WO2014083668A1 - Procédé de mesure de réactions antigène-anticorps utilisant une technique sandwich - Google Patents

Procédé de mesure de réactions antigène-anticorps utilisant une technique sandwich Download PDF

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Publication number
WO2014083668A1
WO2014083668A1 PCT/JP2012/081002 JP2012081002W WO2014083668A1 WO 2014083668 A1 WO2014083668 A1 WO 2014083668A1 JP 2012081002 W JP2012081002 W JP 2012081002W WO 2014083668 A1 WO2014083668 A1 WO 2014083668A1
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antigen
antibody
reaction
solid
solid phase
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PCT/JP2012/081002
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English (en)
Japanese (ja)
Inventor
宣行 笠間
政也 上原
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ミライアル株式会社
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Priority to PCT/JP2012/081002 priority Critical patent/WO2014083668A1/fr
Publication of WO2014083668A1 publication Critical patent/WO2014083668A1/fr

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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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing

Definitions

  • the present invention relates to a method for measuring an antigen-antibody reaction by a sandwich method.
  • a solid phase antibody is prepared in advance on the wall surface of a container such as a microplate or the surface of a spherical bead of about 10 ⁇ m to 40 ⁇ m. Then, the solid phase antibody is reacted with the antigen in the specimen containing the antigen to be measured. Thereafter, the labeled antibody and the solid phase antibody are bound in a state where the antigen is sandwiched by reacting the antibody with a labeled antibody modified with a hydrogen peroxide-degrading enzyme or a fluorescent substrate as a label. At this time, the labeled antibody that is not bound to the antigen remains dispersed in the reaction solution.
  • the labeled antibody not bound to the antigen is washed away by a washing step.
  • the antigen, the solid-phase antibody, and the labeled antibody are bound together so that the antigen is sandwiched (sandwiched) between the solid-phase antibody and the labeled antibody.
  • the labeled antibody in such a state is subjected to a treatment for detecting the label, whereby the number of antigens or the concentration of antigen in the specimen can be measured.
  • HRP hydrogen peroxide-degrading enzyme
  • a luminescent substrate containing luminol and hydrogen peroxide can be used to generate luminescence by the luminol reaction and to obtain luminescence with an intensity proportional to the antigen concentration.
  • fluorescence with an intensity proportional to the antigen concentration can be obtained by irradiating the labeled antibody with excitation light.
  • an antigen concentration higher than the antigen concentration in the specimen may be detected.
  • the antigen concentration in the specimen is low, this phenomenon appears remarkably, the measured value rises with respect to the low concentration antigen, and the low concentration antigen cannot be measured substantially. That is, as shown in FIG. 7, the relationship between the antigen concentration in the specimen and the measured value (light intensity of luminescence or fluorescence) is not a simple increase function, and the measured value decreases as the antigen concentration increases (antigen concentration). Is less than 0.01 ng / mL).
  • the calculation of the antigen concentration from the measured value cannot be uniquely determined, and the concentration cannot be accurately measured for a low concentration antigen.
  • An object of the present invention is to provide a method for measuring an antigen-antibody reaction by a sandwich method capable of measuring a low concentration antigen.
  • the present invention binds the solid phase antibody and the labeled antibody so that the antigen is sandwiched between the solid phase antibody and the labeled antibody having an antibody with a modified identification label in a medium containing a specimen having the antigen.
  • An antigen-antibody reaction measurement method using a sandwich method comprising: an antigen-antibody reaction step to be performed; and an antigen concentration measurement step for measuring the concentration of the antigen by identifying the identification label, wherein the antigen-antibody reaction step includes the step An antigen that causes a solution containing the same antigen as the measurement target of the specimen to flow through the solid-phase antibody at least once before the antigen contained in the specimen binds to the solid-phase antibody and / or the labeled antibody.
