WO2020054300A1 - Method and apparatus for determining quantity of endocrine disruptor and like - Google Patents

Abstract

The purpose of the present invention is to provide a quantity determination method and the like that can be performed in a field in which an environmental sample subject such as an endocrine disruptor is collected. A first aspect of the present invention pertains to a quantity determination method that determines the quantity of a target ingredient contained in a sample solution by using an antigen-antibody reaction, the method comprising: an antigen-antibody reaction step in which an enzyme marker antigen or an enzyme marker antibody which is marked with an enzyme is added to a reacting portion that contains an antibody corresponding to the enzyme marker antigen or an antigen corresponding to the enzyme marker antibody, and an antigen-antibody reaction is performed; a polymerization reaction step in which a monomer having a polymerization reaction promoted by the enzyme is added to the reacting portion, and a polymerization reaction for producing a polymer from the monomer is performed; an absorbance measurement step in which the absorbance of the polymer produced through the polymerization reaction step is measured; and a quantity determination step in which the quantity of the target ingredient is determined from the absorbance obtained through the absorbance measurement step.

Description

Method and device for quantifying endocrine disruptors

(4) The present invention relates to a method and an apparatus for quantifying an endocrine disrupter or the like.

環境 Environmental pollution due to endocrine disrupters (so-called environmental hormones) has attracted attention. The endocrine disrupting substance is a substance in which a chemical substance such as a pesticide discharged into the environment such as soil, river, and ocean is metabolically toxic by microorganisms that live in the environment. In recent years, there has been an increasing demand for measurement and analysis of chemical substances such as pesticides in the environment and endocrine disrupting substances derived from the chemical substances (hereinafter referred to as “endocrine disrupting substances and the like”).

Conventional methods for quantifying endocrine disruptors include gas chromatograph mass spectrometry (eg, high resolution gas chromatographic analysis: HRGC / MS, high resolution mass spectrometry HRGC / HRMS) and liquid chromatograph (eg, high performance liquid chromatograph). Graph: HPLC) method and optical measurement such as ELISA have been used (Patent Document 1).

JP-T-2018-512071

However, the conventional methods for quantifying endocrine disruptors, etc. require high-precision measurement and sensitivity, but require relatively large equipment. However, it was difficult to implement at the site where endocrine disruptors were collected.

The present invention has been made in view of the above circumstances, and an object of the present invention is to provide a quantification method and the like which can be carried out at a site where an environmental sample such as an endocrine disruptor is collected.

A first aspect of the present invention is a quantification method for quantifying a target component contained in a sample solution using an antigen-antibody reaction, wherein the enzyme-labeled antigen or enzyme-labeled antibody labeled with an enzyme is labeled with the enzyme-labeled enzyme. In addition to the reaction section containing the antibody corresponding to the antigen or the antigen corresponding to the enzyme-labeled antibody, an antigen-antibody reaction step of causing an antigen-antibody reaction, and adding a monomer whose polymerization reaction is promoted by the enzyme to the reaction section A polymerization reaction step of performing a polymerization reaction to generate a polymer from the monomer, an absorbance measurement step of measuring the absorbance of the polymer generated in the polymerization reaction step, and from the absorbance obtained in the absorbance measurement step, the target A quantification step of quantifying the components.

第 A second aspect of the present invention is the method for quantification according to the first aspect, wherein the monomer is aniline and the polymer is polyaniline.

A third aspect of the present invention is the quantification method according to the first or second aspect, wherein in the antigen-antibody reaction step, the reaction part containing the antibody is labeled with an enzyme together with the enzyme-labeled antigen. Add the unsampled sample solution.

A fourth aspect of the present invention is a quantification apparatus for quantifying a target component contained in a sample solution by utilizing an antigen-antibody reaction, wherein the reaction section serves as a reaction field of the antigen-antibody reaction, and holds the sample solution. And a second container for holding an enzyme-labeled solution containing an enzyme-labeled product which is labeled with an enzyme and which is an antigen or an antibody in the antigen-antibody reaction, and is promoted by the enzyme. A third container for holding a substrate solution for the polymerization reaction to be performed, a first flow path for guiding the sample solution in the first container to the reaction section, and a solution of the enzyme-labeled substance in the second container. A second flow path leading to the reaction section, a third flow path leading the substrate solution in the third container to the reaction section, a fourth flow path leading the solution in the reaction section to the outside, A light source unit that emits light containing a wavelength that is absorbed by the polymer produced by A light detection unit that detects light, a first light guide path that guides light emitted by the light source unit to a measurement unit that is a part of the fourth flow path, and light from the measurement unit to the light detection unit. A second light guide path for guiding light; and a light absorbing portion that absorbs light and surrounds the first light guide path and the second light guide path.

