Immunoassay method and immunoassay device

Abstract

To provide an immunoassay method and an immunoassay device capable of quantifying a measurement target in a sample even if the concentration is low. [Solution] An immunoassay method comprising a container or substrate containing a sample containing an object to be measured and a plurality of labeled particles carrying antibodies that can bind directly or indirectly to the object to be measured, the method comprising a step of irradiating light onto the container or substrate after the sample containing the object to be measured has come into contact with the plurality of labeled particles carrying antibodies that can bind directly or indirectly to the object to be measured, wherein the light is irradiated into a region of the container or substrate that contains the labeled particles, and a step of detecting the irradiated light that has passed through the region of the container or substrate that contains the labeled particles, the wavelength of the irradiated light being a wavelength that causes Mie scattering by each of the plurality of labeled particles.

Description

[Technical Field]

[0001] The present invention relates to an immunoassay method and an immunoassay device. [Background technology]

[0002] Immunoassays (immunoassays) are methods that utilize specific antigen-antibody reactions to detect or quantify trace components (antigens or antibodies) in a sample. Because of their high sensitivity and selectivity, antigen-antibody reactions are widely used in fields such as medicine, pharmaceuticals, health foods, biotechnology, and the environment, for drugs and foods that act only on specific sites (chemicals) in the body, as well as analytical devices and diagnostic reagents that detect subtle changes in the body. There are various types of immunoassays, each based on different measurement principles. Examples include enzyme immunoassays (ELISAs), radioimmunoassays (RIAs), chemiluminescent immunoassays (CIMAs), fluorescent immunoassays (FIAs), immunoenzymometric assays (IEMAs) that use enzyme-labeled antigens, latex agglutination assays (LAA), latex agglutination inhibition assays (LAA), immunochromatography (ELISA), Western blotting (ELISA), hemagglutination assays (HEAs), and hemagglutination inhibition assays (HEAs). All of these methods share the commonality of utilizing specific antigen-antibody reactions to detect or quantify the target component.

[0003] In immunoassays, a labeling substance is generally attached to either an antibody, an antigen, or an antigen-antibody complex, and the analyte (antigen or antibody) is detected or quantified by measuring the color intensity of the labeling substance, the signal intensity of luminescence, fluorescence, radiation, or the like emitted by the labeling substance, or the turbidity of aggregates formed by crosslinking of the labeling substances. Known labeling substances include color-producing particles such as gold colloid particles, nanoparticles, and quantum dots; signal-producing substances such as radioactive elements, enzymes, chemiluminescent substances, and fluorescent dyes; and agglutinating substances such as latex and red blood cells. Among these labeling substances, colored particles allow the presence or absence and amount of the analyte to be easily determined visually or using a measuring device, and are therefore widely used, particularly when measurements must be performed in a short time, and are primarily used in immunochromatography. Patent Document 1 describes an immunoassay method using a lateral flow chromatography test strip containing, as a labeling substance, a resin-platinum complex in which a plurality of relatively small platinum particles are immobilized on a resin particle. It is described that this resin-platinum complex exhibits good color development and is excellent in durability and visibility. Patent document 2 describes a chromatography measurement device that develops a test solution on a chromatography test piece having at least three or more reagent immobilization sections arranged at intervals on a development layer, and determines the concentration of the substance to be measured in the test solution based on an optical signal obtained by irradiating light onto the chromatography test piece and detecting transmitted or reflected light from the chromatography test piece. Patent Document 3 describes an immunoassay method in which an antibody or antigen labeled with a precious metal colloid particle is prepared, a sample is added to the prepared antibody or antigen, and the antigen or antibody contained in the sample is reacted with the antibody or antigen to form a precious metal colloid-labeled immune complex. In addition, in a cell having at least one surface formed as a total reflection prism, measurement light having a wavelength between visible and infrared is incident on the total reflection prism to cause total reflection, and absorption of the measurement light occurring at the interface between the total reflection prism and a sample mixture containing the labeled immune complex is measured in an absorption region specific to the precious metal colloid-labeled immune complex. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Patent No. 6381642 [Patent Document 2] Japanese Patent Application Laid-Open No. 2006-250787 [Patent Document 3] Patent No. 3436982 Summary of the Invention [Problem to be solved by the invention]

[0005] However, conventional immunoassay methods using colored particles all measure the color intensity (color intensity) of the labeled substance visually or with an optical measurement device. While visual measurement of color intensity has the advantage of being easy and convenient without using an optical measurement device, it is prone to individual judgment errors, and there is a risk that differences in color intensity cannot be visually recognized even when the concentrations of the measured substance are different, resulting in the risk of misjudging the concentrations to be the same when they are actually different. The lower the concentration of the substance being measured, the weaker the color, which increases the risk of making accurate measurements as described above. Furthermore, visual measurement poses problems with reproducibility because test results cannot be quantified. On the other hand, when measuring color intensity using an optical measurement device such as an absorbance meter, a light source is irradiated onto the colored area caused by the labeled substance, and the color intensity of the reflected light is measured using a photodetector. However, there is a limit to the absorbance per labeled particle, and particularly in the low concentration range, the number of labeled particles bound to the analyte is small, resulting in poor sensitivity. As such, conventional methods are limited in the range of measurable concentrations, making it difficult to detect or quantify the analyte with high sensitivity, especially in the low concentration range.

[0006] Furthermore, the immunochromatography optical measurement device described in Patent Document 2 mentions determining the concentration of an analyte in a test solution based on an optical signal obtained by detecting transmitted or reflected light from a chromatography test strip (immunochromatographic strip), but does not provide a detailed description of a measurement method using transmitted light, and the examples do not include components for such a measurement. Patent Document 2 also does not focus on the relationship between the wavelength of irradiated light and the particle size of labeled particles, much less mentions irradiating light under conditions that generate Mie scattering. Furthermore, it does not address the issue of being able to detect or quantify an analyte even in a low concentration range. Patent Document 1 similarly does not focus on the relationship between the wavelength of irradiated light and the particle size of labeled particles, much less mentions irradiating light under conditions that generate Mie scattering. Patent Document 3 does not mention the ability to detect or quantify the object to be measured even in a low concentration range as an issue, but rather clearly describes that methods using Rayleigh scattering or Mie scattering have the problem of low detection sensitivity and measurement accuracy at low concentrations.

[0007] The present invention has been made in view of the above points, and has as its object to provide an immunoassay method and an immunoassay device that can detect or quantify an object to be measured even in a low concentration range. [Means for solving the problem]

[0008] In order to solve the above problems, the inventors have conducted extensive research and found that, instead of irradiating labeled particles with measurement light and measuring the color intensity of the reflected light, it is possible to detect or quantify a measurement target at a lower concentration than conventionally possible by irradiating labeled particles with measurement light under conditions in which Mie scattering occurs and detecting the light intensity of the transmitted light.

[0009] The gist of the present invention is as follows. [1] A step of irradiating a measurement light onto a container or substrate containing a sample containing an analyte and a plurality of labeled particles carrying antibodies capable of directly or indirectly binding to the analyte after the sample containing the analyte and the plurality of labeled particles carrying antibodies capable of directly or indirectly binding to the analyte have come into contact with each other, wherein the measurement light is irradiated onto an area of ​​the container or substrate containing the labeled particles; and a step of detecting light (transmitted light) that has passed through the region out of the irradiated measurement light; Including, An immunoassay method, wherein the wavelength of the irradiated measurement light is a wavelength that causes Mie scattering by each of the plurality of labeled particles. [2] The immunoassay method according to [1], wherein the wavelength at which Mie scattering occurs by each of the plurality of labeled particles satisfies 2<α<10 (where α=π×particle diameter (nm) of labeled particle / wavelength of light (nm)). [3] The immunoassay method according to [1] or [2], further comprising a step of separating, after the contact and before the irradiation of the measurement light, the antibodies carried by the labeled particles that are directly or indirectly bound to the object to be measured from the antibodies carried by the labeled particles that are not bound to the object to be measured. [4] The immunoassay method according to any one of [1] to [3], further comprising a step of determining the concentration or amount of the analyte from the light intensity of the detected transmitted light. [5] The immunoassay method according to any one of [1] to [4], wherein the step of irradiating the measurement light includes irradiating the measurement light into a region of the container or substrate containing the plurality of labeled particles (hereinafter referred to as a first region), and irradiating the measurement light into a second region outside the first region. [6] The immunoassay method according to [5], comprising measuring a light intensity 1 of the measurement light transmitted through the first region and a light intensity 2 of the measurement light transmitted through the second region. [7] The following formula: Light attenuation rate = (light intensity 2 - light intensity 1) x 100 / light intensity 2 The immunoassay method according to [6], further comprising a step of determining the light attenuation rate by the following method. [8] The immunoassay method according to [7], further comprising the step of obtaining a calibration curve or an approximate straight line showing the relationship between the concentration or amount of the target substance and the light attenuation rate using a sample in which the concentration or amount of the target substance is known in advance. [9] The immunoassay method according to [7], further comprising the step of determining a light attenuation rate using a sample containing an analyte whose concentration or amount is unknown, and comparing the light attenuation rate with the calibration curve or approximate line obtained in advance in [8], thereby estimating the concentration or amount of the analyte contained in the sample.

[10] The immunoassay method according to any one of [1] to [9], wherein the antibody capable of directly or indirectly binding to the analyte is an antibody capable of specifically binding to the analyte.

[11] The immunoassay method according to any one of [1] to

[10] , wherein the light is coherent measurement light.

[12] The immunoassay method according to

[11] , wherein the coherent measurement light is laser light.

[13] The immunoassay method according to any one of [1] to

[12] , wherein the labeled particles contain a metal.

[14] The immunoassay method according to any one of [1] to

[13] , wherein the labeled particles are metal-resin composite particles in which a plurality of metal particles are immobilized on a resin particle, or in which a plurality of metal particles are at least partially immobilized within a resin particle.

[15] The immunoassay method according to

[13] or

[14] , wherein the metal is silver, nickel, copper, gold, platinum, or palladium, or an alloy containing any of these.

[16] The immunoassay method according to

[13] or

[14] , wherein the metal comprises platinum.

[17] The immunoassay method according to any one of [1] to

[16] , wherein the container or substrate is a well plate, a test strip, or a membrane.

[18] An immunoassay device, a holder for holding, in or on the device, a container or substrate containing a plurality of labeled particles carrying antibodies capable of directly or indirectly binding to the object to be measured; a light source disposed above the holder for irradiating a measurement light into an area of ​​the container or substrate held by the holder, the area including the plurality of labeled particles; a light detection unit that is disposed on the opposite side of the container or the substrate from the light source and detects transmitted light that has been irradiated from the light source and transmitted through an area of ​​the container or the substrate that contains the labeled particles; and the wavelength of the measurement light emitted by the light source is a wavelength that causes Mie scattering by each of the plurality of label particles; The immunoassay device.

