JPS58225354A - Analysis method of immune and reagent and reaction container used therein - Google Patents

Abstract

PURPOSE:To quantitatively measure an antigen simply and rapidly, by a method wherein a first reagent containing a solidified phase particle and a specimen are mixed and the separated particle and a second reagent containing a labelled substance are mixed to bond a labelled antibody-measuring antigen-solidified phase antibody. CONSTITUTION:A specimen 26 is portionwise injected in a reaction container 11 from a nozzle 28 and a first reagent 30 containing a particle prepared by converting an antibody specifically reacting the antigen in the specimen to a solidified phase is portionwise injected therein from a nozzle 32 to obtain a mixed liquid 33 in which antigen- antibody reaction is in turn carried out for a preset time. After the mixed liquid 33 is discharged through a filter 15 and washed, a second reagent containing an antibody labelled with enzyme is injected from a nozzle 41 to bond the enzyme labelled antibody- the measuring antigen-the solidified phase antibody. After the mixed liquid is discharged and washed, a substrate solution 43 relative to the labelling enzyme is portionwise injected from a nozzle 45 to produce a color forming substance. After a reaction stopping liquid 47 is portionwise injected, the reaction liquid 50 is accommodated in a colorimetric cell 51 and absorbancy is measured by a spectrophotometer 52 to quantitatively measure the antigen in the specimen.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an immunoassay method and reagents and reaction vessels used therein.

The antigen-antibody reaction is a specific reaction that occurs between a specific antigen and an antibody, and using this reaction, trace amounts of antigens (antibodies) in fixed blood or body fluids can be used for diagnosis and treatment. ing. Analysis methods based on this antigen-antibody reaction include radioimmunoassay using radioisotopes,
There are enzyme immunoassays that use enzymes and flow immunoassays that use fluorescent substances, and the simple radioimmunoassays and immunoassays are known as highly sensitive analytical methods. In any analysis method, when quantifying the antigen (antibody) to be measured, the antigen C antibody) and labeled antibody (antigen)
The so-called B, f of the conjugate and the free labeled antibody (antigen)
For example, as an immunoassay device using radioimmunoassay, a reaction vessel (ohamber) having an inlet and an outlet and containing an antibody or antigen that specifically reacts with the antigen or antibody to be measured is used. TeB
, f separation is known. In this device, for example, when quantifying a predetermined antigen in a sample, a reaction vessel in which an antibody that reacts with the antigen to be measured is made in the same phase is used, and the reaction vessel is first connected to the inlet of the antibody ohamber. Mix the sample and radioisotope-labeled antigen as shown in Figure 1A in a prepared tube, and mix the sample and antigen labeled with a radioisotope as shown in Figure 1B.
The sample is sent to a reaction vessel containing an immobilized antibody corresponding to the antigen to be measured as shown in FIG. Free antigens that are not captured by the in-phase labeled antibodies are sent to a scintillation counter via the outlet of the reaction vessel, and free labeled antigens are counted based on their radioactivity.

Next, the bound antigen captured by the immobilized antibody in the reaction vessel is eluted from the immobilized antibody with an elution buffer, and the eluted antigen is scintillated through the outlet of the reaction vessel as shown in D. The labeled antigens are sent to a counter and counted by their radioactivity. In this way, radioactivity characteristics as shown in Figure 2 were obtained, with both peak values, that is, the peak value of the count of free labeled antigen that was not captured by the immobilized antibody, and the peak value of the counted value of the free labeled antigen that was not captured by the immobilized antibody, and the captured and eluted label. The predetermined antigen in the sample is quantified based on a comparison of the antigen count with the peak value.

However, in such an analyzer, the sample and labeled antigen are mixed in a duplex connected to the inlet of the reaction container, so a relatively long tube is required, which tends to cause contamination and the S mixed solution. The drawback is that it takes time to send the sample to the reaction vessel, and the analytical throughput is low. Furthermore, since compounds marked with w4 with a radioactive isotope are usually unstable, it is necessary to repeatedly adjust the labeling substance.

In addition, radioactive isotopes are dangerous to the human body, difficult to dispose of due to environmental pollution, and their handling requires expensive facilities with certain qualifications and approvals based on certain standards. There are drawbacks such as the equipment being expensive.

From the above points of view, recently, enzyme immunoassays using ms in which radioactive isotopes are replaced with enzymes, and 70-immunoassays using fluorescent substances, have sensitivity and accuracy comparable to radioimmunoassays, and are excellent methods that can be performed safely. It has been adopted as an analytical method.