A fifth aspect of the present invention is the quantitative device according to the fourth aspect, wherein the light-absorbing section is made of a pigment-containing silicone member containing a pigment that absorbs light.

A sixth aspect of the present invention is the quantitative device according to the fourth or fifth aspect, wherein the first light guide, the second light guide, and the light absorbing unit are made of silicone resin having the same refractive index. Material.

A seventh aspect of the present invention is the quantitative device according to any one of the fourth to sixth aspects, wherein the first flow path and the second flow path are joined upstream from the reaction unit. ing.

An eighth aspect of the present invention is the quantitative device according to any one of the fourth to seventh aspects, wherein a fourth container for holding an antibody solution used for the antigen-antibody reaction, A fourth flow path for guiding the antibody solution to the reaction section, wherein the enzyme-labeled product is an enzyme-labeled antigen that is an enzyme-labeled antigen.

According to each aspect of the present invention, it becomes possible to measure the absorbance at the site of collecting endocrine disrupting substances and the like to quantify the endocrine disrupting substances and the like.

Further, according to each aspect of the present invention, the reaction catalyzed by the enzyme is a polymerization reaction, so that the reaction is less affected by light or heat as compared with a conventional ELISA using a fluorescent substrate or a chromogenic substrate, and it can be used outdoors. It is possible to provide a quantification method or the like suitable for measurement. In addition, since the reaction time of the polymerization reaction is short, it is possible to quantify endocrine disrupting substances and the like in a short time.

According to the second aspect of the present invention, the quantification can be performed in a short time because the aniline radical polymerization reaction having a short reaction time is used. In addition, aniline is more stable to light and heat than fluorescent substrates used in conventional ELISA, and thus the quantification method of the present invention is suitable for outdoor measurement. Furthermore, since aniline is inexpensive, expensive fluorescent reagents such as conventional ELISA are not required.

According to the third aspect of the present invention, by using an ELISA competitive method, quantification can be performed in a small number of steps, so that the work load of the measurer is reduced, and the time required for quantification is reduced, and the on-site analysis is reduced. It becomes possible to provide a suitable quantification method. For example, in an ELISA sandwich method, after an antibody is adsorbed on a substrate, an antigen is added to cause a first antigen-antibody reaction, and a labeled antibody is further added thereto to cause a second antigen-antibody reaction. On the other hand, in the competition method, as shown in the Examples, the antigen-antibody reaction is performed only once.

According to the fourth aspect of the present invention, it is possible to provide a quantification device that can measure the absorbance at a site where an endocrine disrupting substance or the like is collected to quantify the endocrine disrupting substance or the like. Further, since the stray light is absorbed by the light absorbing portion, it is possible to accurately determine the amount.

According to the fifth aspect of the present invention, the stray light incident on the light absorbing portion is absorbed by the pigment. Therefore, it hardly returns to the transparent resin forming the first light guide path and the second light guide path. Further, no stray light leaks from the light absorbing portion to the outside. Therefore, complicated multiple reflection of stray light hardly occurs.

According to the sixth aspect of the present invention, the transparent resin forming the first light guide path and the second light guide path and the pigment-containing resin forming the light-absorbing portion are made of the same resin, so that both resins are formed. It becomes possible to suppress the reflection and scattering of light at the contacting interface.

According to the seventh aspect of the present invention, it is possible to mix the sample solution in the first container and the enzyme-labeled product solution in the second container, and then guide the mixture to the reaction section.

According to the eighth aspect of the present invention, the antigen to be quantified and the enzyme-labeled antigen can be made to react with the antibody competitively after the antibody is adsorbed on the substrate in the reaction section.

