JPH10246725A - Method for detecting antibody or antigen - Google Patents

Abstract

PROBLEM TO BE SOLVED: To conduct easy and highly accurate measurement by putting a biological liquid to be tested for a fixed time in each hole of an agglutination test plate having a binder for the antigen or antibody adhered to a surface, cleaning the hole, and adding a granular immobilization antigen or antibody. SOLUTION: A sample is put in each hole of an agglutination test plate having a binder for an antigen or antibody adhered to a surface, so that the binder is bonded with the antibody or antigen. After the sample is left stationary for about 15 minutes to one hour or shaken, the sample is disposed of, and the hole is rinsed with water, physiological saline solution or the like, thereby removing the other substance than the antibody or antigen bonded with the binder. Consequently an inhibiter is also removed. When a granular immobilization antigen or antibody is added to the agglutination test plate, if the antibody or antigen is present in the sample, an antigen-antibody reaction is carried out accurately and a mat agglutination image is formed. Accordingly, the antibody or antigen in the sample can be detected highly accurately.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for detecting an antigen or antibody in a biological fluid such as saliva, blood, lymph, or manure by an agglutination reaction based on an antigen-antibody reaction.

[0002]

2. Description of the Related Art As a method of measuring the amount of a specific antibody contained in a specimen (serum, body fluid, etc.), an agglutination reaction using an agglutinating complex particle of an antigen-fixed gelatin, kaolin, synthetic polymer or the like is used. Is conventionally used. A plate having a plurality of holes is used for determination of such an agglutination reaction, and a dilution line of the specimen is prepared. In the judgment, when an agglutination reaction occurs (positive), a mat-like agglutination image in which the agglomerate spreads uniformly on the wall of the hole is formed. It is done by sliding down and gathering in the shape of a circular button at the bottom of the hole. However, in the conventional hemagglutination inhibition test, the specimen, for example,
A complicated operation for removing inhibitors (antigen-antibody reaction inhibitor) such as lipids and glycoproteins in serum was required.
In addition, this inhibitor removal operation generally takes a long time, and sometimes takes a whole day and night.

[0003]

SUMMARY OF THE INVENTION An object of the present invention is to provide a method for detecting an antibody or an antigen which can easily perform a highly accurate determination without requiring an inhibitor removing operation.

[0004]

According to the present invention, a specimen is placed in each hole of an agglutination test plate having a binding agent for an antigen or an antibody attached to the surface thereof, and the binding agent and the antibody or antigen contained in the specimen are separated from each other. After the binding, the sample remaining in each hole is discarded, and the inhibitor or the like attached to the vessel wall of the plate is washed away to achieve the above object.

That is, in the method for detecting an antibody or an antigen according to the present invention, a biological fluid to be tested is placed in each hole of an agglutination test plate having an antigen or antibody binding agent attached to its surface, and after a certain time, The liquid is discarded, the plate is rinsed, and then the particulate immobilized antigen or antibody is added.

The plate for the agglutination test used in the present invention is not limited as long as it has a plurality of holes and a dilution line can be prepared. In general, the bottom of the hole has a V-shaped, U-shaped or cross-sectional shape. A variant is a plate or strip.
As the material, polystyrene, polypropylene, polyvinyl chloride, or the like is used.

[0007] In the present invention, a plate in which an antigen or antibody binding agent is attached to the bottom of each hole of the above-mentioned plate, that is, the inner surface of a V-shaped or U-shaped or a deformed portion thereof is used. . For example, a plate in which protein A and / or protein G is attached to the inner surface of each hole, or a plate in which avidin or streptavidin or a derivative thereof is attached and then reacted with a biotin-labeled antigen or antibody, etc. Can be Examples of the derivative include those obtained by removing a sugar chain portion from avidin, for example, NeutraAvidin, ultraavidin and the like.

[0008] In practicing the present invention, first, a biological liquid to be tested, ie, a specimen, is placed in each hole of an agglutination test plate having an antigen or antibody binding agent attached to its surface, and the above-described binding is performed. The agent is allowed to bind to the antibody or antigen contained in the specimen. In order to perform this binding sufficiently, it is allowed to stand or shake for a certain period of time. The standing or shaking is generally performed for 15 minutes to 1 hour, preferably 20 minutes to 1 hour. Shaking is preferred for efficient binding of the antigen or antibody to the binding agent. Then, as in the conventional method, with the specimen in each hole, an antigen or antibody to the antibody or antigen to be detected is added to the granular immobilized antigen or antibody in which the antigen or antibody is immobilized on the granular carrier, and the antigen-antibody reaction is performed. In this case, an inhibitor such as a lipid or glycoprotein contained in the sample covers the particulate immobilized antigen or antibody, thereby inhibiting the antigen-antibody reaction.

