JP2000321279A - Immunoreagent and kit for immunoassay - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an immunoreagent whose preservation stability is enhanced and to provide a kit for immunoassay. SOLUTION: An insoluble carrier particle reagent in which an antigen or an antibody with reference to a substance, to be measured, in a specimen is immobilized to insoluble carrier particles coexists with insoluble carrier particles which have no specificity with reference to the insuluble carrier particle reagent or the substance to be measured. Then, a unit which contains an immunoreagent and a unit which contains one kind or several kinds of buffers constitute this kit.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an immunoreagent, and more particularly, to an immunoassay method utilizing a particle agglutination reaction caused by immunological binding of an antibody or antigen immobilized on insoluble carrier particles and an antigen or antibody in a sample. The present invention relates to an immunoreagent and an immunoassay kit including the immunoreagent.

[0002]

2. Description of the Related Art An immunoassay method utilizing a reaction in which insoluble carrier particles such as polystyrene latex agglutinate with an immune reaction is a quick, simple, and accurate measurement method. Conventionally, it is widely used (Japanese Patent Publication No. 58-11575). Also, a method using insoluble magnetic carrier particles (Japanese Patent Laid-Open No.
JP 93647, JP-A-3-59459, etc.)
And a method of combining insoluble magnetic carrier particles and fluorescent labeling particles (Japanese Patent Application Laid-Open Nos. 61-128168 and 7-7
No. 2155) has also been developed.

[0003] In order to stably maintain immunological reagents used in these immunoassays, it is generally necessary to keep them refrigerated. However, when a reagent is used, it is usually exposed to room temperature or higher due to the influence of heat inside the measuring device. Therefore, if the remaining reagent is used at a later date, 1) the reagent concentration decreases due to the adsorption of the immunoreagent to the container wall, and the reactivity decreases accordingly. 2) The impurities in the dispersion medium are adsorbed to the immunoreagent, Interference with the reaction. 3) In some cases, the antigen or antibody immobilized on the immunoreagent was detached. The present invention suppresses these and improves the storage stability of the immunological reagent.

[0004]

Means for Solving the Problems The present inventors have conducted intensive studies on the above-mentioned problems, and as a result, in the conventional immunoreagent, by coexisting particles having no specificity with respect to the reagent or the object to be measured by the reagent, The inventors have found that the above problems can be solved, and have reached the present invention. That is, the gist of the present invention is to provide an insoluble carrier particle reagent in which an antigen or an antibody to a measurement target in a sample is immobilized on an insoluble carrier particle, and an insoluble carrier particle reagent or an insoluble carrier having no specificity for the measurement target substance. An immunological reagent characterized in that carrier particles coexist;
And an immunoassay kit comprising a unit containing the immunoreagent and a unit containing one or several buffers.

[0005]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention relates to an immunoreagent comprising an insoluble carrier particle reagent and an "insoluble carrier particle having no specificity for the insoluble carrier particle reagent and the substance to be measured" (hereinafter referred to as "dummy particles"). ), Thereby improving the storage stability of the immunological reagent.

As the insoluble carrier particles used in the reagent of the present invention, organic polymer substances, biological substances, lipid particles, metal colloid particles, inorganic fine powders and the like can be used. When dispersed in
It is desirable that there be no natural sedimentation before the photometry, and that the material be floating. Examples of the organic polymer substance include a latex made of a homopolymer or a copolymer obtained by emulsion polymerization of styrene, vinyl toluene, methyl methacrylate, methacrylic acid, or the like. As a biological substance,
Red blood cells and the like. Lipid particles include liposomes and the like. Examples of the metal colloid particles include gold colloid. Examples of the inorganic fine powder include ceramic fine particles, silica, alumina, silica alumina, and carbon powder. Among them, polystyrene latex is particularly effective. As the particle size of the insoluble carrier particles,
Usually, those having a thickness of 0.01 to 10 μm are used, and preferably 0.1 to 1 μm.