[19] The immunoassay device according to

[18] , for use in the method according to any one of [1] to

[17] .

[20] The immunoassay device according to

[18] or

[19] , wherein the wavelength at which Mie scattering occurs by each of the plurality of labeled particles satisfies 2<α<10 (where α=π×particle diameter (nm) of labeled particle / wavelength of light (nm)).

[21] The immunoassay device according to any one of

[18] to

[20] , further comprising a container or substrate containing a plurality of labeled particles carrying antibodies capable of directly or indirectly binding to the object to be measured.

[22] The immunoassay device according to any one of

[18] to

[21] , wherein the antibody capable of directly or indirectly binding to the object to be measured is an antibody capable of specifically binding to the object to be measured.

[23] The immunoassay device according to any one of

[18] to

[22] , further comprising a display unit that displays the light intensity of the transmitted light detected by the light detection unit.

[24] The immunoassay device according to any one of

[18] to

[23] , further comprising an estimation unit that estimates the amount, concentration, or quantity of the target substance contained in the sample based on the light intensity of the transmitted light detected by the light detection unit.

[25] The immunoassay device is configured to irradiate a measurement light onto a region (first region) of the container or substrate containing the plurality of labeled particles and a region (second region) of the container or substrate outside the first region. The immunoassay device according to any one of

[18] to

[24] .

[26] The immunoassay device according to

[25] , further comprising an estimation unit that estimates the concentration or amount of the target substance contained in the sample based on light intensity 1 when measurement light is irradiated into the first region and light intensity 2 when measurement light is irradiated into the second region.

[27] The immunoassay device according to any one of

[18] to

[26] , wherein the light source is a light source that irradiates coherent light.

[28] The immunoassay device according to

[27] , wherein the coherent measurement light is laser light.

[29] An immunoassay device according to any one of

[18] to

[28] , wherein the light source is a light source that can irradiate measurement light onto an area that is within the area containing labeled particles in the container or substrate and is narrower than the area when viewed from the light source side toward the light detection unit.

[30] An immunoassay device according to any one of

[18] to

[29] , further comprising a slit portion between the light source and the container or substrate, whereby, when viewed from the light source side in the direction of the light detection portion, measurement light from the light source is irradiated onto an area within the container or substrate containing labeled particles, but within a range narrower than the area.

[31] An immunoassay device according to any one of

[18] to

[30] , further comprising a slit portion between the container or substrate and the detection unit, whereby only the measurement light irradiated into the region containing the labeled particles is detected by the light detection unit.

[32] The immunoassay device according to any one of

[18] to

[31] , wherein the label particles contain a metal.

[33] The immunoassay device according to any one of

[18] to

[32] , wherein the labeled particles are metal-resin composite particles in which a plurality of metal particles are immobilized on the surface of a resin particle.

[34] The immunoassay device according to

[32] or

[33] , wherein the metal comprises gold or platinum.

[35] The immunoassay device according to

[32] or

[33] , wherein the metal includes platinum.

[36] The immunoassay device according to any one of

[18] to

[35] , wherein the plurality of labeled particles are present in the region overlapping each other three-dimensionally when viewed from the light source toward the light detection unit.

[37] The immunoassay device according to any one of

[18] to

[36] , wherein the container or substrate is a well plate, a test strip, or a membrane.

[38] The immunoassay device according to any one of

[18] to

[37] , for estimating the concentration or amount of a target substance contained in a sample.

[39] The device according to any one of

[18] to

[38] , a container or substrate containing a plurality of labeled particles carrying antibodies capable of directly or indirectly binding to the object to be measured; A kit for immunoassay comprising:

[40] The device according to any one of

[18] to

[38] , a container or substrate capable of arranging a plurality of labeled particles carrying antibodies capable of directly or indirectly binding to the object to be measured; A kit for immunoassay comprising:

[41] The immunoassay kit according to

[40] , further comprising a plurality of labeled particles carrying antibodies capable of directly or indirectly binding to the object to be measured.

[42] A container or substrate containing a plurality of labeled particles carrying antibodies capable of directly or indirectly binding to the object to be measured, for use in the method according to any one of [1] to

[17] , for use in the device according to any one of

[18] to

[38] , for use in the kit according to

[39] , or for use in the kit according to any one of

[40] to

[41] .

[43] The container or substrate according to

[42] , which is a well plate, a test strip, or a membrane.

[44] The container or substrate according to

[42] , wherein the antibody capable of directly or indirectly binding to the analyte is an antibody capable of specifically binding to the analyte. [Effects of the Invention]

[0010] The immunoassay method and immunoassay device of the present invention can detect or quantify the measurement target with high sensitivity even in a low concentration range. [Brief explanation of the drawings]

[0011] [Figure 1] FIG. 1 is a schematic longitudinal cross-sectional view of an immunochromatographic test strip used in one embodiment of the present invention. [Figure 2] FIG. 2 is a schematic diagram of a housing case used in the test strip of FIG. 1, as viewed from the top side. [Figure 3] FIG. 3 is a schematic diagram of a housing case used in the test strip of FIG. 1, viewed from the bottom side. [Figure 4] FIG. 4 is a schematic longitudinal cross-sectional view of a conventional immunochromatographic test strip. [Figure 5] FIG. 5 is a schematic longitudinal cross-sectional view of an immunoassay device according to one embodiment of the present invention. [Figure 6] FIG. 6 is an external view (perspective view) of an immunoassay device according to one embodiment of the present invention. [Figure 7] FIG. 7 is a schematic longitudinal cross-sectional view of an immunoassay device according to another embodiment of the present invention. [Figure 8] FIG. 8 is a graph showing the relationship between the dilution rate of the measurement object and the light attenuation rate in Example 1. [Figure 9] FIG. 9 is a graph showing the relationship between the dilution ratio of the measurement subject and the color intensity in Comparative Examples 1 and 2. [Figure 10] FIG. 10 is a graph showing the relationship between the dilution ratio of the measurement object and the light attenuation rate in Example 1 and Comparative Example 3. [Figure 11]FIG. 11 is a graph showing the relationship between the dilution rate of the measurement object and the light attenuation rate when light of various wavelengths is irradiated under conditions that cause Mie scattering. [Figure 12] FIG. 12 is a graph showing the relationship between the dilution rate of the measurement target and the light attenuation rate when label particles of various particle sizes are used under conditions that cause Mie scattering. DETAILED DESCRIPTION OF THE INVENTION

[0012] <<Immunoassay method>> A first aspect of the present invention is a method for detecting a target substance in a container or a substrate containing a sample containing a target substance and a plurality of labeled particles carrying antibodies capable of binding directly or indirectly to the target substance, the method comprising: a step of irradiating a measurement light onto the container or substrate after the sample containing the target substance has come into contact with the plurality of labeled particles carrying antibodies capable of binding directly or indirectly to the target substance; wherein the measurement light is irradiated into a region of the container or substrate containing the labeled particles; a step of detecting light (transmitted light) that has passed through the region out of the irradiated measurement light; Including, The immunoassay method is such that the wavelength of the irradiated measurement light is a wavelength that causes Mie scattering by each of the plurality of labeled particles.

[0013] The method of the first aspect can be preferably used to detect an analyte contained in a sample and / or to quantify the analyte (estimate the concentration or amount of the analyte), and particularly preferably used to quantify the analyte (estimate the concentration or amount of the analyte). Each step and component will be described below.

[0014] <Step of irradiating measurement light> The method of the first aspect includes a container or a substrate containing a sample containing an analyte and a plurality of labeled particles carrying antibodies capable of binding directly or indirectly to the analyte, and includes a step of irradiating the container or substrate with measurement light after the sample containing the analyte has come into contact with the plurality of labeled particles carrying antibodies capable of binding directly or indirectly to the analyte, wherein the measurement light is irradiated within a region containing the labeled particles. In the step of irradiating the measurement light, the measurement light is irradiated into an area of ​​the container or the substrate that contains a plurality of label particles. For example, in the case of (lateral flow) immunochromatography, labeled particles that label the measurement target are captured on a test line, and the width of the irradiated measurement light is preferably the same as or narrower than the width of the test line, and more preferably narrower than the width of the test line. By irradiating the area containing the labeled particles, noise can be reduced, signal intensity can be increased, and sensitivity can be improved in the low concentration range. The measurement light may be irradiated into a first region of the container or substrate containing a plurality of labeled particles and into a second region (preferably a region not containing a plurality of labeled particles) different from the first region. The measurement light may be irradiated into the first region and the second region simultaneously or at different times. In the case of simultaneous irradiation, two light sources may be provided to simultaneously irradiate the measurement light into the first region and the second region. The measurement light may be irradiated from the two light sources sequentially, so that the measurement light is irradiated into the first region and the second region at different times. In addition, when the measurement light is irradiated into the first region and the second region at different times, the measurement light may be irradiated into the first region with the light source positioned above the first region, and then the container or substrate may be moved laterally (perpendicular to the direction from the light source to the light detection unit) until the light source is positioned above the second region, and the measurement light may be irradiated into the second region. Alternatively, the light source may be positioned above the first region, and the measurement light may be irradiated onto the first region, and then the light source may be moved laterally (perpendicular to the direction from the light source toward the light detection unit) until the light source is positioned above the second region, and the measurement light may be irradiated onto the second region. The order in which the measurement light is irradiated onto the first and second regions is not particularly limited. The measurement light may be irradiated onto the first region first, and then onto the second region, or the measurement light may be irradiated onto the second region first, and then onto the first region. A sliding device, as described below, may be used as a means for moving the container or substrate.

