JPH05126830A - Manufacturing method of immunological agglutination particle - Google Patents

Abstract

(57) [Summary] [Structure] A method for producing immunological agglutination particles, which comprises sensitizing a carrier with an antigen or an antibody and then treating the carrier with a polysaccharide. [Effect] The immunological agglutination reagent containing the immunological agglutination reaction particles obtained by the production method of the present invention does not cause collapse of the agglutination image and has good sensitivity.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a method for producing immunological agglutination reaction particles.

[0002]

2. Description of the Related Art In the field of clinical examinations, it has been recently emphasized that various diseases are serologically diagnosed. An extremely important issue in this diagnosis is to perform the quantification of the antigen or antibody in serum accurately and quickly, and if possible, simply. As one of methods for solving this problem, a method of using a carrier sensitized with an antigen or an antibody has been widely adopted. When the carrier sensitized with the antigen or the antibody is used, when the antibody or the antigen is present in the sample, the antigen reacts with the antibody to form a complex, and the complex aggregates. This reaction between the antigen and the antibody is called an immunological agglutination reaction. The advantage of the method using the carrier sensitized with the antigen or antibody is that the aggregation of the complex can be observed with the naked eye and the operation performed in the method is relatively simple.

In the above-mentioned method using the carrier sensitized with the antigen or antibody, the following two kinds of carriers are used. That is, a biological carrier such as animal red blood cells and bacterial cells,
It is a non-biological carrier such as latex, kaolin, charcoal powder, silica, alumina and organic-inorganic composite particles.

[0004]

Among the above two types of carriers, when a biological carrier is used, even if a biological carrier collected from the same type of organism is used, the above-mentioned agglutination state will still occur. Differences between solids are often seen. For example, when animal red blood cells are used, there is a problem that the specific antigen present on the surface of the red blood cells does not cause the aggregation when it should occur, that is, the collapse of the aggregation image.

On the other hand, when a non-biological carrier is used, there is a problem that it does not aggregate unless the antibody or antigen is contained in serum in a relatively large amount, that is, the sensitivity is low.

[0006]

Means for Solving the Problems As a result of intensive studies to solve the above problems, the present inventors have found that sensitivity is decreased by treating a carrier sensitized with an antigen or an antibody with a polysaccharide. Further, they have found that the collapse of the aggregate image can be prevented, and have completed the present invention.

That is, the present invention is a method for producing immunological agglutination reaction particles, which comprises sensitizing a carrier with an antigen or an antibody and then treating with a polysaccharide.

The carrier used in the present invention is not particularly limited, and examples thereof include latex particles such as polystyrene particles and polyacrylamide particles, oxides such as kaolin, carbon powder, silica, alumina, titania and zirconia, and the above oxides. Inorganic particles such as complex oxides containing two or more kinds,
The organic compound obtained by treating the surface of inorganic particles such as silica with an organic compound, organic or inorganic composite particles chemically or physically bound to the particle surface, non-biological particles such as gelatin particles, bacterial cells, Included are biological particles such as animal red blood cells. Among these, since the organic-inorganic composite particles are artificial carriers, the surface thereof can be chemically treated according to the purpose, and the collapse of the agglomeration image described above is unlikely to occur, and therefore they are preferably used.

The substance to be sensitized to the above carrier may be any substance that causes an immunological agglutination reaction, and examples of the substance include an antigen and an antibody.

The antigen is not particularly limited as long as it is a substance that induces humoral immunity and cell-mediated immunity by producing antibodies, and examples thereof include proteins, glycoproteins, lipid proteins, lipids and nucleic acids. The antibody is not particularly limited as long as it has an activity of specifically binding to an antigen, and for example, IgG, IgM, Ig
A, IgD, IgE etc. are mentioned.

As a method for sensitizing a carrier with an antigen or an antibody, known methods can be adopted without limitation. For example, in a buffer solution such as a phosphate buffer solution, a Tris buffer solution, a glycine buffer solution, etc., 0.01 to 50 per 1 g of the carrier.
Examples include a method of mixing mg of the antigen or antibody, and then leaving the mixture to stand. The time for leaving the mixture is
It is usually 1 hour or more. The sensitization temperature is not particularly limited, but is usually 4 to 56 ° C., and preferably about room temperature.

In the present invention, the carrier may be treated with a cross-linking agent such as formaldehyde, glutaraldehyde, tannic acid or the like before sensitizing the carrier with the antigen or antibody.

The feature of the present invention resides in that the carrier sensitized with the antigen or antibody obtained by the above-mentioned operation is treated with polysaccharide.