The types of the insoluble carrier particles include magnetic substance-containing particles, labeling agent-containing particles, and non-magnetic non-labeling particles, which are selected according to the type of immunoassay. As the magnetic material-containing particles, those prepared so that iron or magnetic iron oxide or the like is usually contained in the above insoluble carrier particles in an amount of 5 to 100% by weight as a magnetic material are used. Examples of the labeling agent-containing particles include those in which an enzyme, a dye, a fluorescent dye, a chemiluminescent substance, an electrochemiluminescent substance, or the like is contained as a labeling agent in the insoluble carrier particles. As the labeling agent-containing particles, those in which an antigen or an antibody is directly immobilized on the labeling agent without using insoluble carrier particles may be used. In this case, usually, 0.1 to 100 molecules of the labeling agent are bound per antigen or antibody molecule.

[0008] The insoluble carrier particle reagent of the present invention is generally used for immunoassay, and is obtained by immobilizing an antigen or an antibody against an object to be measured in a sample on the insoluble carrier particles. As a method for immobilizing an antigen or an antibody on the insoluble carrier particles, a physical adsorption method, a chemical bonding method using a condensing agent such as carbodiimide, or the like is used. The amount of the immobilized antigen or antibody is usually 50 ng to 500 μg per 1 mg of insoluble carrier particles,
Preferably, it is 500 ng to 200 μg. The insoluble carrier particle reagent is used by being dispersed in the reaction solution at the time of immunoassay so that the weight concentration of the insoluble carrier particles is usually 0.0001 to 1%, preferably 0.0003 to 0.3%.

The dummy particles of the present invention are particles that do not cause an antigen-antibody reaction with the components in the immunoreagent and do not cause an antigen-antibody reaction with the measurement object of the immunoreagent. Examples of such dummy particles include insoluble carrier particles generally used as an insoluble carrier particle reagent in immunoassay and having no specificity for the insoluble carrier particle reagent or the substance to be measured, or the insoluble carrier particles. It is possible to use an insoluble carrier particle reagent or a substance in which a substance having no specificity for the substance to be measured is immobilized on the particles.

The dummy particles or carrier particles thereof of the present invention can be used without particular limitation as long as they are insoluble carrier particles generally used as reagents for insoluble carrier particles in immunoassay. It is preferable to use the same material as the insoluble carrier particle reagent. In order to prevent an interference reaction occurring in the immunological reagent, it is particularly preferable to use one having the same surface properties as the insoluble carrier particle reagent, for example, one having the same synthetic lot. Specific examples include raw material particles before a labeling agent is bound to an insoluble carrier particle reagent, raw material particles before immobilizing an antigen or an antibody, and the like.

The particle size of the dummy particles is not particularly limited, and the particle size (usually 0.01 to 10 μm) of the insoluble carrier particles generally used as an insoluble carrier particle reagent in immunoassay.
However, from the viewpoint of increasing the surface area per weight, a smaller particle size is advantageous. When using a method of measuring the agglutination reaction by changing the turbidity or the permeability without removing the dummy particles from the reaction system, it is preferable to use a particle having a particle size as small as possible to prevent the measurement from being affected. preferable.

When the insoluble carrier particles are immobilized with insoluble carrier particle reagents or substances having no specificity with respect to the substance to be measured, the dummy particles are immobilized on the carrier particles. As a substance,
a) those which do not interfere with the insoluble carrier particle reagent in terms of immunological specificity b) the stability of the dummy particles themselves (storage stability,
C) those which do not reduce the dispersion stability, etc. c) those which have no interaction with the insoluble carrier particle reagent due to non-immunological specificity. The “substance having no specificity” is usually selected from animal-derived proteins, amino acids, high molecular organic substances, and saccharides. The immobilization of these “substances having no specificity” is performed to bring the surface properties of the dummy particles closer to the surface properties of the insoluble carrier particle reagent, or to enhance the dispersion stability of the dummy particles.

It is preferable that the surface properties of the dummy particles are as close as possible to the insoluble carrier particle reagent. In general, the insoluble carrier particle reagent is used to immobilize an antigen or an antibody against an object to be measured, and for the purpose of enhancing dispersion stability, for example, a biological component such as bovine serum albumin (BSA), gelatin, casein, glycine, serine, Amino acids such as alanine,
A hydrophilic polymer organic substance such as sugar or PEG is immobilized and used. Therefore, these substances are preferably immobilized on the dummy particles for the same purpose, and it is particularly practically preferable to immobilize the same substance as the insoluble carrier particle reagent.