  • the present invention relates to a method for measuring an antigen-antibody reaction by a sandwich method, comprising a distribution step. Here, it is preferable to have a washing step of washing the solid phase antibody after circulation of the solution containing the antigen.
  • a method for measuring an antigen-antibody reaction by a sandwich method capable of measuring a low concentration antigen can be provided.
  • FIG. 4 is a schematic diagram showing a state in which an antigen 24 bound to a solid phase antibody 32 is bound to a labeled antibody 23 in an antigen-antibody reaction measurement method by a sandwich method according to the present embodiment.
  • FIG. 1 is a flowchart showing an antigen-antibody reaction measuring method by the sandwich method according to this embodiment.
  • FIG. 2A is a schematic diagram showing a state in which a base material (96-well microplate 3) is modified with a solid phase antibody 32 in the antigen-antibody reaction measurement method by the sandwich method according to the present embodiment.
  • FIG. 2B is a schematic diagram showing a state in which the antigen 24 is bound to the solid phase antibody 32 that modifies the base material in the antigen-antibody reaction measurement method by the sandwich method according to the present embodiment.
  • FIG. 2C is a schematic diagram showing a state in which the antigen 24 bound to the solid phase antibody 32 is bound to the labeled antibody 23 in the antigen-antibody reaction measurement method by the sandwich method according to the present embodiment.
  • the antigen-antibody reaction measurement method by the sandwich method includes a preparation step, an antigen-antibody reaction step, and an antigen concentration measurement step.
  • a solid phase antibody 32 is prepared (step ST11).
  • a specimen having the antigen 24 to be measured is prepared (step ST31).
  • a diluent for diluting the sample to be measured is prepared (step ST32).
  • the antibody 21 is modified with the identification label 22 (see FIG. 2C and the like) to prepare a labeled antibody 23 having the antibody 21 modified with the identification label 22 (step ST21).
  • the preparation step has a solid phase antibody fixing step.
  • the prepared solid phase antibody 32 is modified on a predetermined substrate (96-well microplate 3) (step ST12).
  • a predetermined substrate 96-well microplate 3
  • FIGS. 2A to 2C show a 96-well microplate 3 described later as a base material.
  • the substrate is not limited to the 96-hole microplate 3.
  • spherical beads having an average particle size of about 10 ⁇ m to 40 ⁇ m may be used.
  • a solution containing the same antigen as the antigen to be measured is prepared (step ST111).
  • an antigen distribution process is performed.
  • the solution prepared in step ST111 is circulated around the solid phase antibody 32, and an antigen distribution step for causing an antigen-antibody reaction between the antigen and the solid phase antibody 32 is performed (step ST112).
  • a washing process for washing the solid phase antibody 32 is performed (step ST113). Therefore, in the antigen-antibody reaction step, the solid-phase antibody 32 is washed before causing an antigen-antibody reaction between the antigen 24 contained in the sample to be measured and the solid-phase antibody 32.
  • the above is the preparation process.
  • an antigen-antibody reaction step is performed.
  • a sample to be measured and a diluent are mixed (step ST33), and this mixed solution is supplied to a base material on which a solid phase antibody is fixed, and the antigen 24 in the sample and the solid phase antibody are mixed. 32 are combined (step ST34). After bonding, a cleaning process is performed as necessary.
  • step ST21 a solution containing the labeled antibody 23 composed of the antibody 21 modified with the identification label 22 prepared in step ST21 is supplied to the substrate, and the antigen 24 is sandwiched between the solid phase antibody 32 and the labeled antibody 23.
  • the solid phase antibody 32 and the labeled antibody 23 are bound (step ST13).
  • the above is the antigen-antibody reaction step.
  • an antigen concentration measurement step is performed.
  • the labeled antibody 23 bound to the solid phase antibody 32 in the antigen-antibody reaction step is identified (step ST14). Thereby, the concentration of the antigen 24 is measured.
  • the above is the antigen concentration measurement step.