However, according to the method of the present invention, a sample is put into each hole of the plate, and after a certain period of time, the sample is discarded, and further rinsed to remove all substances other than the antibody or the antigen bound to the antigen or the antibody binding agent. can do. Water, physiological salt solution, physiological saline, or the like can be used for rinsing the hole after discarding the specimen. Since the inhibitor is also removed by this rinsing, there is no need to perform any special inhibitor removal operation, and when the particulate immobilized antigen or antibody is added to this plate, if the antibody or antigen is present in the sample, the antigen- An antibody reaction is performed, and the antibody or antigen in the sample can be detected with high accuracy. When an antibody or an antigen is present in the specimen, a mat-like aggregation image is formed.

[0010] The particulate immobilized antigen or antibody used in the method for detecting an antigen or antibody according to the present invention is not particularly limited, but may be an agglutinated antigen or antibody immobilized on a ceramic-polymer complex, latex, gelatin, kaolin or the like. It is preferably used as a composite particle. As an aggregating composite particle in which an antigen or an antibody is immobilized on a ceramic-polymer composite, for example, the surface of the polymer particle is coated with a calcium phosphate compound, and the polymer particle is dyed or the whole is dyed. An antigen or an antibody is adsorbed and immobilized on the granular polymer complex, and the unadsorbed site is treated with a blocking agent (Japanese Patent Application Laid-Open No.
No. 174762). As the granular polymer composite, one produced by coating a polymer particle surface with a calcium phosphate compound by physically colliding the polymer particles with calcium phosphate compound particles is preferable.

As the above calcium phosphate compound, various calcium phosphate compounds can be used as long as the Ca / P ratio is 1.0 to 2.0.
a ₁₀ (PO ₄ ) ₆ (OH) ₂ , Ca ₁₀ (PO ₄ ) ₆ F ₂ , Ca ₁₀
(PO ₄ ) ₆ Cl ₂ , Ca ₃ (PO ₄ ) ₂ , Ca ₂ P ₂ O ₇ , C
One or more selected from a ₄ O (PO ₄ ) ₂ and CaHPO ₄ can be used. Of these, hydroxyapatite and tricalcium phosphate are preferred, and those containing hydroxyapatite as the main component are most preferred. When using fluorapatite, the content of fluorine in the total calcium phosphate compound is 5% by weight.
It is preferred that: If the fluorine content exceeds 5% by weight, fluorine is eluted, which is not preferable. These calcium phosphate compounds can be synthesized by a known wet synthesis method, dry synthesis method, or the like. The particles of the calcium phosphate-based compound can be prepared by, for example, granulating the slurry of the calcium phosphate-based compound by spray drying and calcining the granulated material, but not limited to this method, and prepared by another granulation method. Is also possible. It is more preferable to select the particle size of the particles within a predetermined range according to the purpose by using a means such as sieving.

[0012]

Next, the present invention will be described in more detail with reference to Examples, but the present invention is not limited thereto.

Reference Example 1 (Production of Ceramic-Polymer Composite Particles Fixed by Japanese Encephalitis Beijing Strain) MITSUI ML Col, a trade name of an anthraquinone-based disperse dye
Ors ML red VF-2 (manufactured by Mitsui Toatsu Dyeing Co., Ltd.) dyed 50 g of nylon beads having an average particle size of 5 μm and 7.5 g of hydroxyapatite particles at a rate of 8,000 rpm using a Nara hybridization system NHS-1 at 8,000 rpm. 50 ℃
For 5 minutes to cover the nylon bead surface with hydroxyapatite. The obtained composite beads had an average thickness of the hydroxyapatite coating layer of 0.44 μm,
The average particle size was 5.8 μm. An equivalent amount of the Japanese encephalitis Beijing strain virus (32 μg / ml) and a 10% solution (w / v) of the above complex beads were mixed, and mixed with a rotator for 1 hour.
The virus antigen was adsorbed on the beads. The beads were spun down in a centrifuge at 1000 rpm for 5 minutes to form a pellet, and the supernatant was discarded. The pellet was loosened, and the antigen was fixed with 0.05% glutaraldehyde [diluted solution of pH 7.2 phosphate buffer (hereinafter sometimes abbreviated as PBS)]. After stirring with a rotator at room temperature, the beads were spun down at 1000 rpm for 5 minutes in a centrifuge to form a pellet, and the supernatant was discarded. While masking the antigen non-adsorbed site of the beads with a 1% Block Ace solution (w / v) solution [Block Ace is a brand name of a masking agent manufactured by Dainippon Pharma Co., Ltd.], the masking is performed for 1 hour with a rotator. went. After stirring with a rotator at room temperature, the beads were spun down in a centrifuge at 1000 rpm for 5 minutes,
A pellet was made and the supernatant was discarded. Fixing was performed with 0.05% glutaraldehyde (a diluted solution of PBS (pH 7.2)) to prevent the detachment of Block Ace. After stirring with a rotator at room temperature, the beads were centrifuged at 1000
The mixture was spun down at rpm for 5 minutes to form a pellet, and the supernatant was discarded. To inactivate the active residues of glutaraldehyde, masking was performed with a 1% Block Ace solution (w / v), and then stored in a 0.4% Block Ace solution.

