JP3230897B2 - Composition for specific binding reaction and composition for specific binding reaction medium - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

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

[0002]

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

[0003]

SUMMARY OF THE INVENTION It is an object of the present invention to provide a composition for a specific binding reaction such as an immunological reaction, which contains a substance different from a substance conventionally used as a stabilizer for an immunoassay reagent as a stabilizer. It is to provide a specific binding reaction medium composition.

[0004]

The present inventors have 0005] is of intensive studies, the o- phenylphenol and its salts and <br/> dehydroacetate and reagent solution for the salt immune response and the immune response of the medium solution The present inventors have found that the present invention exerts an excellent effect on stabilization and completed the present invention.

That is, the present invention provides a composition for a specific binding reaction comprising a specific binding reactant and one or more selected from the group consisting of o-phenylphenol and its salts , and dehydroacetic acid and its salts. I will provide a. Also,
The present invention provides a specific binding reaction medium composition comprising one or more selected from the group consisting of o-phenylphenol and salts thereof , and dehydroacetic acid and salts thereof.

Hereinafter, the present invention will be described in detail.

In the present invention, the “specific binding reaction”
It refers to 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. Among them, a typical one in the present invention is an antigen-antibody reaction. The “specific binding reactant” is a substance that causes the above-described specific binding reaction, and thus includes antibodies, antigens, biotin, avidin, and complementary DNA.

In the present invention, the composition for a specific binding reaction refers to a composition containing the above-mentioned specific binding reactant. The composition contains the above-mentioned specific binding reactant in a liquid medium. Examples of such compositions include compositions containing an antigen or antibody used as a standard,
Examples thereof include a composition containing an 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. The present invention relates to fetal bovine serum (B
Particularly effective when the composition is a liquid composition containing an inactive protein such as SA), casein or animal serum.

[0009] specific binding reaction composition of the present invention, together with the specific binding reactant, as a stabilizer, one or two elements selected from the group consisting of o- phenylphenol and its salts and dehydroacetic acid and its salts The above is included. These concentrations are not particularly limited as long as a desired stabilizing effect is obtained and the concentration does not inhibit the specific binding reaction, but is usually 0.0005 to 5% by weight, preferably 0.05% by weight.
It is about 0.5 to 1% by weight.

In the present invention, the term "specific binding reaction medium composition" refers to a liquid composition that serves as a medium for providing a site for a specific binding reaction. That is, it refers to 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 immunologically active substances (including lyophilized and labeled substances) such as antigens and antibodies. be able to.

The specific binding reaction medium composition of the present invention also contains, as a stabilizer, one or more selected from the group consisting of o-phenylphenol and salts thereof and dehydroacetic acid and salts thereof. It is. These concentrations are not particularly limited as long as a desired stabilizing effect is obtained and the concentration does not inhibit the specific binding reaction.
% By weight, preferably about 0.005 to 1% by weight.

[0012]

According to the present invention, a specific binding reaction composition and a specific binding reaction medium composition capable of stably performing a specific binding reaction with good reproducibility even when stored for a long period of time are provided.

[0013]

The present invention will be described more specifically below with reference to 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-3-2599093
Mouse monoclonal antibody MAb8513 against T was dissolved in 0.1 M carbonate buffer (pH 9.15) in 1 M sodium chloride to a concentration of 10 μg / ml, and polystyrene beads (# 8 manufactured by Sekisui Chemical Co., Ltd.) were added thereto.
0) was immersed to immobilize the antibody 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)
-The sample was immersed in a PBS solution at 37 ° C for 10 hours to prepare anti-GAT antibody-immobilized beads. Thereafter, the beads were stored at 4 ° C. in the immersion liquid.

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 colostrum whey was added to normal human serum at 1/100 volume, and G
An AT standard solution was prepared.