[0015] (measurement light) The measurement light is light emitted from a light source for the purpose of immunoassay (immunoassay). The wavelength of the irradiated measurement light is a wavelength that causes Mie scattering by each of the plurality of label particles. When the measurement light hits a particle, it transmits, reflects, refracts, or scatters. Of these, scattering is classified as light blocking, diffraction, Rayleigh scattering, or Mie scattering depending on the wavelength of the measurement light and the particle size of the particle irradiated with the measurement light. When the particle size parameter α, expressed as α = π × particle size (nm) / light wavelength (nm), is 10 4 <α, shading occurs, and 10<α<10 4It is known that diffraction occurs when α is , Mie scattering occurs when 2<α<10, and Rayleigh scattering occurs when α<2 (for example, see "Fundamental Properties of Powders" published by the Nikkan Kogyo Shimbun, edited by the Society of Powder Technology, p. 25). Note that particle size refers to the diameter of the particle. In the present invention, the wavelength of the irradiated measurement light is a wavelength that causes Mie scattering by each of the multiple labeled particles. The wavelength that causes Mie scattering by each of the multiple labeled particles is preferably a wavelength that satisfies 2<α<10 (where α=π×diameter (nm) of labeled particle) / wavelength of light (nm)). Based on this formula, for example, when labeled particles having a diameter of 450 nm are used as labeled particles, it is preferable to irradiate the measurement light with a wavelength in the range of approximately 141.3 nm to approximately 706.5 nm as the wavelength of light that causes Mie scattering. Furthermore, the irradiated measurement light is preferably coherent light, and more preferably light with coherent properties such as laser light. LED light has a broad wavelength range and is not uniform in phase, and therefore has the characteristic of diverging. Therefore, the scattering characteristics of light in the dispersion medium are also not uniform, and the light has poor penetration. However, LEDs can also be used as long as they have the above characteristics. In contrast, coherent light (particularly laser light) has uniform properties such as wavelength and phase, and is characterized by its high linearity. Using coherent light (particularly laser light) makes it easier to set the relationship between particle size and the wavelength of incident light, which determines particle parameters.

[0016] (Measurement target) The measurement target is not particularly limited, and various targets can be used. The measurement target may be an antigen or an antibody. Examples of measurement targets include bacteria, fungi, viruses, nucleic acids, proteins, sugars, lipids, electrolytes, enzymes, hormones, tumor markers, drugs, and various antibodies produced in vivo. The sample is not particularly limited as long as it can contain the target substance, and various samples can be used. For example, the sample may be one generally used in clinical tests, such as tissues, cells, cell extracts, and body fluids.

[0017] (Labeled particles carrying antibodies that can bind directly or indirectly to the target substance) The labeled particles carrying an antibody capable of binding directly or indirectly to the object to be measured may be labeled particles carrying an antibody capable of binding directly to the object to be measured, or may be labeled particles carrying an antibody capable of binding indirectly to the object to be measured via another substance. The phrase "a labeled particle carries an antibody that can directly bind to the object to be measured" means that the labeled particle is bound to an antibody that directly binds to the object to be measured (for example, if the object to be measured is an antigen, the labeled particle is bound to a monoclonal or polyclonal antibody that specifically binds to the antigen, and if the object to be measured is an antibody (which is also an antigen), the labeled particle is bound to a monoclonal or polyclonal antibody that binds to the antibody). The phrase "a labeled particle carries an antibody capable of indirectly binding to the analyte" means that the labeled particle is bound to an antibody capable of binding to a substance that directly binds to the analyte (for example, if the substance that directly binds to the analyte is a primary antibody, a secondary antibody that binds to the primary antibody). An example of a labeled particle carrying an antibody that can directly bind to the analyte is a labeled particle carrying an antibody that can (specifically) bind to an antigen when the analyte is an antigen. When the analyte is an antibody, an example of a labeled particle carrying an antibody that can (specifically) bind to the antibody is an antibody. The antibody that can (specifically) bind to the analyte may be commercially available or may be prepared in-house. The labeled particles carrying an antibody capable of binding to a substance capable of binding (directly or indirectly) to the analyte can be labeled particles carrying an antibody capable of binding to a substance capable of binding to the analyte via another substance. For example, the antibody capable of binding to the analyte indirectly via another substance may be a secondary antibody. In this case, the secondary antibody carried by the labeled particles binds to an antibody (primary antibody) capable of directly binding to the antigen that is the analyte, thereby labeling the analyte with the labeled particles. Examples of the analyte and an antibody that can bind directly or indirectly to the analyte can be found in the DOJINDO product catalog, "First Antibody Labeling Protocol - 1" (URL) https: / / www.dojindo.co.jp / products / contents / inomu-antibody-enzyme-fluo-protein-fab-labeling-1.html, under the heading "II. Advantages and Disadvantages of Each Immunoassay Method," covering the direct method, indirect method, and sensitization method. Therefore, in the method of the first aspect, the container or substrate contains an object to be measured, a substance that can specifically bind to the object to be measured but is not carried by labeled particles, and a plurality of labeled particles carrying antibodies that can indirectly bind to the object to be measured (for example, a plurality of labeled particles carrying antibodies that can bind to the substance that can specifically bind to the object to be measured), and upon contact between these, the antibodies carried by the labeled particles may indirectly bind to the object to be measured via the substance that specifically binds to the object to be measured.

[0018] (labeled particles) The method of the present invention uses a plurality of labeled particles carrying antibodies capable of binding directly or indirectly to the object to be measured. A plurality of labeled particles carrying antibodies capable of binding directly or indirectly to the analyte refers to a plurality of labeled particles, each of which carries an antibody capable of binding directly or indirectly to the analyte. Each labeled particle may carry one antibody capable of binding directly or indirectly to the analyte, or may carry multiple antibodies. When multiple antibodies are carried, the multiple antibodies may be the same or different. "Supported" refers to immobilization of an antibody capable of directly or indirectly binding to the analyte on the surface of a labeled particle, preferably the surface of the labeled particle. There are no particular limitations on the method for immobilizing an antibody capable of directly or indirectly binding to the analyte on the surface of the labeled particle. For example, an antibody capable of directly or indirectly binding to the analyte can be immobilized on the surface of the labeled particle by a passive adsorption method or a covalent bond via a crosslinker. For information on passive adsorption methods and covalent bond via a crosslinker, see, for example, the following URL (https: / / www.cosmobio.co.jp / support / technology / gold-nanoparticles-technical-note / covalent-conjugation-carboxylated-gold-nanoparticles-ctd.asp). Alternatively, the antibody may be immobilized on the labeled particle via a linker. The particle size of the labeled particles is not particularly limited as long as Mie scattering occurs when irradiated light strikes each labeled particle, and labeled particles of various particle sizes can be used. Among these, labeled particles that satisfy the relationship 2<α<10 (where α=π×particle size (nm) of labeled particles / wavelength of light (nm)) are preferred. Based on this formula, for example, when irradiating with light of a wavelength of 450 nm, the particle size of the labeled particles is preferably within the range of approximately 286.6 nm to approximately 1433.1 nm. Furthermore, for example, when the irradiated light is visible light (e.g., wavelength of approximately 360 nm to approximately 830 nm), the particle size of the labeled particles is preferably within the range of approximately 229.3 nm to approximately 2643.3 nm. Furthermore, in the present invention, the irradiated light is not limited to visible light. For example, even when using ultraviolet or infrared light, the particle size may be selected within the range in which Mie scattering occurs. The particle size (or average particle size) of the labeled particles may be within the range of approximately 528.7 nm to approximately 3.2 mm when irradiated with infrared light (e.g., wavelengths of approximately 830 nm to approximately 1 mm), and may be within the range of approximately 6.4 nm to approximately 1146.5 nm when irradiated with ultraviolet light (approximately 10 nm to approximately 360 nm). It is preferable that the labeled particles are kept in a uniformly dispersed state in the measurement medium. The average particle size can be measured using a centrifugal sedimentation particle size analyzer, laser diffraction / scattering method, dynamic light scattering method, particle counter, field emission scanning electron microscope, etc. The average particle size can be determined by the area average diameter or the like, and is the diameter of the particles.

[0019] The labeled particles are preferably particles through which the irradiated measurement light does not pass. If light does not pass through the labeled particles, the light that passes through the region containing the particles and reaches the detector becomes light derived from Mie scattering, which tends to increase sensitivity. As the labeled particles through which light does not pass, labeled particles containing metal are preferred, and other examples include light-blocking resin particles and particles using silica. For example, resin particles include polyamines, polyamides, polypeptides, polyurethanes, polystyrenes, polyureas, polyimides, polyimidazoles, polyoxazoles, polypyrroles, polyanilines, etc. Furthermore, a wide range of resins can be used, including polyamines such as poly-2-vinylpyridine, poly-3-vinylpyridine, and poly-4-vinylpyridine, acrylic resins, phenolic resins, epoxy resins, cellulose resins, and melamine resins. The labeled particles preferably contain a metal, and more preferably contain a metal on at least a portion or all of the surface of the labeled particles. By including a metal in the labeled particles, it becomes easier to obtain particles that do not transmit the irradiated measurement light through the labeled particles. The labeled particles may be particles made of metal, particles in which a metal is coated on the surface of a non-metallic particle, or particles in which multiple metal particles are immobilized on and / or within a non-metallic particle. Among these, the labeled particles are preferably particles in which a metal is coated on the surface of a non-metallic particle, particles in which multiple metal particles are immobilized on a non-metallic particle, or particles in which multiple metal particles are at least partially immobilized within a non-metallic particle, more preferably particles in which multiple metal particles are immobilized on a non-metallic particle or particles in which multiple metal particles are at least partially immobilized within a non-metallic particle, and even more preferably metal-resin composite particles in which multiple metal particles are immobilized on a resin particle, or metal-resin composite particles in which multiple metal particles are at least partially immobilized within a particle. The phrase "a plurality of metal particles are at least partially fixed within non-metallic particles" means that the plurality of metal particles are at least partially fixed within the non-metallic particles (preferably, some of the plurality of metal particles are exposed outside the non-metallic particles, and the remainder are present in a state encapsulated within the non-metallic particles). The phrase "a plurality of metal particles are at least partially fixed within resin particles" means that the plurality of metal particles are at least partially fixed within the resin particles (preferably, some of the plurality of metal particles are exposed outside the resin particles, and the remainder are present in a state encapsulated within the resin particles). Examples of metal-resin composite particles in which multiple metal particles are fixed to resin particles and methods for producing the same include the metal-resin composite particles and methods for producing the same described in International Publication WO2017 / 010391. Other examples of metal-resin composite particles include those having a structure in which the surface of a metal particle is coated with a resin, and those having a structure in which a metal layer (which may be an aggregate of metal particles) is laminated on the surface of a resin particle. Furthermore, there are no particular limitations on the type of metal, and various metals can be used. Examples include metals such as platinum, gold, iron, iron oxide, nickel, silver, copper, and palladium, or alloys containing any of these. Among these, the labeled particles preferably contain at least one metal selected from the group consisting of platinum, gold, and palladium, more preferably at least one metal selected from the group consisting of platinum and gold, and even more preferably platinum. Furthermore, the metal is preferably a metal such as platinum, gold, iron, iron oxide, nickel, silver, copper, or palladium, or an alloy containing any of these, more preferably at least one metal selected from the group consisting of platinum and gold, and even more preferably platinum. When using particles in which multiple metal particles are fixed to the surface of non-metallic particles, there are no particular restrictions on the average particle size of the multiple metal particles, but for example, the average particle size may be in the range of 0.1 to 50 nm, 0.5 to 30 nm, 1 to 20 nm, 1 to 10 nm, or 2 to 8 nm. When using particles in which multiple metal particles are fixed to non-metal particles, there are no particular restrictions on the proportion of metal fixed to the non-metal particles, but for example, the proportion of metal may be in the range of 10 to 80 mass %, 10 to 60 mass %, 20 to 50 mass %, or 30 to 50 mass %.