FIG. 1 is a flowchart of a method for quantifying an endocrine disrupting substance and the like of the present invention. 1 is an example of the configuration of an apparatus for quantifying an endocrine disrupter or the like of the present invention. It is a figure which shows the absorbance spectrum of aniline and the absorption spectrum after superposition | polymerization reaction. It is a figure which shows the time change of the light absorbency when polymerizing aniline is made to react.

Hereinafter, embodiments of the present invention will be described with reference to the drawings. The embodiments of the present invention are not limited to the following examples.

FIG. 1 shows a flow chart of the method for quantifying an endocrine disrupter or the like of the present invention. In this example, the object of quantification was 3-phenoxybenzoic acid (3-PBA), which is a kind of metabolite of a diphenyl ether herbicide. Horseradish peroxidase (HRP) was used as the enzyme. Note that any chemical substance having a COOH group or an NH ₂ group at the terminal can be labeled with HRP. In addition, a phosphate buffer (Phosphate buffer) was used as a solvent for each solution.

First, an anti-3-PBA antibody that specifically binds to 3-PBA is injected into a reaction cell (an example of a “reaction section” in the claims) and adsorbed on a substrate in the reaction cell (S1). Next, bovine serum albumin (BSA) is injected into the reaction cell (S2). BSA is a blocking agent for suppressing nonspecific adsorption of the labeled antigen to be injected in the next step to the substrate. Subsequently, a 3-PBA solution to be quantified (an example of a “sample solution” in the claims) and a labeled 3-PBA solution (an example of an “enzyme-labeled substance” and an “enzyme-labeled antigen” in the claims) The mixture is injected into the reaction cell (S3, an example of the “antigen-antibody reaction step” in the claims). Here, the labeled 3-PBA solution is a solution containing labeled 3-PBA in which 3-PBA as an antigen is labeled in advance with HRP, and the concentration of labeled 3-PBA is known. When a mixture of the 3-PBA solution and the labeled 3-PBA solution is injected, unlabeled 3-PBA and labeled 3-PBA are competitively captured by the antibody on the substrate, and an antigen-antibody reaction occurs. After the washing, aniline and hydrogen peroxide (H ₂ O ₂ ) are injected into the reaction cell (S4, an example of the “polymerization reaction step” in the claims). Then, the following chemical reaction is catalyzed by HRP of the labeled 3-PBA captured by the antibody on the substrate, and a polymerization reaction in which polyaniline is generated from aniline occurs. After the polymerization reaction, the absorbance of the polyaniline is measured (S5, an example of the "absorbance measurement step" in claims). Finally, the concentration of 3-PBA is calculated based on the obtained absorbance (S6, an example of the "quantitative step" described in claims).

As described above, since the present quantification method utilizes the radical polymerization reaction of aniline, the time required for quantification can be significantly reduced as compared with the conventional ELISA. The time required for the enzyme reaction of the conventional ELISA is several minutes, whereas the time required for the polymerization reaction of the present quantitative method is several seconds. Aniline is suitable for on-site analysis because it is far more stable to light and heat than fluorescent substrates and chromogenic substrates used in conventional ELISA. Furthermore, aniline is less expensive than the substrate used in the conventional ELISA. That is, according to the present quantification method, on-site analysis of endocrine disrupting substances and the like can be performed quickly, safely and inexpensively.

FIG. 2 shows a configuration example of a quantification apparatus 1 for performing the quantification method of the present invention. The quantitative device 1 enables analysis by a flow analysis method (for example, Flow Injection Analysis).

The quantification device 1 includes an antigen container 3 for holding a 3-PBA solution to be quantified (an example of a “first container” in the claims) and a labeled antigen for holding a labeled 3-PBA solution. Container 5 (an example of a “second container” in the claims), an antibody container 7 for holding an antibody solution (an example of a “fourth container” in the claims), and a blocking agent such as BSA A container 9 for blocking agent, a container 11 for H ₂ O ₂ for holding a hydrogen peroxide solution (H ₂ O ₂ ), and a container 13 for an aniline for holding an aniline-containing solution. An example of a “third container”), a container 15 for a running buffer for holding a running buffer, a reaction cell 17 (an example of a “reaction unit” in claims) serving as a reaction field for an antigen-antibody reaction, Guide solution from each container to reaction cell 17 An introduction channel 19, pumps P1-P7 for flowing the solution in the introduction channel 19, a three-way valve V1-V7 provided in the introduction channel 19 and capable of opening and closing, and introducing the solution from the reaction cell 17 to the outside. It has an optical measurement channel 21 (an example of a “fourth channel” in the claims) and an absorbance measurement unit 23 that measures the absorbance of polyaniline. Note that the above-described opening / closing control of the three-way valve includes switching control of the flow direction of the fluid and On / Off control of the flow of the fluid.

