JPH04175657A - Immunological agglutination reagent, method for dissolving immunologically agglutinated particle and solution of immunologially aggulutinated particle - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention provides an immunological agglutination reaction reagent with high storage stability, a method for dissolving immunological agglutination reaction particles aimed at long-term storage stability, and a method thereof. This invention relates to an immunological agglutination reaction particle solution to be used.

[Conventional technology and its problems]

In the field of clinical testing, serological diagnosis of various diseases has recently become important. For this diagnosis, it is extremely important to quantify antigens or antibodies accurately, quickly, and simply. Therefore, the agglutination reaction, which detects antibodies or antigens by sensitizing them to an insoluble carrier, is widely used in clinical testing and research fields because it is easy to operate and the reaction is easy to observe with the naked eye. used.

In principle, such aggregation reactions are of one type or are classified depending on the type of insoluble carrier. That is, as the insoluble carrier, non-biological particles such as latex, kaolin, cooked rice, organic-inorganic composite particles, biological particles such as animal red blood cells, bacterial cells, etc. are used.

Although biological particles have the advantage of having a constant size, the size of the particles is determined depending on the type of organism, and it is not possible to obtain particles of arbitrary size depending on the purpose. For example, animal red blood cells are carriers with a constant size, but have unique antigens on the red blood cell surface.
There is a high possibility that a non-specific reaction will occur and the desired agglutination reaction will be incorrect.

Therefore, recently there has been a tendency to use non-biological particles, which have the advantage of being chemically stable and having no antigenic activity themselves, but they also have the disadvantage of being difficult to adsorb closely to antigens or antibodies. . For example, if non-biological particles sensitized with an antigen or antibody are freeze-dried for storage and then restored to a dispersed solution state after long-term storage, the antigen or antibody is easily released from the particles and restored. It was difficult to store it in a solution state for a long period of time.

[Means to solve the problem]

The present inventors aimed to preserve long-term immunological agglutination reaction particles (hereinafter referred to as agglutination reaction particles) in which an insoluble carrier is sensitized with an antigen or antibody while maintaining their performance in a dispersion solution. As a result of extensive research, the present inventors discovered that the presence of tannic acid maintains the performance of the aggregation reaction particles over a long period of time, leading to the completion of the present invention.

That is, the present invention is an immunological agglutination reaction reagent with excellent storage stability, which is characterized by containing immunological agglutination particles, protein, tannic acid, and water.

Furthermore, the present invention provides immunological aggregation reaction particles characterized in that tannic acid is added at the time of dissolution when the dried immunological agglutination reaction particles are dissolved using a dissolving solution mainly composed of water and protein. This is a method of dissolving.

Furthermore, the present invention is an immunological agglutination reaction particle solution containing water, protein, and tannic acid.

In the present invention, dissolution refers to dispersing dried immunological agglutination reaction particles and restoring them to a solution state, and the dissolution liquid refers to a dispersion liquid used for this restoration.

The agglutination reaction particles used in the present invention are prepared by sensitizing an insoluble carrier with an antigen or antibody.

The insoluble carrier is not particularly limited, and examples include non-biological particles such as latex, kaolin, cooked rice, organic-inorganic composite particles, and gelatin particles, and biological particles such as animal red blood cells and bacterial cells. However, biological particles
Some have specific antigens on their surfaces, which tend to cause non-specific reactions with antibodies, and furthermore, they vary between individual animals, making it difficult to obtain blood cells of constant quality. . It is therefore preferable to use non-biological particles which have the advantage of being chemically stable and having no antigenic activity themselves.

In particular, since organic-inorganic composite particles are artificial carriers, the surface of the carrier can be chemically treated depending on the purpose, and non-specific reactions are extremely unlikely to occur, so they are preferably used.

The substance that sensitizes such an insoluble carrier may be any substance that causes an immunological agglutination reaction, and such substances include antigens and antibodies.

The antigen is not particularly limited as long as it is a substance that produces antibodies and induces humoral immunity or cell-mediated immunity, and examples thereof include proteins, glycoproteins, lipid proteins, lipids, and nucleic acids.

