WO2014083668A1 - Antigen-antibody reaction measurement method using sandwich technique - Google Patents

Antigen-antibody reaction measurement method using sandwich technique 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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French (fr)
Japanese (ja)
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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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    • 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

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  • 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

An antigen-antibody reaction measurement method using a sandwiching technique has: an antigen-antibody reaction step of binding a solid-phase antibody (32) and a labeled antibody (23) that has an antibody (21) modified by an identification label (22) in a medium containing a sample having an antigen (24), such that the antigen (24) is sandwiched between the solid-phase antibody (32) and the labeled antibody (23); and an antigen concentration measurement step of measuring the concentration of the antigen (24) by way of identifying the identification label (22). The method comprises an antigen circulation step wherein a solution containing an antigen that is the same as the antigen (24) that is to be measured in the sample is circulated at least once in the solid-phase antibody (32) before binding of the antigen (24) contained in the sample with the solid-phase antibody (32) and/or the labeled antibody (23) in the antigen-antibody reaction step.

Description

サンドイッチ法による抗原抗体反応測定方法Method for measuring antigen-antibody reaction by sandwich method
　本発明は、サンドイッチ法による抗原抗体反応測定方法に関する。 The present invention relates to a method for measuring an antigen-antibody reaction by a sandwich method.
　サンドイッチ法による抗原抗体反応測定方法では、予めマイクロプレートなどの容器の壁面や１０μｍ～４０μｍ程度の球形ビーズの表面に固相抗体をあらかじめ修飾させて準備する。そして、固相抗体と、測定対象となる抗原を含んだ検体中の当該抗原とを反応させる。
　その後、抗体に過酸化水素分解酵素や蛍光基質を標識として修飾した標識抗体を反応させることによって、抗原をサンドイッチした状態で標識抗体と固相抗体とを結合させる。このとき、抗原と結合していない標識抗体は、反応液中に分散したままの状態となっている。
　その後、洗浄工程により、抗原と結合していない標識抗体を洗い流す。このようにして、抗原を固相抗体と標識抗体とで挟む（サンドイッチする）ように、抗原と固相抗体と標識抗体とが結合されている状態となる。 In the method for measuring an antigen-antibody reaction by the 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 μm to 40 μm. 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.
Thereafter, the labeled antibody not bound to the antigen is washed away by a washing step. In this manner, 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.
　そして、このような状態の標識抗体に、標識を検出するための処理を施すことによって、抗原の数を測定したり、検体中の抗原濃度を測定したりすることができる。
　具体的には、標識抗体の標識として過酸化水素分解酵素（ＨＲＰ：Horseradish
Peroxidase）とルミノールと過酸化水素を含む発光基質とを用いて、ルミノール反応による発光を起こさせ、抗原濃度に比例した強度の発光を得ることができる。また、標識抗体の標識として蛍光基質を用いている場合には、標識抗体に励起光を照射することによって抗原濃度に比例した強度の蛍光を得ることができる。これらにより、抗原の数を測定したり、検体中の抗原濃度を測定したりすることができる（特許文献１、特許文献２参照）。 Then, 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.
Specifically, hydrogen peroxide-degrading enzyme (HRP)
Peroxidase), 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. In addition, when a fluorescent substrate is used as a label for the labeled antibody, fluorescence with an intensity proportional to the antigen concentration can be obtained by irradiating the labeled antibody with excitation light. By these, the number of antigens can be measured, or the antigen concentration in the specimen can be measured (see Patent Document 1 and Patent Document 2).
特開２００９－１５６７６５号公報JP 2009-156765 A 特開２０１０－２１６９４７号公報JP 2010-216947 A
　しかしながら、上述の測定においては、検体中の抗原濃度よりも高い抗原濃度が検出されることがある。検体中の抗原濃度が低い場合には、この現象が顕著に現れて、低濃度抗原に対して測定値が上昇して、実質的には低濃度抗原の測定はできない。つまり、図７に示すように、検体中の抗原濃度と測定値（発光や蛍光の光強度）の関係が単純な増加関数とならず、抗原濃度が増すと測定値が低くなる場合（抗原濃度が０．０１ｎｇ／ｍＬ未満の場合）が存在する。これにより、測定値から抗原濃度を計算することが一意的に決めることができず、低濃度抗原に対しては、その濃度を正確に測定できないこととなる。特に、初期の癌患者を早期発見したい場合には、低濃度抗原の測定ができないことは、大きな問題である。 However, in the above-described measurement, an antigen concentration higher than the antigen concentration in the specimen may be detected. When 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). As a result, 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. In particular, when it is desired to detect an early cancer patient at an early stage, it is a big problem that low concentration antigens cannot be measured.
　本発明は、低濃度抗原の測定が可能なサンドイッチ法による抗原抗体反応測定方法を提供することを目的とする。 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.