On the other hand, the antigen-antibody reaction step in the above-mentioned radioimmunoassay, enzyme immunoassay, and flow immunoassay requires a relatively long reaction time, and in order to shorten this reaction time, uniform antigen-antibody at high concentration is required. A reaction system is required for the reaction. In order to obtain such a homogeneous reaction system, for example, fine cellulose particles are used as a solid phase carrier in an enzyme/assay, and anti-HA
- There is a method in which the interface between the reaction solution, which is the site of antibody reaction, and the surface of the solid phase carrier is increased, and the particles of the same phase carrier are suspended uniformly. This analytical method is carried out manually, for example, α-fetoprotein (α-fetoprotein) in the sample.
When quantifying fetoprotein) antigen,
First, as shown in FIG. 3A, sample 1 and anti-(1-fetoprotein
i-α-fetoprotein) antibody-containing reagent 2 was dispensed into test W8 to perform an antigen-antibody reaction.
Binding fetoprotein and anti-α-fetoprotein. Next, as shown in i81'lB, wash buffer was added to the test tube δ, and aooor, p, m, a
After centrifugation for o seconds, the supernatant is removed by suction to leave the cellulose carrier particles in the test tube 8.

As shown in the apprentice 3FyJO, the enzyme-labeled antibody conjugated anti-α-fetoprotein (CO-nj
ugated anti-α-fetoprotein
) is added to cause an antigen-antibody reaction to form a conjugate of anti-α-fetoprotein-α-fetoprotein-conjugated anti-α-fetoprotein. Next, as shown in the eighth diagram, the washing buffer 4 was added to the test tube 8, and the cells were separated at 3000 r, p, m, 81) seconds, and the supernatant was removed by suction to remove the cellulose carrier particles. is left in the test tube δ. By repeating this washing step shown in Figure 8 eight times, the enzyme-labeled antibody that did not react in the antigen-antibody reaction in Figure 8G is washed away, and separations B and F are performed. Then, as shown in Figure 8E,
Enzyme substrate solution 6 is added to test tube 8 to cause an enzyme-substrate reaction with the labeled enzyme captured on the surface of the cellulose particles, thereby producing a colored substance proportional to the amount of captured labeled enzyme. . After allowing this enzyme-substrate reaction to occur for a predetermined time, the test tube 8 is centrifuged at 800 Or-p, m, for 30 seconds, and the supernatant containing the coloring substance is aspirated and discharged into a colorimetric cell as shown at No. 31 mF. Then, the absorbance is measured using a spectrophotometer 8, and based on this measured value, the α-fetoprotein antigen in the sample 1 is quantified from the calibration line between the absorbance determined from the sample with a known concentration and the α-fetoprotein antigen. do.

In the enzyme immunoassay mentioned above.

Compared to assay threads that use beads or balls that are commonly used as in-phase carrier particles, the use of cellulose particles increases the interface between the reaction solution and in-phase carrier, which is the site of antigen-antibody reaction. Moreover, by uniformly suspending the particles on the surface of the solid phase carrier, a uniform reaction system is obtained. However, in this analysis method, in order to separate cellulose particles and their reaction solution after the antigen-antibody reaction between sample 1 and reagent 2 containing cellulose particles having immobilized antibodies, cellulose particles and an enzyme-labeled antibody are included. In order to separate the cellulose particles and the reaction solution after the antigen-1'1°L reaction with the reagent 5, and after the enzyme-substrate reaction between the cellulose particles and the enzyme substrate solution 6, the reaction solution (containing a coloring substance) is added.
Since centrifugation is required for colorimetric self-separation and fractionation, it has the disadvantage of being cumbersome to operate. Furthermore, there is a technical difficulty in that cellulose particles are also aspirated when the supernatant is aspirated after centrifugation. Furthermore, in the above analysis method, it is difficult to remove the cellulose particles remaining in the test tube 3 after transferring the reaction solution containing the coloring substance to the colorimetric cell 7, so the test tube 3 is made of polystyrene. Although the test tube 3 is disposable, if the test tube 3 is made disposable in this way, the waste such as the reaction liquid after the measurement path r is quenched with body fluids such as serum or 1rl'I serum (the station building does not contain any reaction liquid). Since both the test tube and the test tube must be sterilized, the disadvantage is that disposal becomes extremely complicated.

