JPS6035269A - Microcapsule reagent for immunological reaction and examination using the same - Google Patents

Abstract

PURPOSE:To obtain a sharp and clear agglutination transit image by a mixing a small particle microcapsule and a large particle microcapsule to make a microcapsule reagent for immunological reaction. CONSTITUTION:A microcapsule reagent comprises a smaller particle capsule of the diameter d1 and a larger particle microcapsule of the diameter d2 sensitized with an antigen or an antibody. Here, the d1 and d2 are set at values to meet the formulas of 0.5<=d1<=3.5, 4.0<=d2<=15.0 and 3<=d2/d1<=12. In the antigen-antibody reaction using the microcapsule reagent thus obtained, the gap of a coarse agglutination image formed by the larger particle microcapsule reagent appears to be filled satisfactorily with an agglutination image by the smaller particle microcapsule reagent and thus, a clear agglutination image can be obtained.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention sensitized antigens or antibodies. The present invention relates to a microcapsule reagent for immune reactions consisting of small particle microcapsules and large particle microcapsules, and a testing method for detecting antigens or antibodies using this reagent.

As a simple method for carrying out antigen-antibody reactions, immunological agglutination methods are widely used in which antigens or antibodies are supported on water-insoluble carriers and the state of agglutination based on the antigen-antibody reactions is observed with the naked eye. However, in the conventional method, animal-derived carriers (red blood cells) are used, so non-specific agglutination occurs, performance varies based on individual differences between animals, deterioration over time is likely to occur, and costs are high. There was an inconvenience.

On the other hand, although the method using polystyrene latex as a carrier (latex agglutination reaction) eliminates the above-mentioned disadvantages derived from animals, it is less sensitive than red blood cell agglutination reaction, and has poor long-term storage stability. Furthermore, there are drawbacks such as a tendency to cause spontaneous agglutination reactions that are not based on antigen-antibody reactions.

Instead of such as red blood cells or latex.

A method using microcapsules as a carrier was proposed. (Unexamined Japanese Patent Publication No. 55-94636, No. 57-19 (i6
1. 57-19662, etc.) This method eliminates the above-mentioned drawbacks inherent to animal-derived carriers and the disadvantages of synthetic carriers such as latex, and if this microcapsule carrier method is applied, sensitivity can be increased, and it is easy to use. operation, on/off
In addition to providing much improved effects compared to conventional methods, such as reliable determination of off state, new tests that are practically impossible with conventional methods are also being developed.

The microcapsules used in the above-mentioned microcapsule carrier method usually have a particle size in the range of 0.5 to 20 μm, and in particular, particles of about 5 μm are used. It is sometimes difficult to determine at what dilution ratio an agglutinated image is being formed, as roughness is observed in the area.

According to the present invention, a sharp and clear aggregation transition image can be obtained, non-specific aggregation can be prevented, and sensitivity can be increased.

The present invention sensitized antigens or antibodies. A microcapsule reagent consisting of a small particle microcapsule having a diameter d+ and a large particle microcapsule having a diameter dzt,
The present invention relates to a microcapsule reagent for immunoreaction, characterized in that a diameter d and a diameter d2 satisfy the following formula.

0.5≦d1≦3.5...(],)40≦d2≦
150 (2) 2 3≦−≦12 (3) 1 The present invention also relates to an antibody or antigen testing method characterized by using such a microcapsule reagent for an antigen-antibody reaction.

The diameter a of the small particle microcapsules used in the present invention may be within the range satisfying the formula (1), and the diameter d2 of the large particle microcapsules may be within the range satisfying the formula (1).
Any value within the range that satisfies the formula may be used. However, the diameter d
The ratio of dl to 2 must be 3 or greater than 3 and not 12 or less than 12.

In addition, in practice, when the size distribution of microcapsule particles takes a weight-average distribution, the half width should be less than twice the average value on the large particle side and more than half the average value on the small particle side. It is very effective to mix each microcapsule particle group.

Large-particle microcapsules are advantageous in terms of sensitivity because they can enlarge the blocking surface when an agglutination image is formed, but on the other hand, they agglomerate with a slight stimulus (i.e., non-specific aggregation).
easily cause noise. However, when small-particle microcapsule reagents are mixed, the voids of the rough aggregated image (with jagged edges at the periphery) formed by large-particle microcapsule reagents are moderately filled with aggregated images of small-particle microcapsule reagents. Gives a clear agglomerated image. When using the mixed microcapsule reagent of the present invention, such a clear agglutination image can be clearly obtained, so that it is possible to easily distinguish between an agglutination image and a sedimentation without requiring any skill.

