JPH07151755A - Measuring method of antigen/antibody reaction - Google Patents

Abstract

PURPOSE:To quickly and easily separate unreacted matters form a reaction product and clean the unreacted matters by using magnetic particles and fluorescent particles. CONSTITUTION:A first reagent containing insoluble magnetic particles carrying the antibody or antigen meeting an antigen or antibody to be measured and the antigen or antibody to be measured are caused to react to each other in a liquid medium contained in a reaction vessel. After reaction, the magnetic particles are attracted to the wall of the vessel by utilizing the action of a magnetic field and the liquid medium is removed from the vessel. Then a second reagent containing insoluble fluorescent pigmentlabeled particles carrying an antigen or antibody having the same specificity as the antigen or antibody to be measured has and the magnetic particles which are attracted to the wall of the vessel are caused to react to each other in the liquid medium. Then the magnetic particles are again attracted to the wall of the vessel by utilizing the action of the magnetic field and the liquid medium and unreacted pigment- labeled particles are removed. Thereafter, the fluorescent intensity of the pigment-labeled particles which have reacted to the magnetic particles is measured.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for measuring an antigen-antibody reaction. Specifically, it relates to a method for measuring an antigen-antibody reaction using magnetic particles and fluorescently labeled particles.

[0002]

2. Description of the Related Art Conventionally, an immunoassay method utilizing an antigen-antibody reaction has been known as an early detection method for various diseases and a detection method for a trace amount of a substance. There are various methods for highly sensitive immunoassays, such as radioimmunoassay (RIA) and enzyme immunoassay, in which a radioisotope (RI), an enzyme, a fluorescent substance, a luminescent substance or the like is used as a labeling substance for an antibody or an antigen. (EIA), fluorescent immunoassay (FIA),
It is classified into luminescence immunoassay (LIA) and the like. In addition, a method of reacting an antibody or antigen supported on insoluble carrier particles such as latex and the corresponding antigen or antibody, and measuring the rate of the antigen-antibody reaction from the change in transmitted light of the reaction mixture accompanying the reaction ( LPIA) is known.

[0003]

However, such a conventional technique has various problems. For example, RIA has constraints on RI handling and disposal. LPIA is simple,
Although this method is excellent in operability, improvement in detection sensitivity is desired. EIA, FIA, LIA, etc. are advantageous in handling safety, but their detection sensitivity is slightly inferior to RIA. Also, since EIA involves an enzymatic reaction, handling is FI.
Not as simple as A. LIA also has a drawback in that it is more complicated than FIA because it is necessary to add a reagent such as sodium hydroxide to emit light after separation.

By the way, methods such as EIA, FIA and LIA usually require an operation (B / F separation) of separating and washing a reaction binder and an unreacted material. These B / F separations are the main cause of making it cumbersome and time consuming. For B / F separation, usually, after discarding a reaction solution containing an unreacted substance from a reaction tube such as a tube or a microtiter well, a washing operation of supplying a washing solution, incubating, and discarding the washing solution is repeated several times. Has been done.

[0005] In order to perform B / F separation relatively quickly and easily, there is a filter that uses a filter such as a membrane or glass fiber. Since the flow direction of the liquid is one direction (there is no need to suck it out when discarding the liquid), the time required for the cleaning process can be short, and the device can be easily automated, for example. However, these methods also have various problems. In the case where the primary antibody or the antigen is directly bound to the filter, the filter and the item to be measured need to correspond to each other, and there is a problem in using an automated device in consideration of storage and supply of the filter.

[0006] Further, in the type in which a fine particle such as latex is temporarily bound with an antibody or an antigen and, after the reaction, is collected in a filter, one that collects particles larger than the pore size on the filter and one that collects particles on the filter Some use adsorption. In the former, because the filter is clogged, the labeled substances such as the secondary antibody cannot be completely washed out,
This can cause an increase in background. Also,
In the latter, since the cause of adsorption to the filter has not been clarified, there remain problems such as whether or not particles are completely collected, and the background is increased by using a material that is easily adsorbed. In addition, since it uses adsorption inside the filter, it can be applied to EIA, but FI
In the case of A, the excitation light may not reach and cannot be applied.

