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WO2020054300A1 - Procédé et appareil de détermination de la quantité de perturbateur endocrinien et équivalents - Google Patents

Procédé et appareil de détermination de la quantité de perturbateur endocrinien et équivalents Download PDF

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Publication number
WO2020054300A1
WO2020054300A1 PCT/JP2019/031837 JP2019031837W WO2020054300A1 WO 2020054300 A1 WO2020054300 A1 WO 2020054300A1 JP 2019031837 W JP2019031837 W JP 2019031837W WO 2020054300 A1 WO2020054300 A1 WO 2020054300A1
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Prior art keywords
light
reaction
antigen
antibody
enzyme
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Ceased
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PCT/JP2019/031837
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English (en)
Japanese (ja)
Inventor
亮一 石松
金市 森田
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Kyushu University NUC
Ushio Denki KK
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Kyushu University NUC
Ushio Denki KK
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals

Definitions

  • the present invention relates to a method and an apparatus for quantifying an endocrine disrupter or the like.
  • 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.
  • endocrine disrupting substances and the like chemical substances such as pesticides in the environment and endocrine disrupting substances derived from the chemical substances.
  • 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.
  • 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.
  • 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 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 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.
  • 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.
  • the quantification can be performed in a short time because the aniline radical polymerization reaction having a short reaction time is used.
  • 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.
  • aniline is inexpensive, expensive fluorescent reagents such as conventional ELISA are not required.
  • 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.
  • 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.
  • the competition method as shown in the Examples, the antigen-antibody reaction is performed only once.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • FIG. 1 shows a flow chart of the method for quantifying an endocrine disrupter or the like of the present invention.
  • the object of quantification was 3-phenoxybenzoic acid (3-PBA), which is a kind of metabolite of a diphenyl ether herbicide.
  • HRP horseradish peroxidase
  • 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).
  • BSA bovine serum albumin
  • BSA is a blocking agent for suppressing nonspecific adsorption of the labeled antigen to be injected in the next step to the substrate.
  • a 3-PBA solution to be quantified an example of a “sample solution” in the claims
  • 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).
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • Antibody fixing section is provided in the reaction cell 17.
  • a plastic substrate for example, a PDMS substrate
  • PDMS substrate for example, a plastic substrate
  • the optical measurement channel 21 downstream of the reaction cell 17 is made of a light-transmitting material.
  • 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.
  • noise light such as external light may enter the light-projecting light guide 33 and the light-receiving light guide 35.
  • 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 is made 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.
  • 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.
  • valves V1 to V7 valve control mechanism
  • the pumps P1 to P7 the light source, and the light receiving sensor
  • power supply means not shown
  • control of opening / closing of the valves V1-V7 valve control mechanism
  • control of the pumps P1-P7, the light source, and the light receiving sensor are performed by a control unit (not shown).
  • 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).
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the substrate on which the antibody had been adsorbed in advance may be installed in the reaction cell. .
  • the number of the containers for the running buffer is one.
  • 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.
  • an example using the competitive ELISA method was described, but a direct teaching method, an indirect method, or a sandwich method may be used.
  • a direct teaching method, an indirect method, or a sandwich method may be used.
  • 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.
  • the competition method as shown in the Examples, the antigen-antibody reaction is performed only once.
  • 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

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Abstract

La présente invention vise à fournir un procédé de détermination de quantité et équivalents qui peut être réalisé dans un champ, un sujet d'échantillon environnemental tel qu'un perturbateur endocrinien étant collecté. Un premier aspect de la présente invention concerne un procédé de détermination de quantité qui détermine la quantité d'un ingrédient cible contenu dans un échantillon de solution à l'aide d'une réaction antigène-anticorps, le procédé comprenant : une étape de réaction antigène-anticorps au cours de laquelle un antigène marqueur enzymatique ou un anticorps marqueur enzymatique qui est marqué avec une enzyme est ajouté à une partie de réaction qui contient un anticorps correspondant à l'antigène marqueur enzymatique ou à un antigène correspondant à l'anticorps marqueur enzymatique, et une réaction antigène-anticorps est effectuée ; une étape de réaction de polymérisation au cours de laquelle un monomère ayant une réaction de polymérisation favorisée par l'enzyme est ajouté à la partie de réaction, et une réaction de polymérisation pour produire un polymère à partir du monomère est effectuée ; une étape de mesure d'absorbance dans laquelle l'absorbance du polymère produit par l'étape de réaction de polymérisation est mesurée ; et une étape de détermination de quantité dans laquelle la quantité de l'ingrédient cible est déterminée à partir de l'absorbance obtenue par l'étape de mesure d'absorbance.
PCT/JP2019/031837 2018-09-11 2019-08-13 Procédé et appareil de détermination de la quantité de perturbateur endocrinien et équivalents Ceased WO2020054300A1 (fr)

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JP2018169583A JP2020041909A (ja) 2018-09-11 2018-09-11 内分泌攪乱物質等の定量方法及び定量装置

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Cited By (1)

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WO2021181987A1 (fr) * 2020-03-11 2021-09-16 国立大学法人九州大学 Procédé pour déterminer la quantité de perturbateurs endocriniens et similaires

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WO2021181987A1 (fr) * 2020-03-11 2021-09-16 国立大学法人九州大学 Procédé pour déterminer la quantité de perturbateurs endocriniens et similaires

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