JPH06308124A - Composition for specifically-bonding reaction and composition of specifically-bonding reaction medium - Google Patents

Abstract

PURPOSE:To provide a composition for a specifically-bonding reaction such as an immunoreaction and composition of a specifically-bonding reaction medium which include, as a stabilizer, a different substance from that conventionally used as a stabilizer of a reagent liquid for an immunoassay. CONSTITUTION:The invention provides a composition for a specifically-bonding reaction including a specifically-bonding reaction substance and, one or two or more kinds of o-phenylphenol and its salt, dehydroacetic acid and its salt, sorbic acid and its salt, and benzalconium and its salt.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a composition for a specific binding reaction such as an immunological reaction and a composition for a specific binding reaction medium.

[0002]

2. Description of the Related Art Conventionally, when an antibody solution or the like used for immunoassay is stored for a long period of time, the reproducibility of the measurement result often decreases when used for immunoassay. In order to solve this problem, it has been proposed to add a stabilizer to the liquid containing the substance used for the immune reaction. For example, JP-A-64-6338
It is known from Japanese Patent Laid-Open Publication No. 0-G0100 that addition of alkoxyphenol is used for stabilizing peroxidase used as a labeling enzyme for immune reaction. In addition, JP-A-2-42
In Japanese Patent Publication No. 982, addition of phenol having a substituent defined by a specific Hammett sigma value is described for stabilizing peroxidase and antibody.

[0003]

The object of the present invention is to provide a composition for a specific binding reaction such as an immunoreaction containing a substance different from the one conventionally used as a stabilizer for an immunoassay reagent solution as a stabilizer. It is to provide a specific binding reaction medium composition.

[0004]

As a result of intensive studies, the present inventors have found that o-phenylphenol and its salts, dehydroacetic acid and its salts, sorbic acid and its salts, and benzalkonium and its salts are used for immunoreaction. The present invention has been completed by finding that it exhibits an excellent effect in stabilizing a reagent solution and a medium solution for an immune reaction.

That is, the present invention relates to a specific binding reaction substance and one or more selected from the group consisting of o-phenylphenol and its salts, dehydroacetic acid and its salts, sorbic acid and its salts, and benzalkonium and its salts. Provided is a composition for specific binding reaction containing two or more kinds. Also,
The present invention provides a specific binding reaction medium composition containing one or more selected from the group consisting of o-phenylphenol and salts thereof, dehydroacetic acid and salts thereof, sorbic acid and salts thereof, and benzalkonium and salts thereof. Provide things.

The present invention will be described in detail below.

In the present invention, the "specific binding reaction" means
It means a reaction in which two substances specifically bind, such as an antigen-antibody reaction, a reaction between biotin and avidin, and a reaction between DNA and complementary DNA. Of these, the representative one in the present invention is an antigen-antibody reaction. The "specific binding reaction substance" is a substance that causes the above-mentioned specific binding reaction, and thus includes an antibody, an antigen, biotin, avidin, complementary DNA and the like.

The composition for specific binding reaction in the present invention means a composition containing the above specific binding reaction substance. The composition usually contains the specific binding reaction substance in a liquid medium. As an example of such a composition, a composition containing an antigen or antibody used as a standard substance,
Examples thereof include a composition containing the antibody or antigen immobilized on a carrier such as beads or fine particles, and an antibody or antigen labeled with an enzyme, fluorescence, luminescence, radioisotope, biotin or the like. In addition, the present invention relates to fetal bovine serum (B
SA), casein, or a liquid composition containing an inactive protein such as animal serum is particularly effective.

In the composition for specific binding reaction of the present invention, together with the above specific binding reaction substance, as a stabilizer, o-phenylphenol and its salt, dehydroacetic acid and its salt, sorbic acid and its salt, and benzalkonium and One or more selected from the group consisting of salts thereof are included. These concentrations are not particularly limited as long as the desired stabilizing effect is obtained and the specific binding reaction is not inhibited, but usually 0.0005 to 5% by weight, preferably 0.0
It is about 05 to 1% by weight.