Antibodies are not particularly limited as long as they have the activity of specifically binding to antigens, and include IgG, rgM, [gA, IgD,
Examples include IgE.

The method for producing agglutination reaction particles obtained by sensitizing an antigen or antibody onto an insoluble carrier is not limited in any way, but generally, it is prepared in a buffer such as phosphate buffer, Tris buffer, glycine buffer, physiological saline, etc. and the insoluble carrier and 0 per gram of carrier.
．． Sensitization is carried out by mixing 01 mg to 50 mg of antigen or carrier. Normally, sensitization can be carried out by leaving it at room temperature for about one hour, but when sensitizing to an antigen, sensitization can be carried out over a wide temperature range of 4 to 56°C.

When the aggregation reaction particles produced by the above method are stored in a solution state, they are dispersed and stored in an aqueous medium together with protein and tannic acid. A dispersion containing all these components is called an immunological agglutination reaction reagent (hereinafter also referred to as an agglutination reaction reagent) and is used for diagnosis.

The concentration of agglutination reaction particles in the agglutination reaction reagent is usually 0.3 to
It is 1% by weight. If it is smaller than the lower limit, the agglomerated image becomes unclear and cannot be determined. If it is larger than the upper limit, the sensitivity will deteriorate.

Tannic acid has the effect of preserving the agglomerated reaction particles in a liquid with good performance stability over a long period of time. The tannic acid is not particularly limited as long as it is an aromatic compound having a large number of phenolic hydroxyl groups distributed throughout plants. Usually, it is classified into condensed tannic acid and hydrolyzed tannic acid, but it is more preferable to use hydrolyzed tannic acid. The concentration of tannic acid contained in the agglutination reaction reagent varies depending on the antigen or antibody, but it is 1 to 1000 m/g of carrier.
Preferably, g of tannic acid is present.

If it is less than 1 mg, the presence of tannic acid has no effect, and if it is more than 11,000 rn, a non-specific reaction will occur.

The protein is a necessary component for causing an agglutination reaction, and is not particularly limited as long as it does not inhibit antigen and antibody reactions. Examples of the protein include bovine blood albumin, beef tallow serum, goat serum, rabbit serum, gelatin, skim milk, and the like.

The amount of protein used is usually 1-1000 m/g of carrier.
It is g.

As water, it is preferable to use ultrapure water or distilled water, and furthermore, for example, phosphate buffer, Tris buffer,
It is more preferable to use a buffer solution with a pH of 4 to 9, such as a glycine buffer solution or physiological saline, because the antigen and antibody reactions proceed quickly. Watering is 100-500 per gram of carrier.
Use g.

In order to preserve the agglomerated reaction particles for a longer period of time, they are stored in a dry state. The drying method is not particularly limited, but freeze-drying is preferred.

Such dried agglutinated particles are reconstituted into a dispersed state using water and a protein-based dissolving solution before being used in clinical tests. It is necessary to add tannic acid during this dissolution, and the presence of tannic acid allows the agglutination reaction reagent containing the reconstituted agglutination reaction particles to be more stably stored in a solution state for a long period of time.

Any method may be used to add tannic acid during reconstitution of the dried agglomerated reaction particles, but unless it is added almost simultaneously with dissolution, the effects of the present invention cannot be obtained. Therefore, it is preferable to dissolve tannic acid in the solution in advance in terms of effectiveness and ease of operation.

Whether tannic acid is dissolved in the solution in advance or added during reconstitution, the amount used is 1 to 1 OQO per Ig of insoluble carrier in the final agglutination reaction reagent, as described above.
mg.

Furthermore, as mentioned above, the water and protein contents in the solution used for renaturation are 1 to 1 liter per 1 g of insoluble carrier, respectively.
000 mg and 100 to 500 g.

The agglutination reaction reagent or solution of the present invention may further contain an inorganic salt and, if necessary, an amino acid salt.