　本発明は、抗原を有する検体を含む媒質中で、固相抗体と、識別標識が修飾された抗体を有する標識抗体とで前記抗原を挟むように、前記固相抗体と前記標識抗体とを結合させる抗原抗体反応工程と、前記識別標識を識別することによって前記抗原の濃度を測定する抗原濃度測定工程とを有するサンドイッチ法を用いた抗原抗体反応測定方法であって、前記抗原抗体反応工程において前記検体に含まれる前記抗原と前記固相抗体及び／又は前記標識抗体とが結合する前に、前記検体の測定対象である抗原と同じ抗原を含む溶液を前記固相抗体に少なくとも１回流通させる抗原流通工程を備える、サンドイッチ法による抗原抗体反応測定方法に関する。
　ここで、前記抗原を含む溶液の流通の後、前記固相抗体を洗浄する洗浄工程を有することが好ましい。 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.
　本発明によれば、低濃度抗原の測定が可能なサンドイッチ法による抗原抗体反応測定方法を提供することができる。 According to the present invention, a method for measuring an antigen-antibody reaction by a sandwich method capable of measuring a low concentration antigen can be provided.
本実施形態に係るサンドイッチ法による抗原抗体反応測定方法を示すフローチャートである。It is a flowchart which shows the antigen antibody reaction measuring method by the sandwich method which concerns on this embodiment. 本実施形態に係るサンドイッチ法による抗原抗体反応測定方法において、基材３を固相抗体３２で修飾した様子を示す模式図である。It is a schematic diagram which shows a mode that the base material 3 was modified with the solid-phase antibody 32 in the antigen antibody reaction measuring method by the sandwich method which concerns on this embodiment. 本実施形態に係るサンドイッチ法による抗原抗体反応測定方法において、基材３を修飾する固相抗体３２に抗原２４が結合した様子を示す模式図である。In the antigen antibody reaction measuring method by the sandwich method which concerns on this embodiment, it is a schematic diagram which shows a mode that the antigen 24 couple | bonded with the solid-phase antibody 32 which modifies the base material 3. FIG. 本実施形態に係るサンドイッチ法による抗原抗体反応測定方法において、固相抗体３２に結合した抗原２４が標識抗体２３に結合した様子を示す模式図である。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. 第１実施例に係るサンドイッチ法による抗原抗体反応測定方法で用いられる９６穴マイクロプレート３を示す斜視図である。It is a perspective view which shows 96-well microplate 3 used with the antigen antibody reaction measuring method by the sandwich method based on 1st Example. 第１実施例に係るサンドイッチ法による抗原抗体反応測定方法により得られた結果を示すグラフである。It is a graph which shows the result obtained by the antigen antibody reaction measuring method by the sandwich method concerning a 1st example. 第２実施例に係るサンドイッチ法による抗原抗体反応測定方法で用いられるマイクロ流路チップ１０１を示す平面図である。It is a top view which shows the microchannel chip | tip 101 used with the antigen antibody reaction measuring method by the sandwich method based on 2nd Example. 第２実施例に係るサンドイッチ法による抗原抗体反応測定方法で用いられるマイクロ流路１１０を示す平面図である。It is a top view which shows the microchannel 110 used with the antigen antibody reaction measuring method by the sandwich method based on 2nd Example. 従来のサンドイッチ法による抗原抗体反応測定方法により得られた結果を示すグラフである。It is a graph which shows the result obtained by the antigen antibody reaction measuring method by the conventional sandwich method.
　本発明の実施形態によるサンドイッチ法による抗原抗体反応測定方法について、図面を参照しながら説明する。
　図１は、本実施形態に係るサンドイッチ法による抗原抗体反応測定方法を示すフローチャートである。図２Ａは、本実施形態に係るサンドイッチ法による抗原抗体反応測定方法において、基材（９６穴マイクロプレート３）を固相抗体３２で修飾した様子を示す模式図である。図２Ｂは、本実施形態に係るサンドイッチ法による抗原抗体反応測定方法において、基材を修飾する固相抗体３２に抗原２４が結合した様子を示す模式図である。図２Ｃは、本実施形態に係るサンドイッチ法による抗原抗体反応測定方法において、固相抗体３２に結合した抗原２４が標識抗体２３に結合した様子を示す模式図である。 A method for measuring an antigen-antibody reaction by a sandwich method according to an embodiment of the present invention will be described with reference to the drawings.
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.
　サンドイッチ法による抗原抗体反応測定方法は、準備工程と抗原抗体反応工程と、抗原濃度測定工程とを有する。
　準備工程では、固相抗体３２を用意する（ステップＳＴ１１）。また、測定対象である抗原２４を有する検体を用意する（ステップＳＴ３１）。また、測定対象である検体を希釈するための希釈液を用意する（ステップＳＴ３２）。
　また、識別標識２２（図２Ｃ等参照）で抗体２１を修飾して、識別標識２２で修飾された抗体２１を有する標識抗体２３を用意する（ステップＳＴ２１）。 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.