A preferred embodiment of the present invention will be explained in detail.

Large particle microcapsules with a particle size of 8 μm are prepared. Particle size depends on the type of emulsifier and concentration 9
It can also be controlled during the microcapsule particle formation process by adjusting the emulsification conditions, stirring speed of 1 hour, viscosity balance between oily substances and water-soluble substances, etc.
The obtained microcapsules may be classified to obtain desired microcapsules. The thus obtained large-particle microcapsules are sensitized with an antigen or antibody in a conventional manner. For example, the sensitization technique described in "Immobilized Enzymes" by Kazuto Chibata, Kodansha (1975), Japanese Patent Application Laid-Open No. 1983-19661, etc. may be applied. Small particle microcapsules are prepared in the same manner and sensitized with antigens or antibodies. The thus obtained microcapsule reagents having different particle sizes are mixed so as to satisfy the diameter ratio of formula (3), and more preferably to exhibit the above distribution.

Mixing is preferably carried out after the sensitization operation is completed as described above. This is because if sensitization is performed after mixing large and small particles, the antigen or antibody will be preferentially consumed by binding with small particle microcapsules, which have a large surface area, and the sensitization efficiency of large particle microcapsules will decrease. It is.

In one specific embodiment, the cells were sensitized with antigens or antibodies.

A large particle microcapsule of 8 μm and a small particle microcapsule of 2 μm (dz/dt=4) are mixed to prepare a reagent for an antigen-antibody reaction system. The agglutination image is determined using a conventional method such as the Shimaitaro titer method, and the antibody or antigen is tested.

The microcapsules used as carriers in the present invention consist of a core of an oily substance (core material) and a wall material (insoluble in water and the core material) surrounding the core, and are generally prepared by the following procedure. The oily substance forming the core is mechanically emulsified and dispersed in a liquid with which it is not mixed, forming an oil-in-water emulsion. Liquids that do not mix with oily substances have the effect of assisting emulsification and improving the dispersion of oil droplets, and generally an aqueous polymer solution is used.

Microcapsules are prepared by forming a polymer film wall insoluble in water and oily substance liquid on the surface of the oil droplets thus obtained.

Various emulsifying machines and dispersing machines can be used to emulsify and disperse the oily substance.

Typical methods for forming polymer films include: ■ chemical methods;
There are physicochemical methods and ■physical methods.

The wall material of the microcapsule is not particularly limited as long as it can chemically bond to the antigen or antibody without deactivating it and can be microencapsulated. For example, protein (eg collagen) can be used as a wall material containing amino groups or imino groups.

gelatin, casein, etc.), polyamino acids, polyacrylamide, polyamide, polyurethane, polyurea, and other resins; as wall materials with hydroxyl groups.

Examples include cellulose and its derivatives (eg, methylcellulose, ethylcellulose, calgoxymethylcellulose, etc.), arabicum, denzon, and the like.

Oily substances that serve as core substances for microcapsules include natural mineral oils, animal oils, vegetable oils, and synthetic oils.

Since the surface of these core substances is completely covered with a wall material, they have no direct effect on antigens or antibodies, but it is preferable to avoid biochemically active substances. A specific example of the core material is disclosed in JP-A-56-72347.

57-19662, etc. For details on the production of microcapsules, see 1. For example, "Microcapsules" by Asashi Kondo, published by Nikkan Kogyo Shimbun (1971).
It is described in.

The specific gravity of the microcapsules used as a carrier in the present invention is selected within the range of 0.85 to 1.25. Microcapsules with specific gravity in this range can be easily adjusted by changing the specific gravity of the core material.

Moreover, the average size u of microcapsules is 0.5 μm to 2
It is desirable to select from the range of 0 μm, preferably 1 μm to 10 μm, or 4°.

It is desirable that the solid content of the microcapsule carrier is usually within a range of about 1 to 3 by weight.

In the present invention, antigens or antibodies that can bind to the wall surface of microcapsules and cause antigen-antibody reactions include hormones, drug metabolites, specific proteins,
A variety of substances may be mentioned, including antigens and antibodies of viral, bacterial, cellular and human origin. Specifically, for example, Treponema pallidum antigen, hepatitis B surface antigen (HBs
Antigens) Antibodies against 1HB8 antigen (anti-HB, antibodies), Toxofrazma antigen, Myconolazma antigen, human chorionic connatotropin (HCG), anti-HCG antibodies, nuclear proteins, deoxynucleic acids, antibodies to plasma tongue components (e.g. IgG, IgM, IgA, albumin, α-
7 ethoprotein, C-reactive protein, α2 macroglobulin.