Addition of inorganic salts tends to cause antigen-antibody reactions to occur quickly. Examples of the inorganic salt include sodium chloride, sodium azide, potassium chloride, lithium chloride, calcium chloride, magnesium chloride, and the like. Usually, these inorganic salts are included in the agglutination reaction reagent or solution. O1-1.5 mol/I! Used within the range of 0. If O is less than 1 mol/l, the addition has no effect, and if O is less than 1.5 mol/l. If the amount is higher, it tends to inhibit the antigen-antibody reaction.

Depending on the subject, an antigen-antibody reaction may occur, but the agglutination image may be disrupted. In order to prevent this agglomerated image from collapsing, an amino acid salt can be added. Examples of the amino acid salt include L-. Usually, these amino acid salts are contained in the agglutination reaction reagent or solution in a concentration of 0.5 to
It is used in a range of 10% by weight. If the upper limit is exceeded, the agglutination reaction will not occur.

[Action and effect]

By including tannic acid in the agglutination reaction reagent in advance or during the dissolution and restoration of the agglutination reaction particles that have been stored dry, the agglutination reaction particles can be stored in an aqueous medium for a long period of time without deteriorating their performance. Ta.

The reason for the improved storage stability of the agglutination reaction particles is not clear, but since tannic acid has the ability to astringent proteins, antigens or antibodies sensitized to the carrier may be astringent in an aqueous medium and removed from the carrier. It is presumed that it is difficult to remove.

(Example) The present invention will be specifically explained by the following example.
The present invention is not limited in any way by the examples.

Example In order to create an immunological syphilis diagnostic agent as an agglutination reaction reagent, we first used Trebonema validum, a pathogenic bacterium of syphilis.
Xl0' were dispersed in 1 mp physiological saline. Next, the physiological saline in which Trebonema pallidum was dispersed was crushed using an ultrasonic crusher at 200 W for 30 minutes. 0.0% to the physiological saline containing this crushed Trevoma pallidum. (12) Organic-inorganic composite particles (manufactured by Tokuyama Soda Co., Ltd., product name: Immunoticles HDP) in M phosphate buffer
) 2.5% by weight was added in an equal amount and left at room temperature for 1 hour, then 0.0% by weight was added. (12) 2 with M IJ acid buffer
The particles were washed twice and freeze-dried to prepare aggregation reaction particles consisting of organic-inorganic composite particles sensitized with an antigen derived from Trebonema pallidum. Thereafter, it was left at 4°C for 6 months. A particle dissolution solution was prepared by adding tannic acid (manufactured by Wako Pure Chemical Industries, Ltd.) to a 0.(12)M phosphate buffer containing 5% bovine blood albumin in an amount of 50 mg per gram of agglutination reaction particles. The aggregation reaction particles stored for the above-mentioned period were dissolved in this particle dissolution solution to a concentration of 5% by weight based on the agglutination reaction reagent to prepare an agglutination reaction reagent.

Next, the shelf life of this immunological syphilis diagnostic agent was measured as shown below.

Syphilis patient serum was used as the test solution, and a 10-fold dilution of the serum was used as the stock solution, and a phosphate buffer (p
Dilutions were made using H7,2), and 25 μβ of each diluted solution was added to the wells of a microtiter plate. Next, 25 μβ of the agglutination reaction reagent prepared above was added into the wells, and after stirring for 3 minutes, the wells were allowed to stand at room temperature. 3
After 0 minutes, observe the state of particle aggregation and find the highest dilution factor of the diluted solution in the well where the particle ring in the test solution is clearly large and the agglomerated particles are uniformly spread within the ring. Sensitivity was evaluated.

After the agglutination reaction reagent prepared above was left at room temperature for 7 days and 30 days, the same operation was performed to evaluate the sensitivity.

Table 1 shows these results.

Table 1 Comparative Example 1 The same procedure as in Example 1 was carried out except that tannic acid was not added. Table 2 shows these results.