4) Preparation of Enzyme-Labeled Antibody Solution Anti-GAT mouse monoclonal antibody MAb 8628 that recognizes a site different from the immobilized antibody (JP-A-3-25909)
3) were labeled with peroxidase by the method described in P108, Eiji Ishikawa, Tadashi Kawai, Kiyoshi Miyai, "Enzyme Immunoassay (Third Edition), Medical Shoin, 1987". 1M sodium chloride, 1% BSA, 0.05% p-hydroxybenzoic acid and this peroxidase-labeled anti-GAT antibody were added to 0.1%.
It was dissolved in 02M phosphate buffer (pH 7.2) to prepare an enzyme-labeled antibody solution.

5) Measurement of GAT The beads immobilized with the anti-GAT antibody were removed from the solution, placed in a mixture of 50 μl of GAT standard solution and 200 μl of the incubation solution, and incubated at 45 ° C. for 2 hours. After washing with PBS, 250 μl of enzyme-labeled antibody solution
In addition, the mixture was incubated at room temperature for 1 hour. After washing with PBS, a citrate-phosphate buffer solution containing 0.02% hydrogen peroxide and 3 mg / ml o-phenylenediamine (pH
5.0) 0.3 ml was added, and the color was developed at room temperature for 30 minutes. Thereafter, the color reaction was stopped with 1 ml of 1N sulfuric acid.
The absorbance at 2 nm was measured.

After the preparation, it is stored at 4 ° C. within one month1)
44).

Example 1, Comparative Example 1 Effect of Additive on Anti-GAT-Immobilized Beads A fixed amount of the additive shown in Table 1 was added to the immersion liquid of Reference Example 1-1), and Reference Example 1-1) and Similarly, beads immobilized with anti-GAT antibodies were prepared. Each was stored at 4 ° C. and 42 ° C. for 7 days, and then GAT was carried out in the same manner as in Reference Example 1-5) except that beads were changed.
Was measured. As Comparative Example 1, the one to which no additive was added was used. Table 2 shows the results of five simultaneous measurements.

[0021]

[Table 1] Table 1

[0022]

[Table 2] Table 2

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

Example 2 and Comparative Example 2 Effect of Additives on Incubation Solution Same as Reference Example 1-2) except that a certain amount of additives shown in Table 1 was added to the incubation solution of Reference Example 1-2). A solution for incubation was prepared.

Reference Example 1 except that each was stored at 4 ° C. and 42 ° C. for 10 days, and then the incubation solution was changed.
GAT was measured in the same manner as in -5). As Comparative Example 2, one without an additive was used. Table 3 shows the results of five simultaneous measurements.
Shown in

[0026]

[Table 3] Table 3

[0027] It can be seen that the addition of the additive can suppress a decrease in absorbance at 42 ° C storage and improve the storage stability of the incubation solution.

Example 3, Comparative Example 3 Effect of Additive on GAT Standard Solution To the GAT standard solution of Reference Example 1-3), a certain amount of the additive shown in Table 1 was added, and the same as in Reference Example 1-3) A GAT standard solution was prepared.

After storage 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, one without an additive was used. Table 4 shows the results of five simultaneous measurements.

[0030]

[Table 4] Table 4

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

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

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

[0034]

[Table 5] Table 5

──────────────────────────────────────────────────続 き Continuing from the front page (72) Inventor Seiji Hidaka 1 Sakuracho, Hino-shi, Tokyo Konica Corporation (72) Inventor Masahiko Yamazaki 1 Sakuracho, Hino-shi, Tokyo Konica Corporation (56 References JP-A-62-417 (JP, A) JP-A-61-289024 (JP, A) WO 91/13355 (WO, A1) (58) Fields investigated (Int. Cl. ⁷ , DB name) ) G01N 33/531

Claims (5)

(57) [Claims] 1. A composition for a specific binding reaction comprising a specific binding reactant and one or more selected from the group consisting of o-phenylphenol and its salts , and dehydroacetic acid and its salts. 2. The composition according to claim 1, wherein the specific binding reactant is an antibody or an antigen. 3. The composition according to claim 1, wherein the specific binding reactant is immobilized on a carrier. 4. o- phenylphenol and its salts sequence
A specific binding reaction medium composition comprising one or more selected from the group consisting of dehydroacetic acid and salts thereof. 5. The composition according to claim 4, wherein said specific binding reaction is an immune response.