In the preparation step, a solid phase antibody 32 is prepared (step ST11). In addition, a specimen having the antigen 24 to be measured is prepared (step ST31). In addition, a diluent for diluting the sample to be measured is prepared (step ST32).
Further, 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).
　また、準備工程は、固相抗体固定工程を有する。固相抗体固定工程では、図２Ａに示すように、用意した固相抗体３２を所定の基材（９６穴マイクロプレート３）上に修飾させる（ステップＳＴ１２）。説明の便宜上、図２Ａ～図２Ｃにおいては、基材として後述の９６穴マイクロプレート３を図示する。基材としては、９６穴マイクロプレート３に限定されない。例えば、平均粒径が１０μｍ～４０μｍ程度の球形ビーズを用いてもよい。 Also, the preparation step has a solid phase antibody fixing step. In the solid phase antibody fixing step, as shown in FIG. 2A, the prepared solid phase antibody 32 is modified on a predetermined substrate (96-well microplate 3) (step ST12). For convenience of explanation, 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. For example, spherical beads having an average particle size of about 10 μm to 40 μm may be used.
　また、測定対象である抗原と同じ抗原を含有する溶液を用意する（ステップＳＴ１１１）。
　また、抗原流通工程が行われる。抗原流通工程では、ステップＳＴ１１１で準備した溶液を固相抗体３２の周囲に流通させ、抗原と固相抗体３２との抗原抗体反応を生じさせる抗原流通工程を行う（ステップＳＴ１１２）。
　そして、固相抗体３２を洗浄する洗浄工程を行う（ステップＳＴ１１３）。従って、抗原抗体反応工程において、測定対象である検体に含まれる抗原２４と固相抗体３２との抗原抗体反応を生じさせる前に、固相抗体３２が洗浄される。以上が準備工程である。 In addition, a solution containing the same antigen as the antigen to be measured is prepared (step ST111).
In addition, an antigen distribution process is performed. In the antigen distribution step, 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).
Then, 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.
　次に、抗原抗体反応工程を行う。抗原抗体反応工程では、先ず、測定対象である検体と希釈液を混合させ（ステップＳＴ３３）、この混合液を、固相抗体を固定した基材に供給し，検体中の抗原２４と固相抗体３２を結合させる（ステップＳＴ３４）。結合後、必要に応じて洗浄工程を行う。 Next, an antigen-antibody reaction step is performed. In the antigen-antibody reaction step, first, 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.
　次に、ステップＳＴ２１で用意した識別標識２２が修飾された抗体２１からなる標識抗体２３を含む溶液を基材に供給し、固相抗体３２と、標識抗体２３とで抗原２４を挟むように、固相抗体３２と標識抗体２３とを結合させる（ステップＳＴ１３）。以上が抗原抗体反応工程である。 Next, 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.
　次に、抗原濃度測定工程を行う。抗原濃度測定工程では、抗原抗体反応工程において
固相抗体３２に結合した標識抗体２３を識別する（ステップＳＴ１４）。これにより、抗原２４の濃度を測定する。以上が抗原濃度測定工程である。 Next, an antigen concentration measurement step is performed. In the antigen concentration measurement step, 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.
　本実施形態よれば以下のような効果を発揮することができる。抗原抗体反応工程おいて前記検体に含まれる前記抗原と固相抗体３２とが結合する前に、準備工程として測定対象と同じ抗原を含む溶液を固相抗体３２に１回流通させる抗原流通工程を備える。そして、抗原２４を含む溶液の流通の後、固相抗体３２を洗浄する洗浄工程を有する。このため、抗原濃度測定工程における抗原濃度の測定において、検体中の抗原濃度より高い抗原濃度が検出されることを抑制することができる。 According to this embodiment, the following effects can be exhibited. In the antigen-antibody reaction step, before the antigen contained in the specimen and the solid phase antibody 32 are combined, 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.
（第１実施例）
　本実施例では、抗原抗体反応工程において、癌マーカーＶＥＧＦ（vascular endothelial growth factor）に対するＥＬＩＳＡ（酵素免疫吸着測定法）の基本的方法（バイアル中での操作）を、図３に示す９６穴マイクロプレート３での抗原抗体反応として行う。図３は、第１実施例に係るサンドイッチ法による抗原抗体反応測定方法で用いられる９６穴マイクロプレート３を示す斜視図である。 (First embodiment)
In this example, in the antigen-antibody reaction step, the basic method (operation in a vial) of ELISA (enzyme-linked immunosorbent assay) for the cancer marker VEGF (vascular endothelial growth factor) is shown in FIG. As an antigen-antibody reaction in step 3. 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.