On the other hand, various methods using magnetic particles have been reported as an improved method for B / F separation. This method is characterized in that the operation of collecting magnetic particles using magnetic force can be performed quickly and easily. However, those currently in practical use use secondary antibodies labeled with an enzyme, a fluorescent dye, a luminescent dye, RI and the like, and thus may not have sufficient measurement sensitivity. Particularly, those labeled with a fluorescent dye have a problem in sensitivity.

[0008]

Means for Solving the Problems As a result of intensive studies to solve the above problems, the present inventors have found that B / F separation can be carried out quickly and easily by using magnetic particles, and that fluorescent particles are used. As a result, an immunoassay method has been found in which the measurement sensitivity is higher than that of ordinary FIA. That is, the gist of the present invention lies in a method for measuring an antigen-antibody reaction, which comprises the following steps. (A) a first reagent composed of insoluble magnetic particles carrying an antibody or an antigen against the antigen or antibody to be measured,
The antigen or antibody to be measured is reacted in the liquid medium of the reaction container. (B) The insoluble magnetic particles after the reaction in step (a) are attached to the reaction vessel wall by the action of a magnetic field, and the liquid medium is removed. (C) A second reagent consisting of insoluble fluorescent dye-labeled particles carrying an antigen or antibody having the same specificity as the antigen or antibody to be measured, and the insoluble magnetic particles attached to the reaction vessel wall of step (b) And react in a liquid medium. (D) The insoluble magnetic particles of step (c) are attached to the reaction vessel wall by the action of a magnetic field to remove the liquid medium and the unreacted insoluble fluorescent dye-labeled particles, and then the insoluble fluorescent particles reacted with the insoluble magnetic particles. The fluorescence intensity of the dye-labeled particles is measured.

The present invention will be described in detail below. The insoluble magnetic particles used in the present invention are, for example, ferric tetroxide (F
e ₃ O _4), iron sesquioxide _{(γ-Fe 2 O 3)} , various ferrites, iron, manganese, nickel, cobalt, metals such as chromium, cobalt, nickel, particles or these magnetic particles made of an alloy such as manganese Hydrophobic polymer such as polystyrene, polyacrylonitrile, polymethacrylonitrile, polymethylmethacrylate, polycapramide, polyethylene terephthalate, etc. containing therein, polyacrylamide, polymethacrylamide, polyvinylpyrrolidone, polyvinyl alcohol, poly (2-oxyethyl) Acrylate), poly (2-oxyethyl methacrylate), poly (2,3-dioxypropyl acrylate), poly (2,3-dioxypropyl methacrylate), cross-linked hydrophilic polymer such as polyethylene glycol methacrylate, The other is of each of the monomer 2-
Latex such as about 4 kinds of copolymers, gelatin, liposome, or particles obtained by immobilizing the above magnetic fine particles on the surface of latex, gelatin, liposome, or the like is used.

The particle size of the insoluble magnetic particles is 0.05 μm-5.
The insoluble magnetic particles having a particle size in the range of 0.1 μm to 1 μm are preferable. As a method for supporting the antibody or antigen on the insoluble magnetic particles, the method is carried out by physically adsorbing or chemically supporting the antigen or antibody to be measured or the antigen or antibody in the analyte. Be seen.

Examples of the physically adsorbing method include a method of directly immobilizing an antibody or an antigen on insoluble magnetic particles, and a method of chemically adsorbing to another protein such as albumin and then adsorbing and immobilizing it. To be As a method of chemically supporting, an amino group, a carboxyl group, a mercapto group, a hydroxyl group, an aldehyde group, an epoxy group or the like existing on the surface of magnetic particles is chemically modified to bind to an antibody or an antigen molecule. Using a functional group capable of forming, a method of directly immobilizing on a particle, a method of immobilizing by introducing a spacer molecule by a chemical bond between the particle and an antibody or an antigen molecule, an antibody to another protein such as albumin or One method is to chemically bond the antigen and then chemically bond the protein to the particles.

