JPH0384461A - Immunological agglutination reagent and production thereof - Google Patents

Abstract

PURPOSE:To improve the preservable stability of the above reagent by consisting the reagent of insoluble artificial carrier particles sensitized with an antigen or antibody, incorporating at least one kind of the compd. selected from albumin and dextran as a stabilizer therein and freeze-drying the same. CONSTITUTION:Inorg. compd./dye composite particles having 1.2 to 2.0mum grain size and 1.2 to 2.0sp.gr. are preferable as the insoluble artificial carrier particles. Customary methods of physically adsorbing the antigen or antibody to the carrier particles, etc., are used for the sensitization of the antigen or antibody to the carrier particles. The albumins derived from man and bovine are more preferable as albumin to be used as the stabilizer. Dextrans of various mol.wt. are usable as the dextran if these dextrans are soluble in water. The content of the albumin and dextran is specified to about 3 to 20pts.wt. total of both per 5pts.wt. insoluble artificial carrier particles. The freeze-drying of the suspension of the immune material sensitized carrier particles added with the above- mentioned stabilizer is executed by the customary method.

Description

[Detailed description of the invention]

[Industrial Application Field] The present invention relates to an immunological agglutination reaction reagent and a method for producing the same. More specifically, the present invention relates to a freeze-dried preparation of an immunological agglutination reaction reagent comprising insoluble artificial carrier particles sensitized with an antigen or antibody, and a method for producing the same. [Prior art and problems to be solved by the invention] Immunological agglutination test methods using antigen-antibody reactions, such as indirect agglutination test methods and agglutination inhibition test methods, are used in the fields of clinical testing, pharmaceutical manufacturing, etc. Accordingly, antigens, antibodies, etc. contained in samples such as serum and blood products are measured. The most common indirect agglutination test method involves adding sensitized carrier particles, in which antigens or antibodies are bound or adsorbed to insoluble carrier particles of an appropriate size, to a specimen appropriately diluted with a diluent, and then This is a method for measuring antibodies or antigens in a specimen by utilizing a reaction in which sensitized carrier particles aggregate when antibodies or antigens corresponding to the antigen or antibody, respectively, are present in the specimen. The presence or absence of antibodies in the specimen is determined by the presence or absence of an agglutination reaction when the antigen-sensitized carrier particles obtained by sensitizing the obtained antigen to insoluble carrier particles are mixed with the specimen under appropriate conditions. I can do it,
Furthermore, by comparing with a positive control, the amount of antibody in the sample can be semi-quantitated. Similarly, if antibody-sensitized carrier particles are prepared by sensitizing purified polyclonal or monoclonal antibodies to insoluble carrier particles, the antigen in the specimen can be measured. The immunological agglutination test method does not require special equipment;
It is also widely used because it has the advantages of being easy to operate, completing measurements in a short time, and being able to quickly process large amounts of specimens. Since the immunological agglutination reaction is carried out in an aqueous medium, insoluble carrier particles sensitized with an antigen or antibody are used in a suspended state in an aqueous medium. Even if particles are stored in a cool place (for example, 2 to 8°C), nonspecific aggregation tends to occur and a decrease in sensitivity is observed after about one month. In particular, when measuring antigens and antibodies of viral diseases (e.g., hepatitis B, AIDS, human T-cell leukemia, etc.) that require high sensitivity, Although measurement with high precision is required, when sensitized carrier particles stored in a suspended state are used, there is a problem that the measurement results lack reliability due to a decrease in sensitivity and the like. In order to solve this problem and maintain long-term storage stability, a suspension of sensitized carrier particles is usually freeze-dried to form a freeze-dried preparation. However, when comparing lyophilized sensitized carrier particles and sensitized carrier particles before lyophilization, lyophilized sensitized carrier particles are significantly inferior in sensitivity, specificity, clarity of judgment image, etc. In some cases, it may become unusable for measurement. The present invention was devised to solve the above-mentioned problems of the prior art, and as a result of various studies by the present inventors on immunological agglutination reaction reagents with excellent storage stability, a suspension of sensitized carrier particles was developed. They found that by adding a specific stabilizer to the product, the stability during freeze-drying and storage stability were significantly improved. That is, an object of the present invention is to provide a freeze-dried preparation of an immunological agglutination reaction reagent that can maintain required performances such as sensitivity, specificity, and clarity of judgment images for a long period of time, and a method for producing the same. [Means for Solving the Problems] The immunological agglutination reaction reagent of the present invention, which was made to solve the above problems, consists of insoluble artificial carrier particles sensitized with antigens or antibodies, and contains albumin and stabilizers as stabilizers. It is characterized by containing at least one compound selected from dextran and being freeze-dried. Furthermore, the above immunological agglutination reaction reagent may additionally contain at least one compound selected from the group consisting of mannitol, sorbitol, inositol, citric acid, and amino acids, and by containing such a compound, Storage stability (especially at high temperatures) can be improved within the layer. Further, the method for producing an immunological agglutination reaction reagent of the present invention is the above-mentioned method for producing an immunological agglutination reaction reagent, in which a suspension of insoluble artificial carrier particles sensitized with an antigen or an antibody is added to a suspension selected from albumin and dextran. It is characterized by adding at least one kind of compound and then freeze-drying it,