Reference Example 2 (Production of a plate for agglutination test) Protein A having a concentration of 1.0 × 10 ⁻³ mg / ml was added to each hole of a V-type polystyrene plate having 96 holes (capacity: 0.3 ml). Put 0.05 ml of PBS solution containing
After a minute, the solution was discarded, 0.05 ml of PBS solution was put into each well of the plate, the solution was discarded to remove the released protein A, and the plate was dried to obtain a protein A-attached plate.

Reference Example 3
Production of polymer composite particles) Except for using paragonimites antigen at an antigen protein amount of 25 μg / ml, ceramic-polymer composite particles immobilized with paragonimites antigen were produced in the same manner as in Reference Example 1.

Example 1 100 μl of Japanese encephalitis patient serum (sample) diluted 10-fold with PBS was placed in each well of the first row of the protein A-attached plate prepared in Reference Example 2, and 10% was placed in each well of the other rows. Add 50 μl of PBS solution containing Block Ace, and add 5 μl from the first row.
After performing a 0-μl-fold dilution and shaking for 1 hour, the sample was removed from each well, the plate was washed with water, and then drained.
Thereto was added 100 μl of a 0.1% solution of the ceramic-polymer composite particles fixed in Japan Encephalitis Beijing strain prepared in Reference Example 1. Two hours later, when the judgment was made, the serum dilution rate was 10
From 2 times a clear positive image was observed.

Example 2 (1) Preparation of IgG-measuring plate specific for Paragonimiasis neutravidin (manufactured by PIERCE) at 25 μg /
50 μl of a solution diluted with a carbonate buffer (pH 9.6) was added to each well of a microtiter plate having 96 (12 × 8) V-shaped holes from Greiner, and the mixture was allowed to stand at room temperature for 1 hour. Later, the solution was discarded. Each hole of the avidin-fixed plate thus obtained was washed with ion-exchanged water, and then diluted 4 times with Block Ace (Dainippon Pharmaceutical Co., Ltd.) (P
1% by weight / solution in BS] was added to each well, and allowed to stand at room temperature for 1 hour. After the Block Ace diluent was discarded,
It was air-dried to obtain an avidin-fixed plate. In each well of the avidin-fixed plate, 0.5 mg / ml of an anti-human IgG biotin-labeled antibody diluted 400 times was added to 50 μl.
1 each. The mixture was allowed to stand at room temperature for 1 hour to bind avidin and biotin, thereby preparing an IgG measurement plate specific for fluke parasitosis.

(2) Measurement of human serum Each of the positive serum and the negative serum of Paragonimiasis lung was diluted 10-fold, and the plate prepared in the above (1) was diluted twice to the first to twelfth wells. , 50 μl of the test serum dilution was placed. After allowing to stand at room temperature for 1 hour in this state, the plate was washed with ion-exchanged water.
A 100% solution was added to each well, and allowed to stand. After one hour, the aggregation image was determined. As a result, in the case of the paragonimiasis positive serum, a clear positive image was shown up to 10 to 20480 times.
On the other hand, with the negative serum, clear negative images were obtained up to 10 to 20480 times.

Comparative Example 1 100 μl of the same specimen as in Example 1 was placed in each hole of the first row of the protein A-attached plate prepared in Reference Example 2, and 50% of a PBS solution containing 10% Block Ace was placed in each hole of the other rows.
After adding 1 μl and performing a 50-fold dilution from the first row, the mixture was shaken for 1 hour. Then, in each hole, 0.1% of the ceramic-polymer composite particles fixed in Beijing, Japan Encephalitis produced in Reference Example 1.
50 μl of a 1% solution was added. Two hours later, when judgment was performed, a false negative reaction image was observed from a serum dilution ratio of 10 to 80 times, and the dilution ratio turned to a positive reaction image at a subsequent dilution ratio.

[0020]

According to the method for detecting an antibody or antigen according to the present invention, an inhibitor removing operation is not required in a detection method using an agglutination method, and a pseudo-agglutination image appearing at low dilution of a sample such as serum can be prevented from being seen. Can be easily determined.

Claims (3)

[Claims] 1. A test biological liquid is placed in each hole of an agglutination test plate having an antigen or antibody binding agent attached to its surface, and after a certain time, the liquid is discarded, and the plate is rinsed. A method for detecting an antibody or antigen, comprising adding an immobilized antigen or antibody. 2. The method for detecting an antibody or an antigen according to claim 1, wherein the plate for agglutination test having a surface to which an antigen or an antibody binding agent is adhered is a plate to which protein A and / or protein G are adhered. 3. The agglutination test plate having an antigen or antibody binding agent attached to the surface thereof is a plate on which avidin or streptavidin or a derivative thereof is attached and then a biotin-labeled antigen or antibody is reacted. The method for detecting an antibody or antigen according to claim 1.