In addition, after physically or chemically binding a substance that specifically binds to the antibody or antigen to be immobilized (for example, antibody, protein A, etc.) to the particle surface,
There is also a method of immobilizing the antibody or antigen of interest on the particle surface. IgG is usually used as an antibody to be carried on the insoluble magnetic particles, but it is possible to use F by using digestive enzymes such as pepsin and papain or reducing agents such as dithiothreitol and mercaptoethanol.
Low molecular weight compounds such as (ab ′) ₂ , Fab ′, and Fab may be used. Further, not only IgG but also IgM or a fragment obtained by lowering the molecular weight of IgM by the same treatment as IgG may be used. Further, both a monoclonal antibody and a polyclonal antibody can be applied. When using a monoclonal antibody, one or more types of monoclonal antibodies can be used for a protein having a repetitive structure such as hepatitis B virus surface antigen or an antigen having a plurality of epitopes in the molecule such as CA19-9 antigen. Can be used. Also, two or more kinds having different recognition epitopes can be used in combination.

On the other hand, examples of the antigen include proteins, polypeptides, steroids, polysaccharides, lipids, pollen,
A variety of recombinant proteins and drugs produced by genetic engineering can be used. That is, the "antigen" referred to in the present invention
The term refers to a single substance or a plurality of substances selected for special purposes such as diagnosis among all substances capable of inducing antibody production in humans or animals, or a mixture containing them. .

Generally, the amount of antibody or antigen to be carried varies depending on the surface area of the insoluble carrier particles used, the amount of functional groups, etc., but is usually 1 mg to 500 m per 1 g of carrier particles.
g, preferably 10 mg to 100 mg. In the insoluble fluorescent dye-labeled particles used in the present invention, any fluorescent dye can be used as the labeling dye. For example, rare earth chelates such as europium (Eu), terbium (Tb) and samarium (Sm), phycobiliproteins such as phycocyanin and phycoerythrin, fluorescein, tetramethylrhodamine, Texas red, 4-methylumbelliferin. , 7-amino-4-
Methylcoumarin or the like is used.

As the insoluble carrier particles for labeling the dye, those which are substantially insoluble in the liquid medium used for the reaction and have an average particle diameter of 0.05 to 5 μm, preferably 0.1 to 1 μm are used. The material of the carrier particles are the same as those described for the insoluble magnetic particles, polystyrene, polyacrylonitrile, polymethacrylonitrile,
Hydrophobic polymers such as polymethylmethacrylate, polycapramide, polyethylene terephthalate, polyacrylamide, polymethacrylamide, polyvinylpyrrolidone,
Polyvinyl alcohol, poly (2-oxyethyl acrylate), poly (2-oxyethyl methacrylate),
Crosslinked hydrophilic polymer such as poly (2,3-dioxypropyl acrylate), poly (2,3-dioxypropyl methacrylate), polyethylene glycol methacrylate, or a copolymer of about 2-4 kinds of each monomer. In addition to latex, seratin, liposome, etc., biological components such as red blood cells, metal colloid particles such as gold colloid, and the like are used.

The method for supporting the fluorescent dye on the insoluble carrier particles is to chemically bond the fluorescent dye to the functional group on the surface of the particle, or to add the dye when the particles are polymerized and synthesized. A method of encapsulating inside the particle, a method of physically adsorbing and encapsulating inside or on the surface of the particle, a dye is physically or chemically bound to a protein or peptide in advance, and then the protein or peptide is fixed to the particle. There is a method to make it. For example, JP-A-54-101439 discloses that a rare earth chelate is TOP.
A method of entrapment inside an organic polymer latex using a cooperative extraction method with O (tri-n-octylphosphine oxide) is described. Labeled particles produced according to this have good labeling strength and stability.

An antigen or antibody having the same specificity as the antigen or antibody to be measured is physically or chemically immobilized on the fluorescent particles as described above. In the present invention, "having specificity" means that when the substance to be measured is an antigen, it is recognized as non-self by the living body as biologically or physiologically identical to the aforementioned antigen,
Refers to all substances that induce the production of antibodies. When the substance to be measured is an antibody, it reacts with the immunoglobulin class of such an antibody, that is, IgG, IgA, Ig
M, IgD, IgE or antibodies against their antibody light chain portion, protein A or C which is one of the complement components
It refers to a substance having the ability to selectively recognize and bind to the characteristics of certain antibody molecules, such as lq.