The purpose of the present invention is to eliminate the various drawbacks mentioned above, to provide an immunoassay method that can easily and quickly determine the antigen or antibody to be measured, and is particularly suitable for automation. The analysis method involves mixing a sample with a first reagent containing an antibody that specifically reacts with the antigen or antibody to be measured, or particles that are in phase with the antigen, causing an antigen-antibody reaction. The separated particles are mixed with a second reagent containing an antibody that specifically reacts with the antigen or antibody to be measured, or a labeling substance bound to the antigen. A step of performing an antibody reaction and then separating the mixed solution and the particles using a filter, and mixing the separated particles with an eighth reagent capable of producing a specific substance in the presence of the labeling substance. The method is characterized in that the following steps are performed next: a step of carrying out a reaction, and a step of colorimetrically measuring the specific substance obtained by this reaction to quantify the antigen or antibody to be measured in the sample. It is something.

An object of the present invention is to provide a reagent containing particles immobilized with an antibody or antigen that specifically reacts with the antigen or antibody to be measured, which is suitable for use in the above-mentioned immunoassay method. .

The reagent of the present invention is characterized by comprising particles having a predetermined specific gravity, shape, and size, and having a predetermined antibody or antigen that specifically reacts with the antigen or antibody to be measured on the surface thereof. It is something to do.

A further object of the present invention is to provide a reaction vessel suitable for use in the above-mentioned immunoassay method.

The reaction container of the present invention has an inlet at one end and an outlet at the end of the pond, and is characterized in that a filter is provided between the inlet and the outlet to prevent particles of the first reagent from passing through. It is.

The present invention will be described in detail below with reference to the drawings.

FIG. 4 is a sectional view showing the structure of an example of the reaction vessel of the present invention. This reaction vessel 11 has an inlet 12 and an outlet 13. A part of the passage 14 connecting the inlet 12 and the outlet 18 is provided with a passage 141 so as to block the passage of particles immobilized with an antibody or antigen that specifically reacts with the antigen or antibody to be measured. A filter 15 made of glass, for example, is provided to allow the reaction liquid and the like to pass through. Further, a stopper 16 made of, for example, a stainless steel wire mesh is provided near the filter 15 on the side of the discharge port 18, and a plate valve 17 made of, for example, silicone rubber is arranged between the filter 15 and the stopper 16, so that the reaction Depending on the pressure direction in the container 11, the plate valve 17 is separated from the filter 15 as shown in FIG.
8, and the plate valve 1 as shown in FIG. 5B.
7 in close contact with the filter 15 to remove the reaction liquid, etc. from the filter 15.
be configured to prevent the passage of. Note that it is preferable that the thickness of the filter 15 be made as thin as possible in terms of strength.

6A to 6D are diagrams for explaining the sequential steps of an example of the immunoassay method of the present invention using the reaction container 11 shown in FIG. 4, and the reaction container 11 is schematically shown. In this J+lj, the outlet 13 of the reaction vessel 11 is opened to the air via the two-way valve 21, and the two-way valve 22 and the pump 2 are
3 and open to air through a two-way valve 24. First, in FIG. 6A, close the C two-way valve 21 and the two-way valve 22,
After opening the two-way valve 24 and operating the pump 23 to suck air, the two-way valve 22 is opened, the two-way valve 24 is closed, the pump 23 is operated to discharge, and the sucked air is transferred to the exhaust port 13.
is supplied into the reaction vessel 11, whereby the plate valve 17 is brought into close contact with the filter 15 (as shown in FIG. At the same time, the sample 26 contained in the Yampuru cup 25 is dispensed from the injection port 12 into the reaction vessel 11 via the pump 27 and the sample nozzle 28, and the sample 26 contained in the reagent tank 29 is dispensed into the reaction vessel 11. antigen(
A first reagent 8o containing particles immobilized with an antibody (antigen) that specifically reacts with the antibody (antibody) is pumped through a pump 81 and a dispensing nozzle 8.
2 into the reaction container 11 through the injection port 12 to obtain a mixed solution 88, and the antigen-antibody reaction between the antigen (antibody) in the sample 26 and the in-phase antibody (antigen) is carried out at room temperature or at a constant temperature. Let the process take place in the tank for a certain period of time.