  • the preparation step includes an antigen distribution step in which a solution containing the same antigen as the measurement target is distributed to the solid phase antibody 32 once. Prepare. Then, after the solution containing the antigen 24 is distributed, the solid phase antibody 32 is washed. For this reason, in the measurement of the antigen concentration in the antigen concentration measurement step, detection of an antigen concentration higher than the antigen concentration in the specimen can be suppressed.
  • FIG. 3 is a perspective view showing a 96-well microplate 3 used in the antigen-antibody reaction measurement method by the sandwich method according to the first embodiment.
  • a 96-well microplate 3 is used as a base material on which the solid phase antibody 32 is fixed.
  • HRP hydrogen peroxide-degrading enzyme
  • a luminol solution is used as a luminescent substrate.
  • VEGF is used as the antigen 24. Details are as follows.
  • the necessary reagents in this example are as follows.
  • Water soluble carbodiimide (WSC) WSC 1mg + HCl aqueous solution (pH5) 1mL Carbonate buffer (CB) 25mM NaHCO3, 25mM Na2C3 (ph9.7)
  • Tris buffer (TB) 0.1M Tris-HCl (ph8.6), 0.1M NaCl (Wash Buffer) WB 0.05% Tween in PBS Blocking agent Block Ace 400ng / mL (ion exchange water) VEGF antigen 100 ⁇ g / ml 0.1% BSA (Bovine Serum Albumin) Reconstituted in PBS.
  • BSA Bovine Serum Albumin
  • an anti-VEGF monoclonal antibody is prepared (step ST11), and the anti-VEGF monoclonal antibody is modified on the bottom surface of each well 31 of the 96-well microplate 3 (step ST12).
  • an anti-VEGF monoclonal antibody (100 ⁇ g / ml, reconstituted with PBS) is diluted with CB, and an anti-VEGF antibody solution having a concentration of 20 ⁇ g / ml is applied to the bottom surface of each well 31 of the 96-well microplate 3. Inject and incubate overnight at 4 ° C, then wash. If necessary, a blocking process is performed to prevent antigens and antibodies from adhering to the bottom surface of each well 31. As a result, an antibody modified plate in which the solid phase antibody 32 is modified on the bottom surface of each well 31 of the 96-well microplate 3 is generated.
  • a specimen having the antigen 24 (VEGF antigen) to be measured is prepared (step ST31). Further, a phosphate buffer containing 0.1% BSA as a diluent is prepared (step ST32).
  • a phosphate buffer containing 0.1% BSA as a diluent is prepared (step ST32).
  • the above-described buffer is used as a diluent, the present invention is not limited to this, and other types of buffers, ion-exchanged water, and the like can be used.
  • an anti-VEGF polyclonal antibody with HRP label is prepared (step ST21).
  • a solution prepared by diluting the same antigen as the antigen to be measured with a phosphate buffer containing 0.1% BSA is prepared (step ST111).
  • the concentration of the antigen was 0.01 ng / ml.
  • the antigen 24 diluted with the prepared 0.1% BSA-containing phosphate buffer of the antibody-modified plate in which the solid-phase antibody 32 is modified on the bottom surface of each well 31 of the 96-well microplate 3 is prepared. Are allowed to react for 2 hours (step ST112). Thereafter, the solid phase antigen is washed with a phosphate buffer (step ST113). The above is the preparation process.
  • the specimen is diluted twice with a diluent (step ST33).
  • a diluent a phosphate buffer containing 0.1% BSA is used.
  • 200 ⁇ L of the diluted specimen is put into each well of the antibody-modified plate, and antigen-antibody reaction is performed (step ST34).
  • the reaction time is 2 hours.
  • the antibody-modified plate is washed with a phosphate buffer, and all the solution is discarded.
  • 200 ⁇ L each of HRP-labeled anti-VEGF antibody solution is added to cause an antigen-antibody reaction (step ST13).
  • the reaction time is 2 hours.
  • an antigen concentration measurement step is performed.
  • the antibody-modified plate is washed with a phosphate buffer, and the entire solution is discarded.
  • a luminescent substrate is inserted to emit light, and the luminescence intensity is detected by a high-sensitivity photodetector such as photomal (step ST14).
  • FIG. 4 is a graph showing the results obtained by the antigen-antibody reaction measurement method by the sandwich method according to the first example.
  • the antigen concentration increases from 0.00 ng / mL to 100 ng / mL
  • the value of the luminescence amount increases. Therefore, it can be seen that even when the antigen concentration is between 0.00 ng / mL and 0.01 ng / mL, the amount of luminescence can be accurately detected in the antigen concentration measurement step.
  • FIG. 5 is a plan view showing the microchannel chip 101 used in the antigen-antibody reaction measuring method by the sandwich method according to the second embodiment.