The order in which the insoluble carrier particles carry the labeling dye, the antibody having the above specificity, the antigen (hereinafter abbreviated as "special antibody" and "special antigen"), etc., is not particularly limited. After carrying the labeling dye, it is preferable to carry the special-antibody, the special-antigen and the like. Generally, the amount of the specific antibody or specific antigen to be carried varies depending on the surface area of the insoluble carrier particles to be used, the amount of functional groups, etc., as in the case of the above insoluble magnetic particles, but is usually 1 mg to 500 m per 1 g of the carrier particles.
g, preferably 10 mg to 100 mg. When the substance to be measured is an antigen, the following may be mentioned as an example of a combination of substances to be carried by the insoluble magnetic particles and the insoluble fluorescent dye-labeled particles.

[0019]

[Table 1]

On the other hand, when the substance to be measured is an antibody, the following combinations are given as examples.

[0021]

[Table 2]

Next, a specific measuring method of the present invention will be described. First, a sample solution considered to contain the antigen or antibody to be measured and an antibody against the antigen or antibody or insoluble magnetic particles carrying the antigen (first reagent) are mixed and reacted in a liquid medium of a reaction container. (First reaction). The mixing at the start of the reaction needs to be sufficiently performed, but after the uniform mixing, the mixing may be stopped and allowed to react. The reaction is carried out at pH 5 to 10, preferably pH 7 to 9 as in the general immunochemical reaction. Regarding the temperature, it can be carried out in the range of 2 to 50 ° C., preferably room temperature to 37 to
React at 40 ° C. The reaction time is arbitrary from immediately after the reaction to one day and night, but is usually 3 to 3 in consideration of sensitivity and operability.
It is set within the range of 60 minutes. These reaction conditions are the same for the subsequent steps.

A buffer is usually used to maintain the desired pH. As the buffer solution, for example, phosphoric acid, tris (hydroxymethyl) aminomethane, etc. are used,
Most neutral to weakly alkaline buffers can be used. Often, to avoid non-specific reactions,
Salts such as sodium chloride and proteins such as bovine serum albumin are added. At this time, the insoluble magnetic particles are contained in the reaction solution in an amount of 0.0001 to 1% by weight, preferably 0.001% by weight.
It is used so as to be 1 to 0.1% by weight. When the insoluble magnetic particles and the sample are mixed, the antigen or antibody contained in the sample binds to the antibody or antigen on the particle surface.

Then, by the action of the magnetic field, the insoluble magnetic particles are separated from the reaction solution and attached to the wall of the reaction vessel. After removing the remaining reaction solution, if necessary, a washing step (adding an appropriate washing solution, stirring, immediately applying a magnetic field for separation,
Repeat (except solution) several times. Then, the separated insoluble magnetic particles and insoluble fluorescent dye-labeled particles (second reagent) carrying an antigen or antibody (special-antigen or special-antibody) having the same specificity as the antigen or antibody to be measured
And are mixed and reacted in a liquid medium in the same manner as the first reaction (second reaction). The insoluble fluorescent dye-labeled particles are also used by suspending them in a buffer solution like the above-mentioned insoluble magnetic particles.
At this time, the insoluble fluorescent dye-labeled particles are contained in the reaction solution at 0.00
01-0.1% by weight, preferably 0.0001-0.0
Used at 1% by weight.

When the insoluble magnetic particles and the insoluble fluorescent dye-labeled particles are mixed, the antibody or antigen on the magnetic particles reacts with the special-antigen or special-antibody on the fluorescent dye-labeled particles to cause particle binding. At this time, the antigen or the antibody in the sample bound to the antibody or the antigen on the insoluble magnetic particles in the first reaction is the specific antigen or the specific antigen on the surface of the fluorescent dye-labeled particle.
Prevents the antibody from binding to the antibody or antigen on the magnetic particles.

Next, the insoluble magnetic particles are separated from the reaction solution by the action of the magnetic field and attached to the wall of the reaction vessel. After removing the remaining reaction solution, the washing step is repeated several times if necessary. After that, the final dispersion liquid is added to the separated insoluble magnetic particles, and as a suspension, the amount of the insoluble fluorescent dye-labeled particles contained in the solution is irradiated with excitation light specific to the fluorescent dye, and the emitted fluorescence intensity is adjusted. Measure by measuring. For example, when Eu chelate is used as the labeling dye, the excitation light is 30
The ultraviolet light is 0 to 380 nm or 240 to 270 mm, and the fluorescence is 600 to 630 nm (having a maximum value at 615 nm).