　本実施例では、固相抗体３２を固定する基材として、９６穴マイクロプレート３を用いる。また、標識としてＨＲＰ（過酸化水素分解酵素：Horseradish peroxidase）を用い、発光基質としてルミノール溶液を用いる。また、抗原２４としては、ＶＥＧＦを用いる。詳細には以下のとおりである。 In this example, a 96-well microplate 3 is used as a base material on which the solid phase antibody 32 is fixed. In addition, HRP (hydrogen peroxide-degrading enzyme) is used as a label, and a luminol solution is used as a luminescent substrate. As the antigen 24, VEGF is used. Details are as follows.
　本実施例において必要な試薬は以下の通りである。
Water soluble carbodiimide (ＷＳＣ)　　　ＷＳＣ1mg+HCl水溶液(pH5)1mL
カーボネートバッファー（ＣＢ）　　　　25mM NaHCO3, 25mM Na2C3
(ph9.7)
リン酸バッファー（ＰＢＳ）　　　　　　0.02Mリン酸緩衝液(ph7.0)
トリスバッファー（ＴＢ）　　　　　　　0.1M Tris-HCl(ph8.6),
0.1M NaCl
(Wash Buffer)　ＷＢ　　　　　　　　　0.05％ Tween in ＰＢＳ
ブロッキング剤　ブロックエース　　　400ng/mL (イオン交換水)
ＶＥＧＦ抗原　　　100μg/ml　0.1％ＢＳＡ(Bovine Serum Albumin) ＰＢＳ中で復元。
　　　　　　　　　　　　　使用時には、0.1％ＢＳＡ　in　ＰＢＳで必要濃度に希釈。
酵素標識抗ＶＥＧＦ ＨＲＰ(Horseradish Peroxidase)標識付き抗ＶＥＧＦポリクロナール抗体（1mg/mLにイオン交換水で復元）　　　　　　　　希釈液0.1％ＢＳＡ in ＰＢＳ in 0.15MnaClで1000～5000倍希釈。
希釈溶液　　　　　　　　　　　　　　0.1％ ＢＳＡ in ＰＢＳ 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)
Phosphate buffer (PBS) 0.02M phosphate buffer (ph7.0)
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 μg / ml 0.1% BSA (Bovine Serum Albumin) Reconstituted in PBS.
When using, dilute to the required concentration with 0.1% BSA in PBS.
Enzyme-labeled anti-VEGF HRP (Horseradish Peroxidase) -labeled anti-VEGF polyclonal antibody (reconstituted with ion-exchanged water to 1 mg / mL) Diluted 1000-5000 times with 0.1% BSA in PBS in 0.15 MnaCl.
Diluted solution 0.1% BSA in PBS
　準備工程では、抗ＶＥＧＦモノクロナール抗体を用意し（ステップＳＴ１１）、抗ＶＥＧＦモノクロナール抗体を、９６穴マイクロプレート３の各ウエル３１の底面に修飾させる（ステップＳＴ１２）。具体的には、抗ＶＥＧＦモノクロナール抗体（１００μｇ／ｍｌ、ＰＢＳで再構成したもの)をＣＢで希釈し、２０μｇ／ｍｌにした抗ＶＥＧＦ抗体溶液を９６穴マイクロプレート３の各ウエル３１の底面に注入し、４℃で一晩インキュベート後、洗浄する。
　必要に応じて、各ウエル３１の底面に抗原や抗体が付かないようにするためのブロッキング処理を行う。これにより、９６穴マイクロプレート３の各ウエル３１の底面に固相抗体３２が修飾された抗体修飾プレートが生成される。 In the preparation step, 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). Specifically, an anti-VEGF monoclonal antibody (100 μg / ml, reconstituted with PBS) is diluted with CB, and an anti-VEGF antibody solution having a concentration of 20 μg / 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.
　また、準備工程では、測定対象である抗原２４（ＶＥＧＦ抗原）を有する検体を用意する（ステップＳＴ３１）。また、希釈液としての０．１％ＢＳＡ入りリン酸バッファーを用意する（ステップＳＴ３２）。希釈液として上記のバッファーを用いたが、これに限定されず、他の種類のバッファーやイオン交換水などを用いることもできる。 In the preparation step, 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). Although 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.