Antibody components such as transferrin and fibrinogen (C1q, C1r, C1s, C3, C
There are antibodies against 4).

The amount of antigen or antibody to be sensitized to the microcassicle carrier is appropriately selected depending on the type of antigen or antibody and various conditions of the precision tube for the intended measurement, but in general, it is 0% to the solid content of the microcassette. .01 to 10 weight category is hereditary.

The present invention will be explained in more detail with reference to Examples below.

Example 1 Creation of microcapsules A with large particle size Mixed oil of 11.89 diisozolopyrnaphthalene and 1.3.2 g of chlorinated crude oil (degree of chlorination 50%) (specific gravity approximately 1.10)
0.259 fc of oil-soluble red dye Oleosole Red BB (manufactured by Sumitomo Chemical) was dissolved in the solution.

This oily substance liquid was mixed with 2 g of maleic anhydride-methyl vinyl ether copolymer (GANTREZ AN-1,49, manufactured by General Aniline & Film Co.) and 1 part water.
00ml, added to the solution dissolved in l. Approximately 12.00 Orp using Ultra Disno Yasu (manufactured by Yamato Scientific)
Emulsify for about 40 seconds at
-The average size of the oil droplets obtained in Type II was measured. The above emulsification and measurement were repeated to prepare an average particle size of 8 μmK. Add to this urea 25I, resorcinol 0.25, ji
' and ammonium chloride' 13 g 'k water 25 ml
A solution dissolved in was added. Furthermore, 7 dk of a 37% formaldehyde aqueous solution was added, and the mixture was reacted at 60° C. for 2 hours to perform microencapsulation. after that.

The PHg was adjusted to 0 by adding an IN sodium hydroxide aqueous solution, and microcapsules were prepared. The microcapsules thus prepared were centrifuged and washed with physiological saline to remove unreacted residues. The microcapsules thus created were dissolved in a sucrose-11 hereditary aqueous solution (specific gravity approximately 1.04.)
suspended in. Microcapsules with an average particle size of 8 μm and a particle size distribution of 4 to 16 μm were collected by centrifuging at 1.00 Orpm to remove small particles and +40 Orpm to remove large particles. The microcapsule particles were dispersed in physiological saline so that the concentration of the particles was 10%, and this was designated as microcapsule A.

One of the small particles is the microcar Iseno and B-9.
-B In the same manner as microcapsule A, the oily substance liquid was added to a solution in which 3 g of maleic anhydride-methyl vinyl ether copolymer was dissolved in 50 ml of water. Emulsification was carried out using an ultra disperser at 18.00 rpm for about 3 minutes, and additional emulsification was carried out while monitoring the average particle size using a Coulter counter to adjust the average particle size to 2 μm.

Microencapsulation was performed in the same manner as in Microcapsule A.

After removing unreacted residues, the mixture was suspended in a sucrose 11-fold aqueous solution. Large and small particles were removed by centrifugation at 2.50 Orpm and 800 rpm+n to prepare microcapsules B with an average particle size of 2 μm and a particle size distribution of 1 to 4 μm.

Preparation of comparative microcapsules C: In the same manner as microcapsules A, an oily substance solution was added to a solution in which 2.51 parts of maleic anhydride-methyl vinyl ether copolymer was dissolved in 75 ml of water. Emulsify for about 80 seconds at 18.00 rpm using an Ultra Decoder and perform additional emulsification while monitoring the average particle size with a Coulter counter.

The average particle size was adjusted to 5 μm. Microencapsulation was performed in the same manner as in Microcapsule A.

This microcapsule was used as Comparative Microcapsule C without any special operation for removing large and small particles.

Example 2 Rhesotosvira autumnalis desiccant A strain was grown in Colt's medium (containing 10% normal rabbit serum), and cultured from 6 to
The culture solution on day 10 was centrifuged at fI: 9.00 rpm for 20 minutes (5°C). After washing Shenjoo twice with physiological saline, the cells were redispersed in physiological saline.

The mixture was disrupted for 10 minutes using a 20 kHz sonicator (manufactured by Inugake Seisakusho), and the optical density at a wavelength of 280 nrn was adjusted to 0.02 using a 1 spectrophotometer, and this was used as an antigen solution.