Furthermore, in this manufacturing method, based on the same reasons as above,
At least one compound selected from the group consisting of mannitol, sorbitol, inositol, citric acid, and amino acids may be added together with albumin and/or dextran. As the insoluble artificial carrier particles used in the present invention having the above structure, any of those conventionally used as insoluble artificial carrier particles for immunological agglutination reactions can be used, such as polystyrene latex, gelatin particles, etc. , epoxy resin particles, cellulose particles, kaolin particles, charcoal powder, etc., and preferably inorganic compound/dye composite particles (see, for example, JP-A-63-184056). The particle size of these insoluble artificial carrier particles is about 0.5 to 5 μm, preferably 1.2 to 5 μm.
Examples include those having a specific gravity of about 2.0 μm and a specific gravity of about 1 to 265, preferably about 1.2 to 2.0. Sensitization of antigens or antibodies to the above-mentioned insoluble artificial carrier particles can be carried out by conventional methods, such as by physically adsorbing them to the carrier particles, or by using a coupling agent such as glutaraldehyde or tolylene diisocyanate. Examples include a method of chemically bonding. Insoluble artificial carrier particles sensitized with antigens or antibodies are coated with a blocking agent (e.g., skim milk powder, bovine serum albumin, animal serum,
gelatin and its hydrolyzate, casein and its hydrolyzate, dextran, polyethylene glycol, etc.). The origin of albumin used as a stabilizer in the present invention is not particularly limited, and examples thereof include albumin derived from humans, cows, rabbits, sheep, horses, and goats, and albumin derived from humans or cows is preferably used. These albumins are preferably heat-treated, for example,
Albumin heated at 60° C. for 1 hour (hereinafter referred to as heat-treated albumin) is used. The heat treatment has the effect that the judgment image at the time of aggregation becomes clear. In addition, as for dextran, if it is water-soluble,
Any of various molecular weights can be used. The content of albumin and dextran in the agglutination reaction reagent according to the present invention is
The total amount of both is about 3 to 20 parts by weight per 5 parts by weight of soluble artificial carrier particles (hereinafter referred to as immune substance sensitized carrier particles). If the total content of albumin and dextran is less than 3 parts by weight, stability during freeze-drying cannot be ensured sufficiently, and the immunosubstance-sensitized carrier particles after freeze-drying have poor sensitivity, specificity, and clarity of judgment images. Although there is a possibility that the total content exceeds 208 parts by weight, there is no particular problem, but since a sufficient effect is achieved with up to 20 parts by weight, there is no particular need for an amount exceeding that. Furthermore, the content of mannitol, sorbitol, inositol, and amino acids used as stabilizers together with albumin and/or dextran in the agglutination reaction reagent of the present invention is 1 to 2 parts by weight, respectively, per 5 parts by weight of immune substance-sensitized carrier particles.
The amount of citrate is approximately 0 parts by weight, and the content of citrate is 1 to 50 parts by weight.
The amount is approximately 1i parts (in terms of citric acid). In addition,
Examples of the citrate include alkali metal citrates such as sodium citrate and potassium citrate, ammonium citrate, and the like. Furthermore, various amino acids can be used as the amino acid, but basic amino acids are preferably used, such as arginine, lysine, asparagine, and the like. The immunological agglutination reaction reagent of the present invention can be prepared, for example, by suspending immunosubstance-sensitized carrier particles in a suitable aqueous solvent, adding a predetermined amount of albumin and/or dextran, and further adding mannitol as necessary. , sorbitol, inositol, citric acid and/or amino acids in predetermined amounts, and then freeze-drying. In the above method, an appropriate aqueous solvent is used as the aqueous solvent in which the immune substance-sensitized carrier particles are suspended, but usually a buffer solution with a pH of 5 to 9, more preferably a pH of 6 to 8;
For example, a phosphate buffer, a saline phosphate buffer, etc. are used. The amount of stabilizer such as albumin added to the immune substance sensitized carrier particle suspension is within the above range, for example,
The concentration of immune substance sensitized carrier particles in the suspension is 5% (W/V
(hereinafter the same unless otherwise specified), albumin and/or dextran is added so that the total amount thereof is about 3 to 20%, and if necessary, mannitol, sorbitol, inositol and/or amino acids are added. 1
Add citrate to about 0.05 to 0.25M. Freeze-drying of the immunosubstance-sensitized carrier particle suspension to which the above-mentioned stabilizer has been added is carried out in a conventional manner. For example, after dispensing the suspension into appropriate vials, -45
Place in a freeze dryer cooled to about 24-48 °C.
This is carried out by vacuum freeze-drying for about an hour, then purging with dry air or nitrogen, and then sealing the vial. When using the thus obtained immunological agglutination reaction reagent of the present invention, it is resuspended by adding water or an appropriate diluent, and the immunological substance sensitized carrier particle concentration is 0.3 to 0.6% ( The suspension is preferably adjusted to a concentration of about 0.5%. The substance to be measured using the immunoagglutination reagent of the present invention is not particularly limited, and it can be used to measure various antigens, antibodies, habten, drugs, and the like. In addition, antibodies corresponding to these measurement target substances are sensitized on insoluble artificial carrier particles.