[0020] (Container or substrate) The container or substrate used in the method of the first aspect contains a sample containing the analyte and a plurality of labeled particles carrying antibodies capable of binding directly or indirectly to the analyte, and the sample containing the analyte and the plurality of labeled particles carrying antibodies capable of binding directly or indirectly to the analyte are in contact with each other. Measurement light is irradiated onto the container or substrate in which the sample containing the analyte and the labeled particles have already come into contact. There are no limitations on the type of container or substrate, and various containers or substrates can be used. However, the container or substrate must be configured so that when measurement light from a light source is irradiated onto an area containing labeled particles (and an area not containing labeled particles) in the container or substrate, the light can pass through the area containing labeled particles in the container or substrate (or each of the area containing labeled particles and other areas (preferably areas not containing labeled particles)) and reach the photodetector. This is expected to improve sensitivity. For example, the container or substrate may be made of a transparent material or a porous material at least in the portion through which the measurement light passes, so that the measurement light can pass through. Examples of porous materials include porous membranes such as nitrocellulose membranes, woven fabrics, and nonwoven fabrics. Furthermore, when a substrate is used that is housed in a container, the container that houses the substrate may have a hollow at least in the portion of its bottom through which light passes (i.e., a hole in the light-transmitting portion of the container). Examples of containers or substrates include well plates such as 96-well plates, test strips (for immunochromatography), membranes, spitz, test tubes, and preparations. Furthermore, test strips and membranes may be housed in housing cases. Among these, well plates such as 96-well plates, test strips such as immunochromatography test strips, and membranes for test strips are preferably used as containers. The well plate preferably has a transparent bottom so that light can pass through the well plate when irradiated from above. It is preferable that at least the light-transmitting portion of the backing sheet of the test strip be removed so that light can pass through when irradiated from above. Furthermore, when the test strip or membrane is housed in a housing case, it is preferable that at least the light-transmitting portion of the front and back surfaces of the housing case be removed (opened).

[0021] The container or substrate after contacting a sample containing the analyte with a plurality of labeled particles carrying antibodies capable of directly or indirectly binding to the analyte is the container or substrate after contacting a sample containing the analyte with a plurality of labeled particles carrying antibodies capable of directly or indirectly binding to the analyte (after causing an antigen-antibody reaction). Because the analyte is labeled with labeled particles by the antigen-antibody reaction, the presence of the analyte can be detected or the concentration or amount of the analyte can be estimated by irradiating light into the area where the labeled particles are present and measuring the light intensity of the transmitted light. There are no particular limitations on the method for contacting a sample containing an analyte with a plurality of labeled particles carrying antibodies capable of directly or indirectly binding to the analyte. For example, in the case of immunochromatography, a plurality of labeled particles carrying antibodies capable of directly or indirectly binding to the analyte can be placed in the conjugate pad 4 of the test strip 1, and a (liquid) sample containing the analyte can be dropped into the sample pad 3. After dropping, the sample is transferred from the sample pad 3 to the conjugate pad 4, where the sample containing the analyte comes into contact with a plurality of labeled particles carrying antibodies capable of directly or indirectly binding to the analyte, thereby labeling the analyte with the labeled particles. The sample containing the analyte particles labeled with the labeled particles then spreads across the membrane 5 and passes through the test line 6. Since the test line 6 has antibodies immobilized thereon that can bind to the analyte, the analyte labeled with the labeled particles is captured by the test line 6. This test line 6 is the area where the measurement object labeled with the labeling particles exists, and by irradiating light into this area, the light intensity of the transmitted light irradiated into the area where the measurement object labeled with the labeling particles exists can be measured. Other substances not captured by the test line 6 are developed along with the sample to the control line 7. Since the control line 7 has fixed thereto antibodies capable of binding to antibodies capable of binding directly or indirectly to the analyte, the plurality of labeled particles carrying antibodies capable of binding directly or indirectly to the analyte that have not bound to the analyte are captured by the control line 7. The remaining sample is then absorbed by the absorbent pad 8.

[0022] (Area where the measurement target labeled with labeled particles exists) After a sample containing the analyte is brought into contact with a plurality of labeled particles carrying antibodies capable of directly or indirectly binding to the analyte, the labeled particles that have labeled the analyte exist or move in a region where the analyte labeled with the labeled particles exists. The region where the analyte labeled with the labeled particles exists preferably has a thickness in the direction from the light source to the detector. The thicker the region, the more labeled particles are present throughout that thickness, resulting in more labeled particles being hit by the light before reaching the photodetector. Since the amount of light attenuation increases exponentially (depending on the frequency of light hitting labeled particles) with thickness, adjusting the thickness facilitates sensitive measurement of the concentration and amount of the analyte. For example, in the case of immunochromatography, the analyte labeled with labeled particles is present on the membrane of the test line, so the thickness of the region corresponds to the thickness of the membrane of the test line. In the case of ELISA, the analyte labeled with labeled particles is present in the liquid in each well of a well plate, so the thickness of the region corresponds to the thickness of the liquid (or the thickness of the region in the liquid where the analyte labeled with labeled particles is present). The region where the analyte labeled with labeled particles is present may also be referred to as the medium, since it transmits light irradiated from the light source side to the detector side. Similarly, the thickness of the region may also be referred to as the thickness of the medium. In this case, there are no particular limitations on the type of medium, and various media can be used. Examples include membranes of test strips used in immunochromatography, liquids (developing solvents, buffer solutions, water, organic solvents) contained in the wells of well plates used in ELISA, gels and thin layers (silica particle gels, ceramic particles, resin particles, cellulose particles), woven fabrics, nonwoven fabrics, and paper. For reference, when light travels through the thickness of a homogeneous, semitransparent medium, the attenuation is exponential and is expressed by the Beer-Lambert law: JPEG0007756407000002.jpg658 (in the formula, F A (λ) is the spectral radiant flux (unit: [W / nm]), x is the thickness, and F B (λ) is the spectral radiant flux at the x-axis (unit: [W / nm]), and α(λ) is the spectral absorption coefficient of the medium.

[0023] There is no particular limitation on the thickness of the region where the measurement target exists or the thickness of the medium, and the optimum thickness may be selected depending on the type of measurement target and its expected concentration.

[0024] <Separation process> The method of the first aspect may further include a step of separating the antibodies carried by the labeled particles that are directly or indirectly bound to the analyte from the antibodies carried by the labeled particles that are not bound to the analyte after contacting the sample containing the analyte with a plurality of labeled particles that carry antibodies that can directly or indirectly bind to the analyte and before irradiating the measurement light. By including a separation process, it becomes easier for only the antibodies carried by the labeled particles that are directly or indirectly bound to the object to be measured to be contained within the region containing the labeled particles onto which the measurement light is irradiated, thereby further improving sensitivity. The separation step may be configured, as in a lateral flow immunochromatographic assay, so that while the sample and labeled particles flow through the test strip, only antibodies carried by labeled particles that are directly or indirectly bound to the analyte are captured in a predetermined area, while antibodies carried by labeled particles that are not bound to the analyte are not captured in the above area, thereby (automatically) separating antibodies carried by labeled particles that are directly or indirectly bound to the analyte from antibodies carried by labeled particles that are not bound to the analyte. Alternatively, the two can be separated manually by providing an area on the bottom of the container that captures antibodies carried by labeled particles that are directly or indirectly bound to the analyte, and discarding the liquid containing antibodies carried by labeled particles that are not bound to the analyte.

[0025] <Light detection process> The method of the first aspect includes a step of detecting transmitted light of the measurement light that has passed through a region of the container or substrate containing labeled particles. There are no particular limitations on the means for detecting the transmitted light, and the transmitted light may be detected using, for example, a photodiode, a photomultiplier tube, a photoconductive element, an imaging element, or the like. In addition, when the measurement light is irradiated into a first region containing a plurality of labeled particles of the container or substrate and into a second region outside the first region in the step of irradiating the measurement light, the step of detecting the transmitted light not only detects the transmitted light that has passed through the first region, but also detects the transmitted light that has passed through the second region. In this specification, light emitted from a light source toward a container or a substrate is called measurement light, and light of the measurement light that has passed through the container or the substrate is called transmitted light.

[0026] <Step of detecting the target substance / estimating the concentration or amount of the target substance> The method of the first aspect of the present invention may include a step of detecting the analyte from the intensity of the detected light, and may further include a step of estimating the concentration or amount of the analyte from the intensity of the detected light. The light intensity is Values ​​(e.g., lux) obtained using a photodiode-based illuminance meter are used.

[0027] Furthermore, when estimating the concentration or amount of a target substance, a first region containing a plurality of labeled particles in a container or a substrate and a second region different from the first region (preferably a region not containing a plurality of labeled particles) may be irradiated with light, and light intensity 1 of the light transmitted through the first region and light intensity 2 of the light transmitted through the second region may be measured. Furthermore, the method of the first aspect of the present invention may further include a step of determining the light attenuation rate using the following formula: Light attenuation rate = (light intensity 2 - light intensity 1) x 100 / light intensity 2 The light attenuation rate is an index that indicates the extent to which the light intensity of transmitted light when light is irradiated into a first region containing a measurement target labeled with multiple labeled particles is reduced compared to the light intensity of transmitted light when light is irradiated into a second region, and allows for understanding the degree of attenuation of transmitted light caused by Mie scattering.

[0028] <Steps for determining the calibration curve> The method of the first aspect may further include a step of obtaining a calibration curve showing the relationship between the concentration or amount of the analyte and the light attenuation rate using a sample whose concentration or amount is known in advance. The calibration curve is preferably linear, and more preferably an approximated straight line. The calibration curve or the approximated straight line may be, for example, a curve in which the vertical axis (y) represents the light attenuation rate (%) and the horizontal axis (x) represents the dilution rate (%), with both axes plotted as Log 10The values ​​obtained above are plotted on a double logarithmic scale graph, and a calibration curve or an approximate straight line can be obtained by the regression equation (y = ax + b) of simple regression analysis using the least squares method. By obtaining the calibration line, the method of the first aspect can be performed using a sample containing an analyte whose concentration or amount is unknown to calculate its light attenuation rate, and the calculated light attenuation rate can be compared with the calibration curve to estimate the concentration or amount of the analyte.