  • FIG. 6 is a plan view showing the microchannel 110 used in the antigen-antibody reaction measurement method by the sandwich method according to the second embodiment.
  • the microchannel chip 101 is constituted by a disk-shaped front disk-shaped plate in which a plurality of microchannels 110 having the same shape through which a fluid can flow are formed.
  • a later-described liquid reservoir 116 of the microchannel 110 is arranged in the radial direction of the microchannel chip 101 so that the later-described input port 111 of the microchannel 110 is closest to the center of the microchannel chip 101.
  • the microchannels 110 are arranged radially from the center of the microchannel chip 101 so as to be farthest from the center of the channel chip 101.
  • the back side disk-like plate (not shown) is affixed on the surface of the front side disk-like plate on which the micro flow path 110 is formed, and a two-layer structure inspection disk is formed by these.
  • the front disk-shaped plate is made of silicone resin
  • the back disk-shaped plate (not shown) is made of glass.
  • the disc-shaped inspection disk can be driven to rotate about the axis of the inspection disk as a rotation axis so that centrifugal force acts.
  • each of the plurality of micro flow paths 110 includes an input port 111, a reaction tank 113, a liquid reservoir 116, a first flow path 121, and a second flow path 122.
  • the input port 111 is configured by a chamber formed at a position closest to the center of the microchannel chip 101 in the microchannel chip 101 in which the microchannel 110 is formed. As shown in FIG. 6, the input port 111 has a circular shape in plan view, and communicates with the outside through a hole (not shown) formed in the front disk-like plate (not shown).
  • the first flow path 121 is outward in the radial direction of the micro flow path chip 101 as a direction in which the centrifugal force acts on the input port 111 when the inspection disk is rotated and the micro flow path chip 101 is rotated. , Extending from the input port 111.
  • the reaction tank 113 is composed of a chamber formed in a position radially outward of the microchannel chip 101 from the input port 111 in the microchannel chip 101 in which the microchannel 110 is formed. As shown in FIG. 6, the reaction tank 113 has an oval shape in plan view. The reaction tank 113 communicates with the input port 111 via the first flow path 121.
  • the second flow path 122 extends from the reaction tank 113 outward in the radial direction of the micro flow path chip 101 as a direction in which centrifugal force acts in the reaction tank 113 when the micro flow path chip 101 is rotationally driven. Put out.
  • the liquid reservoir 116 is configured by a chamber formed at a position radially outward of the microchannel chip 101 from the reaction tank 113 in the microchannel chip 101 in which the microchannel 110 is formed. As shown in FIG. 5, the liquid reservoir 116 has a rectangular shape in plan view. The liquid reservoir 116 is connected to the extending end of the second flow path 122. The liquid reservoir 116 communicates with the reaction tank 113 via the second flow path 122.
  • the channel width of the first channel 121 is 100 ⁇ m. Further, the channel depth of the first channel 121, that is, the depth in the normal direction of the paper surface of FIG. 6 is 60 ⁇ m.
  • the channel width of the second channel 122 is 100 ⁇ m, and the channel height of the second channel 122 is 6 ⁇ m.
  • This flow path height is a value of the minimum dimension of the cross section of the second flow path 122. Therefore, solid-phase antibody-modified beads having an average particle diameter of 20 ⁇ m described later cannot pass through.
  • the diameter of the input port 111 having a circular shape in plan view is 1 mm.
  • the reaction tank 113 having an oval shape in plan view has a major axis of 1000 ⁇ m and a minor axis of 500 ⁇ m.
  • the antigen-antibody reaction measurement method by the sandwich method using the microchannel chip 101 described above is as follows.
  • the labeled antibody 23 is prepared as in the first embodiment (step ST21).
  • a specimen having antigen 24 (VEGF antigen) as a measurement target is prepared (step ST31).
  • a phosphate buffer containing 0.1% BSA as a diluent is prepared (step ST32).
  • a solution is prepared by diluting the same antigen as the antigen to be measured with a phosphate buffer containing 0.1% BSA (step ST111). In this example, the concentration of the antigen was 0.01 ng / ml.