[Polyclonal antibodies or monoclonal antibodies or their F(ab-)2 components] or antigens can be prepared by conventional methods, and if the target substance to be measured is a non-immunogenic substance such as a hapten, After preparing an immunizing antigen by binding it to a commonly used carrier such as , albumin, globulin, hemoglobin, etc., antibodies can be obtained by a conventional antibody production method. [Operations and Effects of the Invention] According to the immunological agglutination reaction reagent and manufacturing method of the present invention, albumin and/or dextran are added as a stabilizer, thereby preventing the deterioration of immune substance-sensitized carrier particles during freeze-drying and storage. etc., the same sensitivity, specificity, reproducibility, and
The clarity of the judgment image can be maintained for a long time, and the storage stability is significantly improved. In particular, mannitol together with albumin and/or dextran,
When sorbitol, inositol, citric acid and/or amino acids are used, the effect is that storage stability at high temperatures can be improved. [Examples] Hereinafter, the present invention will be explained in more detail based on production examples and examples, but the present invention is not limited to these examples. Production Example 1 Using high specific gravity composite particles (manufactured by Tokuyama Soda ■, diameter 1.8 μm, specific gravity 1.5) as a sensitizing carrier, h) HBsAg
(Hepatitis B virus surface antigen) positive plasma, garlic, 60℃
, 10-hour virus inactivation treatment, ammonium sulfate/Aerosil◆
Appropriate amounts of HBsAg purified by PEG fractionation, gel chromatography, and density gradient ultracentrifugation were mixed and sensitized for 10 minutes in a constant temperature water bath at 37°C. Next, centrifugal washing was performed with 0.15M salt-containing phosphate buffer to remove unsensitized HBsAg, and then albumin (or animal serum) was added to block the unreacted surface. In this way H
BsAg sensitized particles were prepared. Example 1 A 5% suspension of HBsAg sensitized particles obtained in Production Example 1 (
Solvent: 0.15M saline phosphate buffer, pH 7.0, hereinafter simply referred to as rPBSJ), heat-treated bovine albumin (5% final concentration) and mannitol (5% final concentration)
After addition, the pH was readjusted to 7□0±0.2 as necessary. After dispensing the resulting suspension into vials, -
It was placed in a freeze dryer cooled to 40°C to -45°C and freeze-dried under reduced pressure for 24 hours. Next, the sensitivity, specificity, reproducibility, and clarity of the judgment image of the antigen-sensitized carrier particles before and after freeze-drying were compared and tested using an indirect agglutination test method using HBs antibody-positive specimens and HBs antibody-negative specimens. No difference was observed before and after freeze-drying. In the sensitivity test, a dilution series was prepared in which a positive sample was diluted several times in accordance with a conventional method, and the reaction with sensitized carrier particles was evaluated at the highest dilution ratio that showed agglutination. In the specificity test, non-specific reactions were evaluated using negative samples. The reproducibility test evaluated the reproducibility of test results in multiple tests. The clarity of the judgment image was evaluated by visually observing the clarity of the aggregate image. Furthermore, the storage stability at room temperature was tested and it was found that the quality could be maintained for more than 3 months, and furthermore, the solution state at the time of dissolution was good, and no turbidity, precipitates, etc. were observed. Example 2 A 5% suspension of HBsAg sensitized particles prepared in Production Example 1 (
After adding dextran (5% final concentration) and arginine (0.05 M final concentration) to the solvent (PBS), the pH was readjusted to 7.0±0.2 as necessary. The resulting suspension was dispensed into vials and freeze-dried in the same manner as in Example 1. Next, in the same manner as in Example 1, the sensitivity of the antigen-sensitized carrier particles before and after freeze-drying was determined by indirect agglutination test method.
