JPS6217709B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a microcapsule for antigen-antibody reaction and a method for detecting antigen-antibody reaction using the same.

A specific biochemical reaction in which a first protein, called an antigen, binds to a second protein, called an antibody, that has a specific relationship with it, forming a complex.
Generally called an immunological reaction. When a foreign protein, that is, an antigen, exists in a living body, antibodies specific to it are produced, and the two bind at specific binding sites to form a complex. This specific reaction is responsible for maintaining the animal's life and for disease. plays an extremely important role.

For this reason, antigen-antibody reaction tests are performed to determine whether or not antibodies exist in a specimen against a certain type of antigen, or to detect an antigen specific to a certain type of antibody.

The above antigen-antibody reaction occurs as a precipitation reaction when the antigen is a soluble substance, but when the antigen is particulate, such as red blood cells or bacteria, or when the antigen is present in polystyrene latex, kaolin particles, etc. When adsorbed, an agglutination reaction appears that can be seen with the naked eye. This is because when an antibody corresponding to a particulate antigen reacts, interparticle crosslinking occurs due to the antibody, and the particles aggregate one after another.

Among these antigen-antibody reactions, agglutination reactions are generally about 100 to 1000 times more sensitive than precipitation reactions, so
It is used in clinical tests such as pregnancy tests and rheumatology tests.

Conventionally, sheep red blood cells are the most common carrier particles for antigen and antibody binding in the agglutination reaction used in antigen-antibody reaction tests, but other inorganic particles such as polystyrene latex, polyester, nylon, and kaolin particles are also used. etc. are used. When using these carrier particles, antigens and antibodies are bonded to the carrier particles chemically using aldehydes such as glutaraldehyde for red blood cells, while for polystyrene latex, polyester, nylon, and inorganic particles, They were bonded using physical adsorption.

However, when sheep red blood cells are used as carrier particles, since they are obtained from living organisms, there are many variations in quality, the accuracy of determining antigen-antibody reactions is insufficient, and the carrier particles themselves contain antigens or antibodies. Therefore, there are disadvantages such as non-specific reactions, low reproducibility of antigen-antibody reactions, difficulty in long-term storage, and high cost.
On the other hand, polystyrene latex, polyester,
When using nylon, inorganic particles, etc., in addition to high costs, the binding of antigens and antibodies to the carrier particles is weak due to physical adsorption, and therefore the antigens and antibodies are likely to be released and the antigen - The drawbacks were that the sensitivity of the antibody reaction was reduced and the reproducibility of the antigen-antibody reaction was poor. This tendency was particularly noticeable when stored for a long period of time.

The present invention provides antigen-
The object of the present invention is to provide an antigen for antibody reaction, antibody-binding particles, and a method for detecting an antigen-antibody reaction using the particles.

This object of the present invention is achieved by using microcapsule particles as carrier particles for binding antigens and antibodies.

Methods for binding antigens and antibodies to microcapsules in the present invention include aldehyde crosslinking method,
Various conventional treatment methods such as the cyanogen bromide method, carbodiimide crosslinking method, and alkylation method were used (Ichiro Chibata, "Immobilized Enzymes", Kodansha (1975)).
(see 2015), etc.), although not particularly limited,
It is important that the activities of the bound antigen and bound antibody are not lost. A common bonding method is to use aldehydes such as glutaraldehyde.

When binding antigens and antibodies to microcapsules, it is necessary to select the composition of the microcapsule wall depending on the binding method. For example, when bonding is performed using an aldehyde such as glutamic aldehyde, it is necessary that a functional group containing an active proton, such as an amino group, an imino group, or a hydroxyl group, be present on the microcapsule wall.

The wall material of the microcapsule in the present invention is not particularly limited as long as it can chemically bind antigens or antibodies without losing their activity and can be encapsulated. For example, wall materials having amino groups or imino groups include proteins (e.g. collagen, gelatin, casein, etc.), polyamino acids, resins such as polyacrylamide, polyamide, polyurethane, polyurea, etc.; wall materials having hydroxyl groups include cellulose and its derivatives. (for example, methylcellulose, ethylcellulose, carboxymethylcellulose, etc.), gum arabic, starch, and the like.

For various wall materials and encapsulation methods, see, for example, "Microcapsule" by Asashi Kondo, Nikkan Kogyo Shimbun (1971), "Microcapsule" by Tamotsu Kondo and Masumi Koishi, Sankyo Publishing Co., Ltd. (1978)
It is described in etc.

Oily substances that serve as capsule core materials include natural mineral oils, animal oils, vegetable oils, and synthetic oils. Since the surface of these core substances is completely covered by the capsule wall, they do not seem to have a direct effect on antigens or antibodies, but it is preferable to avoid biochemically active substances.