  • the same anti-VEGF monoclonal antibody as in the first embodiment is prepared (step ST11), and the surface is modified with polystyrene spherical beads having an average particle diameter of 20 ⁇ m (step ST12). If necessary, the surface of the spherical bead is subjected to a blocking treatment to prevent the antibody from adhering to the surface of the spherical bead. As a result, solid-phase antibody-modified beads in which the solid-phase antibody 32 is modified on the surface of spherical beads having an average particle diameter of 20 ⁇ m are generated.
  • the antigen 24 diluted with the phosphate buffer containing 0.1% BSA prepared in step ST111 is circulated through the solid phase antibody-modified beads and reacted for 15 minutes (step ST112). Thereafter, the solid phase antigen is washed with a phosphate buffer (step ST113).
  • the same luminescent substrate as in the first embodiment is prepared.
  • the preparation step 2 ⁇ L of the solid phase antibody-modified bead solution is added from the input port 111 of the microchannel 110, and the centrifugal force is applied by increasing the rotation speed to 5000 rpm. Thereby, the solution of the solid-phase antibody-modified beads passes through the first flow path 121 and is held in the reaction tank 113 in a state where the solid-phase antibody-modified beads remain.
  • the above is the preparation process.
  • the prepared specimen to be measured and the prepared diluent are mixed in equal amounts (step ST33) and injected into the microchannel 110 from the input port 111. Then, the microchannel chip 101 is rotated at 5000 rpm for 30 seconds, the mixed solution is sent to the reaction tank 113, and the mixed solution and the solid phase antibody-modified beads are incubated for 15 minutes to be reacted (step ST34).
  • the reaction tank 113 is washed to remove the remaining sample components. Specifically, a phosphate buffer is injected from the input port, and the rotating body is rotated at 5000 to 12000 rpm. The injected phosphate buffer flows in the order of the reaction tank 113, the second flow path 122, and the liquid reservoir 116. When all of the phosphate buffer is discharged to the liquid reservoir 116, the first washing of the solid phase antibody-modified beads is completed. Similarly, injection of phosphate buffer is repeated twice, and washing of the solid phase antibody-modified beads is repeated twice, for a total of three washes.
  • the anti-VEGF antibody solution with HRP label is injected into the input port 111 to the input port, and the anti-VEGF antibody solution with HRP label is sent to the reaction tank 113 while rotating the microchannel chip 101.
  • the HRP-labeled anti-VEGF antibody solution and the solid-phase antibody-modified beads are reacted by incubating for 15 minutes (step ST13).
  • the same washing as the washing of the solid-phase antibody-modified beads described above is performed three times on the solid-phase antibody-modified beads.
  • the luminescent substrate is inserted from the input port 111, the microchannel chip 101 is rotated, and the solution is sent to the reaction tank 113. Thereafter, the antigen concentration is measured by measuring the luminescence intensity (step ST14).
  • step ST112 of the second embodiment the antigen 24 diluted with the diluent and the solid phase antibody-modified beads are reacted before entering the reaction vessel 113, but the present invention is not limited to this.
  • the antigen 24 diluted with the diluent in the reaction tank 113 may be reacted with the solid-phase antibody-modified beads.
  • the solid phase antibody-modified beads are sufficiently washed with a phosphate buffer.
  • the channel height of the second channel 122 is 6 ⁇ m, but is not limited to this. The height may be such that the solid phase antibody-modified beads cannot pass.
  • the solution containing the same antigen 24 as the measurement target diluted with the diluent is circulated once through the solid phase antibody 32, but the number of times is not limited to one. What is necessary is just to distribute
  • the label of the labeled antibody 23 is a luminescent label, but is not limited thereto.
  • a fluorescent label may be used.
  • the label is a fluorescent protein such as an APC protein
  • the amount of the antigen 24 can be measured by irradiating the reaction tank 113 with excitation light and measuring the fluorescence.
  • the cleaning solution is not limited to the phosphate buffer.
  • the solution prepared in step ST111 is distributed around the solid phase antibody 32, although the antigen-antibody reaction between the antigen and the solid phase antibody 32 is caused, the present invention is not limited to this.
  • an antigen distribution step of circulating a solution containing the same antigen as the measurement target through the solid phase antibody once is provided. That's fine.