Take 1.5g of each of the microcapsules A and B prepared in Example 1, and add 10ml of physiological saline. Distributed to ri, ri.

Next, 10 ml of a 100-fold dilution with 25% glutaraldehyde saline was added to each sample. After 45 minutes of reaction at 37°C. The mixture was washed by centrifugation and redispersed in 10 ml of physiological saline.

2 each of aldehyde-treated microcuff 0 cells A and B
1.5 ml and 3 ml of antigen solution were added to each ml.

After incubating at 37°C for 90 minutes, it was left standing in a refrigerator at 4°C for 18 hours. Then, after washing twice with physiological saline containing 0.2% guinosine, 2 ml of 0.2 ml of 3% bovine serum albumin (BSA) was added. Each was redispersed in 15M IJ acid-buffered physiological water (PBS, pH=7.2).

Both were mixed to obtain reagent A.

Comparative Reagent Color Antigen was bound under the same conditions as in Example 2, except that 2ml and 14f of antigen solution was added to Comparative Microcassicle C to obtain Comparative Reagent B.

Example 3 The reagent prepared in Example 2 was tested using the microtiter method using the antiserum described below (preparation of antiserum). Serum was prepared. Centrifuge the Kortov medium culture of Desire A strain. The precipitated bacterial cells were suspended in physiological saline. This was subcutaneously injected into rabbits twice at intervals of 4 to 5 days, and intravenously injected 9 times over a further 4 to 5 days. Seven to eight weeks had passed since the first subcutaneous injection, and after confirming that the mice had the prescribed antibody titer, all blood was collected and antiserum was prepared.

(Test using microtiter method) Antigen-antibody reactions were carried out using the microtiter method using the reagent prepared in Example 2. All tubes with obvious agglutination were considered positive, and the highest dilution of the positive serum was calculated and used as the antibody titer.

Add 100 μl of 25 μl of antiserum to each well of the microplate.
The 0-fold diluted solution was further diluted with 0.0-fold diluted solution containing bovine serum albumin. 1
A multiple dilution series was prepared with 5M IJ acid buffered saline at 2-fold intervals.

Next, add 25 μl of the reagent A prepared in Example 2.

・Collect the samples with a tube and drop them into the tube holes of the antiserum dilution series of the micrograte. The microplate was shaken for 5 minutes to advance the antigen-antibody reaction, and then left in a refrigerator at 4°C for 18 hours. After that, the microplate was placed on a light table and the agglomerated image at the bottom of the tube was observed.

The same operation was performed for the comparative reagent color and compared with reagent A.

Table 1 shows the antibody titers obtained at the highest dilution of serum that showed agglutination.

Table 1: Reagent A, which is made by separately sensitizing small particle size microcassicles and then mixing them, aggregates to the pores 4 times diluted compared to the comparative reagent color with the same average particle size, and has high detection sensitivity. . Furthermore, the agglutination image produced with reagent A is a clear, easy-to-see pattern with less roughness compared to the comparative reagent color, which prevents misjudgment.

Patent applicant, Fuji Photo Film Co., Ltd. Agent: Patent attorney Gobe Sunakawa (and other persons) Procedural amendment (voluntary) Patent application (4) 3, filed on August 5, 1980. Make amendments. Relationship with the case: Patent applicant representative Minoru Tainishi 4, agent address Takafuto 2-2-39-4175 Jingumae, Shibuya-ku,
Date of amendment order Vol. 8, Contents of amendment? ? After page 5, line 12, insert a new line with a missing paragraph. Preferably l: is in the range of 2 to 10,000 ° 3.]

Claims (1)

[Claims] 1. Sensitized with an antigen or antibody. A microcassicle reagent consisting of a small particle microcapsule having a diameter d and a large particle microcapsule having a diameter d2e, the microcassicle reagent having a diameter d.
A microcapsule reagent for immunoreaction, characterized in that 1 and diameter d2 satisfy the following formula. 0.5≦d1≦3.5 40≦d2≦15. O 12 antigen or antibody total sensitization was performed. Small ψ of diameter dl”−7−
A microcassicle reagent consisting of a half-candy microcapsule, which has a diameter d1 and a diameter d2 satisfying the following formula, is used for antigen-antibody reaction. A method for testing antibodies or antigens characterized by using: 0.5≦d, ≦3.5 40≦d2≦150