In this example, the first reagent 30 is a solid phase carrier particle of an antibody (antigen) with an average diameter of 1 (lμ111.specific gravity 1°o3~
Using 1.05 spherical nylon particles, INHOI
Hydrolyzed in a solution at 6+1"C for 8 hours, washed with pure water, then reacted in 1% glutaraldehyde at 24"C for 1 hour, and then 0.25M sodium phosphate buffer (1)
Wash with I(7,5) and then 1. An antibody (antibody) that specifically reacts with the antigen (antibody) to be measured at a ratio of OQ/lnl
Antigen) was added to the antibody (antigen) by reacting it at 4'C for 24 hours in 0.25M sodium phosphate buffer, and
Next, the reaction solution was removed and washed with +1.25M sodium phosphate buffer. IM Na07.

1 mM MgC7, 0.1% bovine serum albumin, O,
It is suspended in a 0.01M sodium phosphate buffer containing X% Na, N8. This first attempt 118o is 4
Store at °cj.

In this way, as solid phase carrier particles, the average diameter is 100 μm.
, specific gravity 1. (If spherical nylon particles of 18 to 1.05 are used, the interface between the mixed liquid, which is the site of antigen-antibody reaction, and the surface of the solid phase carrier can be made large, and the particles can be suspended uniformly in the mixed liquid. Therefore, a homogeneous reaction system can be obtained.

Next, in FIG. 6B, the two-way valve 22 is opened and the two-way valve 24 is opened.
When the pump 23 is closed, the pump 23 is operated for suction, thereby opening the plate valve 17 and allowing the mixed liquid 38 in the reaction vessel 11 to pass through the filter 15 to obtain four grains.

After separating the mixed liquid from the liquid and sucking it, the two-way valve 21 is opened and the pump 23 is operated to discharge the mixed liquid, thereby discharging the sucked mixed liquid.

Thereafter, in FIG. 6C, the two-way valves 21, 22, 24 and the pump 28 are operated so that the plate valve 17 is closed, and the cleaning liquid 85 contained in the cleaning liquid tank 34 is pumped through the pump 86 and the pump 28. After dispensing into the reaction vessel 11 from the injection port 12 through the injection nozzle 87, the G two-way valves 21, 22, 24 and the pump 2 are injected as explained in FIG. 6B.
8 to open the plate valve 17 and discharge the dispensed cleaning liquid, thereby cleaning the filter 15 and the buiron particles. This washing step is performed one or more times.

Next, in the sixth diagram, the plate valve] 7 is closed and the second sample g [9, which is an enzyme-labeled antibody (antigen) against the antigen (antibody) to be measured stored in the reagent tank 88, is transferred to the pump 40.
and the reaction volume 6 from the injection port 12 via the dispensing nozzle 41.
11 and perform antigen-antibody incubation for a certain period of time at room temperature or in a thermostatic bath.This allows the enzyme-labeled antibody (antigen) - the antigen to be measured (antibody) - and the immobilized antibody (antigen) to bind. . Examples of the labeling enzyme for the second reagent 89 include malate dehydrogenase, glucose 6-phosphate dehydrogenase, glucose oxidase, horseradish peroxidase, acetylcholine esterase, alkaline phosphatase, peroxidase, colicoamylase, and lysozyme. , β-D galactosidase, etc., which have high activity, use relatively inexpensive substrates, and are stable enzymes can be used.

After the above antigen-antibody reaction, the filter 15 and nylon particles are washed with a cleaning solution by performing the operations explained in FIGS. 6B and 0, and then, as shown in FIG. 6E, the plate valve 17 is closed and the reagent is The third reagent 43, which is a substrate solution for the labeled enzyme stored in the tank 42, is injected from the reaction container 1 through the pump 44 and the dispensing nozzle 45.
The substrate solution of the reagent 43 is dispensed into the substrate solution of the second reagent 89, and the enzyme-substrate reaction is carried out for a certain period of time at room temperature or in a constant temperature bath, thereby producing a coloring substance. For alkaline phosphatase, phenyl phosphate,
2.Sodium and imaminoantipyrine, for peroxidase ()-phenylenediamine, β-D
- In the case of galactosidase, C) - ditrophenol.
β-D-galactoside, β in the case of darcose oxidase
-D-glucose etc. can be used.

After carrying out the enzyme-substrate reaction for a certain period of time, Fig. 6F
As shown in FIG. 2, with the plate valve 17 closed, the reaction stop liquid 47 contained in the reaction stop liquid tank 46 is injected into the reaction vessel 11 from the injection port 12 via the pump 48 and the dispensing nozzle 4g.
Thereby, a reaction solution 50 is obtained in which the enzyme-substrate reaction is stopped and the increase in coloring substance is stopped. Since the enzyme 2-substrate reaction can be easily stopped by changing p)I, H is used as the reaction stop solution 47.
Cjl, H, So, a solution, etc. can be used.