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Abstract

L'invention concerne un procédé de mesure de réactions antigène-anticorps utilisant une technique de prise en sandwich et comprenant: une étape de réaction antigène-anticorps consistant à lier un anticorps (32) en phase solide et un anticorps marqué (23) qui comprend un anticorps (21) modifié par un marqueur (22) d'identification dans un milieu contenant un échantillon comportant un antigène (24), de telle façon que l'antigène (24) soit pris en sandwich entre l'anticorps (32) en phase solide et l'anticorps marqué (23); et une étape de mesure de la concentration d'antigène consistant à mesurer la concentration de l'antigène (24) en identifiant le marqueur (22) d'identification. Le procédé comporte une étape de circulation d'antigène lors de laquelle une solution, contenant un antigène qui est identique à l'antigène (24) à mesurer dans l'échantillon, est mise en circulation au moins une fois dans l'anticorps (32) en phase solide avant de lier l'antigène (24) contenu dans l'échantillon avec l'anticorps (32) en phase solide et/ou avec l'anticorps marqué (23) lors de l'étape de réaction antigène-anticorps.
PCT/JP2012/081002 2012-11-29 2012-11-29 Procédé de mesure de réactions antigène-anticorps utilisant une technique sandwich WO2014083668A1 (fr)

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Citations (6)

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Publication number Priority date Publication date Assignee Title
DK52483A (da) * 1982-02-10 1983-08-11 Baker Terence S Immunoanalytisk fremgangsmaade til bestemmelse af antigen-koncentra tioner i oploesning, samt materialer og udstyr til anvendelse ved remgangsmaaden
GB2165046A (en) * 1982-02-10 1986-04-03 Baker Terence S Ligand molecule
JPH0694716A (ja) * 1992-02-05 1994-04-08 Kanebo Ltd 免疫測定法
JP2002365298A (ja) * 2001-06-08 2002-12-18 Tosoh Corp 測定妨害を低減する方法及び試薬組成物
JP2004205504A (ja) * 2002-12-13 2004-07-22 Meidensha Corp 内分泌撹乱化学物質の酵素免疫測定方法
JP2009300280A (ja) * 2008-06-13 2009-12-24 Sumika Enviro-Science Co Ltd 高感度酵素免疫測定法

Patent Citations (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DK52483A (da) * 1982-02-10 1983-08-11 Baker Terence S Immunoanalytisk fremgangsmaade til bestemmelse af antigen-koncentra tioner i oploesning, samt materialer og udstyr til anvendelse ved remgangsmaaden
EP0086095A2 (fr) * 1982-02-10 1983-08-17 Boots-Celltech Diagnostics Limited Essai
GB2116318A (en) * 1982-02-10 1983-09-21 Baker Terence S Immunoassay method
BR8300656A (pt) * 1982-02-10 1983-11-08 Baker Terence S Ensaio para determinar a concentracao de antigenos, processo para determinar o periodo fertil do ciclo menstrual, conjunto de reagente, e molecula ligante
AR231477A1 (es) * 1982-02-10 1984-11-30 Baker Terence S Una molecula ligando para ensayos y conjunto de reactivos que la contiene
US4508830A (en) * 1982-02-10 1985-04-02 Baker Terence S Assay
GB2165046A (en) * 1982-02-10 1986-04-03 Baker Terence S Ligand molecule
SG75486G (en) * 1982-02-10 1987-02-27 Boots Celltech Diagnostics Assay
ATE29595T1 (de) * 1982-02-10 1987-09-15 Boots Celltech Diagnostics Pruefverfahren.
DE3373543D1 (en) * 1982-02-10 1987-10-15 Boots Celltech Diagnostics Assay
IE54109B1 (en) * 1982-02-10 1989-06-21 Boots Celltech Diagnostics Assay
JPH0314140B2 (fr) * 1982-02-10 1991-02-26 Buutsu Serutetsuku Daiagunosuteitsukusu Ltd
JPH0694716A (ja) * 1992-02-05 1994-04-08 Kanebo Ltd 免疫測定法
JP2002365298A (ja) * 2001-06-08 2002-12-18 Tosoh Corp 測定妨害を低減する方法及び試薬組成物
JP2004205504A (ja) * 2002-12-13 2004-07-22 Meidensha Corp 内分泌撹乱化学物質の酵素免疫測定方法
JP2009300280A (ja) * 2008-06-13 2009-12-24 Sumika Enviro-Science Co Ltd 高感度酵素免疫測定法

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