[0029] <Step of estimating the concentration or amount of the measurement target> The method of the first aspect may further comprise a step of comparing the light attenuation rate obtained using a sample in which the concentration or amount of the analyte is unknown with a calibration curve obtained in advance, to estimate the concentration or amount of the analyte contained in the sample in which the concentration or amount of the analyte is unknown. A calibration curve has the concentration or amount (dilution rate) of the analyte on the horizontal axis and the light attenuation rate on the vertical axis. Therefore, by comparing the light attenuation rate obtained using a sample whose concentration or amount of the analyte is unknown with the calibration curve, the concentration or amount of the analyte on the calibration curve corresponding to the light attenuation rate obtained using the sample whose concentration or amount of the analyte is unknown can be determined. The determined concentration or amount can be estimated to be the concentration or amount of the analyte in the sample.

[0030] <<Immunoassay device>> The device according to the second aspect of the present invention is an immunoassay device, a holder for holding a container or a substrate containing a plurality of labeled particles carrying antibodies capable of directly or indirectly binding to the object to be measured; a light source disposed above the holder for irradiating light into an area of ​​the container or substrate held by the holder, the area including the plurality of label particles; a light detection unit that is disposed on the opposite side of the container or the substrate from the light source and detects light that is irradiated from the light source and transmitted through a region of the container or the substrate that contains the labeled particles; and the wavelength of the light emitted by the light source is a wavelength that causes Mie scattering by each of the plurality of label particles; The immunoassay device. The device of the second embodiment can be preferably used to detect an analyte contained in a sample, or to estimate the concentration or amount of the analyte. Furthermore, the device of the second aspect can be suitably used in the method of the first aspect. Therefore, the device of the second aspect is preferably a device for use in the method of the first aspect. The measurement object, the antibody capable of binding directly or indirectly to the measurement object, the labeled particles, and the like can be those listed in the first embodiment.

[0031] (light source) The device of the second embodiment has a light source positioned above the holder and for irradiating light into an area of ​​the container or substrate held by the holder that contains a plurality of label particles. The measurement light irradiated from the light source preferably has a wavelength that causes Mie scattering by each of the multiple labeled particles, and more preferably has a wavelength that satisfies 2<α<10 (where α=π×labeled particle diameter (nm) / light wavelength (nm)). Although there is no particular limitation on the type of light source, it is preferable that the light source has coherent characteristics and generates a light amount that can withstand the exponential attenuation of the light amount due to Mie scattering, and laser light is more preferable. Note that measurement light other than laser light can also be used as long as it has the above properties. Furthermore, the light source is preferably a light source that can irradiate light onto an area that is the same as or narrower than the area containing the labeled particles in the container or substrate when viewed from the light source side toward the light detection unit, and more preferably a light source that can irradiate light onto an area that is narrower than the area containing the labeled particles in the container or substrate. Furthermore, the device of the second aspect is preferably configured to irradiate light into a first region of the container or substrate containing the plurality of labeled particles and a second region of the container or substrate not containing the plurality of labeled particles. The light irradiation into the first region and the second region may be simultaneous or at different times. In the case of simultaneous irradiation, two light sources can be provided to simultaneously irradiate the first region and the second region with light. In this case, it is preferable to provide two light detection units. That is, it is preferable that light source A irradiates light into the first region and light detection unit A detects the transmitted light, and light source B irradiates light into the second region and light detection unit B detects the transmitted light. When irradiating light into the first region and the second region at different times, the light source may be positioned above the first region to irradiate the first region with light, and then the container or substrate may be moved laterally (perpendicular to the direction from the light source to the light detection unit) until the light source is positioned above the second region, and light is then irradiated into the second region. Alternatively, the light source may be positioned above the first region and irradiated with light into the first region, and then the light source may be moved laterally (perpendicular to the direction from the light source toward the light detection unit) until the light source is positioned above the second region, and then the second region may be irradiated with light. There are no particular restrictions on the order in which the first and second regions are irradiated with light; the first region may be irradiated with light before the second region, or the second region may be irradiated with light before the first region. Examples of a means for moving the container or substrate include a sliding device, which will be described later.

[0032] (Slit part) The device of the second aspect may have a slit section between the light source and the container or substrate. This makes it easier to irradiate light onto an area narrower than the area containing labeled particles in the container or substrate when viewed from the light source side toward the light detection section. The slit section is a plate-shaped member with a small slit hole, and light from the light source passes through the slit hole, allowing light to be irradiated onto a narrower area. There are no particular restrictions on the shape of the slit hole, and it may be circular, square, rectangular, triangular, or any other shape. A slit hole of an appropriate shape may be selected according to the shape of the area containing labeled particles. It is preferable that the maximum width of the slit hole is smaller than the width of light irradiated from the light source onto the slit. Furthermore, the device of the second aspect may have a slit between the container or substrate and the detection unit, instead of or in addition to the slit between the light source and the container or substrate. This makes it easier for the light detection unit to detect only the light irradiated into the region containing the labeled particles. The shape of the slit is not particularly limited and may be circular, square, rectangular, triangular, or any other shape. An appropriate shape for the slit may be selected according to the shape of the region containing the labeled particles. The maximum width of the slit is preferably smaller than the width of the light irradiated from the light source into the region containing the labeled particles and transmitted through the slit.

[0033] (holding part) The device of the second embodiment has a holding unit for holding a container or substrate containing a plurality of labeled particles carrying antibodies capable of directly or indirectly binding to the analyte. The shape of the holding unit is not particularly limited as long as it can hold the container or substrate. For example, the immunoassay device may have a holding unit with a concave shape extending in one direction, and the container or substrate may be held by accommodating the container or substrate in the concave space formed by the concave shape extending in one direction. Having a holding unit with a concave shape extending in one direction makes it easier to slide the container or substrate in either the front-to-back or left-to-right direction. Alternatively, the immunoassay device may have a holding unit with a mechanism for clamping and fixing the container or substrate. In this case, it is preferable that the holding unit has the function of moving the container or substrate in the front-to-back and / or left-to-right directions.

[0034] (Container or substrate) The device of the second embodiment may further comprise a container or substrate containing a plurality of labeled particles carrying antibodies capable of directly or indirectly binding to the analyte. The device of the second embodiment may further include a container or a substrate. Alternatively, the container or substrate may be prepared separately from the device, and a sample containing the analyte may be contacted in the container or substrate with a plurality of labeled particles carrying antibodies capable of directly or indirectly binding to the analyte, and then the sample may be held in a holding section within the device. For example, in the case of an ELISA method, a plurality of labeled particles carrying antibodies capable of directly or indirectly binding to the analyte are not pre-contained in a container but are added during testing. Therefore, the device of the second embodiment may further include the container or substrate in a form that does not contain a plurality of labeled particles carrying antibodies capable of directly or indirectly binding to the analyte. Examples of the container or substrate include those listed in the first embodiment. When the container or substrate is irradiated with light from a light source, it is preferable that the labeled particles are present in an overlapping manner within a region containing the labeled particles when viewed from the light source toward the light detection unit. The overlapping of the labeled particles means that the labeled particles are densely present in the region of the container or substrate in all of the X-axis direction (left-right direction), Y-axis direction (front-back direction), and Z-axis direction (thickness direction), and the labeled particles appear to overlap each other when viewed from the light source toward the light detection unit.

[0035] (Photodetector) The device of the second aspect has a light detection unit that is disposed on the opposite side of the container or substrate from the light source and detects light that is irradiated from the light source and transmitted through a region of the container or substrate containing labeled particles. The light detection unit may be composed of, for example, a photodiode. In addition, when the light source irradiates light into a first region of the container or substrate containing a plurality of labeled particles and into a second region different from the first region (preferably a region not containing a plurality of labeled particles), the light detection unit not only detects light that has passed through the first region, but also detects light that has passed through the second region.

[0036] (Display) The device of the second aspect may further include a display unit that displays the light intensity of light detected by the light detection unit, etc. The display unit is connected to the light detection unit by wire or wirelessly, and displays the light intensity detected by the light detection unit, the light attenuation rate calculated from the light intensity, and / or the concentration or amount of the target substance estimated from the light intensity or the light attenuation rate.

[0037] (Estimation Department) The device of the second aspect may further include an estimation unit that estimates the amount, concentration, or quantity of the analyte contained in the sample based on the light intensity of the light detected by the light detection unit. The estimation unit may be incorporated into the display unit or may be separate from the display unit. Furthermore, the device of the second aspect may be configured to irradiate light into a first region of a container or substrate containing a plurality of labeled particles and into a second region of the container or substrate not containing the plurality of labeled particles, and may further include an estimation unit that estimates the concentration or amount of the analyte contained in the sample based on a light intensity 1 when the light is irradiated into the first region and a light intensity 2 when the light is irradiated into the second region. The method described in the first aspect can be used as a method for estimating the concentration or amount of the analyte.

[0038] (Slide part) The device of the second aspect may further include a sliding portion for sliding the container or substrate. The sliding portion may slide so as to push the container or substrate away from the sliding portion, or so as to pull the container or substrate toward the sliding portion, or may include both of these mechanisms. Furthermore, the sliding part may be capable of sliding the container or substrate only in the left-right direction, or may be capable of sliding not only in the left-right direction but also in the front-back direction. By being able to slide the container or substrate in the left-right and front-back directions, even in the case of a 96-well plate having multiple wells in both the front-back and left-right directions, the well plate can be moved by the sliding part, and the light intensities of the measurement targets present in the multiple wells can be measured continuously.

[0039] <<Immunoassay kit 1>> A kit according to a third aspect of the present invention is an immunoassay kit comprising the device according to the second aspect and a container or substrate containing a plurality of labeled particles carrying antibodies capable of directly or indirectly binding to the object to be measured. As the measurement target, the antibody capable of binding directly or indirectly to the measurement target, the labeled particle, the container or the substrate, etc., those described in the first and second embodiments can be used. The kit of the third embodiment can be preferably used in the method of the first embodiment.

[0040] <<Immunoassay kit 2>> A kit according to a fourth aspect of the present invention is an immunoassay kit comprising the device according to the second aspect and a container or substrate on which a plurality of labeled particles carrying antibodies capable of binding directly or indirectly to the object to be measured can be placed. The kit of the fourth embodiment preferably further comprises a plurality of labeled particles carrying antibodies capable of binding directly or indirectly to the analyte. As the measurement target, the antibody capable of binding directly or indirectly to the measurement target, the labeled particle, the container or the substrate, etc., those described in the first and second embodiments can be used. The kit of the fourth embodiment can be preferably used in the method of the first embodiment.