By the separation after the second reaction, the fluorescent dye-labeled particles bound to the magnetic particles by the special-antigen or special-antibody on the insoluble fluorescent dye-labeled particles reacted with the antibody or antigen on the insoluble magnetic particles are also collected. Since they are separated, the degree of reaction between the antibody or antigen carried on the insoluble magnetic particles and the antigen or antibody in the sample can be easily known by measuring the amount of the fluorescent dye-labeled particles. That is,
The stronger the reaction of the antigen or antibody in the sample solution that specifically reacts with the antibody or antigen carried on the magnetic particles, the smaller the fluorescence intensity.

When quantifying an antigen or an antibody, an antigen or antibody concentration known product or an antigen or antibody reference product is measured in advance as a sample, and the obtained quantitative value is compared with the antigen or antibody concentration of the sample. If shown, a calibration curve of the antigen or antibody can be obtained, and thus the concentration of the antigen or antibody can be determined from the reaction quantitative value of the sample of unknown concentration.

Regarding the method of applying a magnetic field, the insoluble magnetic particles after the first reaction take about 1 to 3 minutes, and similarly, the insoluble magnetic particles bound to the insoluble fluorescent dye labeled particles after the second reaction also take about 1 minute. The strength of the magnetic field and the shape of the reaction system are preferably such that they can be separated in about 3 minutes. If the time required for separation is too short, sensitivity and reproducibility are generally deteriorated, and if too long, operability is deteriorated. For this reason, it is easier to handle the reaction system with a smaller size. In 96-well microplates, the size of individual wells is small, and if a small magnet is placed in the gap between the wells, the antigen or antibody can be easily quantified using a microplate reader as in EIA using microplates. A material for a reaction vessel suitable for carrying out the invention. Further, plastics such as polystyrene and acrylic, tubes made of glass, cuvettes and the like are also materials for the reaction vessel suitable for the present invention.
A permanent magnet, an electromagnet, or the like is used as the magnet.

[0030]

The present invention will be described in more detail with reference to examples, but the present invention is not limited thereto.
The compositions of the washing liquid and the dispersion liquid used in the following experiments were 20 mM Tris, 0.5 M in Example 1.
Consists of NaCl, 0.05% Tween 20, pH 7.5, and in Examples 2 and 3, 50 mM Tris, 0.9%.
Consists of NaCl, 0.1% Tween 20, pH 9.0.

Example 1 A magnetic substance-containing polystyrene latex having an average particle size of 0.7 μm (hereinafter referred to as Mg latex, manufactured by Rhone Poulenc).
After the hepatitis B virus surface antigen (HBsAg) produced by the gene recombination method was immobilized by the chemical coupling method using carbodiimide, bovine serum albumin (BS
The particles were stabilized by treatment with A) and suspended in a buffer solution at a concentration of 0.05% to prepare a Mg latex reagent.

Eu-NTA (β-naphthoyltrifluoroacetone) compound of a rare earth chelate (made by itself) 1 × 10
^-4 mol and TOPO (trioctylphosphine oxide)
(Dojindo Co., Ltd.) After dissolving 2 × 10 ⁻⁴ mol in 40 g of acetone, 3 g of polystyrene latex having an average particle size of 0.653 μm (Nippon Synthetic Rubber Co., Ltd.) suspended in 40 ml of water was mixed and mixed with acetone by an evaporator. The Eu-chelate compound was co-extracted with TOPO in the latex particles by removing the above to prepare an Eu chelate-labeled latex (hereinafter referred to as Eu latex).

An anti-HBsAg antibody (rabbit) was immobilized on Eu latex by a chemical binding method using carbodiimide, treated with BSA, and then suspended in a buffer solution at a concentration of 0.003%, followed by Eu latex reagent. Was produced. Anti-H
BsAg antibody standard solution (human, self-made) was diluted with Tris buffer to prepare 0, 10, 100 and 1000 mIU / ml standard solutions.

