JPH05240859A - Aggregation immunoassay method and device - Google Patents

Abstract

(57) [Abstract] [Purpose] To provide an agglutination immunoassay method and a device therefor, which have sensitivity as high as that of the endpoint method and can perform measurement in a short time. [Structure] After reacting a specimen with an antibody or a magnetic particle bound with an antigen that specifically binds to an antigen or an antibody to be measured, (a) the particles are forcibly aggregated in a container by a magnetic force, (B) agglutination immunoassay method comprising releasing the particles from forced aggregation due to magnetic force, and (c) measuring the dispersed state of the released particles; and applying a magnetic field to the mixture of the specimen and the magnetic particles, Thus, an agglutination immunoassay device having means for forcibly aggregating the particles by magnetic force, means for releasing the agglomeration, and means for measuring the dispersed state of the released particles was provided.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an immunoaggregation measuring method and an apparatus used therefor.

[0002]

2. Description of the Related Art Latex agglutination assay, which is one of the immunoassays, utilizes an antigen-antibody reaction to detect an antigen to be measured or an antibody that specifically reacts with the antibody or latex particles to which an antigen is bound and a specimen. This is a method of measuring the aggregation process caused by mixing and with increasing sensitivity as a change in light transmittance or turbidity. This agglutination reaction is rapid because it uses fine latex particles, and is widely put to practical use as a simple immunoassay method. The immunoassay based on this agglutination reaction is an enzyme immunoassay (EIA) or a radioimmunoassay (RIA) that is widely used in immunoassays.
Compared with labeled immunoassays such as those described above, it has features such as easy operation and easy automation of the measuring device.

Examples of the measuring method by the latex agglutination reaction include an end point method for measuring the net light scattering intensity and a rate method (kinetic method) for measuring the maximum change rate of the light scattering intensity. Among them, the endpoint method has a problem that the time required for the measurement is long although the sensitivity to the immune reaction is high. Further, the rate method has an advantage that the time required for measurement is shorter than the end point method, but on the contrary, it has a problem that the sensitivity cannot be increased.

In the latex agglutination reaction measuring method, polyethylene glycol (PEG) is added to the reagent for the purpose of accelerating the reaction rate and improving the sensitivity, but the addition of PEG is a non-specific reaction. It has been known that the amount of the compound used is also increased, and there are many problems such as inevitably limiting the amount used in the reaction that emphasizes specificity.

As described above, in the conventional latex agglutination method, the end point method has high sensitivity but takes a long time for measurement, and the rate method can perform measurement in a short time, but has a problem in sensitivity.

[0006]

SUMMARY OF THE INVENTION Therefore, an object of the present invention is to provide an agglutination immunoassay method and an apparatus therefor which are as sensitive as the endpoint method and can be measured in a short time. Is.

[0007]

Means for Solving the Problems As a result of earnest research, the inventors of the present invention have used magnetic particles as carrier particles in an agglutination immunoassay method, reacting a sample with particles, and then forcibly aggregating the particles by magnetic force, Then, the inventors have found that the particles can be released from the forced aggregation due to the magnetic force and the dispersed state of the released particles can be measured, whereby the immunoassay can be performed with high sensitivity in a short time, and the present invention has been completed.

That is, according to the present invention, after reacting an analyte with an antigen or an antibody which binds specifically to an antigen to be measured or a magnetic particle bound with the antigen, (a) the particle is magnetically charged in a container. To provide a method for agglutination immunoassay, which comprises: (b) releasing the particles from forced agglomeration by magnetic force, and (c) measuring the dispersed state of the released particles.

Furthermore, the present invention applies a magnetic field to a mixture of an analyte and the above magnetic particles, thereby forcibly aggregating the particles by magnetic force, means for releasing the agglomeration, and the released particles. And an agglutination immunoassay device having a means for measuring the dispersion state of.

In the agglutination immunoassay method of the present invention, first, a specimen is allowed to react with magnetic particles bound with an antigen or an antibody, and then the particles are forcibly agglutinated by magnetic force. In order to agglomerate these particles, a commercially available and easily available electromagnet, permanent magnet or the like can be used. As the permanent magnet, for example, a REC magnet (rare earth magnet; TD)
Electromagnetic holder KE-A
Molds (made by Kanetsu Kogyo Co., Ltd.) and the like can be used.

Then, in order to release the agglomeration, it can be carried out by stopping the energization in the electromagnet. With a permanent magnet, it can be easily performed by separating the reaction container and the permanent magnet. Further, in order to measure the dispersion state, changes in light transmittance (absorbance) and turbidity scattered light may be measured. For example, L using a monochromatic light emitting diode as a light source.
Devices such as a PIA system (manufactured by Mitsubishi Kasei) and an LA system (manufactured by Alphatech) using a tungsten lamp can be used.

