptic by its bactericidal action.

[Brief description of drawings]

FIG. 1 shows an indicator [2] in Example 1,
2 shows the relationship between the POD density and the degree of color development of [ratio 1] and [ratio 2].

FIG. 2 shows an indicator [4] in Example 2,
4 shows the relationship between the POD density and the degree of color development of [ratio 3] and [ratio 4].

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[Procedure amendment]

[Submission date] November 10, 1993

[Procedure Amendment 1]

[Document name to be corrected] Drawing

[Name of item to be corrected] Figure 1

[Correction method] Change

[Correction content]

[Figure 1]

[Procedure Amendment 2]

[Document name to be corrected] Drawing

[Name of item to be corrected] Figure 2

[Correction method] Change

[Correction content]

[Fig. 2]

Claims (7)

[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,
Further, an indicator for measuring a peroxide decomposable substance, which contains γ-cyclodextrin (γ-CD). 2. The content of γ-CD is 0.1 to 50 mg /
The indicator according to claim 1, which is ml. 3. The indicator according to claim 1, wherein (B) is 3,3 ′, 5,5′-tetramethylbenzidine. 4. The content of (B) is 0.01 to 5 mg / m.
The indicator according to any one of claims 1 to 3, which is l. 5. The indicator according to claim 1, wherein (C) is peroxidase. 6. The indicator according to any one of claims 1 to 5, which further contains a cationic surfactant (D) together with γ-CD. 7. A reagent kit comprising a peroxide decomposable substance (C) or a compound labeled with (C) as a constituent, at least the indicator according to any one of claims 1 to 6 or each component of the indicator. A reagent kit, characterized in that both are components.