After that, in FIG. 6G, the two-way valve 22 is opened, and the two-way valve 22 is opened.
4 is closed to operate the pump 23, thereby opening the plate valve 17 and sucking the reaction liquid 50 in the reaction container 11 through the filter 15, separating it from nylon particles.The two-way valve 21 is then opened to operate the pump 23. ; 3 is discharged and the reaction liquid 5+1 sucked is stored in the colorimetric cell 51.

Next, as shown in FIG. 6H, the absorbance of the reaction liquid 60 contained in the colorimetric cell 51 is measured using a spectrophotometer a152,
Based on this measurement value, the antigen to be measured (antibody) in the sample 22 is quantified from a calibration curve between the absorbance obtained from the known concentration of `lampurus and the measured anti-1j;< (antibody). Further, after the reaction vessel 11 is cleaned with the cleaning liquid by performing the operations explained in FIGS. 6B and 6C, the cleaning liquid and nylon particles remaining in the reaction vessel 11 are removed by the suction nozzle 58 and the nylon particles as shown in FIG. 6. It is discharged via the suction pump 54 and prepared for the next "v-amble analysis."

The above-described suction nozzle 68 has a notch 55 formed at the end surface of the nozzle tip through which nylon particles can pass, as shown in FIGS. 7A and 7B, for example, in front and bottom views, respectively. When such a suction nozzle 58 is used, by bringing its tip into contact with the filter 15 and suctioning, the nylon particles and the remaining cleaning liquid can be effectively suctioned and removed.

FIG. 8 is a sectional view, but does not show the structure, of an example of the pond of the reaction vessel of the present invention. This reaction vessel 61 is the inlet 1 of the filter 15.
The only difference from the one shown in FIG. 1 is that the second side surface is processed into a concave shape. If this reaction vessel 61 is used, the sixth
In the suction step shown in the drawing, the nylon particles gather at the center of the concave surface of the filter 15, so that they can be easily and reliably removed by suction.

Note that the present invention is not limited to the above-mentioned example, and can be modified or changed in many ways.

For example, the plate valve 17 and the stopper 16 are provided in the reaction vessels 11.61 described above, but the filter 11.61 is provided with the plate valve 17 and the stopper 16.
If the hole diameter and area of 5, the inner diameter of the reaction vessel, the amount of reaction (mixing) liquid, etc. are set appropriately, the reaction (mixing) can be carried out by air pressure from the outlet 18 side without using these plate valves 17 and stoppers 16. Liquids and the like can be effectively retained within the reaction container. In addition, in the above example, the antigen (antibody) to be measured
Nylon particles were used as solid phase carrier particles for antibodies (antigens) that specifically react with cellulose, ABS.

It is also possible to use particles of polystyrene, styrene-butadiene resin, or the like, or microcapsules whose specific gravity is controlled by coating the surface of a core substance with a high specific gravity such as iron powder with a film with a low specific gravity. Furthermore, in the above-mentioned analysis method, the enzyme-substrate reaction was stopped by dispensing the reaction stop solution 47 in FIG. By discharging the labeled enzyme into the colorimetric cell 51 through the filter 15, the labeled enzyme captured by the nylon particles is separated into the nylon particles and the substrate liquid by the filter 15, whereby the substrate is no longer subjected to enzyme action and the reaction is stopped. From this, the process shown in FIG. 6F can be eliminated. Furthermore, in the example described in 1 and F, the reaction liquid 50 was transferred to the colorimetric cell 51 for photometry, but the reaction vessel 1
1 can also be used for direct photometry.

As explained in the examples above, in the immunoassay method according to the present invention, a mixed solution and a solid phase are used as in-phase carrier particles that immobilize antibodies (antigens) that specifically react with the antigen (antibody) to be measured. A uniform reaction system can be obtained because the phase carrier has a large interface with the surface and its specific gravity, shape, and size are controlled so that it can float uniformly.

Furthermore, by using a reaction vessel having a filter that does not allow the particles to pass through, the particles can be separated without centrifugation, which simplifies the analytical operation and facilitates automation. Furthermore, since the reaction container can be used repeatedly by cleaning, it has the advantage that disinfection and disposal after analysis are very easy.

