JP2584924B2 - Method for producing immunological agglutination particles - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for producing immunological agglutinated particles having a high storage stability.

[0002]

2. Description of the Related Art In the field of clinical examination, serological examination of various diseases has recently been regarded as important. And
For this test, it is extremely important to accurately, quickly and easily quantify the antigen or antibody. Therefore, a method using an agglutination reaction for detecting an antibody or an antigen by sensitizing the antigen or the antibody to an insoluble carrier is not only simple in operation, but also because the reaction is easily observed with the naked eye. Widely used in the field.

Although the method utilizing this agglutination reaction is only one in principle, it is classified according to the type of the insoluble carrier. That is, as an insoluble carrier, latex, kaolin, charcoal powder, non-biological particles such as organic-inorganic composite particles are used, and animal erythrocytes, and biological particles such as bacterial cells are used. You.

[0004] The above-mentioned biological particles have the advantage that their respective sizes are constant, but the size of the particles is determined by the type of living organism, and it is possible to obtain particles of any size according to the purpose. Can not. For example, animal erythrocytes are carriers of a fixed size, but since animal erythrocytes have a unique antigen on their surface, they may cause nonspecific reactions and give errors to the target agglutination reaction. Is big.

[0005] Accordingly, recently, there has been a tendency to use the above-mentioned non-biological particles which have advantages such as being chemically stable and having no antigenic activity by themselves.

[0006]

However, the above-mentioned non-biological particles have a drawback that antigens or antibodies are hardly adsorbed densely. For example, non-biological particles sensitized by an antigen or an antibody are freeze-dried for storage, stored for a long time in a dry state, and then restored to a dispersion state. Therefore, it was difficult to store the particles in a dispersion state for a long period of time in a reconstituted state.

[0007]

Means for Solving the Problems The inventors of the present invention have eagerly aimed at solving the above-mentioned problems, in particular, for long-term storage while maintaining the sensitivity of a carrier sensitized with an antigen or an antibody. As a result of repeated studies, they have found that the above object can be achieved by treating the above-mentioned carrier with tannic acid, and have completed the present invention.

That is, the present invention is a method for producing immunological agglutinated particles, comprising treating a carrier sensitized with an antigen or an antibody with tannic acid.

In the present invention, a carrier obtained by sensitizing an antigen or antibody to a carrier and obtained by sensitizing the carrier with the antigen or antibody is used.

In the present invention, the antigen is not particularly limited as long as it produces antibodies to induce humoral immunity and cellular immunity. For example, proteins, glycoproteins, lipid proteins, lipids,
Nucleic acids and the like.

The antibody is not particularly limited as long as it has an activity of specifically binding to an antigen, and examples thereof include immunoglobulin IgG, IgM, IgA, IgE, and IgD.

The carrier used in the present invention is not particularly limited as long as it is generally used for obtaining immunological agglutinated particles. For example, latex, kaolin, charcoal powder, gelatin particles, silica, alumina, titania , Inorganic particles such as zirconia or composite oxides containing these as a main component, an organic-inorganic composite in which the surface of the inorganic particles such as silica is treated with an organic compound and the organic compound is chemically or physically bonded to the surface of the inorganic particles. Examples include non-biological particles such as particles, and biological particles such as animal red blood cells and bacterial cells. Among the various carriers exemplified herein, non-biological particles are preferable because of their advantages such as being chemically stable and having no antigenic activity by themselves. In particular, the organic-inorganic composite particles, which are a kind of the non-biological particles, are preferably used because they are artificial carriers, so that the surface of the carrier can be treated according to the purpose, and a non-specific reaction hardly occurs. .

The method of sensitizing a carrier with an antigen or an antibody is not particularly limited, but generally, the carrier and the carrier are sensitized in a buffer such as a phosphate buffer, a Tris buffer, a glycine buffer, or a physiological saline. A method is employed in which sensitization is carried out by mixing 0.01 to 50 mg of antigen or antibody per 1 g of carrier. In addition, when sensitizing a carrier to an antigen, a cell producing the antigen may be used as it is. In such a case, the number of cells to be used is not particularly limited, but is usually 1 × 10 ⁶ to 1 × 10 ¹³ per 1 g of the carrier. The sensitization temperature at the time of sensitization of the carrier with the antigen or antibody is not particularly limited, but is usually in the range of 4 to 56 ° C, and room temperature is more preferable because of easy handling.