30 μl of the standard solution, 200 μl of BSA-containing Tris buffer and 40 μl of the Mg latex reagent were added and stirred, and an immunoreaction was carried out for 10 minutes. Next, the Mg latex in the reaction cell was separated from the reaction solution using a magnet, and after removing the supernatant, 200 μl of the buffer solution and 40 μl of the Eu latex reagent were added and stirred to carry out an immunoreaction for 10 minutes. .

The Mg latex in the reaction cell is separated from the reaction solution by using a magnet, the remaining reaction solution is removed, 200 μl of the washing solution is added, stirred, and immediately separated by the magnet. Discard the supernatant, add 200 μl of the washing solution, and stir. After repeating this separation and washing step three times, 200 μl of the final dispersion liquid was added to the separated Mg latex and stirred, and then 100 μm.
1 was collected and used as a binding sample.

The amount of Eu latex contained in the bound sample was measured by counting fluorescent dye labeled particles at 615 nm. Time-resolved fluorescence measurement system CyberFlu
The results measured using or615 (CyberFluor) are shown in FIG. A decrease in fluorescence intensity was observed as the anti-HBsAg antibody concentration in the standard solution increased.

Example 2 A magnetic substance-containing polystyrene latex having an average particle size of 0.7 μm (hereinafter referred to as Mg latex, manufactured by Rhone Poulenc Co.).
After immobilizing the hepatitis B virus core antigen (HBcAg) (Chemical and Serum Therapy Research Institute) produced by the gene recombination method by carbodiimide by the chemical coupling method,
The particles are stabilized by treatment with bovine serum albumin (BSA) and suspended in a buffer solution at a concentration of 0.05% to form Mg.
A latex reagent was made.

Eu-of the rare earth chelate used in Example 1
After the NTA Compound 3 × 10 ^-4 mol and TOPO6 × 10 ^-4 mol was dissolved in acetone 40 g, average particle size 0.459μ
3 g of polystyrene latex (manufactured by Japan Synthetic Rubber Co., Ltd.) in 40 ml of water was mixed, and acetone was removed by an evaporator to allow the Eu chelate to be contained in the latex particles. It is referred to as latex).

Carbodiimide was used for Eu latex, and anti-HBcAg monoclonal antibody (mouse, Fit
zgerald) was immobilized by the chemical bonding method,
Eu latex reagent was prepared by treating with BSA and suspending it in a buffer solution at a concentration of 0.003%. Anti-HB by RIA method
20 positive samples and 2 negative samples for which cAg antibody has been measured
Specificity was examined using 0 specimens.

Sample 10 μl, BSA-containing Tris buffer 2
After adding 60 μl and 40 μl of the above-mentioned Mg latex reagent, the mixture was stirred and immunoreacted for 5 minutes. Next, the Mg latex in the reaction cell was separated from the reaction solution using a magnet, the supernatant was removed, 250 μl of the buffer solution and 40 μl of the Eu latex reagent were added and stirred, and an immune reaction was carried out for 10 minutes. . The magnet latex was used to separate the Mg latex in the reaction cell from the reaction solution, and the remaining reaction solution was removed.
Add μl. After repeating this separation and washing step three times, 300 μl of the final dispersion liquid was added to the separated Mg latex,
After stirring and dispersing, the fluorescence intensity is measured.

The amount of Eu latex contained in the bound sample was measured by counting fluorescent dye labeled particles at 615 nm. FIG. 2 shows the result of measurement using a time-resolved fluorescence measuring device using a self-made Xe flash lamp (Hamamatsu Photonics KK). A low fluorescence intensity was observed in the positive sample, and a high fluorescence intensity was observed in the negative sample.

Example 3 A magnetic substance-containing polystyrene latex having an average particle size of 0.7 μm (hereinafter referred to as Mg latex, manufactured by Rhone Poulin).
And anti-T3 monoclonal antibody (mouse, BiosPa
(Cific) was immobilized by a chemical bonding method using carbodiimide, and then treated with bovine serum albumin (BSA) to stabilize the particles to 0.012 in a buffer solution.
A Mg latex reagent was suspended at a concentration of 5%.