Each of the containers (the antigen container 3, the labeled antigen container 5, the antibody container 7, the blocking agent container 9, the H ₂ O ₂ container 11, the aniline container 13, and the running buffer container 15) _{(19 1,} 19 _{2, 19 3,} 19 4, 19 _5, 19 6, 19 ₇₎ connected to a pump P1-P7 through the solution in each container by driving the pump P1-P7 is the introduction into channel 19 Flow enters reaction cell 17. Downstream of the pumps P1-P6, three-way valves V1-V6 that can be controlled to open and close are provided. The introduction flow path 19 ₁₀ between the merge point C3 and reaction cell 17 to be described later, the valve V7 is provided and the other is connected to the waste insertion 25. In FIG. 2, each valve is shown individually for easy understanding. However, a valve control mechanism that integrates each valve to form one structure may be used.

An introduction channel 19 ₁ through which the 3-PBA solution flows from the antigen container 3 (an example of the “first channel” in the claims), and an introduction channel 19 ₂ through which the labeled 3-PBA solution flows from the labeled antigen container 5. (An example of the “second flow path” in the claims) merges at the junction C1. That is, the 3-PBA solution that flows when the pumps P1 and P2 operate and the valve V1 is open only in the horizontal direction of the drawing and the 3-PBA solution that flows when the valve V2 is open only in the horizontal direction of the drawing merge. When flow into the section C1, the introduction channel 19 ₈ on the downstream side of the merging portion C1 flows the mixture of 3-PBA solution and labeled 3-PBA solution.

The introduction channel 19 ₈ that mixture flows, is connected to the upstream of the first mixing coil unit 27. The mixed solution of the 3-PBA solution and the labeled 3-PBA solution is sufficiently mixed by flowing through the first mixing coil unit 27. By providing the first mixing coil unit 27, the 3-PBA solution and the labeled 3-PBA solution can be mixed in a short flow path, so that it is possible to provide a compact quantitative device 1.

Introduction flow path 19 ₈ on the downstream side of the first mixing coil 27, the merging point C3, the pump P3 is operated, introducing flowing antibody solution than antibodies container 7 when in the open state the valve V3 is only drawings lateral a road 19 _3, the pump P4 is operated and the introduction flow path 19 ₄ the valve V4 is flowing BSA than the blocking agent container 9 when only opened drawings lateral, as described _below, H 2 _{O 2} and merges with introduction flow path 19 ₉ mixture of the aniline solution flows. The introduction flow path _{19 10} of the downstream side of the merging portion C3 is connected to the reaction cell 17.

The introduction flow path 19 ₅ than H ₂ O ₂ for container 11 _H 2 _{O 2} flows, an example of the "third channel" of the introduction flow path 19 ₆ (claimed flowing aniline-containing solution from an aniline solution container 13 ) Merge at the junction C2. That is, H ₂ O ₂ flowing when the pumps P5 and P6 operate and the valve V5 is open only in the horizontal direction of the drawing, and the aniline solution flowing when the valve V6 is open only in the horizontal direction of the drawing are connected to the junction C3. It flows when, in the introduction channel 19 ₉ downstream of the merging portion C2 is a mixture of H ₂ O ₂ and the aniline solution flows.

The introduction channel 19 ₉ downstream of the merging portion C2, the second mixing coil unit 29 is provided downstream of the second mixing coil portion 29, joins the junction C3. That is, the mixed solution of the H ₂ O ₂ and the aniline solution that has passed through the junction C2 flows through the second mixing coil unit 29, and is sufficiently mixed.

抗体 Antibody fixing section is provided in the reaction cell 17. As the antibody fixing part, for example, a plastic substrate (for example, a PDMS substrate) is used.