Table 2 Example 2 The antigen was changed to one derived from hepatitis B virus, and a particle dissolution solution containing tannic acid was added in an amount of 0.(12) g per 1 g of agglutination reaction particles. The same procedure as in Example 1 was carried out except that hepatitis B patient serum was used as the test solution and the serum was used as the stock solution.Table 3 shows the results.

Table 3 Comparative Example 2 The same procedure as in Example 2 was carried out except that tannic acid was not added. Table 4 shows these results.

Table 4 Example 3 To prepare an immunological adult T-cell leukemia diagnostic agent as an immunological agglutination reaction reagent, an antigen derived from adult T-cell leukemia virus was added to physiological saline containing 0.1% by weight of sodium dodecyl sulfate. 100 μg was dissolved per 1 m47. 0.0% to the lysate. (12) Organic-inorganic composite particles (manufactured by Tokuyama Soda Co., Ltd., product name: Immunoticles @
An equal volume of a solution in which 2.5% HDP) was dispersed was added, left at room temperature for 1 hour, and then 0.0% HDP) was added. (12) 2 with M phosphate buffer
Organic-inorganic composite particles sensitized with an antigen derived from adult cell leukemia virus were prepared by washing twice. Next, after removing the supernatant, 0, (12) M IJ acid containing 0.5% rabbit serum to which tannic acid was added so that the amount was 0.08 g per 1 g of antigen-sensitized particles. Using a buffer solution,
The antigen-sensitized particles were prepared to have a concentration of 0.5% by weight, and used as an agglutination reaction reagent.

Adult T-cell leukemia patient serum was used as the test solution, and the serum was used as a stock solution and diluted to 0. (12) Perform dilution using M phosphate buffer, add 25 μl of each diluted solution into the wells of a microtiter plate, then add 25 μβ of the agglutination reaction reagent prepared above into the wells for 3 minutes. After stirring, the mixture was allowed to stand at room temperature.

After 30 minutes, observe the state of aggregation of the particles, and determine the maximum dilution ratio of the diluted solution in the well where the particle ring in the test solution is clearly large and the aggregated particles are uniformly spread within the ring. evaluated.

After the agglutination reaction reagent prepared above was left at room temperature for 7 days and 30 days, the same operation was performed to evaluate the sensitivity.

Table 5 Comparative Example 3 The same procedure as in Example 3 was carried out except that tannic acid was not added. Table 6 shows these results.

Table 6 Comparative Example 4 The same method as Example 1 was carried out except that the protein crosslinking agent shown in Table 7 was used instead of tannic acid. In Table 7, agglutination reaction particles in a freeze-dried state were restored to a solution state using a particle dissolving solution, and those that maintained performance as a diagnostic agent for 3 days were marked as x, and those that did not maintain O1 performance were marked as x.

Table 7

Claims (1)