　また、準備工程では、ＨＲＰ標識付き抗ＶＥＧＦポリクロナール抗体を用意する（ステップＳＴ２１）。また、発光基質を用意する。具体的には、Ｌｕｍｉｎｏｌ
（Ｃ８Ｈ７Ｎ３Ｏ２＝１７７．１６）　８．９ｍｇ　をＴＢ４．８ｍＬ、 ＮａＯＨ
０．２ｍＬに溶かして液体１とする（１０ｍＭ　Ｌｕｍｉｎｏｌ）。また、Ｐ－ｐ－ｉｏｐｈｅｎｏｎｏｌ （ＰＩＰ＝Ｃ６Ｈ６Ｏ２＝２２０．１）１１ｍｇをエタノール５ｍＬに溶かして液体２とする（１０ｍＭ　ＰＩＰ）。そして、ＴＢ１８４２μＬに液体１を７５μＬ、液体２を８０μＬ加えて、更に３０％Ｈ２Ｏ２の１２倍希釈溶液３μＬを加えて調整することにより発光基質が生成される。 In the preparation step, an anti-VEGF polyclonal antibody with HRP label is prepared (step ST21). A luminescent substrate is also prepared. Specifically, Luminol
(C 8 H 7 N 3 O 2 = 177.16) 8.9 mg of TB 4.8 mL, NaOH
Dissolve in 0.2 mL to make Liquid 1 (10 mM Luminol). In addition, 11 mg of Pp-iophenolol (PIP = C 6 H 6 O 2 = 220.1) is dissolved in 5 mL of ethanol to obtain liquid 2 (10 mM PIP). Then, 75 μL of liquid 1 and 80 μL of liquid 2 are added to 1842 μL of TB, and 3 μL of a 12-fold diluted solution of 30% H 2 O 2 is further added to adjust the luminescent substrate.
　また、準備工程では、測定対象である抗原と同じ抗原を０．１％ＢＳＡ入りリン酸バッファーで希釈した溶液を用意する（ステップＳＴ１１１）。本実施例では、その抗原の濃度は０．０１ｎｇ／ｍｌとした。そして、９６穴マイクロプレート３の各ウエル３１の底面に固相抗体３２が修飾された抗体修飾プレートの、当該固相抗体３２に、用意した０．１％ＢＳＡ入りリン酸バッファーで希釈した抗原２４を流通させて２時間反応させる（ステップＳＴ１１２）。その後リン酸バッファーで固相抗原を洗浄する（ステップＳＴ１１３）。以上が準備工程である。 In the preparation step, 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). In this example, the concentration of the antigen was 0.01 ng / ml. Then, 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.
　抗原抗体反応工程では、先ず、検体を希釈液で２倍に希釈する（ステップＳＴ３３）。希釈液としては、０．１％ＢＳＡ入りリン酸バッファーを用いる。次に、希釈した検体を抗体修飾プレートの各ウエルに２００μＬずつ入れ、抗原抗体反応させる（ステップＳＴ３４）。反応時間は２時間である。反応後に、抗体修飾プレートをリン酸バッファーで洗浄し、溶液を全て捨てる。その後、ＨＲＰ標識抗ＶＥＧＦ抗体溶液を２００μＬずつ入れ、抗原抗体反応させる（ステップＳＴ１３）。反応時間は２時間である。反応後、抗原濃度測定工程を行う。 In the antigen-antibody reaction step, first, the specimen is diluted twice with a diluent (step ST33). As a diluent, a phosphate buffer containing 0.1% BSA is used. Next, 200 μL 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. After the reaction, the antibody-modified plate is washed with a phosphate buffer, and all the solution is discarded. Thereafter, 200 μL each of HRP-labeled anti-VEGF antibody solution is added to cause an antigen-antibody reaction (step ST13). The reaction time is 2 hours. After the reaction, an antigen concentration measurement step is performed.
　抗原濃度測定工程では、抗体修飾プレートをリン酸バッファーで洗浄し、溶液を全て捨てる。発光基質を入れて発光させ、発光強度をフォトマルなどの高感度光検出器で検出する（ステップＳＴ１４）。 In the antigen concentration measurement step, 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).
　本実施例において、測定対象である検体の抗原濃度を０．００ｎｇ／ｍＬ～１００ｎｇ／ｍＬと変えた場合の、発光量の値の変化は、図４に示すとおりである。図４は、第１実施例に係るサンドイッチ法による抗原抗体反応測定方法により得られた結果を示すグラフである。 In this example, when the antigen concentration of the sample to be measured is changed from 0.00 ng / mL to 100 ng / mL, the change in the luminescence value is as shown in FIG. 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.
　図４に示すように、抗原濃度が０．００ｎｇ／ｍＬから１００ｎｇ／ｍＬへと値が大きくなるにつれて、発光量の値も大きくなる増加関数となっている。従って、抗原濃度が０．００ｎｇ／ｍＬ～０．０１ｎｇ／ｍＬまでの間においても、抗原濃度測定工程において、正確に発光量を検出することができることが分かる。 As shown in FIG. 4, as 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.
（第２実施例）
　本実施例では、上述の抗原抗体反応工程において、９６穴マイクロプレート３に代えて、図５に示すマイクロ流路チップ１０１を用いる。図５は、第２実施例に係るサンドイッチ法による抗原抗体反応測定方法で用いられるマイクロ流路チップ１０１を示す平面図である。図６は、第２実施例に係るサンドイッチ法による抗原抗体反応測定方法で用いられるマイクロ流路１１０を示す平面図である。 (Second embodiment)
In this embodiment, in the above-described antigen-antibody reaction step, the microchannel chip 101 shown in FIG. 5 is used in place of the 96-well microplate 3. 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.