[Brief explanation of the drawing]

11321IA-D is a diagram for explaining the successive two-stage process by radioimmunoassay; Figure 2 is a diagram showing an example of counts by radioimmunoassay shown in Figure 1; Diagram for explaining conventional sequential steps by assay; FIG. 4 is a sectional view showing the configuration of an example of the reaction vessel of the present invention; FIGS. 5A and B are plates of the reaction vessel shown in FIG. 4. Diagrams illustrating the operation of the valve; FIGS. 6A-6 are diagrams illustrating the sequential steps of the immunoassay method of the present invention; FIGS. 7A and B are diagrams 6-D; Suction nozzle shown in
FIG. 8 is a front view and a bottom view showing the configuration of an example of the reaction vessel according to the present invention, and FIG. 11, -1... Reaction container 12... Inlet 18
...Discharge 1''14...Passage 15...Filter 16...Stopper 17
... Plate valve 21.22.24 ... Two-way valve 28, 27.81.86.4 (+, 44.48.5
4...Pump 25...Sample cup 26...
・Sample 28...Sample nozzle 21)* 88.42...Reagent tank 80...First
Reagent 82, 37.41.45.49...dispensing nozzle 88.
・Mixed liquid 84 ・Cleaning liquid tank 85 ・
...Washing liquid 89...Second reagent 48...
8th reagent 46... Reaction stop solution tank 47.
...Reaction stop solution 1lIO...Reaction solution 51...
・Colorimetric Cal 52...Spectrophotometer 58...
Suction nozzle 55...Notch No. 11゛4 A II CI) Diaphragm #1 Soup No.: I's diagram dtM (intermediate) Figure 8 Figure 4 f Figure 5 A I (Σ q)

Claims (1)

[Scope of Claims] L A first reagent containing an antibody that specifically reacts with the antigen or antibody to be measured or a particle immobilized with the antigen is mixed with the sample to perform an antigen-antibody reaction, and then A step of separating the mixed liquid and the particles using a filter, and mixing the separated particles with a second reagent containing a labeling substance bound to an antibody or antigen that specifically reacts with the antigen or antibody to be measured. a third reagent capable of producing a specific substance in the presence of the separated particles and the labeling substance; and a step of colorimetrically measuring the specific substance obtained by this reaction to determine the antigen or antibody to be measured in the sample, sequentially. Characteristic immunoassay method. λ The immunoassay method according to claim 1, characterized in that a labeled enzyme is used as the labeling substance of the second reagent, and a substrate solution for the labeled enzyme is used as the third reagent. 8. Mixing the sample with a first reagent containing an antibody that specifically reacts with the antigen or antibody to be measured, or a particle in which the antigen is in phase, causing an antigen-antibody reaction, and then combining the mixed solution with the particles. The separated particles are mixed with a second reagent containing an antibody that specifically reacts with the antigen or antibody to be measured, or a labeling substance that binds the antigen to perform an antigen-antibody reaction. and then separating the mixed solution and the particles using a filter, and mixing and reacting the separated particles with an eighth reagent capable of producing a specific substance in the presence of the labeling substance. and a step of colorimetrically measuring the specific substance obtained by this reaction to immobilize the antigen or antibody to be measured in the sample. The reagent particles have a predetermined specific gravity, shape, and size, and are characterized in that they are composed of particles having a predetermined antibody or antigen that specifically reacts with the antigen or antibody to be measured on the surface thereof. reagent. 4 A first reagent containing an antibody that specifically reacts with the antigen or antibody to be measured, or particles on which the antigen is immobilized, and a sample are mixed in a reaction container to perform an antigen-antibody reaction, and then the mixed solution is prepared. The particles remaining in the reaction container are mixed with a reagent containing an antibody that specifically reacts with the antigen or antibody to be measured, or a labeling substance bound to the antigen. a step of performing an antibody reaction and then discharging the mixture through a filter;
A step of mixing the particles remaining in the reaction container with an eighth reagent capable of producing a specific substance in the presence of the labeling substance to cause a reaction, and colorimetrically comparing the specific substance obtained by this reaction. A reaction container used in an immunoassay method in which the steps of measuring and quantifying the antigen or antibody to be measured in the sample are sequentially performed, the reaction container having one injected into one end and discharged into the other end. 1. A reaction vessel characterized in that a filter is provided between the inlet and the outlet to prevent particles of the first reagent from passing through.