In the present invention, the specific binding reaction medium composition means a liquid composition serving as a medium for providing a place for a specific binding reaction. That is, it means an incubation solution or the like for performing an immune reaction. Examples include a buffer solution for diluting a sample, a buffer solution for diluting a standard substance, and a buffer solution for diluting an immunoactive substance such as an antigen or an antibody (including a freeze-dried substance and a labeled substance). be able to.

Also in the specific binding reaction medium composition of the present invention, a stabilizer is selected from the group consisting of o-phenylphenol and its salts, dehydroacetic acid and its salts, sorbic acid and its salts, and benzalkonium and its salts. One or two or more types are included. These concentrations are not particularly limited as long as the desired stabilizing effect is obtained and the specific binding reaction is not inhibited, but usually 0.0005 to 5
The weight is about 0.005 to 1% by weight.

[0012]

INDUSTRIAL APPLICABILITY The present invention provides a specific binding reaction composition and a specific binding reaction medium composition capable of stably performing a specific binding reaction with good reproducibility even after long-term storage.

[0013]

EXAMPLES The present invention will be described more specifically below based on examples. However, the present invention is not limited to the following examples.

Reference Example 1 Measurement of GAT 1) Preparation of anti-GAT antibody-immobilized beads GA obtained by the method described in JP-A-3-2590903
The mouse monoclonal antibody MAb8513 against T was dissolved in 0.1 M carbonate buffer (pH 9.15) in which 1 M sodium chloride was dissolved so as to have a concentration of 10 μg / ml, and polystyrene beads (Sekisui Chemical # 8.
0) was immersed and the antibody was immobilized at 4 ° C. for 24 hours. After washing with phosphate buffered saline (hereinafter referred to as PBS), 1% bovine serum albumin (hereinafter referred to as BSA)
-Immersed in PBS solution at 37 ° C. for 10 hours to prepare anti-GAT antibody-immobilized beads. After that, the beads were stored as they were in the immersion liquid at 4 ° C.

2) Preparation of Incubation Solution 1M sodium chloride and 1% BSA were added to 0.02M.
It was dissolved in a phosphate buffer (pH 6.5) to prepare an incubation solution.

3) Preparation of GAT standard solution Human colostral whey was added to normal human serum in an amount of 1/100 to prepare GAT standard solution.
An AT standard solution was prepared.

4) Preparation of enzyme-labeled antibody solution Anti-GAT mouse monoclonal antibody MAb8628 which recognizes a site different from the immobilized antibody (Japanese Patent Laid-Open No. 25909/1993)
3) was labeled with peroxidase by the method described in "Physical Enzyme Immunoassay (3rd Edition) Medical Book 1987" edited by Eiji Ishikawa, Tadashi Kawai, Kiyoshi Miyai, P108. 1M sodium chloride, 1% BSA, 0.05% p-hydroxybenzoic acid and the peroxidase-labeled anti-GAT antibody were added to 0.
It was dissolved in 02M phosphate buffer (pH 7.2) to prepare an enzyme-labeled antibody solution.

5) Measurement of GAT The anti-GAT antibody-immobilized beads were taken out of the solution, put in a solution prepared by mixing 50 μl of GAT standard solution and 200 μl of incubation solution, and incubated at 45 ° C. for 2 hours. After washing with PBS, 250 μl of enzyme-labeled antibody solution
In addition, it was incubated at room temperature for 1 hour. After washing with PBS, a citric acid-phosphate buffer solution (pH: 0.02% hydrogen peroxide and 3 mg / ml o-phenylenediamine) was added.
5.0) 0.3 ml was added and color was developed for 30 minutes at room temperature. After this, the color reaction was stopped with 1 ml of 1N sulfuric acid,
Absorbance at 2 nm was measured.

Stored at 4 ° C for less than 1 month after production 1)
~ 4) was used to measure.