When the dummy particles are used for the purpose of suppressing the detachment of the antigen or antibody immobilized on the immunoreagent, a substance similar to the antigen or antibody immobilized on the insoluble carrier particle reagent may be used. Immobilize non-specific substances on dummy particles. With such dummy particles, it becomes possible to shift the desorption / adsorption equilibrium between the carrier particles and the dispersion medium of the antigen or antibody immobilized on the insoluble carrier particle reagent to the adsorption side. Here, as the "substance similar to the antigen or antibody immobilized on the insoluble carrier particle reagent", the antigen or antibody immobilized on the reagent and the physical properties on the particle surface, specifically, isoelectric point, hydrophilicity, Substances that are similar in hydrophobicity and the like and do not have the same immunospecificity can be used. For example, when the rabbit-derived antibody to the measurement target is immobilized on the insoluble carrier particle reagent,
It is effective to immobilize rabbit-derived IgG that has not been immunized or a rabbit-derived antibody against a substance different from the measurement target to the dummy particles. When the antigen is immobilized on the insoluble carrier particle reagent, it is effective to immobilize a substance having a similar isoelectric point, hydrophilicity, hydrophobicity, and the like to the antigen.

As a method for immobilizing these substances having no specificity on the carrier of the dummy particles of the present invention, a physical adsorption method, a chemical bonding method using a condensing agent such as carbodiimide, or the like is used. . The amount of the substance having no specificity to be immobilized is usually 50 ng to 1 mg per carrier particle.
500 μg, preferably 500 ng to 200 μg
It is. The compounding concentration of the dummy particles of the present invention is usually 0.1 to 300% by weight, preferably 1 to 100% by weight, based on the insoluble carrier particle reagent.

The immunoreagent of the present invention usually contains a dispersion medium in addition to the insoluble carrier particle reagent and the dummy particles. As the dispersion medium, water containing a buffer, a biological component, salts, a surfactant and a combination thereof is usually used. When a buffer is used, it is usually used at 1 to 500 mM with respect to water. Further, the pH is usually 5 to 10, preferably 6 to 9.
Adjust so that Examples of the buffer include trishydroxymethylaminomethane, phosphoric acid and the like. When a biological component is used, it is usually contained in an amount of 0.01% by weight to 10% by weight based on water. As biological components,
Bovine serum albumin, gelatin and the like. When salts are used, they are usually contained in an amount of 0.1 to 10% by weight based on water. Examples of the salts include sodium chloride. When using a surfactant, usually 0.0
001 to 1%. Surfactants include nonionic surfactants, anionic surfactants, cationic surfactants, amphoteric surfactants, and combinations thereof. The immunoreagent of the present invention is usually insoluble carrier particle reagent and dummy particles are dispersed in a dispersion medium by an appropriate means,
If necessary, it is produced by freeze-drying.

In the immunoassay using the immunoreagent of the present invention,
Usually a reaction buffer is used. As a reaction buffer, usually 1 to 500 mM such as trishydroxymethylaminomethane, phosphoric acid, sodium acetate, glycine and boric acid are added to water, and inorganic salts such as sodium chloride are added to the buffer adjusted to pH 1 to 10. 0.5 to 20% by weight, 0.1 to 10% of biological components such as bovine serum albumin and normal rabbit serum
What contains 0.0003 to 1% by weight of a surfactant and a surfactant is used. Depending on the reaction system, one or more reaction buffers are used. If only one buffer is used, the pH
Is often used near the neutrality of 6-9, but when using two or more types, pH 2 is added at the beginning, and another buffer is added afterwards,
Eventually it may be near neutral. Since the salt concentration, protein concentration, and the like also vary considerably depending on the reaction system, the range of use is wide.