By treating the carrier sensitized with the above antigen or antibody with tannic acid, the obtained immunological agglutination particles can be stored in a liquid for a long time with good performance stability. The tannic acid is not particularly limited,
For example, tannic acid, which is an aromatic compound having a large number of phenolic hydroxyl groups distributed in plants, may be mentioned. More specifically, examples thereof include condensed tannic acid and hydrolyzed tannic acid, and among them, hydrolyzed tannic acid is more preferable.

In the present invention, when the treatment with tannic acid is carried out, the carrier obtained by sensitizing the carrier with the antigen or antibody, which is obtained by sensitizing the antigen or antibody, is treated in a wet state from the time of the sensitization. It is preferable to carry out the treatment with tannic acid while keeping the temperature. If the treatment with tannic acid is performed while maintaining the wet state, the storage stability of the obtained immunological agglutination particles in the liquid is further improved.

The above-mentioned method of treatment with tannic acid is as follows:
Although not particularly limited, for example, a solution in which tannic acid is dissolved by dissolving tannic acid in an aqueous solvent such as a buffer solution such as a phosphate buffer solution, a Tris buffer solution, or a glycine buffer solution, or a physiological saline solution or distilled water is prepared. Then, a method in which a solution in which the tannic acid is dissolved and a carrier in which the antigen or the antibody is sensitized is blended.

In the treatment with tannic acid in the present invention, the amount of tannic acid used is not particularly limited, but may be from 1 to 20 per gram of the carrier sensitized with the antigen or antibody.
The content of 00 mg is preferable because the effect of the treatment with tannic acid is further enhanced, and the nonspecific reaction can be more reliably prevented. In addition, when using a solution in which tannic acid is dissolved as described above, the concentration of tannic acid in the solution is not particularly limited, but is preferably 1 ×.
10 ^-4 to 1% by weight is preferred. When the concentration of tannic acid is within the above range, the amount of the liquid to be handled may be appropriate, and the effect of the treatment with tannic acid may appear more quickly.

The immunological agglutination particles obtained by the treatment with tannic acid described above can be stored in a liquid, but if they are to be stored for a longer period of time, they can be stored in a dry state. The method for drying the immunological agglutination particles may be any known drying method without limitation.
For example, there is a method by freeze-drying.

When the immunological agglutinated particles obtained by the method of the present invention are dried, the dried immunological agglutinated particles are dispersed and reconstituted in an aqueous medium before being used for clinical examination. The aqueous medium may contain a protein.

[0020]

The tannic acid used for treating a carrier sensitized with an antigen or an antibody has the effect of astringent proteins. It is presumed that the antigen or antibody sensitized to the carrier is converged and hardly released from the carrier due to the action of tannic acid.

[0021]

The immunological agglutinated particles produced by the method of the present invention can be stored for a long period of time in an aqueous medium without deteriorating its performance.

[0022]

EXAMPLES The present invention will be described in detail with reference to the following examples, but the present invention is not limited to the examples.