Examples of mineral oils include petroleum, kerosene, gasoline, naphtha, and paraffin oil; examples of animal oils include fish oil and lard oil. Examples of vegetable oils include castor oil, linseed oil, soybean oil, castor oil and corn oil. Examples of synthetic oils include biphenyl compounds (e.g. isopropyl biphenyl, isoamyl biphenyl) and terphenyl compounds (e.g.
OLS-2, 153, 635), naphthalene compounds (e.g.
Diisopropylnaphthalene, US-4, 003,
589), alkylated diphenyl alkanes (e.g. 2,
4-dimethyldiphenylmethane, US-3, 836,
383), phthalic acid compounds (e.g. diethyl phthalate, dibutyl phthalate, dioctyl phthalate), etc.

The capsule inner core material used in the present invention is not limited to those described above.

The core material may be colored by adding an oil-soluble coloring dye to improve the contrast of the aggregation reaction. Although the oil-soluble coloring dyes are not particularly limited, examples include Color Index Solvent Red 1, 3, 8, 23,
24, 25, 27, 30, 49, 81, 82, 83, 84, 100,
109, 121 etc. are used.

It is possible to color carrier particles to improve the contrast of the antigen-antibody agglutination reaction, for example when using polystyrene latex, polyester, nylon, etc.; - It is not possible to color with dyes that have any effect on antibody reactions, and the dyes that can be used are extremely limited.
Selection of the dye was not only troublesome but also economically problematic, but when using microcapsules as carrier particles, it is sufficient to add the dye to the core material, and the dye affects the antigen-antibody reaction. Because there is nothing,
It becomes possible to select dyes solely from the standpoint of improving contrast and economical efficiency.

Furthermore, since sheep red blood cells are a natural substance, their specific gravity and particle size cannot be controlled, nor can they be easily controlled in the case of polystyrene latex, polyester, nylon, inorganic particles, etc. When using microcapsules as carrier particles, the specific gravity can be easily controlled by mixing two types of core materials with different specific gravity or by dispersing inert solid particles in the core material. However, the particle size can also be controlled very easily. Therefore, it is possible to respond flexibly to future changes in test methods.

The specific gravity of the microcapsules is 1.05 to 1.20, preferably 1.10 to 1.20 under current test methods.
It is desirable to keep it within the range of 1.17 from the viewpoint of speeding up the antigen-antibody reaction and improving sensitivity.

Also, the average particle size of microcapsules is 0.1
It is desirable that it be in the range of ~30μ, preferably 0.5-10μ.

The method for producing microcapsules according to the present invention includes:
There are no particular limitations, and known methods can be used.

According to the present invention, the following novel effects can be obtained.

(i) Since it is possible to select the wall material of the microcapsule that is suitable for the antigen and antibody to be bound, it is easy to firmly bind the antigen and antibody to the carrier, improving storage stability and facilitating the antigen-antibody reaction. Judgment accuracy and reproducibility can be significantly improved.

(ii) Unlike conventional sheep red blood cells, there is no variation in quality, and there is no antigenicity in itself;
It is possible to sufficiently improve the determination accuracy, reproducibility, and specificity of the antigen-antibody reaction.

(iii) By containing a colored dye in the core substance, it is possible to color the microcapsule particles, which improves the accuracy of determining antigen-antibody reactions. Dyes that adversely affect the reaction can also be widely used.

(iv) Since it is easy to control the particle size and specific gravity of microcapsules, it is possible to flexibly respond to future changes in antigen-antibody reaction test methods.

(v) Carrier particles for binding antigens and antibodies can be provided at a lower cost than in the past.

Examples will be given below in order to make the effects of the present invention even clearer. In the following, parts indicate parts by weight, and percentages indicate weight %.

Example 1 5 parts of acid-treated gelatin having an isoelectric point of 7 or 8 and 5 parts of gum arabic were dissolved in 40 parts of warm water at 40°C, and then a dye represented by the following structural formula was dissolved. Diisopropylnaphthalene/chlorinated paraffin mixed oil with a specific gravity of 1.10 at a concentration of 1% (mixing ratio 14:11)
50 parts were added with vigorous stirring to obtain an oil-in-water emulsion with an average drop size of 6.0 microns. This emulsion was diluted by adding 213 parts of warm water at 40°C, and then acetic acid was added dropwise under constant stirring to dilute the system.
The pH was lowered to 4.6 to cause coacervation.

Afterwards, the system was cooled to 10°C, the coacervate was gelled, and 37% formalin 2 was added for dura mater.
part was added. Furthermore, 40 parts of a 10% aqueous solution of carboxymethylcellulose (average degree of polymerization 220) was added to the system. Next, in order to enhance the hardening effect, a 10% caustic soda aqueous solution was added dropwise to bring the pH to 10, and then the temperature was further heated at 50°C.
The temperature rose to The microcapsules thus prepared were washed with water and filtered to remove residual formalin. The specific gravity of this microcapsule particle is 1.10,
The average particle size was 6.3μ.