The immunoreagent of the present invention is suitably used in the following immunoassay. (1) After dispensing a specimen, a reaction buffer, and an immunoreagent in which an antibody or antigen is immobilized on insoluble carrier particles in a reaction container, stirring, and measuring the agglutination reaction of the particles by the antigen-antibody reaction by absorbance or scattered light. (2) a sample, a reaction buffer, a first immunoreagent having an antibody or antigen immobilized on magnetic particles, and a second immunoreagent containing a labeled antibody or antigen are dispensed and stirred. A method of performing magnetic force separation and washing after the reaction, and measuring the concentration of the labeling agent bound or not bound to the magnetic particles by the antigen-antibody reaction. (3) The above method, wherein the second immunoreagent is one in which a labeling agent is directly bound to an antibody or an antigen, or insoluble particles containing a labeling agent. (4) The above immunoassay wherein a separation and washing step is inserted between the first reaction with the first immunoreagent and the second reaction with the second immunoreagent. (5) The immunoassay described above, wherein the method for detecting the labeling agent is absorbance, fluorescence, chemiluminescence, or electrochemiluminescence.

[0019]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to the following examples.

Example 1 In a fluorescent immunoreagent for measuring hepatitis B surface antigen (HBsAg) in a sample, the following were used as a first immunoreagent and a second immunoreagent. ) Storage at 4 ° C., (2) Storage at 10 ° C., (3) Storage on the device for 8 hours every day, storage at 4 ° C. for 3 weeks, and H
The fluorescence intensity of a standard solution having a BsAg concentration of 0 IU / ml and 1 IU / ml was measured. As an immunoassay device, LPIA
-A700 [manufactured by Mitsubishi Chemical Corporation] was used. The measurement results are shown in Table 1.

(First immunoreagent) Dispersion medium: 10 mM Tris, 0.01% Tween 20, pH 8.0 Insoluble carrier particle Reagent: Anti-HB to polystyrene particles containing a europium complex (particle size: 0.4 μm) [manufactured by Seradyne Co., Ltd.]
A sAg antibody immobilized by a chemical bonding method was added in an amount of 0.003% by weight based on the dispersion medium. Dummy particles: 0.4 μm polystyrene latex,
Immobilized antibody (F (ab ') 2) derived from normal rabbit and bovine serum albumin, 0.1% by weight based on the dispersion medium

(Second immunoreagent) Dispersion medium: 10 mM Tris, 0.01% Tween 20, pH 8.0 Insoluble carrier particle Reagent: Anti-HBsAg antibody was added to polystyrene particles (1 μm in diameter) containing a magnetic substance (produced by Prolab). What was immobilized by the chemical bonding method, 0.05% by weight based on the dispersion medium

Comparative Example 1 Measurement was performed in the same manner as in Example 1 except that no dummy particles were used as the first immunological reagent. The measurement results are shown in Table 1.

[0024]

[Table 1]

In the comparative example containing no dummy particles,
Three weeks later, the blank value (fluorescence intensity at HBsAg concentration 0) of the reagent set on the apparatus was higher than that of the reagent under other storage conditions, and the value of the standard solution of 1 IU / ml also increased. On the other hand, in the reagent containing the dummy particles, there is no difference under any storage conditions, and it can be seen that the storage stability is excellent.

[0026]

By coexisting the dummy particles of the present invention at the time of storage, the reagent concentration is reduced by the adsorption of the immunoreagent to the container wall, the interference in the dispersion medium is caused by the interference reaction due to the adsorption, or immobilized on the immunoreagent. It is possible to suppress the detachment or the like of the antigen or antibody, and to improve the storage stability of the immunological reagent.

Claims (5)

[Claims] 1. An insoluble carrier particle reagent in which an antigen or an antibody to an object to be measured in a sample is immobilized on the insoluble carrier particle, and an insoluble carrier particle having no specificity for the insoluble carrier particle reagent or the substance to be measured. An immunological reagent characterized by coexistence. 2. The insoluble carrier particle having no specificity is characterized in that the insoluble carrier particle reagent or the substance having no specificity for the substance to be measured is immobilized on the carrier particle. The immunological reagent according to claim 1. 3. The immunoreagent according to claim 2, wherein the substance having no specificity is selected from animal-derived proteins, amino acids, high molecular weight organic substances, and saccharides. 4. The immunoreagent according to claim 1, wherein the insoluble carrier particles of the insoluble carrier particle reagent are insoluble magnetic carrier particles or particles containing an insoluble labeling agent. 5. An immunoassay kit comprising a unit containing the immunoreagent according to any one of claims 1 to 4 and a unit containing one or several buffers.