光学 The optical measurement channel 21 downstream of the reaction cell 17 is made of a light-transmitting material. Around the optical measurement channel 21, an absorbance measuring section 23 for measuring the absorbance of polyaniline is provided. The absorbance measurement unit 23 includes a light source 31 (an example of a “light source unit”) that emits light for measuring absorbance, and a light projection that guides light emitted from the light source 31 to the optical measurement channel 21. Light guide path 33 (an example of the “first light guide path” in the claims), and a light receiving light guide path 35 (the “first light guide path” in the claims) that guides light that has passed through the polyaniline-containing solution in the optical measurement channel 21. 2) includes a light receiving sensor 37 (an example of a “light detecting unit”) that receives light guided by the light receiving light guiding path 35.

Here, noise light (stray light) such as external light may enter the light-projecting light guide 33 and the light-receiving light guide 35. In order to suppress the influence of the external light, the light source 31, the light guide path 33 for light projection, the light guide path 35 for light reception, and the light reception sensor 37 are made of a light absorbing material (for example, a silicone resin containing a pigment such as carbon black). The light-shielding member 39 (an example of the “light-absorbing section” described in the claims) is made of

Of the noise light that enters the light-projecting light guide 33 or the light-receiving light guide 35, the component that travels in the same direction as the optical axis of the light-projecting light guide 33 and the light-receiving light guide 35 is very small, and most of them are Light enters the light blocking member 39 from the interface between the light projecting light guide path 33 and the light blocking member 39 and the interface between the light receiving light guide path 35 and the light blocking member 39. At this time, the noise light incident on the light shielding member 39 is absorbed by the light absorbing member contained in the light shielding member 39. Therefore, it is possible to effectively reduce the influence of noise light (stray light) on the measurement result of the optical measurement.

The light-projecting light guide 33 and the light-receiving light guide 35 are configured as cavities provided in the light-blocking member 39, and the light-projecting light guide 33 is transparent to light emitted from the light source 31. It can also be constituted by a resin (for example, a silicone resin). Similarly, the light receiving light guide path 35 can be made of a resin (for example, a silicone resin) transparent to light emitted from polyaniline.

Here, if the transparent resin forming the light-projecting light guide path 33 and the light-receiving light guide path 35 and the resin containing the pigment forming the light shielding member 39 are made of the same material, the refractive indices of the two resins are the same. Therefore, reflection and scattering at the interface between the two resins are suppressed. That is, in this case, like the case where the light-projecting light guide path 33 and the light-receiving light guide path 35 are hollow, the stray light incident on the light shielding member 39 is absorbed by the pigment contained in the light shielding member 39. Since reflection and scattering at the interface between the light-projecting light guide path 33, the light-receiving light guide path 35, and the light-shielding member 39 are suppressed, stray light that has entered the light-shielding member 39 receives the light-projecting light guide path 33, the light-receiving light guide path 35. Hardly ever returns to Therefore, complicated multiple reflection of stray light hardly occurs.

The power supply to the valves V1 to V7 (valve control mechanism), the pumps P1 to P7, the light source, and the light receiving sensor is performed by power supply means (not shown). Similarly, control of opening / closing of the valves V1-V7 (valve control mechanism) and control of the pumps P1-P7, the light source, and the light receiving sensor are performed by a control unit (not shown).

定量 The quantification of 3-PBA in the quantitative device 1 is performed as follows. First, the pump P3 is operated, the valve V3 is opened only in the horizontal direction of the drawing, and the antibody solution is introduced from the antibody container 7 into the reaction cell 17 through the junction C3. The antibody in the antibody solution introduced into the reaction cell 17 is adsorbed on the plastic substrate (S1).

(4) After the antibody is adsorbed on the substrate, the operation of the pump P3 is stopped, and the valve V3 is closed. Thereafter, the pump P4 is operated to open the valve V4 only in the lateral direction in the drawing, and BSA is introduced into the reaction cell 17 from the blocking agent container 9 through the junction C3 (S2). The reason why BSA is introduced into the reaction cell 17 is to suppress non-specific adsorption of the labeled 3-PBA, which is subsequently introduced into the reaction cell 17, onto the substrate without being recognized by the antibody.