Comparative tests were conducted on specificity, reproducibility, and clarity of judgment images.
No difference was observed before and after freeze-drying. Example 3 A 5% suspension of HBsAg sensitized particles prepared in Production Example 1 (
After adding dextran (7% final concentration) and inositol (1% final concentration) to solvent: PBS, the pH was readjusted to 760±0.2 as necessary. The resulting suspension was dispensed into vials and freeze-dried in the same manner as in Example 1. Next, in the same manner as in Example 1, the sensitivity of the antigen-sensitized carrier particles before and after freeze-drying was determined by indirect agglutination test method.
Comparative tests were conducted on specificity, reproducibility, and clarity of judgment images.
No difference was observed before and after freeze-drying. Example 4 A 5% suspension of HBsAg sensitized particles prepared in Production Example 1 (
After adding the stabilizer shown in Table 1 below to a predetermined final concentration to PBS (solvent: PBS), add pBS as necessary.
H was readjusted to 7.0th0.2. Thereafter, freeze-drying was carried out in the same manner as in Example 1. Next, in the same manner as in Example 1, the sensitivity, specificity, reproducibility, clarity of the determined image, and solubility of the antigen-sensitized carrier particles after freeze-drying were comparatively tested using the indirect agglutination reaction test method. The results are shown in Table 1. As is clear from Table 1, in the system without the addition of a stabilizer, everything agglomerated during the freeze-drying operation, making it impossible to judge, whereas the agglutination reaction reagent of the present invention It showed the same performance as before. (Left below) Example 5 A 5% suspension of HBsAg sensitized particles prepared in Production Example 1 (
After adding the stabilizer shown in Table 2 below to a predetermined final concentration to PBS (solvent: PBS), add PBS as necessary.
H was readjusted to 7.0±0.2. Next, it was freeze-dried in the same manner as in Example 1, filled with nitrogen gas, and then sealed. The lyophilized preparation thus obtained was stored at room temperature for 3 months. Next, in the same manner as in Example 1, the sensitivity, specificity, reproducibility, clarity of the determined image, and solubility of the antigen-sensitized carrier particles immediately after freeze-drying and after storage for 3 months were compared using the indirect agglutination test method. Tested. The results are shown in Table 2. In addition, after resuspending the lyophilized preparation, store it in a cold place (2-8℃) for 3 days.
The sensitivity of the antigen-sensitized carrier particles stored for a week and the clarity of the judgment image of the antigen-sensitized carrier particles after the lyophilized preparation was stored at 40° C. for 3 months were tested. The results are also shown in Table 2. (Left below) As is clear from Table 2, the agglutination reaction reagent of the present invention has extremely good storage stability, and in particular, the addition of mannitol, sorbitol, inositol, citric acid, and amino acids improves storage stability at high temperatures. was found to be significantly improved.

Claims (1)

[Claims] 1. It is characterized by being made of insoluble artificial carrier particles sensitized with an antigen or antibody, containing at least one compound selected from albumin and dextran as a stabilizer, and being lyophilized. Immunological agglutination reagent. 2. The immunological agglutination reaction reagent according to claim 1, which additionally contains at least one compound selected from the group consisting of mannitol, sorbitol, inositol, citric acid, and amino acids. 3. Immunological agglutination reaction characterized by mixing a suspension of insoluble artificial carrier particles sensitized with an antigen or antibody and at least one compound selected from albumin and dextran, and then freeze-drying the mixture. Reagent manufacturing method. 4. The method for producing an immunological agglutination reaction reagent according to claim 3, which uses at least one compound selected from the group consisting of mannitol, sorbitol, inositol, citric acid, and amino acids together with at least one compound selected from albumin and dextran. .