[0041] <<Immunoassay containers or substrates>> The container or substrate of the fifth aspect of the present invention is a container or substrate containing a plurality of labeled particles carrying antibodies capable of directly or indirectly binding to the object to be measured, for use in the method of the first aspect, for use in the device of the second aspect, for use in the kit of the third aspect, or for use in the kit of the fourth aspect. As the measurement target, the antibody capable of binding directly or indirectly to the measurement target, the labeled particle, the container or the substrate, etc., those described in the first and second embodiments can be used.

[0042] <Application to various immunoassay methods> The method, device, kit, and container or substrate of the present invention can be applied to various types of immunoassays, including enzyme immunoassay, radioimmunoassay, chemiluminescent immunoassay, fluorescent immunoassay, immunoradiometric assay (IRMA), enzyme-labeled antigen immunoenzymometric assay (IEMA), latex agglutination, latex agglutination inhibition, immunochromatography, ELISA, Western blotting, hemagglutination, and hemagglutination inhibition.

[0043] (Immunochromatography) Immunochromatography is a technique for detecting antigens in a sample using an immunochromatography test strip coated with a reagent containing an antibody that induces an antigen-antibody reaction at the measurement location. When detecting these, detection sensitivity can be increased by binding a label to the antibody, antigen, or complex. A typical immunochromatography method uses the sandwich method. In sandwich immunochromatography, a test strip is prepared with three types of antibodies that recognize different regions of the target substance. The first uses a labeled antibody with metal and metal oxide colloidal particles as the labeling particles on the conjugate pad of the test strip. Here, we will explain using a commonly used gold colloid-labeled antibody. A second antigen-specific antibody (first capture antibody) is immobilized on the test line on the test strip membrane, and a third labeled antibody (second capture antibody) is immobilized on the control line downstream of the test line.

[0044] When a test solution containing the analyte is dropped onto the sample pad, an analyte-colloidal gold-labeled antibody complex is formed on the conjugate pad. As this complex passes through the test line, it is captured by the first capture antibody, forming a first capture antibody-analyte-colloidal gold-labeled complex and causing a colored line to appear on the test line. The colloidal gold-labeled antibody that did not form a complex with the analyte is captured by the second capture antibody on the control line, causing a colored line to appear on the control line, indicating that the test was performed correctly. In conventional methods, the color intensity of the colored test line is measured visually or with an optical measurement device to detect and quantify the analyte. To replace this measurement method with the method of the present invention, instead of measuring the color intensity of the colored test line, light such as laser light is irradiated onto the test line under conditions that cause Mie scattering, the intensity of the light transmitted through the test strip (membrane) is measured, and the amount and concentration of the analyte can be estimated from this light intensity. This makes it possible to quantify the analyte down to a lower concentration range than conventional methods.

[0045] (Enzyme immunoassay) ELISA is a widely known enzyme immunoassay method. In ELISA, the analyte (target protein or target antibody) is immobilized in each well of a well plate, and if the analyte is a protein, an enzyme-labeled antibody that specifically binds to the protein is added. A substrate that reacts with the enzyme is then added, causing an enzymatic reaction, and the analyte is detected or quantified based on the enzymatic activity. By replacing the labeled enzyme with the labeled particles used in the present invention, the analyte can be detected or quantified using the method of the present invention. In this case, the addition of a substrate that reacts with the enzyme is unnecessary. In addition to enzyme immunoassays, radioimmunoassays, chemiluminescent immunoassays, and fluorescent immunoassays are also known, depending on the type of label used for the labeled antigen. While these methods differ from enzyme immunoassays in that the antigen is labeled with a radioisotope in radioimmunoassays, the antigen is labeled with a chemiluminescent substance in chemiluminescent immunoassays, and the antigen is labeled with a fluorescent dye in fluorescent immunoassays, they share the same measurement principle. To replace these measurement methods with the method of the present invention, the above-mentioned labeling substance may be replaced with the labeling particles used in the present invention, and measurement may be performed using the method of the present invention. Also, by installing membranes or the like that fit the shape of the bottom of the wells of a well plate and fixing the measurement target to each membrane, the region where the measurement target labeled with the labeling particles exists has a thickness in the direction from the light source to the detector, enabling measurement with higher sensitivity.

[0046] (Immunometric assay method) In immunometric assays, the target antigen (or antibody) is reacted with an excess amount of labeled antibody (or labeled antigen) to form a complex, after which the unreacted labeled antibody (or labeled antigen) is removed. The greater the amount of complex formed (the greater the amount of target), the greater the signal intensity of the labeled substance. Therefore, the amount and concentration of the target can be estimated by measuring the signal intensity upon addition of a sample containing the target. A calibration curve is then prepared, which shows the relationship between the antigen (or antibody) whose concentration is already known and the signal intensity. By comparing the signal intensity upon addition of a sample containing the target with the calibration curve, the amount and concentration of the target in the sample can be estimated. Depending on the type of label, there are variations, such as immunoradiometric assays, in which the antibody (or antigen) is labeled with a radioisotope, and immunoenzymometric assays, in which the antibody (or antigen) is labeled with an enzyme. When using enzyme labeling, a substrate is added to initiate a substrate-enzyme reaction, and the intensity of the resulting signal, such as luminescence or fluorescence, is measured. To replace these measurement methods with the method of the present invention, the antibody (or antigen) labeled with the above-mentioned radioisotope or enzyme can be replaced with an antibody (or antigen) labeled with the labeled particles used in the present invention, and measurement can be performed using the method of the present invention.

[0047] (Sandwich assay method) Sandwich assays are known as variants of immunoradiometric assays, immunoenzymometric assays, or ELISAs. Immunochromatography is also a sandwich assay. In a sandwich assay, an antibody or antigen is immobilized on a well or other surface, and a sample containing the analyte (antigen or antibody) is added to react with the antibody or antigen. A labeled antibody or antigen that recognizes and binds to a different analyte site than the immobilized antibody or antigen is then added, sandwiching the analyte between the antibody (or antigen) and the labeled antibody (or antigen). Any unreacted analyte or labeled antibody (or labeled antigen) is removed. The greater the amount of analyte, the stronger the labeled antibody or antigen binds to the analyte, resulting in a stronger signal intensity. By calculating a calibration curve that shows the relationship between the signal intensity and the analyte whose concentration is already known, the signal intensity obtained when a sample containing an unknown analyte is added can be compared with the calibration curve to estimate the concentration and amount of the analyte in the sample. To replace these measurement methods with the method of the present invention, the labeled antibody (or labeled antigen) may be replaced with an antibody (or antigen) labeled with the labeled particles used in the present invention, and measurement may be performed using the method of the present invention. Furthermore, it is also possible to replace the labeled substance used in conventional immunoassays, such as latex particles used in latex agglutination assays and latex agglutination inhibition assays, with the labeled particles used in the present invention, and perform measurement using the method of the present invention. Furthermore, by installing a membrane or the like that matches the shape of the bottom surface of the well plate and fixing the measurement target to each membrane rather than to each well, the area where the measurement target labeled with labeled particles is present will have a thickness in the direction from the light source to the detector, allowing for more sensitive measurements.

[0048] (One embodiment when applied to immunochromatography) Hereinafter, one embodiment of the method of the present invention applied to immunochromatography, which is one of the immunoassay methods, will be described with reference to FIGS. 1 is a schematic longitudinal cross-sectional view of a test strip 1 used in this embodiment. Test strip 1 is a lateral flow type test strip, and is composed of a backing sheet 2, a sample pad 3, a conjugate pad 4, a membrane 5, an absorbent pad 8, etc., and is housed in a housing case 9. The backing sheet is equipped with a sample pad 3, a conjugate pad 4, a membrane 5, and an absorbent pad 8, arranged in this order. A hole is formed in the housing case 9 above the sample pad 3, through which a (liquid) sample containing the analyte is dropped onto the sample pad 3. The conjugate pad 4 contains a plurality of labeled particles carrying antibodies capable of directly or indirectly binding to the analyte. When the sample is dropped onto the sample pad 3, the sample liquid spreads to the conjugate pad 4, where the plurality of labeled particles carrying antibodies capable of directly or indirectly binding to the analyte bind to the analyte in the sample, forming a complex. The sample liquid spreads onto the membrane, and the sample liquid containing the complex migrates to the test line 6. A substance (e.g., an antibody) that specifically binds to the analyte is arranged on the test line 6, and the analyte (complex) labeled with the labeled particles is captured at the test line 6. Meanwhile, labeled particles that are not bound to the analyte pass through the test line with the sample liquid and migrate to the control line 7. An antibody (such as an IgG antibody) against an antibody that can directly or indirectly bind to the analyte is placed in the control line 7, where labeled particles that are not bound to the analyte are captured. The sample liquid is finally spread to the absorbent pad 7 and absorbed. FIG. 5 is a longitudinal cross-sectional schematic diagram of an embodiment of an immunoassay device that can be used in this embodiment, showing the case where light 12 is irradiated from a light source 10 onto an immunochromatographic strip 1 held in a holder (not shown) of the immunoassay device. FIG. 6 is an outline diagram (perspective view) of an embodiment of an immunoassay device that can be used in this embodiment, showing the immunochromatographic strip 1 held in a holder configured with a concave shape extending in one direction. Note that in FIGS. 5 and 6, the slit section 11 is disposed between the light source and the test strip 1, but the slit section 11 does not have to be disposed. Furthermore, the slit section 11 may be disposed between the test strip 1 and the light detection section 13, rather than between the light source and the test strip 1, or may be disposed both between the light source 10 and the test strip 1 and between the test strip 1 and the light detection section 13. In FIG. 6, the cover next to the slit section 11 is shown open to more clearly show the location of the slit section 11; however, this cover is closed during use to prevent the intrusion of external light. Unlike conventional test strips (FIG. 4), the test strip 1 used in the present invention does not have a backing sheet 2 or housing case 9 on a portion of the back surface of the membrane 5 (see FIGS. 1, 3, and 5). This allows light irradiated onto the membrane 5 or test line 6 to reach the light detection unit 13, located on the opposite side of the light source 10, via the test strip 1, and the intensity of the light that reaches it can be measured. The measured light intensity and other information can be displayed on the display unit 14. In this case, light 12 emitted from light source 10 is not only irradiated onto test line 6, but may also be irradiated onto membrane 5 other than test line 6 (areas not containing labeled particles). When irradiating test line 6 and membrane 5 with light, test strip 1 may be manually slid to sequentially irradiate each area, or test strip 1 may be slid laterally using sliding device 16. In this embodiment, housing case 9 containing the test strip is placed on a base connected to sliding device 16. The base, like housing case 9, has a cavity, allowing transmitted light to reach light detection unit 13 on the opposite side of light source 10, and the light intensity of the transmitted light can be measured. Furthermore, a sliding device handle 17 is provided, and by rotating the handle 17, the base of sliding device 16 connected to the handle 17 is slid leftward using a feed screw system. Alternatively, sliding device 16 may be provided with a member (e.g., a hook, magnet, spring, etc.) for connecting test strip 1. In this case, by rotating the handle 17, the screw inside the sliding device 16 connected to the handle 17 rotates and extends to the right or contracts to the left, thereby allowing the test strip 1 connected to the sliding device 16 to also slide to the right or left. Light 12 transmitted through the region of test line 6 containing the labeling particles is detected by photodetector 13. The intensity of the detected light is determined by the estimation unit. The light intensity may be displayed on display unit 14. The estimation unit and the display unit may be integrated together. Similarly, light 12 transmitted through membrane 5 is detected by photodetector 13. The intensity of the detected light is determined by the estimation unit. The light intensity may be displayed on display unit 14. The estimation unit and the display unit may be integrated together. Next, the light attenuation rate is calculated from the light intensity of the light transmitted through the test line and the light intensity of the light transmitted through membrane 5 and / or control line 7. The calculated light attenuation rate may be displayed on display unit 14. In addition to the above measurement, it is preferable to obtain a calibration curve (or an approximate straight line) showing the relationship between the concentration or amount of the target substance and the light attenuation rate using a sample whose concentration or amount is known in advance. By determining a calibration curve in advance, the concentration or amount of the analyte contained in the sample can be determined by comparing the light attenuation rate measured using a sample containing the analyte of unknown concentration or amount with the calibration curve. This allows the concentration or amount of the analyte contained in the sample to be estimated with high accuracy. The estimated concentration or amount of the analyte contained in the sample may be displayed on display unit 14.