The microcapsules thus obtained were dissolved in phosphate buffer (8 g of NaCl, 0.2 g of KCl, Na ₂ HPO ₄ .
Dissolve 12H ₂ O2.9g and KH ₂ PO ₄ 0.2g in water and dissolve 1
Take 0.5g of the product washed with
of phosphate buffer. To this dispersion, 1 ml of ovalbumin 10 mg/ml was added as an antigen, and then 100 μ of glutaraldehyde (25%) was added.
The reaction was allowed to proceed for 1 hour at room temperature. Each sample is then spun down and washed with phosphate buffer again.
Disperse 0.5g in 5ml of phosphate buffer to give a concentration of approximately 10%.
An antigen-binding microcapsule (sample #1) was obtained.

Next, one drop (25μ) of phosphate buffer was added to each well of the microplate using a dropper over 12 rows.

Next, take 25μ of rabbit antiserum against ovalbumin using a diluter, add it to the diluent in the first row, and stir thoroughly. Take 25μ of the rabbit antiserum against ovalbumin in the diluter, add it to the diluent in the second row, mix well, and add it to the diluent in the second row. A 25μ portion of the solution was taken and added to the diluent in the third column, followed by thorough stirring. Continue this operation and multiply by 212 (4096
diluted up to 2 times).

In addition, 1% antigen-binding microcapsules 25μ
was added dropwise to the diluted antiserum in each row using a dropper, and the microplate was shaken well to thoroughly mix the antigen and antiserum, and then left at room temperature for 3 hours. Afterwards, the image of the tube base was read to determine whether it was positive or negative.
Here, it was determined that the microcapsule particles were spread on the bottom surface as a positive test, and the case that the microcapsule particles were gathered in the center was considered a negative test.

Commercially available sheep red blood cells were treated in the same manner, ovalbumin was bound as an antigen in the same manner, and positive and negative results were determined in the same manner.

Furthermore, polystyrene latex with a particle size of 0.8μ and a specific gravity of 1.05 was subjected to the same treatment, and ovalbumin was used as an antigen to determine whether it was positive or negative using the same method.

Each of the above experiments was conducted in 10 cases.

As a result of the evaluation, the agglutination sensitivity when using sheep red blood cells was the same as that using microcapsules, but nonspecific reactions were observed in 2 out of 10 cases, and when using polystyrene latex, Although no nonspecific reactions were observed, the agglutination sensitivity was 1/16 of that of microcapsules. On the other hand, when microcapsules were used, no nonspecific reactions were observed.

In this way, when microcapsules are used as carrier particles, they can strongly bind antigens, resulting in high agglutination sensitivity, and also, unlike sheep red blood cells, which do not themselves have antigens or antibodies, non-specific reactions can occur. It has been found that the reproducibility is extremely high, with no occurrence of this problem.

Example 2 A test was carried out in the same manner as in Example 1 except that only the antigen was changed to human denatured γ globulin, and the RA-like factor was measured. Experiments were conducted on 10 cases each.

As a result of the evaluation, when sheep red blood cells were used, the agglutination sensitivity was the same as that of microcapsules, but nonspecific reactions were observed in 3 out of 10 cases, and when polystyrene latex was used, Although no non-specific reaction was observed, the agglutination sensitivity was 1/8 that of microcapsules. On the other hand, when microcapsules were used, no nonspecific reactions were observed.

In this way, when microcapsules are used as carrier particles, they can strongly bind antigens, resulting in high agglutination sensitivity.Also, unlike sheep red blood cells, which do not themselves have antigens or antibodies, non-specific reactions can occur. It has become clear that no problems occur and the reproducibility is extremely high.

Example 3 A test was conducted in the same manner as in Example 1, except that only the antigen was changed to TP antigen, and syphilis antibodies were measured.

Each experiment was conducted on 10 cases.

As a result of the evaluation, when sheep red blood cells were used, the agglutination sensitivity was the same as that of microcapsules, but nonspecific reactions were observed in 3 out of 10 cases, and when polystyrene latex was used, Although no nonspecific reaction was observed, the agglutination sensitivity was 1/64 of that of microcapsules. On the other hand, when microcapsules were used, no nonspecific reactions were observed.

In this way, when microcapsules are used as carrier particles, they can strongly bind antigens, resulting in high agglutination sensitivity, and also, unlike sheep red blood cells, which do not themselves have antigens or antibodies, non-specific reactions can occur. It has been found that the reproducibility is extremely high, with no occurrence of this problem.

Claims (1)

[Claims] 1. A microcapsule having an antigen or antibody chemically bonded to its wall surface. 2. In a method for detecting an antibody or antigen in a specimen by reacting an antigen or antibody bound to the surface of a carrier particle with an antibody or antigen in a specimen and aggregating the reacted carrier particles, a wall surface as a carrier particle is used. 1. A method for detecting an antigen-antibody reaction, comprising using microcapsules in which an antigen or an antibody is chemically bonded to a microcapsule.