Example 1, Comparative Example 1 Effect of Additives on Anti-GAT-Immobilized Beads To the immersion liquid of Reference Example 1-1), a fixed amount of the additive shown in Table 1 was added, and Reference Example 1-1) and Similarly, anti-GAT antibody-immobilized beads were prepared. Each was stored at 4 ° C and 42 ° C for 7 days, and then GAT was prepared in the same manner as in Reference Example 1-5) except that the beads were replaced.
Was measured. As Comparative Example 1, one without any additive was used. The results of 5 simultaneous measurements are shown in Table 2.

[0021]

[Table 1]

[0022]

[Table 2]

It can be seen that the addition of the additive suppressed the decrease in absorbance and improved the storage stability of the anti-GAT antibody-immobilized beads.

Example 2, Comparative Example 2 Effect of Additives on Incubation Solution The same amount of additive as shown in Table 1 was added to the incubation solution of Reference Example 1-2) and the same as Reference Example 1-2). An incubation solution was prepared.

Reference Example 1 except that the incubation solution was changed after each of them was stored at 4 ° C. and 42 ° C. for 10 days
GAT was measured in the same manner as in (5). As Comparative Example 2, one in which no additive was added was used. Table 3 shows the results of five simultaneous measurements.
Shown in.

[0026]

[Table 3]

It can be seen that the addition of the additive suppresses the decrease in absorbance at 42 ° C. storage and improves the storage stability of the incubation solution.

Example 3, Comparative Example 3 Effect of Additive on GAT Standard Solution The same amount as the additive in Table 1 was added to the GAT standard solution of Example 1-3), and the same procedure as in Example 1-3) was performed. A GAT standard solution was prepared.

After each of them was stored at 4 ° C. and 37 ° C. for 3 days, GAT was measured in the same manner as in Reference Example 1-5) except that the GAT standard solution was changed. As Comparative Example 3, the one to which no additive was added was used. The results of 5 simultaneous measurements are shown in Table 4.

[0030]

[Table 4]

It can be seen that, by adding the additive, the rapid decrease in absorbance at 37 ° C. was suppressed and the storage stability of the GAT standard solution was improved.

Example 4, Comparative Example 4 Effect of Additives on Enzyme-Labeled Antibody Solution To the enzyme-labeled antibody solution of Reference Example 1-4), a certain amount of the additive shown in Table 1 was added, and then Reference Example 1-4). An enzyme-labeled antibody solution was prepared in the same manner as in.

After storage at 4 ° C. and 42 ° C. for 7 days, GAT was measured in the same manner as in Examples 1-5) except that the enzyme-labeled antibody solution was changed. As a comparative example, one without any additive was used. The results of 5 simultaneous measurements are shown in Table 5.

[0034]

[Table 5] It can be seen that the addition of the additive can reduce the decrease in the absorbance at 42 ° C. and improve the storage stability of the enzyme-labeled antibody solution.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Seiji Hidaka No. 1 Sakura-cho, Hino City, Tokyo Konica Stock Company (72) Inventor Nobuhiko Yamazaki No. 1 Sakura-cho, Hino City, Tokyo Konica Stock Company

Claims (5)

[Claims] 1. A specific binding reaction substance, and one or more selected from the group consisting of o-phenylphenol and its salts, dehydroacetic acid and its salts, sorbic acid and its salts, and benzalkonium and its salts. A composition for specific binding reaction comprising: 2. The composition according to claim 1, wherein the specific binding reaction substance is an antibody or an antigen. 3. The composition according to claim 1, wherein the specific binding reaction substance is immobilized on a carrier. 4. A specific binding reaction medium containing one or more selected from the group consisting of o-phenylphenol and salts thereof, dehydroacetic acid and salts thereof, sorbic acid and salts thereof, and benzalkonium and salts thereof. Composition. 5. The composition according to claim 4, wherein the specific binding reaction is an immune reaction.