JPH06148192A - Manufacture of antibody or antigen fixing carrier - Google Patents

Abstract

PURPOSE:To provide an antibody or antigen fixing carrier capable of improving the reactivity between antigen and antibody in immunological measurement. CONSTITUTION:A method for manufacturing an antibody or antigen fixing carrier comprises adhering a magnetic material to a thermoplastic resin having a particle size of 0.05-20mm, and washing it with a solvent selected from the group consisting of water containing surfactant, organic solvents containing surfactant, and organic solvents, whereby the above object can be attained.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for producing a carrier for immobilizing an antibody or an antigen, and more specifically, when the antibody or the antigen is immobilized, the antibody or the antigen exhibits excellent immunoreactivity. The present invention relates to a method for producing a carrier that can exert its effect.

[0002]

2. Description of the Related Art The content of trace substances, such as proteins, contained in biological materials such as serum and urine can be known by carrying out immunoassays using antibodies and antigens.

In an assay kit for carrying out immunoassays, an insoluble carrier may be used for immobilizing an antibody or an antigen, and plastic carriers are often used.

[0004]

Generally, there are two methods of immunoassay, a sandwich method and a competition method, but the measuring reagent contains an immobilized antibody or antigen. Since the reaction rate of the antigen and the antibody is extremely high and their binding constants are as high as 10 ¹² l / mole, they can be measured rapidly and quantitatively even if the concentration of the antigen or antibody to be measured is low. Should be.

However, the lower limit of measurement of an antigen or antibody in actual immunoassay is limited by various factors. A typical cause of this is that immunoreactivity is low and the amount detected by the reaction is small.

[0006]

The present invention aims to improve the above-mentioned lower limit of measurement of an antigen or antibody in an immunoassay, and a method for producing a carrier for immobilizing an antibody or an antigen having improved immunoreactivity. Is provided. That is, the present invention is
Attaching a magnetic substance to a thermoplastic resin having a particle size of 0.05 to 20 mm, and further washing with a solvent selected from the group consisting of water containing a surfactant, an organic solvent containing a surfactant, and an organic solvent. A method for producing a carrier for immobilizing an antibody or an antigen, which comprises:

The present invention also has a particle size of 0.05 to 20 m.
The magnetic substance is attached to the thermoplastic resin of m, the surface thereof is covered with a polymer, and if necessary, a chemical treatment is performed so that at least one of --CHO, --OH or NH ₂ groups appears in the terminal group of the polymer. Further, the method for producing a carrier for immobilizing an antibody or an antigen is characterized by washing with a solvent selected from the group consisting of water containing a surfactant, an organic solvent containing a surfactant and an organic solvent. Hereinafter, the present invention will be described in detail.

The thermoplastic resin has a particle size of 0.05 mm to
It is selected to be within the range of 20 mm, but it may be adapted to the measurement system using the carrier produced by the present invention. Examples of the material include ethylene-vinyl acetate copolymer (EVA) and polystyrene, and Japanese Patent Application No. 61-3827.
Examples thereof include the thermoplastic resins disclosed in No. 9 and the like. Further, its shape is not particularly limited, in the measurement system in which the reaction is carried out in a state where the produced carrier is placed in a container, while minimizing the contact surface with the container, a spherical shape capable of maximizing the contact surface with the liquid,
Particularly, the spherical shape is particularly preferable. To produce a thermoplastic resin having a true spherical shape, for example, a dispersant is added to a thermoplastic resin having an approximate spherical shape, and the resin is heated at a temperature not higher than + 30 ° C. from the melting point of the resin in a medium that does not dissolve the resin. The method of doing can be illustrated.

A magnetic substance is attached to the thermoplastic resin as described above. Magnetic substances are powdery or lumpy ferrite,
Examples thereof include iron, cobalt, nickel, and oxides thereof that are thermally fused and adhered. For example, it is also possible to put the magnetic substance in a cobalt solution or the like and deposit cobalt or the like on the surface thereof. .

As described above, a thermoplastic resin to which a magnetic substance is adhered can be moved and stirred by putting it in a container and applying a magnetic force from the outside, so that it can be separated from a solution and immune reaction is caused. It is a carrier material which is excellent in promoting property.
Such a carrier material can be used as a carrier by physically adsorbing the antibody or antigen after washing as described below or by binding the magnetic substance and the antibody or antigen with an appropriate coupling agent. In the present invention, the surface of this carrier material is coated with glycidyl methacrylate (GM).
A) and the like, covered with a polymer disclosed in Japanese Patent Application No. 61-38279, etc., and subjected to a chemical treatment if necessary to give at least one of --CHO, --OH or NH ₂ group to the terminal group of the polymer. Those that have emerged can also be used as carrier materials.
In the case where the thermoplastic resin to which the magnetic substance is attached is further covered with a polymer, the loss of the magnetic substance is reduced, and the surface area is further increased.
It is preferable for a measurement system that requires more vigorous stirring operation or a measurement system that requires reuse. Here, at least one of —CHO, —OH or NH ₂ group is made to appear in the terminal group of the polymer means that the terminal group of the polymer is converted into these functional groups by treatment with a suitable chemical reagent, Or, it means that a chemical reagent having these functional groups can be introduced into the polymer end by an appropriate treatment. Regarding this carrier material, as a method of manufacturing the material described later, for example, Japanese Patent Application No. 61-3827.
No. 9 can be exemplified.

Subsequently, these carrier materials are washed with a solvent selected from the group consisting of water containing a surfactant, an organic solvent containing a surfactant and an organic solvent. Here, the organic solvent is not particularly limited as long as it becomes a poor solvent for the carrier material, but ethanol, methanol, acetone and the like are preferable in view of price, easiness of storage and the like. In addition, since organic solvents such as ethanol, methanol, and acetone are easily dried after carrying out the present invention without special treatment of the produced carrier, when considering storage or the like, Preferred as compared to water.