When the antigen is measured by the immunoassay method of the present invention, for example, the following method can be used. First, an appropriately diluted sample is added to a reaction vessel, and then magnetic particles bound with an antibody that specifically reacts with the antigen are added and stirred to measure the turbidity. Then 0.5 minutes ~
After continuing stirring for 5 minutes, aggregation is forcibly performed by the aggregating means. The magnetic field required for forced aggregation is not particularly limited, and the time required for aggregation can be changed depending on the strength of this magnetic field. The time required for this aggregation can be changed depending on the magnetic particles used, for example, 0.5.
It can be performed for 5 minutes to 5 minutes.

After that, forced coagulation is released, the turbidity of the particles in the container after 0.5 to 1 minute is measured, and the change in the turbidity immediately after the start of the reaction and after the reaction is measured.

When the forced aggregation is released, when the sample does not contain the antigen or antibody to be measured, the aggregated particles are dispersed again, so that the turbidity of the liquid is rapidly reduced. On the other hand, when the sample contains a sufficient amount of the antigen or antibody to be measured, the particles that are forcibly aggregated are in a state of being crosslinked by the antigen or antibody, and thus even if forced aggregation is released. It remains aggregated and has a turbidity corresponding to the antigen or antibody in the sample. When the amount of antigen or antibody to be measured is in the middle of these, when forced aggregation is released, some of the aggregated particles are dispersed, and the amount, and thus the amount of change in turbidity, should be measured in the sample. Depends on the amount of antigen or antibody. Therefore, it is possible to qualitatively detect and quantify the antigen or antibody to be measured in the sample by measuring the change in turbidity after releasing the forced aggregation.

The measurement can be carried out in a suitable aqueous solution,
For example, a phosphate buffer may be mentioned. The pH of this buffer solution is preferably 6.0 to 8.0.

The magnetic particles used in the method of the present invention include magnetic particles or particles containing a magnetic material. As the particles of the magnetic material, particles having ferromagnetism, such as iron oxide-based cobalt ferrite and nickel ferrite, can be preferably used. Examples of the particles containing a magnetic material include gelatin particles containing a ferromagnetic material (Japanese Patent Laid-Open No. 59-196161), particles in which a magnetic material is coated with serum albumin, particles similarly coated with a synthetic polymer, and a synthetic polymer. Particles coated with a ferromagnetic material (see, for example, JP-A-3-1155862), particles coated with a magnetic material with silane (see, for example, JP-A-55-141670 and 56-122997), and a magnetic material in a synthetic polymer are used. These include particles and the like. As the magnetic substance, it is preferable to use a substance having ferromagnetism and having a low residual magnetism that is easily magnetized and demagnetized. Particles having a particle size of 0.01 to 0.6 μm can be used as the magnetic particles or particles containing a magnetic material. Among them, particles having a particle size of 0.2 to 0.4 μm can be used for efficient reaction. Is preferred.

The magnetic particles to which the antigen or the antibody is bound can be produced by a method of binding the antibody or the antigen to the object to be measured by a known chemical binding method or physical adsorption method. This chemical coupling method is a known method, and a coupling reagent such as tannic acid, glutaraldehyde, bisdiazobenzidine, tolylene diisocyanate, difluoronitrobenzene, carbodiimides, quinones, chromium chloride, and a silane coupling agent is used. It can be carried out by reacting an antigen or an antibody in a buffer using a ring agent. The physical adsorption method is carried out by reacting the above-mentioned particles with an antigen or antibody in a suitable buffer. The buffer solution used in this reaction is a phosphate buffer solution, a Tris-hydrochloric acid buffer solution, a carbonate buffer solution, or the like. The reaction proceeds easily by mixing both, and in the physical adsorption method, the target product can be obtained by coating the nonspecific adsorption site with serum protein or the like. The binding method by the chemical binding method or the physical adsorption method can be appropriately selected and used depending on the type of particles used.

The antigen or antibody to be bound to the particles used in the present invention can be determined depending on the substance to be measured. For example, cancer markers such as CEA and AFP, HIV and HTL.
Examples include viruses such as V, HBV, and HCV, antigens such as hormones, haptens, drugs, and various antibodies against these antigens. These antibodies may be monoclonal antibodies or polyclonal antibodies, and further are F (ab ′) ₂ , Fab ′, Fa which are degradation products of the antibodies.
It may be b or the like. Examples of the sample include body fluids containing the measurement target, for example, whole blood, serum,
Examples include urine.