As the polysaccharide used in the present invention, known polysaccharides can be used without limitation. Examples of this polysaccharide include simple polysaccharides such as cellulose, agarose, and inulin, glucomannan, galactoglucomannoglycan,
Complex polysaccharides such as arabinogalactoglycan, gum arabic, alginic acid, acid polysaccharides such as tragacanth acid, neutral polysaccharides such as cellulose, amylose and laminaran, anionic polysaccharides such as carboxymethylcellulose, carboxycellulose and cellulose phthalate acetate, where Examples include the mixture of polysaccharides.

Some or all of the hydrogen atoms of the carboxyl group in the above-mentioned polysaccharide are replaced with atoms such as sodium atom and calcium atom, and the above-mentioned polysaccharide may be in the form of salt such as sodium salt and calcium salt. Good.

Among the above-exemplified polysaccharides, the above-mentioned anionic polysaccharides are preferably used.

In the present invention, the method of treating the carrier with the polysaccharide is not particularly limited. As an example of the method of treating with the polysaccharide, (A) a method of bringing a buffer solution containing a carrier sensitized with an antigen or an antibody into contact with the polysaccharide or a solution thereof, (B)
Examples include a method in which a carrier sensitized with an antigen or an antibody is isolated from a buffer solution, and the isolated carrier is brought into contact with a solution of a polysaccharide.

The solvent used for preparing the above-mentioned polysaccharide solution may be any known solvent without limitation. Examples of this solvent include water such as distilled water and ion-exchanged water, a buffer solution such as a phosphate buffer solution, a glycine buffer solution, and an acetate buffer solution. In the method (B) of treating with the above-mentioned polysaccharide, as the method of isolating the carrier sensitized with the antigen or the antibody, a known isolation method can be adopted without limitation. For example, a method using centrifugation and the like can be mentioned.
Then, before isolating the carrier, the carrier to which the antigen or antibody has been sensitized in advance is washed with a buffer solution such as a phosphate buffer solution, a glycine buffer solution, a borate buffer solution, or a phthalate buffer solution.
It is preferable because the effect of the treatment with the polysaccharide is further enhanced.

The amount of the polysaccharide used in the present invention is not particularly limited, but is 1 to 1 mg / mg of the antigen or antibody used for sensitization.
It is preferably 1000 mg. When the amount of the polysaccharide used is within the above range, the sensitivity can be increased and the collapse of the agglutination image can be prevented more reliably. In the present invention, the temperature for treating the carrier sensitized with the antigen or antibody with the polysaccharide is not particularly limited, but is usually 4 to 56 ° C, preferably 4 to 10 ° C.
℃.

In the present invention, the time for treating the carrier with the polysaccharide is not particularly limited, but is preferably 2 to 24 hours. When the treatment time with the polysaccharide is within the above range, the sensitivity can be enhanced and the collapse of the agglutination image can be prevented more reliably.

In the present invention, if the carrier sensitized with the antigen or the antibody is blocked with a protein such as bovine serum albumin, casein, gelatin or the like before the carrier is treated with the polysaccharide, the sensitivity is improved, and It is preferable because the aggregation image is less likely to collapse. The blocking method with the above-mentioned protein can be adopted without limiting the blocking method with a known protein.

Immunological agglutination reaction particles are obtained by the production method of the present invention described above. The immunological agglutination reaction particles are usually dispersed in an aqueous medium and used as an immunological agglutination reaction reagent (hereinafter also simply referred to as "aggregation reaction reagent").

When the immunological agglutination reaction particles of the present invention are used as an active ingredient of an agglutination reaction reagent, the immunological agglutination reaction particles are usually preliminarily treated with a buffer solution such as a phosphate buffer, a glycine buffer or an acetate buffer. Etc. at least once, and then the immunological agglutination reaction particles are isolated by centrifugation.

As described above, when the immunological agglutination reaction particles obtained by the production method of the present invention are dispersed in an aqueous medium to be used as an agglutination reaction reagent, the concentration of the immunological agglutination reaction particles is not particularly limited. However, based on the total amount of immunological agglutination reaction particles and the aqueous medium, 0.3 to 1% by weight
Is preferable because the agglomeration image becomes clear and the sensitivity is good.

The agglutination reagent is a protein, an inorganic salt,
Amino acid salts and the like may be added. The protein is not particularly limited as long as it does not inhibit the antigen-antibody reaction in the immunological agglutination reaction particles. Examples of the protein include bovine serum albumin, goat serum, rabbit serum gelatin, skim milk and the like. Examples of the inorganic salt include sodium chloride, potassium chloride, sodium azide and the like. Further, examples of the amino acid salt include sodium aspartate, sodium glutamate and the like.

Preferred examples of the aqueous medium usually used in obtaining the agglutination reagent are ultrapure water, water such as distilled water, buffer solutions such as phosphate buffer solution, Tris buffer solution and glycine buffer solution. , An aqueous medium having a pH of 4 to 9 such as physiological saline. Of these, phosphate buffer,
Tris buffer, glycine buffer, and physiological saline are preferable because the immunological agglutination reaction in the immunological agglutination reaction particles rapidly proceeds.