　マイクロ流路チップ１０１は、流体が流通可能な同一形状の複数のマイクロ流路１１０が形成された円盤状の表側ディスク状プレートにより構成されている。マイクロ流路１１０の後述の入力ポート１１１がマイクロ流路チップ１０１の中央に最も近くなるように、且つ、マイクロ流路１１０の後述の液溜１１６が、マイクロ流路チップ１０１の半径方向において、マイクロ流路チップ１０１の中央から最も遠くなるように、マイクロ流路１１０は、マイクロ流路チップ１０１の中央から放射状に配置されている。 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.
　マイクロ流路１１０が形成された表側ディスク状プレートの面には、裏側ディスク状プレート（図示せず）が、貼り付けられており、これらにより二層構造の検査ディスクが構成される。マイクロ流路チップ１０１は、表側ディスク状プレートはシリコーン樹脂により構成されており、裏側ディスク状プレート（図示せず）は、ガラスにより構成されている。円盤状の検査ディスクは、遠心力が作用するように、検査ディスクの軸心を回転軸心として回転駆動可能である。 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. In the microchannel chip 101, the front disk-shaped plate is made of silicone resin, and 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.
　図６に示すように、複数のマイクロ流路１１０は、それぞれ、入力ポート１１１と、反応槽１１３と、液溜１１６と、第１の流路１２１と、第２の流路１２２とを有する。 As shown in FIG. 6, 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.
　入力ポート１１１は、マイクロ流路１１０が形成されたマイクロ流路チップ１０１において、最もマイクロ流路チップ１０１の中心に近い位置に形成された室により構成されている。図６に示すように、入力ポート１１１は、平面視で円形を有しており、表側ディスク状プレート（図示せず）に形成された穴（図示せず）を通して、外部と連通している。
　第１の流路１２１は、検査ディスクが回転駆動されてマイクロ流路チップ１０１が回転させられるときに、入力ポート１１１において遠心力が作用する方向としてのマイクロ流路チップ１０１の半径方向外方へ、入力ポート１１１から延出する。 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.
　反応槽１１３は、マイクロ流路１１０が形成されたマイクロ流路チップ１０１において、入力ポート１１１よりも、マイクロ流路チップ１０１の半径方向外方の位置に形成された室により構成されている。図６に示すように、反応槽１１３は、平面視で長円形状を有している。反応槽１１３は、第１の流路１２１を介して、入力ポート１１１に連通する。 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.
　第２の流路１２２は、マイクロ流路チップ１０１が回転駆動されるときに、反応槽１１３において遠心力が作用する方向としてのマイクロ流路チップ１０１の半径方向外方へ、反応槽１１３から延出する。 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.
　液溜１１６は、マイクロ流路１１０が形成されたマイクロ流路チップ１０１において、反応槽１１３よりも、マイクロ流路チップ１０１の半径方向外方の位置に形成された室により構成されている。図５に示すように、液溜１１６は、平面視で長方形を有している。液溜１１６は、第２の流路１２２の延出端部に接続されている。液溜１１６は、第２の流路１２２を介して、反応槽１１３に連通する。 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.
　第１の流路１２１の流路幅、即ち、図６の紙面に平行な方向における幅は、１００μｍである。また、第１の流路１２１の流路深さ、即ち、図６の紙面の法線方向における深さは、６０μｍである。また、第２の流路１２２の流路幅は、１００μｍであり、第２の流路１２２の流路高さは、６μｍである。この流路高さは、第２の流路１２２の断面の最小寸法の値である。従って、後述の平均粒径２０μｍの固相抗体修飾ビーズは通り抜けることができない。
　また、平面視で円形状を有する入力ポート１１１の直径は、１ｍｍである。また、平面視で長円形状を有する反応槽１１３は、長軸が１０００μｍ、短軸が５００μｍの寸法を有している。 The channel width of the first channel 121, that is, the width in the direction parallel to the paper surface of FIG. 6, is 100 μm. 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 μm. The channel width of the second channel 122 is 100 μm, and the channel height of the second channel 122 is 6 μm. 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 μm 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 μm and a minor axis of 500 μm.
　上述のマイクロ流路チップ１０１を用いる、サンドイッチ法による抗原抗体反応測定方法は以下の通りである。
　準備工程では、第１実施例と同様に標識抗体２３を用意する（ステップＳＴ２１）。また、準備工程では、測定対象として抗原２４（ＶＥＧＦ抗原）を有する検体を用意する（ステップＳＴ３１）。また、準備工程では、希釈液としての０．１％ＢＳＡ入りリン酸バッファーを用意する（ステップＳＴ３２）。また、さらに、測定対象である抗原と同じ抗原を０．１％ＢＳＡ入りリン酸バッファーで希釈した溶液を用意する（ステップＳＴ１１１）。本実施例では、その抗原の濃度は０．０１ｎｇ／ｍｌとした。 The antigen-antibody reaction measurement method by the sandwich method using the microchannel chip 101 described above is as follows.