ＢＳ After introducing BSA into the reaction cell 17, the operation of the pump P4 is stopped and the valve V4 is closed. Thereafter, the pumps P1 and P2 are operated to open the valves V1 and V2 only in the horizontal direction in the drawing, and the mixed solution of the 3-PBA solution from the antigen container 3 and the labeled 3-PBA solution from the labeled antigen container 5 is opened. Is sufficiently mixed by the first mixing coil unit 27 and passes through the junction C3, and then is introduced into the reaction cell 17. The introduced liquid mixture is adsorbed on the substrate and captured by the immobilized antibody (S3).

After the antigen-antibody reaction occurs, the operations of the pumps P1 and P2 are stopped, and the valves V1 and V2 are closed. Then, by operating the pump P5 and P6, the valves V5 and V6 are opened _state, a mixture of aniline-containing solution from _H 2 _{O 2} and the aniline solution container 13 from the H 2 _{O 2} container 11, the After being sufficiently mixed by the two mixing coil sections 29 and passing through the junction C3, they are introduced into the reaction cell 17. Then, polyaniline is generated in the reaction cell 17 (S4), and the polyaniline-containing solution flows through the optical measurement channel 21 downstream of the reaction cell 17.

Then, the absorbance of the polyaniline is measured in an absorbance measurement section 23 provided around the optical measurement flow path 21 on the downstream side of the reaction cell 17 (S5), and the concentration of 3-PBA is calculated based on the absorbance. (S6). The introduction channel 19 and the reaction cell 17 are washed by appropriately flowing a running buffer between each step. For example, it washed as follows: for some outlet passageway _{19 6,} 19 9, _{19 10} aniline solution flows. When the pump P7 is operated and only the plug connected to the aniline container 13 of the valve V6 is closed and the other two plugs are opened, the running buffer is moved from the running buffer container 15 to the junction C2, The liquid is guided to pass through the second mixing coil 29 and the junction 3 and flows into the waste liquid container below the valve V7.

As described above, according to the quantitative device 1 of the present invention, the analysis by the flow analysis method is enabled, so that a competitive antigen-antibody reaction and polyaniline generation can be performed in the flow system, and the analysis can be performed quickly. -Convenience and reduction of sample (reagent) can be realized. Further, in the absorbance measuring section 23, the light guide path 33 for light projection and the light guide path 35 for light reception are configured to be surrounded by the light blocking member 39, so that noise light can be easily suppressed, and the noise as in the related art can be reduced. There is no need for a large-scale structure for light control. As a result, the quantitative device 1 of the present invention can be reduced in size and portability, and can realize relatively stable and quick measurement in an outdoor measurement environment.

FIG. 3 shows (a) an absorption spectrum of a 1 mM aniline solution, and (b) an absorbance spectrum obtained by reacting 0.1 to 100 ppm of HRP, 1 mM of H ₂ O ₂ , and 10 mM of aniline in a batch system. Show. In (a), there is an absorption peak in the ultraviolet, whereas in (b) after the polymerization reaction, broad absorption is seen in the visible light region. In (b), the absorbance increases as the HRP concentration increases. As described above, since the absorption spectra of the monomer and the polymer are significantly different, the concentration of the polymer and the concentration of the target component can be accurately calculated from the absorbance after the polymerization reaction.

FIG. 4 is a diagram showing a time change of absorbance at 410 nm when HRP, H ₂ O ₂ and aniline are reacted in a batch system. The absorbance reaches almost constant in several tens of seconds. That is, in the polymerization reaction of aniline, it can be said that stable data can be obtained in a relatively short time.

Note that the target components to be quantified are not limited to endocrine disrupters contained in soil, but may be those present in environments such as rivers and oceans.

Further, in the above embodiment, the antibody solution was flowed from the antibody container to the reaction cell, and the antibody was adsorbed on the substrate in the reaction cell. However, the substrate on which the antibody had been adsorbed in advance may be installed in the reaction cell. .

In the quantitative apparatus of the embodiment, the number of the containers for the running buffer is one. However, in order to adjust the pH in the reaction cell to the optimum pH for each of the enzyme reaction and the antigen-antibody reaction, a plurality of containers for the running buffer are provided. You may.

The measurement wavelength may be any wavelength that can appropriately measure the absorbance of polyaniline, preferably 400 nm to 430 nm, and more preferably 405 nm to 410 nm.