When the immunological agglutination reaction particles obtained by the production method of the present invention are to be stored for a long period of time, it is usually preferable to store the immunological agglutination reaction particles by drying. The method for drying the immunological agglutination reaction particles is not particularly limited, but a freeze-drying method is preferred.

The above dried immunological agglutination reaction particles are
When one component of the agglutination reagent is used, it is dispersed in the above aqueous medium.

[0029]

The agglutination reaction reagent containing immunological agglutination reaction particles obtained by the production method of the present invention does not cause the collapse of the agglutination image and has good sensitivity. Although the reason for this is not clear, it is considered that suitable immunological agglutination reaction particles and thus high-quality agglutination reaction reagents can be obtained due to the viscosity and stability of the polysaccharide itself.

[0030]

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

Example 1 To prepare an immunological syphilis diagnostic agent as an agglutination reagent, first, Treponema pallidum 1 which is a pathogenic bacterium of syphilis.
× 10 ⁹ pieces were dispersed in 1 ml of 0.1 M sodium chloride aqueous solution. Next, add 49 ml of 0.1 M to the aqueous sodium chloride solution in which this Treponema pallidum was dispersed.
Add an aqueous solution of sodium chloride and use an ultrasonic crusher to add 20
It was crushed at 0 W for 30 minutes. Next, organic-inorganic composite particles (Tokuyama Soda Co., Ltd. product name: Immunoticles HD
P) was washed twice with a 0.02 M phosphate buffer (pH 7.2) containing 0.1 M sodium chloride, and then the above particles were dispersed in 1 ml of the buffer so that the concentration became 2.5% by weight. Let To 1 ml of an aqueous sodium chloride solution containing the crushed Treponema pallidum, the buffer solution containing the organic-inorganic composite particles was added, and the mixture was allowed to stand at room temperature for 1 hour.
After centrifuging at 0 rpm for 3 minutes to isolate the organic-inorganic composite particles sensitized with the Treponema pallidum-derived antigen, the sensitized organic-inorganic composite particles were dissolved in distilled water to prepare a 1 wt% carboxymethylcellulose solution (1 ml). The mixture was allowed to stand at 4 ° C. for 4 hours to obtain a liquid containing immunological agglutination reaction particles. The amount of carboxymethyl cellulose, which is a polysaccharide, used per 20 mg of antigen was 20 mg. Then, the immunological agglutination reaction particles were treated with 0.02M phosphate buffer (pH
7.2) Washing with 1 ml was performed twice and freeze-drying. After standing at 4 ° C. for 2 months, 5 ml of 0.02M phosphate buffer containing 5% by weight of bovine serum albumin
The freeze-dried immunological agglutination reaction particles were added to the agglutination reaction reagent at a concentration of 0.5% by weight to prepare an agglutination reaction reagent.

Next, the performance of this agglutination reagent as an immunological syphilis diagnostic agent was measured as follows.

Serum of syphilis patient was previously used as a test liquid in an amount of 0.02.
A 10-fold dilution with M phosphate buffer (pH 7.2) was used as a stock solution, and diluted with the phosphate buffer according to the multiple dilution method to obtain 10-fold, 20-fold, 40-fold, 80-fold.
Dilutions of 1-fold, 160-fold, 320-fold, 640-fold and 1280-fold were prepared. Then add 2 from each of the above dilutions.
Aliquot 5 μl into each well of a microtiter plate, and add 25 μl of the agglutination reagent to each well.
After adding 1 each (the above-mentioned dilution ratios are each 2 times), the mixture was stirred for 1 minute and allowed to stand. After 30 minutes, the aggregation state of the particles was observed, and the highest dilution multiple of the dilution solution in the well where the particle ring was clearly large and the particles were uniformly spread in the ring was taken as the sensitivity (antibody value). evaluated. In addition, in the above observation, when the particle links were small or the particles did not spread uniformly in the ring, the number of aggregated images was considered to have collapsed, and the number was counted. The collapse of the agglutination image occurs from the lower dilution ratio. On the other hand, according to the above-mentioned method, a diluted test serum was used as a test liquid, and the antibody titer and the number of collapse of the agglutination image were measured. The results are shown in Table 1.

[0034]

[Table 1]

Comparative Example 1 The procedure of Example 1 was repeated except that the treatment with the carboxymethyl cellulose solution was omitted. The results are shown in Table 1.

Comparative Example 2 The same procedure as in Example 1 was carried out except that in Example 1, 1 ml of a 1% by weight bovine serum albumin aqueous solution was used instead of the carboxymethyl cellulose solution. The results are shown in Table 1.