As the surfactant, ionic or nonionic surfactants can be used, and specific examples thereof include Triton and Tween. There is no particular limitation on the concentration, but 0.01% to
About 1% by weight is effective.

In the present invention, the number of washings is not particularly limited,
It is preferable to perform 1 to 3 times when using a cleaning liquid containing a relatively high concentration of surfactant, and about 1 to 10 times when using a cleaning liquid containing a relatively low concentration of surfactant. Moreover, it is preferable to perform the washing in consideration of the shape and material of the carrier material. That is, it is only necessary to adopt a temperature condition in which the carrier material is not denatured or a stirring condition in which the attached magnetic substance does not drop off.

The carrier for immobilizing the antibody or antigen of the present invention can be produced by completing the washing, but when the produced carrier is stored, it can be prevented from deterioration by keeping it in a dry state.

[0015]

EXAMPLES In order to explain the present invention in more detail, examples are described below, but the present invention is not limited to these examples.

Example 1 Average diameter of 1.4 m obtained by water strand method
m, average length 1.5 mm EVA pellets (Tosoh
Manufactured by Tosoh Corp. according to the method described in Japanese Patent Application No. 61-38279.
Was heat-sealed and further covered with GMA. The polymer coating carrier material thus obtained was treated with a caustic soda / methanol solution to open the epoxy group of the surface layer thereof to form a di-
Converted to le. After putting 2.5 g of the carrier material into a 15 ml plastic container and adding 5 ml of deionized water,
The container was rotated at about 120 rpm for 3 hours at room temperature.
After that, water in the container was removed, and the same operation was repeated 10 times.

Washing was performed by adding 5 ml of methanol and performing the same operation. Then, the methanol in the container was removed, and vacuum drying was performed using a vacuum pump. For comparison, vacuum drying was performed without washing with methanol to obtain a comparative carrier.

Example 2 Carrier obtained in Example 1 (hereinafter simply referred to as carrier)
Also, a mouse anti-human TSH monoclonal antibody was immobilized on a comparative carrier and immunoassay was performed.

50 mg of N, N'-carbonyldiimidazole (CDI, manufactured by Tokyo Kasei Kogyo Co., Ltd.) was added to each of 1000 carriers and comparative carrier according to the method described in Japanese Patent Application No. 61-38279. 2.5 ml of dry acetone containing was vigorously stirred at room temperature under a nitrogen atmosphere for 30 minutes. After washing the activated carrier and the comparative carrier,
0.25 mg / 2 ml mouse anti-human TSH monochrome-
The null antibody was added and shaken at room temperature for 16 hours to bind the antibody to the particles. After washing this, a phosphate buffer (pH 7.0) containing 1.0% bovine serum albumin (BSA) was added for blocking treatment.

Using the obtained antibody-immobilized carrier and antibody-immobilized comparison carrier, TSH was subjected to enzyme immunoassay. Put 12 carriers in a plastic cup and put 50μ
1 of alkaline phosphatase-labeled anti-TSH mouse monoclonal antibody (about 4 μg / ml) was added, an antigen solution was added, and the mixture was set in an automatic enzyme immunoassay device (AIA-600, manufactured by Tosoh Corp.). The antigen solution is 0 or 47.6.
A solution of human TSH at μIU / ml was used.

The measuring device is operated to dispense 100 μl of human TSH solution into a plastic cup at 37 ° C.
Incubated for 40 minutes. At this time, an incubate operation was carried out in which the magnet installed under the reaction vessel of the above apparatus was moved to stir the carrier. After removing the liquid in the container by suction, the container is washed with a cleaning solution (B / F separation), and 4-methylumbelliferone phosphate, which is a substrate of alkaline phosphatase, is dispensed to cause an enzymatic reaction, which appears by the reaction. The rate of increase of 4-methylumbelliferone (4MU) was measured. The results are shown in Table 1.

As is clear from Table 1, the immunoreactivity when the carrier immobilized with the antigen or the antibody produced by the present invention was higher than that when the comparative carrier was used. This indicates that the present invention is a method suitable for producing a carrier for immobilizing an antigen or antibody to be used for immune reaction and the like.

[0023]

[Table 1]

[0024]

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to produce a carrier for immobilizing an antigen or an antibody, which can improve the reactivity between the antigen and the antibody in the immunoassay utilizing the immune reaction. Moreover, the present invention can be used without significantly changing the conventional measurement system, unlike the method of modifying the antigen or antibody itself.

Claims (3)

[Claims] 1. A magnetic substance is adhered to a thermoplastic resin having a particle diameter of 0.05 to 20 mm, and further selected from the group consisting of water containing a surfactant, an organic solvent containing a surfactant, and an organic solvent. A method for producing a carrier for immobilizing an antibody or an antigen, which comprises washing with a solvent. 2. A magnetic substance is attached to a thermoplastic resin having a particle diameter of 0.05 to 20 mm, the surface of the magnetic substance is covered with a polymer, and if necessary, a chemical treatment is applied to the end group of the polymer to form --CHO, -OH or an NH ₂ group, at least one of which appears, and further, washing with a solvent selected from the group consisting of water containing a surfactant, an organic solvent containing a surfactant, and an organic solvent, or A method for producing a carrier for immobilizing an antigen. 3. A thermoplastic resin, which is obtained by adding a dispersant to a thermoplastic resin having an approximately spherical shape, and heating the resin at a temperature not higher than + 30 ° C. from the melting point of the resin in a medium that does not dissolve the resin. A thermoplastic resin having a spherical shape is used.
Or the method for producing the antibody or antigen-immobilizing carrier according to 2).