Also, in the above-described embodiment, an example using the competitive ELISA method was described, but a direct teaching method, an indirect method, or a sandwich method may be used. For example, in an ELISA sandwich method, after an antibody is adsorbed on a substrate, an antigen is added to cause a first antigen-antibody reaction, and a labeled antibody is further added thereto to cause a second antigen-antibody reaction. On the other hand, in the competition method, as shown in the Examples, the antigen-antibody reaction is performed only once. Therefore, by using an ELISA competitive method, quantification can be performed in a small number of steps, so that the work load of the measurer can be reduced, and the time required for quantification can be reduced, and a quantification method suitable for on-site analysis can be provided. Will be possible.

1 ... quantification device, 3 ... antigen container, container 5 ... labeled antigen, 7 ... antibodies container, 9 ... blocking agent container, for 11 ... _H 2 _{O 2} Vessel, 13: Vessel for aniline, 15: Vessel for running buffer, 17: Reaction cell, 19: Introductory channel, P1-P7 ... Pump, V1-V7 ... Three-way valve , 21 ... Optical measurement channel, 23 ... Absorbance measurement unit, 25 ... Waste liquid container, 27 ... First mixed coil unit, 29 ... Second mixed coil unit, 31 ... Light source, 33: light guide path for projecting light, 35: light guide path for light reception, 37: light receiving sensor, 39: light shielding member

Claims (8)

A quantitative method for quantifying a target component contained in a sample solution using an antigen-antibody reaction,
An antigen-antibody reaction step of adding an enzyme-labeled antigen or an enzyme-labeled antibody labeled with an enzyme to a reaction section containing an antibody corresponding to the enzyme-labeled antigen or an antigen corresponding to the enzyme-labeled antibody, and causing an antigen-antibody reaction When,
A polymerization reaction step of adding a monomer whose polymerization reaction is promoted by the enzyme to the reaction section and causing a polymerization reaction to generate a polymer from the monomer,
Absorbance measurement step of measuring the absorbance of the polymer generated in the polymerization reaction step,
A quantification step of quantifying the target component from the absorbance obtained in the absorbance measurement step. The method according to claim 1, wherein the monomer is aniline and the polymer is polyaniline. The method according to claim 1 or 2, wherein in the antigen-antibody reaction step, the sample solution not labeled with an enzyme is added to the reaction section containing the antibody together with the enzyme-labeled antigen. A quantitative device for quantifying a target component contained in a sample solution using an antigen-antibody reaction,
A reaction unit that serves as a reaction field for the antigen-antibody reaction,
A first container for holding the sample solution;
A second container for holding an enzyme-labeled product solution containing an enzyme-labeled product that is labeled with an enzyme, and that is an antigen or an antibody in the antigen-antibody reaction,
A third container for holding a substrate solution for the polymerization reaction promoted by the enzyme;
A first channel for guiding the sample solution in the first container to the reaction section,
A second flow path for guiding the enzyme-labeled product solution in the second container to the reaction section,
A third channel for guiding the substrate solution in the third container to the reaction section,
A fourth channel for guiding the solution in the reaction section to the outside,
A light source unit that emits light including a wavelength that is absorbed by the polymer generated by the polymerization reaction,
A light detection unit for detecting light,
A first light guide path that guides light emitted by the light source unit to a measurement unit that is a part of the fourth flow path;
A second light guide path for guiding light from the measurement unit to the light detection unit,
A light-absorbing unit that surrounds the first light guide and the second light guide and that absorbs light. (5) The metering device according to (4), wherein the light absorbing portion is made of a pigment-containing silicone member containing a pigment that absorbs light. The quantitative measurement device according to claim 4, wherein the first light guide, the second light guide, and the light absorbing portion are made of a silicone resin having the same refractive index. The quantitative device according to any one of claims 4 to 6, wherein the first flow path and the second flow path merge upstream of the reaction unit. A fourth container for holding an antibody solution used for the antigen-antibody reaction,
A fourth flow path that guides the antibody solution in the fourth container to the reaction section,
The quantification device according to claim 4, wherein the enzyme-labeled product is an enzyme-labeled antigen that is an enzyme-labeled antigen.

Images