In the preparation step, the labeled antibody 23 is prepared as in the first embodiment (step ST21). In the preparation step, a specimen having antigen 24 (VEGF antigen) as a measurement target is prepared (step ST31). In the preparation step, a phosphate buffer containing 0.1% BSA as a diluent is prepared (step ST32). Further, 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.
　また、準備工程では、第１実施例と同様の抗ＶＥＧＦモノクロナール抗体を用意し（ステップＳＴ１１）、平均粒径２０μｍのポリスチレン球形ビーズの表面に修飾させる（ステップＳＴ１２）。必要に応じて、球形ビーズの表面に抗体が付かないようにするためのブロッキング処理を、球形ビーズの表面に行う。これにより、平均粒径２０μｍの球形ビーズの表面に固相抗体３２が修飾された固相抗体修飾ビーズが生成される。 Also, in the preparation step, 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 μm (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 μm are generated.
　また、準備工程では、固相抗体修飾ビーズに、ステップＳＴ１１１で用意した０．１％ＢＳＡ入りリン酸バッファーで希釈した抗原２４を流通させて１５分間反応させる（ステップＳＴ１１２）。その後リン酸バッファーで固相抗原を洗浄する（ステップＳＴ１１３）。また、準備工程では、第１実施例と同様の発光基質を用意する。 In the preparation step, 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). In the preparation step, the same luminescent substrate as in the first embodiment is prepared.
　また、準備工程では、マイクロ流路１１０の入力ポート１１１から、固相抗体修飾ビーズの溶液を２μＬ入れ、５０００ｒｐｍまで回転数を上げて、遠心力を加える。これにより、固相抗体修飾ビーズの溶液は、第１の流路１２１を通過し、反応槽１１３において固相抗体修飾ビーズが留まった状態で保持される。以上が準備工程である。 In addition, in the preparation step, 2 μL 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.
　抗原抗体反応工程では、先ず、測定対象である用意した検体と、用意した希釈液を同量ずつ混ぜて（ステップＳＴ３３）、入力ポート１１１からマイクロ流路１１０に注入する。そして、マイクロ流路チップ１０１を５０００ｒｐｍで３０秒回転させ、反応槽１１３まで上記混合液を送液し、混合液と固相抗体修飾ビーズとを１５分間インキュベートして反応させる（ステップＳＴ３４）。 In the antigen-antibody reaction step, first, 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).
　次に反応槽１１３を洗浄して、残留している検体成分を除去する。具体的には、入力ポートからリン酸バッファーを注入し、回転体を５０００～１２０００ｒｐｍで回転させる。注入されたリン酸バッファーは、反応槽１１３、第２の流路１２２、液溜１１６の順に流れる。全てのリン酸バッファーが全て液溜１１６に排出されたときに、固相抗体修飾ビーズの１回目の洗浄が終了する。同様にしてリン酸バッファーの注入を２回繰返して行い、固相抗体修飾ビーズの洗浄を２回繰返して、合計３回の洗浄を行う。 Next, 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.
　次に、入力ポートにＨＲＰ標識付き抗ＶＥＧＦ抗体溶液を入力ポート１１１へ注入し、マイクロ流路チップ１０１を回転させながら、ＨＲＰ標識付き抗ＶＥＧＦ抗体溶液を反応槽１１３まで送液する。これにより、ＨＲＰ標識付き抗ＶＥＧＦ抗体溶液と固相抗体修飾ビーズを１５分間インキュベートすることにより反応させる（ステップＳＴ１３）。次に前述した計３回の固相抗体修飾ビーズの洗浄と同様の洗浄を固相抗体修飾ビーズに対して行う。 Next, 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. Thus, the HRP-labeled anti-VEGF antibody solution and the solid-phase antibody-modified beads are reacted by incubating for 15 minutes (step ST13). Next, 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.
　抗原濃度測定工程では、入力ポート１１１から、発光基質を入れて、マイクロ流路チップ１０１を回転させて、反応槽１１３まで送液する。その後、発光強度を測定することで、抗原濃度を測定する（ステップＳＴ１４）。以上の工程により、第１実施例と同様の効果を得る。 In the antigen concentration measurement step, 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). Through the above steps, the same effect as in the first embodiment is obtained.
　本発明は、上述した実施形態及び実施例に限定されることはなく、特許請求の範囲に記載された技術的範囲において変形が可能である。
　例えば、第２実施例のステップＳＴ１１２では、反応槽１１３に入れる前に、希釈液で希釈した抗原２４と固相抗体修飾ビーズとを反応させているが、これに限定されない。固相抗体修飾ビーズを反応槽１１３に保持させた後に、反応槽１１３で希釈液で希釈した抗原２４と固相抗体修飾ビーズとを反応させてよい。この場合には、反応後に、固相抗体修飾ビーズをリン酸バッファーで十分に洗浄する。
　また、第２実施例では、第２の流路１２２の流路高さは６μｍであったが、これに限定されない。固相抗体修飾ビーズが通過できない程度の高さであればよい。 The present invention is not limited to the above-described embodiments and examples, and can be modified within the technical scope described in the claims.