Example 1 In order to prepare an immunological syphilis diagnostic reagent as an agglutination reagent, 1 × 10 ⁹ Treponema pallidum, which is a bacterium of syphilis, was dispersed in 1 ml of a 0.1 M aqueous sodium chloride solution. . Next, the sodium chloride aqueous solution in which Treponema paridum was dispersed was subjected to ultrasonic crushing for 20 minutes.
Crushed at 0 W for 30 minutes. Separately, a 0.02 M phosphate buffer containing 0.1 M sodium chloride (pH 7.
In 2), 2.5% by weight of bromostyrene particles (manufactured by Nippon Synthetic Rubber Co., Ltd.) as latex particles (particle concentration with respect to the dispersion) was dispersed to prepare a phosphate buffer in which bromostyrene particles were dispersed. Then, in 1 ml of the aqueous sodium chloride solution in which the above-mentioned Treponema paridum is dispersed,
An equal amount of the above-mentioned phosphate buffer in which the bromostyrene particles are dispersed is blended and left for 1 hour at room temperature.
centrifugation at 20 m for 20 minutes to collect the bromostyrene particles.
2 g of tannic acid (manufactured by Wako Pure Chemical Industries, Ltd.) was dissolved in 1 ml of 0.02 M phosphate buffer (pH 7.2), and the bromostyrene particles recovered above were added to the tannic acid solution, and the solution was added at 37 ° C. for 15 minutes.
Let stand for a minute. Then, a 0.02 M phosphate buffer (pH
The mixture was centrifugally washed twice in 7.2) and freeze-dried to prepare agglutination reaction particles comprising bromostyrene particles to which an antigen derived from Treponema pallidum was sensitized. Then, for six months,
It was left at 4 ° C. 5% by weight in 0.02M phosphate buffer
Then, bovine blood albumin was added to prepare a particle solution. The agglutination reaction particles stored for the above period were dissolved in this particle dissolving solution so as to have a concentration of 0.5% by weight based on the agglutination reaction reagent to prepare an agglutination reaction reagent.

Next, the performance of the immunological syphilis diagnostic agent was measured as described below.

Using a serum of a syphilis patient as a test solution, using a 10-fold diluted solution of the serum as a stock solution and diluting with the above phosphate buffer (pH 7.2) according to a multiple dilution method, and diluting each diluted solution with a microfluidic solution. 25 μl in the wells of a titer plate
Was added. Next, 25 μl of the prepared agglutination reagent was added to the wells, and after stirring for 1 minute,
Leave at room temperature. After 30 minutes, observe the state of aggregation of the particles,
The maximum dilution factor of the diluent in the well where the particle ring was clearly large in the test solution and the aggregated particles were uniformly spread in the ring was determined, and the sensitivity was evaluated. Similarly, an agglutination reagent is prepared according to the method described above to obtain 7
The sensitivity was evaluated using those on day 30 and those on day 30. Table 1 shows these results.

[0026]

[Table 1]

Comparative Example 1 The procedure of Example 1 was repeated except that bromostyrene particles sensitized with the antigen derived from Treponema pallidum were not treated with tannic acid. The syphilis patient serum used for the evaluation of sensitivity was the same as that used in Example 1. Table 1 shows these results.

Example 2 In order to prepare an adult T-cell leukemia diagnostic agent as an immunological agglutination reagent, an antigen derived from an adult T-cell leukemia virus was added to 1 ml of physiological saline containing 0.1% by weight of sodium dodecyl sulfate. 100 μg was dissolved to prepare a physiological saline solution containing an antigen derived from adult T-cell leukemia virus.
Separately, 2.5% by weight of organic-inorganic composite particles (manufactured by Tokuyama Soda Co., Ltd.) was dispersed in a 0.02M phosphate buffer to prepare a phosphate buffer in which the organic-inorganic composite particles were dispersed. Then, equal amounts of the physiological saline containing the antigen derived from the adult T-cell leukemia virus and the phosphate buffer in which the organic-inorganic composite particles are dispersed are left to stand for 1 hour at room temperature, and then the supernatant is added. After removing the antigen, 1 g of the antigen-sensitized particles
1 ml of a 0.02 M phosphate buffer to which tannic acid was added so that the amount became 0.08 g with respect to
Stirred slowly at 1 ° C. for 1 hour. Thereafter, the plate is washed twice with a 0.02 M phosphate buffer, and the concentration of the antigen-sensitized particles is reduced to 0.5% by weight using a 0.02 M phosphate buffer containing 0.5% rabbit serum. And used as an agglutination reagent.

Next, the performance of the immunological syphilis diagnostic agent was measured as described below.

Using a serum of an adult T-cell leukemia patient as a test solution, a two-fold dilution of the serum was used as a stock solution, and dilution was performed using the above-mentioned phosphate buffer (pH 7.2) according to a multiple dilution method. The solution was added to each well of the microtiter plate in an amount of 25 μl. Next, 25 μl of the prepared agglutination reagent was added to the wells, and the mixture was stirred for 1 minute and left at room temperature. After 30 minutes, the state of aggregation of the particles is observed, and the maximum dilution factor of the diluent in the well where the particle ring is clearly large in the test solution and the aggregated particles are uniformly spread in the ring is observed. And the sensitivity was evaluated. Similarly, agglutination reagents were prepared according to the method described above, and the sensitivity was evaluated using those on day 7 and those on day 30. Table 1 shows these results.