Carbodiimide was used for the Eu-labeled latex prepared in Example 2 (hereinafter referred to as Eu latex) to immobilize triiodothyronine (T3) (Sigma Co.) by the chemical bonding method, followed by BSA. It was treated and suspended in a buffer solution at a concentration of 0.003% to prepare an Eu latex reagent. T3 was dissolved in Tris buffer containing BSA to prepare standard solutions of 0, 1, 4, 16, and 64 ng / ml.

10 μl of the standard solution, 250 μl of BSA-containing Tris buffer, and 40 μl of the Mg latex reagent were added and stirred, and an immunoreaction was carried out for 5 minutes. Next, using a magnet, the Mg latex in the reaction cell was separated from the reaction solution,
After removing the supernatant, 250 μl of a buffer solution and 40 μl of the above Eu latex reagent were added and stirred to carry out an immunoreaction for 10 minutes.

The Mg latex in the reaction cell was separated from the reaction solution using a magnet. The remaining reaction solution is removed, 200 μl of the washing solution is added, stirred, and immediately separated by a magnet. Discard the supernatant, add 200 μl of the washing solution, and stir. After repeating this separation and washing process three times, 200 μl of the final dispersion was added to the separated Mg latex, and after stirring 100 μl
Was collected and used as a binding sample. The amount of Eu latex contained in the bound sample was measured by counting fluorescent dye labeled particles at 615 nm. Time-resolved fluorescence measurement system CyberFluor 615 (CyberFluor
3) shows the result of measurement using the same company. A decrease in fluorescence intensity was observed as the T3 concentration in the standard solution increased.

[0046]

INDUSTRIAL APPLICABILITY According to the method of the present invention, by using magnetic particles and fluorescent particles, B / F separation can be carried out quickly and simply, and the measurement sensitivity can be increased as compared with ordinary FIA, so that antigens and antibodies can be obtained. The reaction can be measured.

[Brief description of drawings]

FIG. 1 shows the fluorescence intensity of particles (bound sample) separated by applying a magnetic field after the second reaction and anti-HBs in Example 1.
It is the figure which showed the relationship with the density | concentration of Ag antibody.

FIG. 2 shows the fluorescence intensity of particles (bound sample) separated by applying a magnetic field after the second reaction and anti-HBc in Example 2.
It is a figure showing the relation with the RIA measurement result of Ag antibody.

FIG. 3 is a diagram showing the relationship between the fluorescence intensity of particles (bound sample) separated by applying a magnetic field after the second reaction and the T3 concentration in Example 3.

Claims (5)

[Claims] 1. A method for measuring an antigen-antibody reaction comprising the following steps. (A) a first reagent composed of insoluble magnetic particles carrying an antibody or an antigen against the antigen or antibody to be measured,
The antigen or antibody to be measured is reacted in the liquid medium of the reaction container. (B) The insoluble magnetic particles after the reaction in step (a) are attached to the reaction vessel wall by the action of a magnetic field, and the liquid medium is removed. (C) A second reagent consisting of insoluble fluorescent dye-labeled particles carrying an antigen or antibody having the same specificity as the antigen or antibody to be measured, and the insoluble magnetic particles attached to the reaction vessel wall of step (b) And react in a liquid medium. (D) The insoluble magnetic particles of step (c) are attached to the reaction vessel wall by the action of a magnetic field to remove the liquid medium and the unreacted insoluble fluorescent dye-labeled particles, and then the insoluble fluorescent particles reacted with the insoluble magnetic particles. The fluorescence intensity of the dye-labeled particles is measured. 2. The measuring method according to claim 1, wherein insoluble magnetic particles carrying a polyclonal antibody and insoluble fluorescent dye-labeled particles carrying an antigen are used. 3. The measuring method according to claim 1, wherein insoluble magnetic particles carrying a monoclonal antibody and insoluble fluorescent dye-labeled particles carrying an antigen are used. 4. The measuring method according to claim 1, wherein insoluble magnetic particles carrying an antigen and insoluble fluorescent dye-labeled particles carrying a polyclonal antibody are used. 5. The measuring method according to claim 1, wherein insoluble magnetic particles carrying an antigen and insoluble fluorescent dye-labeled particles carrying a monoclonal antibody are used.