[0049] (One embodiment when applied to ELISA) Next, with reference to FIG. 7, an embodiment in which the method of the present invention is applied to ELISA, which is another immunoassay method, will be described. FIG. 7 is a schematic longitudinal cross-sectional view of a well plate 18 held in a holder (not shown) of the immunoassay device used in this embodiment, irradiated with light 12 from a light source 10. Well plate 18 has one or more wells, preferably a plurality of wells (e.g., 96 wells, etc.), each of which is preferably made of a transparent material so that light 12 can pass through the bottom of the well when irradiated with the well. A sample containing the analyte is added to a test sample well 20 with a solid-phase antibody adsorbed to its surface. After the reaction is complete, excess liquid is discarded and the well is washed, allowing the analyte to bind to the solid-phase antibody. Next, a sample containing multiple labeled particles carrying antibodies capable of directly or indirectly binding to the analyte is added, allowing the reaction to proceed, and excess standard particles are washed away, resulting in the test sample. Alternatively, a membrane or other device conforming to the shape of the well's bottom is placed on the bottom, the solid-phase antibody is adsorbed onto the membrane, and the sample containing the analyte is added. After the reaction is complete, excess liquid is discarded and the well with the membrane or other device installed is washed, allowing the analyte to bind to the solid-phase antibody. Next, a sample containing multiple labeled particles carrying antibodies capable of directly or indirectly binding to the analyte is added, allowing the reaction to proceed, and excess standard particles are washed away. The region containing the analyte labeled with labeled particles has a thickness from the light source to the detector, enabling more sensitive measurements. The control sample well 19 has an antibody for immobilization adsorbed on its surface. Alternatively, when using a test sample with a membrane or the like placed on the bottom of the well, a membrane or the like matching the shape of the bottom of the well is placed, and the antibody for immobilization adsorbed on the membrane is used as the test sample. Light 12 is irradiated onto the test sample well 20 from a light source 10. The irradiated light passes through the test sample well and is detected by a light detection unit 13 located on the opposite side of the test sample well 20 from the light source 10. This makes it possible to measure the intensity of the light that reaches the well. The measured light intensity and other information may be displayed on a display unit 14. At this time, a housing case 9 containing a test strip is placed on a base connected to the slide device 16. The base, like the housing case 9, has a cavity, allowing transmitted light to reach the light detection unit 13 located on the opposite side of the light source 10, and the intensity of the transmitted light can be measured. Furthermore, a slide device handle 17 is provided, and by rotating the handle 17, the base of the slide device 16 connected to the handle 17 is slid leftward using a feed screw system. Alternatively, the slide device 16 may be provided with a member (e.g., a hook, a magnet, a spring, etc.) for connecting the well plate 18. In this case, by rotating the handle 17, a screw in the slide device 16 connected to the handle 17 rotates, extending rightward or retracting leftward, thereby allowing the well plate 18 connected to the slide device 16 to slide rightward or leftward. Light 12 transmitted through the area of ​​test sample well 20 is detected by photodetector 13. The intensity of the detected light is determined by an estimation unit. The light intensity may be displayed on display unit 14. The estimation unit and display unit may be integrated together. Similarly, light 12 transmitted through control sample well 19 is detected by photodetector 13. The intensity of the detected light is determined by an estimation unit. The light intensity may be displayed on display unit 14. The estimation unit and display unit may be integrated together. Next, the light attenuation rate is calculated from the light intensity transmitted through test sample well 20 and the light intensity transmitted through control sample well 19. The calculated light attenuation rate may be displayed on display unit 14. In addition to the above measurement, it is preferable to obtain a calibration curve (or an approximate straight line) showing the relationship between the concentration or amount of the target substance and the light attenuation rate using a sample whose concentration or amount is known in advance. By determining a calibration curve in advance, the concentration or amount of the analyte contained in the sample can be determined by comparing the light attenuation rate measured using a sample containing the analyte of unknown concentration or amount with the calibration curve. This allows the concentration or amount of the analyte contained in the sample to be estimated with high accuracy. The estimated concentration or amount of the analyte contained in the sample may be displayed on display unit 14.

[0050] The above describes in detail an embodiment in which the method of the first aspect (and the device of the second aspect) is applied to immunochromatography and ELISA as representative examples of immunoassay methods. However, the method, device, kit, and container or substrate of the present invention are not limited to application to these two immunoassay methods, and can be applied to various other immunoassay methods.

[0051] <Effects> The present invention utilizes the principle that light scattering is increased by combining the wavelength of light used as measurement light in an immunoassay with the particle size of labeled particles carrying the analyte under conditions that cause Mie scattering. As the measurement light passes through an area containing the analyte, the measurement light strikes the labeled particles carrying the analyte, and the amount of light scattering increases exponentially through repeated scattering and transmission. As a result, the transmitted light attenuates exponentially, and it has been discovered that the attenuation of light intensity can be measured even if the concentration of labeled particles carrying the analyte is low. The method of the present invention irradiates labeled particles with light under conditions that cause Mie scattering, and detects the transmitted light, thereby enabling the quantification of an object to be measured down to a concentration range lower than conventionally possible. The method of the present invention is a measurement method that uses a novel measurement principle that utilizes light, preferably coherent laser light with uniform properties such as wavelength and phase, as the input wavelength, and utilizes the characteristic that the linearity of light in the Mie scattering region increases depending on the particle size combination of the labeled particles, enabling quantitative measurement of the amount of light attenuation in a dispersion medium, and is different from the existing measurement principle that uses reflected light.A feature of this measurement method is that it not only achieves sensitivity that cannot be measured by the reflection method, but also has reproducibility that is important for quantitative measurement. This measurement method can be preferably used in a measurement system in which labeled particles that label the measurement target through an antigen-antibody reaction exist in the measurement region (or medium) with a certain thickness not only in two dimensions but also in the optical axis direction. It can be applied to various media that satisfy the above conditions, such as membranes, liquid phases, gel phases, and solid phases. Furthermore, one embodiment in which the method of the first aspect is applied to immunochromatography is a method that is completely different from conventional immunochromatography using light reflection, and has the characteristics of rapid testing, specificity, and high sensitivity in the low concentration range of the analyte. The present invention provides linear results even in the lower concentration range of the analyte, making rapid quantitative testing possible in immunochromatography. Conventionally, the commonly used optical reflectance method measures the reflected light obtained as a result of attenuation due to two-dimensional (planar) absorption and scattering of incident light by particles present on the optical axis. In contrast, the present invention does not use the absorption method that two-dimensionally measures the reflected light from particles near the surface on the test line, but rather solves the problem by applying the relationship between particle size and the wavelength of incident light and the optical effect of labeled particles to three-dimensionally measure the attenuation of detected light relative to incident light through the effect of Mie scattering by all particles present on the optical vertical axis in the test line zone. As described above, the method and device of the present invention can be applied to various immunoassay methods, and is not limited to immunochromatography or ELISA. [Example]

[0052] Test examples relating to the present invention will now be described, but these are not intended to limit the scope of the present invention.

[0053] [Example 1] <Measurement of light attenuation rate of transmitted light under Mie scattering conditions> The materials used in Example 1 were prepared as follows, and the optical attenuation rate of transmitted light was determined when the C-reactive protein (CRP) to be measured was diluted to various concentrations, and the relationship between the dilution rate and the optical attenuation rate was investigated. When the wavelength of Example 1 is 450 nm and the particle size of the labeled particles is 454 nm, α is approximately 3.2, and the wavelength of the measurement light is a wavelength that causes Mie scattering by each of the labeled particles. (1) Synthesis of resin particles Labeled particles (platinum-resin composite particles, average particle size 454 nm) were synthesized according to the following procedure. Aliquat 336 (Aldrich) (1.50 g) and polyethylene glycol methyl ether methacrylate (PEGMA, 10.00 g) were dissolved in 300 g of purified water, followed by the addition of 2-vinylpyridine (2-VP, 49.50 g) and divinylbenzene (DVB, 0.50 g). The mixture was stirred under a nitrogen stream at 30°C for 50 minutes, then at 60°C for 30 minutes. After stirring, 2,2-azobis(2-methylpropionamidine) dihydrochloride (AIBA, 0.50 g) dissolved in 18.00 g of purified water was added dropwise and stirred at 60°C for 3.5 hours to obtain resin particles with an average particle size of 430 nm. The resulting mixture was precipitated by centrifugation (9000 rpm, 45 minutes). The supernatant was removed, and impurities were removed by dialysis. The concentration was then adjusted to obtain a 10 wt% resin particle dispersion. 245 ml of pure water was added to the above resin particle dispersion (90 ml), followed by 400 mM chloroplatinic acid aqueous solution (90 ml). The mixture was stirred at 30°C for 3 hours and then left at room temperature for 24 hours. The resin particles were then precipitated by centrifugation (3100 rpm, 30 minutes), and the supernatant was removed to remove excess chloroplatinic acid. The concentration was then adjusted to prepare a 5 wt% platinum ion-adsorbing resin particle dispersion. Next, the 5 wt% platinum ion-adsorbing resin particle dispersion (55 ml) was added to 3825 ml of pure water, and while stirring at 3°C, 132 mM dimethylamine borane aqueous solution (110 ml) was added dropwise, followed by stirring at 3°C ​​for 1 hour. Stirring was then continued at 25°C for 3 hours to obtain a platinum-resin composite with an average particle size of 454 nm. The platinum-resin composite was precipitated by centrifugation (3100 rpm, 60 minutes), the supernatant was removed, and then purified by dialysis to remove impurities. The concentration was then adjusted to obtain a 1 wt% platinum-resin composite dispersion. The formed platinum particles had an average particle size of 5 nm, and the platinum loading was 37.7 wt%. <Measurement of the average particle size of metal particles> A substrate was prepared by dropping a platinum-resin composite particle dispersion onto a metal mesh with a carbon support film, and the area-average diameter of 100 randomly selected metal particles was measured from the image observed using a field emission scanning electron microscope (FE-SEM; Hitachi High-Technologies Corporation, SU-9000) to determine the average particle size.