[Scope of Claims] (1) An immunological agglutination reaction reagent with excellent storage stability, characterized by containing immunological agglutination reaction particles, protein, tannic acid, and water. (2) The immunological agglutination reaction reagent according to claim (1), which contains 0.01 to 1.5 mol/l of an inorganic salt. (3) The inorganic salt is sodium chloride, sodium azide,
The immunological agglutination reaction reagent according to claim (2), which is at least one member selected from the group consisting of potassium chloride, calcium chloride, magnesium chloride, and lithium chloride. (4) The immunological agglutination reaction reagent according to claim (2), which contains 0.5 to 10% by weight of an amino acid salt. (5) The amino acid salt is sodium L-glutamate, L-sodium glutamate,
The immunological agglutination reaction reagent according to claim 4, which is at least one selected from the group consisting of -lysine hydrochloride, L-histidine hydrochloride, L-arginine hydrochloride, and sodium L-aspartate. (6) Claims (1) to (5) wherein the immunological agglutination reaction particles are antigen-sensitized particles obtained by sensitizing an insoluble carrier with an antigen.
The immunological agglutination reaction reagent described in Section 1. (7) The immunological agglutination reaction reagent according to claim (6), wherein the insoluble carrier is a non-biological carrier particle. (8) The immunological agglutination reaction reagent according to claim (7), wherein the tannic acid is a condensed tannic acid or a hydrolyzed tannic acid. (9) Tannic acid is 1 to 10% per 1 g of insoluble carrier.
The immunological agglutination reaction reagent according to claim (8), which is in an amount of 0.00 mg. (10) The immunological agglutination reaction reagent according to claim (9), wherein the protein is at least one animal serum selected from the group consisting of bovine blood albumin, beef tallow serum, goat serum, and rabbit serum. (11) Claim No. 1, wherein the water is a buffer solution with a pH of 4 to 9.
0) The immunological agglutination reaction reagent described in section 0). (12) Dissolution of immunological agglutination reaction particles characterized by adding tannic acid at the time of dissolving the dried immunological agglutination reaction particles using a dissolving solution mainly composed of water and protein. Method. (13) The method for dissolving immunological agglutination reaction particles according to claim (12), wherein the dissolving solution contains 0.01 to 1.5 mol/l of an inorganic salt. (14) The immunological agglutination reaction according to claim (13), wherein the inorganic salt is at least one selected from the group consisting of sodium chloride, sodium azide, potassium chloride, calcium chloride, magnesium chloride, and lithium chloride. Method of dissolving particles. (15) The method for dissolving immunological agglutination reaction particles according to item (13), wherein the dissolving solution contains 0.5 to 10% by weight of an amino acid salt. (16) The amino acid salt is sodium L-glutamate,
Claim (15) which is at least one selected from the group consisting of L-lysine hydrochloride, L-histidine hydrochloride, L-arginine hydrochloride and sodium L-aspartate.
A method for dissolving immunological agglutination reaction particles as described in . (17) The method for dissolving immunological agglutination particles according to items (12) to (16), wherein the immunoagglutination particles are antigen-sensitized particles obtained by sensitizing an insoluble carrier with an antigen. . (18) The method for dissolving immunological agglutination reaction particles according to item (17), wherein the insoluble carrier is a non-biological particle. (19) The method for dissolving immunological agglutination reaction particles according to item (18), wherein the tannic acid is a condensed tannic acid or a hydrolyzed tannic acid. (20) Tannic acid is 1 to 1 per 1 g of insoluble carrier.
The method for dissolving immunological agglutination reaction particles according to claim 19, wherein the amount is 000 mg. (21) Dissolution of the immunological agglutination reaction particles according to claim (20), wherein the protein is at least one kind of animal serum selected from the group consisting of bovine blood albumin, beef tallow serum, goat serum, and rabbit serum. Method. (22) Claim No. 21, wherein the water is a buffer solution with a pH of 4 to 9.
) The method for dissolving the immunological agglutination reaction particles described in section 2. (23) An immunological agglutination reaction particle solution containing water, protein and tannic acid. (24) The immunological agglutination reaction particle solution according to claim (23), which contains 0.01 to 1.5 mol/l of an inorganic salt. (25) The immunological agglutination reaction according to claim (24), wherein the inorganic salt is at least one selected from the group consisting of sodium chloride, sodium azide, potassium chloride, calcium chloride, magnesium chloride, and lithium chloride. Particle solution. (26) The immunological agglutination reaction particle solution according to claim (24), which contains 0.5 to 10% by weight of an amino acid salt. (27) The amino acid salt is sodium L-glutamate,
Claim (26) which is at least one selected from the group consisting of L-lysine hydrochloride, L-histidine hydrochloride, L-arginine hydrochloride, and sodium L-aspartate.
Immunological agglutination reaction particle dissolution solution described in . (28) The immunological agglutination reaction particle solution according to items (23) to (27), wherein the tannic acid is a condensed tannic acid or a hydrolyzed tannic acid. (29) The immunological agglutination reaction particle solution according to claim (28), wherein the protein is at least one kind of animal serum selected from the group consisting of bovine blood albumin, beef tallow serum, goat serum, and rabbit serum. . (30) Claim No. 2, wherein the water is a buffer solution with a pH of 4 to 9.
9) The immunological agglutination reaction particle solution described in section 9).