For example, in 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. After the solid-phase antibody-modified beads are held in the reaction tank 113, the antigen 24 diluted with the diluent in the reaction tank 113 may be reacted with the solid-phase antibody-modified beads. In this case, after the reaction, the solid phase antibody-modified beads are sufficiently washed with a phosphate buffer.
In the second embodiment, the channel height of the second channel 122 is 6 μm, but is not limited to this. The height may be such that the solid phase antibody-modified beads cannot pass.
　また、上述の実施形態及び実施例では、希釈液で希釈した測定対象と同じ抗原２４を含む溶液を固相抗体３２に１回流通させたが、回数は１回に限定されない。希釈液で希釈した測定対象と同じ抗原２４を含む溶液を固相抗体３２に複数回流通させればよい。 In the above-described embodiment and examples, 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 | circulate the solution containing the same antigen 24 as the measuring object diluted with the dilution liquid to the solid-phase antibody 32 several times.
　また、標識抗体２３の標識は発光標識であったが、これに限定されない。例えば、蛍光標識であってもよい。標識がＡＰＣたんぱく質等の蛍光タンパク質である場合には、反応槽１１３に励起光を照射して蛍光を測定することによって、抗原２４の量を測定することができる。また、洗浄液は、リン酸バッファーに限られない。 Further, the label of the labeled antibody 23 is a luminescent label, but is not limited thereto. For example, a fluorescent label may be used. When 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. Further, the cleaning solution is not limited to the phosphate buffer.
　また、抗原流通工程では、測定対象である検体に含まれる抗原２４と固相抗体３２との抗原抗体反応を生じさせる前に、ステップＳＴ１１１で準備した溶液を固相抗体３２の周囲に流通させ、抗原と固相抗体３２との抗原抗体反応を生じさせたが、これに限定されない。前記検体に含まれる前記抗原と固相抗体及び／又は標識抗体とが結合する前に、準備工程として測定対象と同じ抗原を含む溶液を固相抗体に１回流通させる抗原流通工程を備えていればよい。 Further, in the antigen distribution step, before causing the antigen-antibody reaction between the antigen 24 contained in the sample to be measured and the solid phase antibody 32, 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. Before the antigen contained in the sample is bound to the solid phase antibody and / or the labeled antibody, as a preparation step, 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.
２２　識別標識
２３　標識抗体
２４　抗原
３２　固相抗体 22 Identification label 23 Labeled antibody 24 Antigen 32 Solid phase antibody

Claims (2)

  1. 　抗原を有する検体を含む媒質中で、固相抗体と、識別標識が修飾された抗体を有する標識抗体とで前記抗原を挟むように、前記固相抗体と前記標識抗体とを結合させる抗原抗体反応工程と、
    　前記識別標識を識別することによって前記抗原の濃度を測定する抗原濃度測定工程とを有するサンドイッチ法を用いた抗原抗体反応測定方法であって、
    　前記抗原抗体反応工程において前記検体に含まれる前記抗原と固相抗体及び／又は前記標識抗体とが結合する前に、前記検体の測定対象である抗原と同じ抗原を含む溶液を前記固相抗体に少なくとも１回流通させる抗原流通工程を備える、サンドイッチ法による抗原抗体反応測定方法。     Antigen-antibody reaction that binds the solid-phase antibody and the labeled antibody so that the antigen is sandwiched between a solid-phase antibody and a labeled antibody having an antibody with a modified identification label in a medium containing a specimen having an antigen Process,
    An antigen-antibody reaction measurement method using a sandwich method comprising an antigen concentration measurement step of measuring the concentration of the antigen by identifying the identification label,
    In the antigen-antibody reaction step, before the antigen contained in the specimen is bound to the solid phase antibody and / or the labeled antibody, a solution containing the same antigen as the antigen to be measured of the specimen is added to the solid phase antibody. A method for measuring an antigen-antibody reaction by a sandwich method, comprising an antigen distribution step of distributing at least once.
  2. 　前記抗原を含む溶液の流通の後、前記固相抗体を洗浄する洗浄工程を有する請求項１に記載のサンドイッチ法による抗原抗体反応測定方法。 The method for measuring an antigen-antibody reaction by a sandwich method according to claim 1, further comprising a washing step of washing the solid phase antibody after the solution containing the antigen is distributed.
PCT/JP2012/081002 2012-11-29 2012-11-29 Antigen-antibody reaction measurement method using sandwich technique WO2014083668A1 (en)

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