Comparative Example 2 The same procedure as in Example 2 was carried out except that the organic-inorganic composite particles sensitized with the antigen derived from the adult T-cell leukemia virus were not treated with tannic acid. The serum used in Example 2 was used as the serum for an adult T-cell leukemia patient for evaluation of sensitivity. Table 1 shows these results.

Example 3 In Example 2, the antigen was changed to that derived from hepatitis B virus, and a phosphate buffer solution containing tannic acid was used in an amount of 0.02 g per 1 g of the agglutinated particles. The procedure was performed in the same manner as in Example 2 except that the sample was left at room temperature for 30 minutes, and the serum of a hepatitis B patient was used as a test solution. Table 1 shows these results.

Comparative Example 3 The procedure of Example 3 was repeated except that no tannic acid treatment was performed. Table 1 shows these results.

Example 4 To prepare a diagnostic reagent for immunological colorectal cancer as an agglutination reagent, a rabbit-derived anti-human hemoglobin antibody (manufactured by Kappel) was added to a concentration of 0.1 mg / ml. 05M
It was dissolved in 1 ml of an aqueous sodium chloride solution to prepare an aqueous sodium chloride solution containing an anti-human hemoglobin antibody.
Separately, a solution containing 0.05M sodium chloride containing 0.1M.
02M phosphate buffer (pH 7.2) and organic-inorganic composite particles (trade name: Immunoticulus H, manufactured by Tokuyama Soda Co., Ltd.)
DP) 0.5% by weight was dispersed to prepare a phosphate buffer in which organic-inorganic composite particles were dispersed. Then, an aqueous sodium chloride solution 1 containing the anti-human hemoglobin antibody
ml and 1 ml of the phosphate buffer in which the organic-inorganic composite particles were dispersed, and allowed to stand at room temperature for 1 hour.
Wash twice with 2M phosphate buffer (pH 7.2)
After removing the supernatant by centrifugation at 000 rpm for 3 minutes, 1 ml of a solution obtained by dissolving tannic acid in the above phosphate buffer to a concentration of 0.01% by weight was added to the carrier. After being left at 4 ° C. for 30 minutes, centrifugal washing was performed once. Furthermore, freeze-drying was performed to prepare agglutination particles comprising organic-inorganic composite particles sensitized with an anti-human hemoglobin antibody. Then, it was left at 4 ° C. for 3 months. The agglutination reaction particles stored for the above period were dissolved in distilled water to a concentration of 0.5% by weight based on the agglutination reaction reagent to prepare an agglutination reaction reagent.

Next, the performance of the immunological colorectal cancer diagnostic agent was measured as described below.

As a test solution, 3 mg of a stool of a colon cancer patient was added to 0.01 mg.
0.01 M with weight% sodium deoxycholate
Using a solution dissolved in a glycine buffer (pH 8.0), the test solution is used as a stock solution, and the dilution is performed using the glycine buffer according to the multiple dilution method. Each diluted solution is placed in a well of a microtiter plate. Added 25 μl each. Then, 25 μl of the prepared agglutination reagent was added to the wells.
After stirring for 1 minute, the mixture was left at room temperature. After 30 minutes, the state of aggregation of the particles is observed, and the highest dilution factor of the diluent in the well where the particle ring is clearly large in the test solution and the aggregated particles are uniformly spread in the ring is determined. The sensitivity was evaluated. Similarly, according to the method described above, the agglutination reagent is prepared on the 7th day,
And the sensitivity on day 30 were evaluated. Table 1 shows these results.

Comparative Example 4 The procedure of Example 4 was repeated except that no tannic acid treatment was performed. Table 1 shows these results.

Claims (1)

(57) [Claims] 1. A method for producing immunological agglutinated particles, comprising treating a carrier sensitized with an antigen or an antibody with tannic acid.