The container for receiving the sample and the reagent for carrying out the method of the present invention is made of a material such as plastic, such as polystyrene, ABS resin, or glass, which is light transmissive. A sample containing a measurement object by adding a suitable stirring device to the container,
It is preferable for promoting the reaction with the magnetic particles to which the antigen or the antibody is bound and efficiently carrying out the reaction. When the magnetic particles are particles that are easily magnetized and have residual magnetism, a demagnetization device can be added. By adding this device, the aggregation of particles can be measured only by the aggregation due to the binding of the antigen-antibody reaction.

The agglutination immunoassay apparatus of the present invention comprises a container for receiving a sample containing a measurement object and a measurement reagent as described above, means for forcibly aggregating magnetic particles of the reagent by magnetic force,
A means for releasing the forced aggregation and a light detecting means for measuring the transmittance or turbidity of the container using a light source can be used. A conceptual diagram of this device is shown in FIG. FIG. 2 shows a conceptual diagram of an apparatus for measuring scattered light as a means for measuring the dispersion state. 1 and 2, 1 is a light source, 2 is a photodetector, 3 is a container, and 4 is a magnet.

When these devices are used for fully automatic agglutination immunization, as shown in FIG. 3, for example, a reaction container supply device 5, a sample dispensing device 6, a reagent dispensing device 7, a sample and reagent stirring device 8, Forced agglomeration device 9, magnetic release device 10,
A device 11 for measuring the light transmittance, turbidity or scattered light in the container, a recovery device 12 for recovering the container after use, and the like can be combined.

[0022]

【Example】

[Preparation of AFP-sensitized iron-coated polystyrene particles] An anti-human AFP (Rabbit, DACO) was used for Barna in iron-coated polystyrene latex having a particle size of about 0.3 micron.
rd et al. (Clin. Chem., 27 (6) 83).
2 (1981), anti-human AFP-sensitized iron-coated polystyrene particles were prepared.

[Aggregation measurement] Sample serum was diluted with a serum dilution solution to 1
Dilute to 0-fold, transfer to a turbidity measuring cell, add 50 μl of a dispersion containing 0.1% of the above-mentioned anti-human AFP-sensitized iron-coated polystyrene latex particles to the buffer 40
Add 0 μl and stir, and after 2 minutes, leave it on a forced aggregating table (magnetic field: 5000 gauss) using a coil for 1 minute, and then place it on an open table of magnetic force, and from the dispersion state of the agglomerated particles, the presence or absence of immune reaction Was judged from the turbidity. The particles in which the particles were completely dispersed were defined as negative, and the particles in which the particles were not dispersed were defined as positive.

The above-mentioned immunoassay method according to the present invention was compared with the result by EIA using the conventional Imzain AFPmono (a reagent for measuring α-fetoprotein, manufactured by Fujirebio Co., Ltd.), and the correlation is shown in FIG. .. It can be seen from FIG. 4 that the method of the present invention enables highly sensitive measurement as high as EIA.

[0025]

According to the method of the present invention, since the particles are forcibly aggregated by using the magnetic force, the final state in the conventional end point method can be realized in a short time. Therefore, highly sensitive measurement equivalent to the end point method can be performed in a short time.

[Brief description of drawings]

FIG. 1 is a measurement conceptual diagram when a detection means for measuring the permeability and turbidity of a container is used as a means for measuring the dispersed state of an agglutination immunoassay device.

FIG. 2 is a measurement conceptual diagram when measuring scattered light as a detection means.

FIG. 3 is a block diagram showing a configuration example of an automatic agglutination immunoassay device using the device of the present invention.

FIG. 4 is a correlation diagram of titers between the agglutination immunoassay method of the present invention and the conventional latex agglutination method in AFP measurement.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 light source 2 photodetector 3 container 4 magnet 5 reaction container supply device 6 sample dispensing device 7 reagent dispensing device 8 sample and reagent stirring device 9 forced aggregating device 10 magnetic release device 11 turbidity measuring device 12 reaction container recovery device

Claims (2)

[Claims] 1. After reacting a sample with an antibody or an antigen-binding magnetic particle that specifically binds to an antigen or an antibody to be measured, (a) the particles are forcibly aggregated in a container by a magnetic force. (B) An agglutination immunoassay method which comprises releasing the particles from forced aggregation due to magnetic force and (c) measuring the dispersed state of the released particles. 2. A magnetic field is applied to a mixture of the specimen and the magnetic particles according to claim 1, whereby the particles are forcibly agglomerated by magnetic force, means for releasing the agglomeration, and the released particles An agglutination immunoassay device having a means for measuring a dispersed state.