Example 2 As an agglutination reagent, in order to prepare an immunological colorectal cancer diagnostic agent, an anti-human hemoglobin antibody of rabbit origin (manufactured by Kappel) was added at a concentration of 0.1 mg / ml. 05
It was dissolved in 1 ml of an aqueous solution of sodium chloride M. Then
1 ml of an aqueous sodium chloride solution containing this human hemoglobin antibody contained 0.0 M of 0.05 M sodium chloride.
A solution prepared by dispersing 1 ml of 0.5% by weight sheep red blood cells immobilized with formalin to a 2M phosphate buffer (pH 7.2) was added and left at room temperature for 1 hour for sensitization. Then 300
After the sensitized sheep red blood cells were isolated by centrifugation at 0 rpm for 3 minutes, 0.2 M containing 0.05 M sodium chloride was isolated.
After centrifugal washing once with 1 ml of M phosphate buffer (pH 7.2), the mixture was allowed to stand in 1 ml of a 2% by weight cellulose acetate phthalate solution dissolved in distilled water at 4 ° C. overnight to contain immunological agglutination reaction particles. A liquid was obtained. The amount of cellulose acetate phthalate, a polysaccharide, used per 1 mg of antibody is 200 mg.
Met. Then, the immunological agglutination reaction particles are added to 0.02.
Washing with 1 ml of M phosphate buffer (pH 7.2) was performed twice and freeze-dried. After leaving it at 4 ° C for 3 months,
The freeze-dried immunological agglutination reaction particles were added to distilled water 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 this agglutination reagent as an immunological colorectal cancer diagnostic agent was measured as shown below. That is, in Example 1, 3 mg of colon cancer patient stool was used as a stock solution, and 0.01 M glycine buffer solution (p) containing 0.01% by weight of sodium deoxycholate (p
H8.0) was used and diluted by the multiple dilution method with the glycine buffer, and the stool of healthy subjects was diluted as the stock solution of the other test solution. The measurement was performed in the same manner as 1. The sensitivity measured at this time is also called the antigen titer. The results are shown in Table 1.

Comparative Example 3 The procedure of Example 2 was repeated except that the cellulose acetate phthalate solution was not used.
The results are shown in Table 1.

Comparative Example 4 A 2 wt% dairy casein (manufactured by Wako Pure Chemical Industries) aqueous solution 1 was used in place of the cellulose acetate phthalate solution in Example 2.
The same procedure as in Example 2 was carried out except that the treatment was performed with ml. The results are shown in Table 1.

Example 3 As an agglutination reagent, in order to prepare an immunological adult T-cell leukemia diagnostic agent, an adult T-cell leukemia virus-derived antigen was used in an amount of 10 per 1 ml of a 0.075 M sodium chloride aqueous solution.
0 μg was dissolved. Then, the zirconia particles prepared from zirconium oxychloride by the usual sol-gel method in the presence of sodium hydroxide were washed twice with a 0.02M phosphate buffer solution (pH 7.2) containing 0.075M sodium chloride, It was dispersed in the above buffer solution so as to be 2.5% by weight. A solution prepared by dispersing 1 ml of the above-mentioned inorganic particles was added to 1 ml of the above-mentioned antigen solution, and the mixture was allowed to stand at room temperature for 1 hour, and then 0.02
It was washed twice with 1 ml of M phosphate buffer (pH 7.2) to prepare agglutination reaction particles sensitized with the antigen derived from adult T cell leukemia virus. Then, in the phosphate buffer solution in which the above-mentioned agglutination reaction particles are dispersed, 0.02
g of sodium alginate was added, and the mixture was treated at 4 ° C. overnight to obtain a liquid containing immunological agglutination reaction particles.
The amount of polysaccharide sodium alginate used per 1 mg of the antigen was 200 mg. Thereafter, the immunological agglutination reaction particles were added to 5 ml of a 0.02M phosphate buffer containing 1% by weight of bovine serum albumin so that the concentration of the immunological agglutination reaction was 0.5% by weight based on the agglutination reagent, and the agglutination was performed. The reaction reagents were prepared.

Next, the immunological adult T of this agglutination reagent was used.
The performance as a diagnostic agent for cell leukemia was measured as shown below. That is, the measurement was performed in the same manner as in Example 1 except that adult T-cell leukemia patient serum was used as the stock solution and serum of a healthy individual was used as the stock solution of the other test solution. The results are shown in Table 1.

Comparative Example 5 The procedure of Example 3 was repeated except that sodium alginate was not added. The results are shown in Table 1.

Comparative Example 6 In Example 3, a 2% by weight aqueous gelatin (Nitta Gelatin Co., Ltd.) aqueous solution 1 was used in place of the sodium alginate solution.
The same procedure as in Example 3 was performed except that the treatment was performed with ml. The results are shown in Table 1.

Claims (1)

[Claims] 1. A method for producing immunological agglutination reaction particles, which comprises sensitizing a carrier with an antigen or an antibody and then treating the carrier with a polysaccharide.