[0054] <Measurement of the average particle size of resin particles and platinum-resin composite particles> Measurement was performed using a centrifugal sedimentation particle size distribution analyzer (LUMiSizer610 manufactured by LUM GmbH) with the particles dispersed in water or a solution.

[0055] (2) Binding of labeled particles to antibodies After mixing 25 μg of anti-CRP antibody with 0.45 mL of 50 mM HEPES buffer (pH 7), 0.05 mL of the 1 wt % platinum-resin composite particle dispersion (solvent: water) prepared in (1) above was added, and the mixture was stirred end-over-end at room temperature for 1 hour to obtain labeled antibody dispersion A containing anti-CRP antibody labeled with platinum-resin composite particles.

[0056] (3) Blocking process The labeled antibody dispersion A obtained in (2) was centrifuged at 3000 rpm for 5 minutes, and the supernatant was removed. Then, 0.5 mL of HEPES buffer (pH 7) containing 1 wt% sodium caseinate was added to the sediment, which was then ultrasonically dispersed. After that, the mixture was further stirred by end-over-end stirring at room temperature for 1 hour to obtain labeled antibody dispersion B.

[0057] (4) Cleaning process Labeled antibody dispersion B was centrifuged at 3000 rpm for 5 minutes, and the supernatant was removed. Then, 0.5 mL of a 5 mM Tris aqueous solution (pH 8.5) containing less than 0.1 wt% surfactant was added to the sediment, followed by ultrasonic dispersion. This procedure was repeated three times to obtain labeled antibody dispersion C.

[0058] (5) Preparation of conjugate pad Labeled antibody dispersion C was centrifuged at 3000 rpm for 5 minutes, and the supernatant was removed. A 5 mM Tris aqueous solution (pH 8.5) containing 5 wt% sucrose and 2.5 wt% BSA was then added to the sediment, followed by ultrasonic dispersion to obtain labeled antibody dispersion D. Labeled antibody dispersion D was uniformly impregnated into a glass fiber nonwoven fabric, which was then dried at 50°C for 1 hour to produce conjugate pad 4. The concentration of labeled antibody dispersion D and the amount applied to the glass fiber nonwoven fabric were adjusted so that the platinum-resin complex particle content in conjugate pad 4 was 3 μg per test evaluated by the immunochromatography method described below.

[0059] (6) Preparation of test strips As shown in Figure 1, a 1 mg / mL anti-CRP antibody solution was applied to a 25 mm wide, 100 μm thick nitrocellulose membrane 5 to create a test line 6. A 0.5 mg / mL anti-mouse IgG antibody solution was applied downstream of the test line 6 to create a control line 7. After drying the nitrocellulose membrane 5 at 50°C for 1 hour, a backing sheet 2, a conjugate pad 4, a sample pad 3 (glass fiber nonwoven fabric), and an absorbent pad 8 (cotton nonwoven fabric) were laminated on top of each other, with a portion removed to allow light to pass through, as shown in the cross-sectional view of the test strip 1 in Figure 1. Finally, the membrane was cut to a width of 3.5 mm to create the test strip 1, which was then placed in a housing case 9. As shown in FIGS. 2 and 3, the housing case 9 has openings 21 not only on the top surface but also on the bottom surface so that light transmitted through the membrane can reach the light detection unit without being blocked. (7) Preparation of developing solution An aqueous solution (pH 7.5) containing 50 mM Tris, 150 mM NaCl, 1.0 wt % BSA, and 1.0 wt % surfactant Triton X-100 was prepared as a developing solution.

[0060] (8) Preparation of immunoassay device (immunochromatography reader) As shown in Figures 5 and 6, the immunochromatographic reader device is composed of a light source 10, a slit 11, a holder for the light source 10 and the slit 11, a light detection unit 13, a slide table 15, a slide device 16, a handle 17 for the slide device, and a light attenuation rate measurement result display unit 14. The housings for the holders of the light source 10 and slit 11, the housing for the slide table 15, the housing for the slide device 16, and the handle 17 for the slide device were made using a 3D printer (manufactured by FRASHFORGE, product name Adventurer5M Pro). Specifically, the light source 10 uses a semiconductor laser (manufactured by Civil Laser, output 10 mW, wavelength 450 nm) with a line-shaped beam, and the slit 11 is parallel to the test line 6 of the immunochromatographic strip (for example, when the long side is 3.5 mm and the short side is 1 mm), with an opening of 2 mm in the long side direction of the test line 6 and 0.3 mm in the short side direction of the test line 6. The slit 11 is adjusted so that the laser light emitted from the light source 10 is irradiated only onto the test line 6 on the membrane 5 of the test strip 1, and is fixed with a holder. The holder is designed so that the laser light 12 emitted from the light source 10 can pass through the slit 11, further pass through the membrane 5 of the immunochromatographic strip 1, and reach the light detection unit 13. The slide base 15 has two bar-shaped protrusions along the long side on the upper side of the housing of the slide base 15 so that it can move while holding the housing case 9 containing the immunochromatographic strip 1, and the housing case 9 containing the test strip 1 is held between the two protrusions and designed so that it can move only in the long side direction. The light detection unit 13 uses a photodiode matched to the light intensity of the light source 10, and is installed in a groove on the slide base 15 so that it can receive the laser light 12 emitted from the light source 10 and passed through the slit 11 directly below. The holder that holds the light source 10 and the slit 11 is designed to be perpendicular to the slide base 15. The light detection unit 13 is connected to the measurement result display unit 14. An illuminance meter (manufactured by Zhangzhou WeiHua Electronic Co., Ltd., product name LX-1010B) is used as the measurement result display unit 14. 5 and 6, the slide device 16 is designed using a feed screw mechanism so that it can slide precisely in the long side direction while holding the housing case 9. The slide device 16 is designed so that it can be moved by turning the slide device handle 17.

[0061] (9) Measurement and evaluation by immunochromatography A positive sample and a negative control (antigen-free) were prepared by diluting 3.12 mg / mL of CRP antigen with the developer as shown in Tables 1 and 2. 50 μL of the sample solution was dropped onto sample pad 3 of the test strip. The CRP antigen concentration (pg / mL) at each dilution rate is shown in Tables 1 and 2.

[0062] [Table 1]

[0063] [Table 2]

[0064] After 30 minutes, the light intensity of the light transmitted through the membrane (background) on the sample pad side and the test line was measured using the immunochromatographic reader. The light attenuation rate (%) was calculated by subtracting the light intensity of the test line from the light intensity of the membrane and dividing the result by the light intensity of the membrane according to the following formula: Light attenuation rate (%) = (BA) / B × 100 A: Light intensity (unit: lx) detected by the photodiode (light detection part) when light was irradiated onto the membrane. B: Light intensity (unit: lx) detected by the photodiode (light detection part) when light is irradiated onto the test line. The results are shown in Figure 8.

[0065] [Comparative Example 1] <Conventional method of measuring color intensity using reflected light (part 1)>

[0066] In Comparative Example 1, the color intensity was measured using reflected light, which is a conventional method, instead of transmitted light, to examine the relationship between the dilution ratio and the color intensity.

[0067] Specifically, as shown in Figure 4, in (6) of Example 1, a test strip 1 was prepared in which the backing sheet 2 was not partially removed, no opening was provided at the bottom of the housing case 9 that stores the immunochromatographic strip 1, in (8) and (9), a commercially available product C10066-10 manufactured by Hamamatsu Photonics KK was used as the immunochromatographic reader, and in the evaluation by immunochromatographic method in (9), the color intensity (mABS) of reflected light was measured instead of transmitted light. The immunochromatographic evaluation was performed in the same manner as in Example 1, except for the following. The results are shown in Figure 9.

[0068] Comparative Example 2 <Conventional method of measuring color intensity using reflected light (part 2)>

[0069] In Comparative Example 2, gold colloid particles were used instead of platinum-resin composite particles, and the same test as in Comparative Example 1 was carried out. That is, as in Comparative Example 1, color intensity was measured using reflected light, which is the conventional method, rather than transmitted light, and the relationship between dilution ratio and color intensity was investigated.

[0070] Specifically, immunochromatographic evaluation was performed in the same manner as in Comparative Example 1, except that gold colloid (Tanaka Kikinzoku Kogyo, Au colloid solution-SC, particle size 30 nm) was used as the labeling particle, 5 mM Tris aqueous solution (pH 9.5) was used as the binding buffer, and 5 mM Tris aqueous solution (pH 9.5) containing 1 wt% BSA was used as the blocking buffer. The results are shown in Figure 9.

[0071] Comparative Example 3 <Measurement of the optical attenuation rate of transmitted light under conditions that do not cause Mie scattering> In Comparative Example 3, the attenuation rate of transmitted light was measured in the same manner as in Example 1 under conditions that did not cause Mie scattering (specifically, conditions that caused Rayleigh scattering), and the relationship between the dilution rate and the color intensity was examined.

[0072] Specifically, immunochromatographic evaluation was performed in the same manner as in Example 1, except that gold colloid (Tanaka Kikinzoku Kogyo, Au colloid solution-SC, particle size 30 nm) was used as the labeling particle, 5 mM Tris aqueous solution (pH 9.5) was used as the binding buffer, and 5 mM Tris aqueous solution (pH 9.5) containing 1 wt% BSA was used as the blocking buffer. The results are shown in Figure 10.

[0073] <Result> In Example 1, in which the optical attenuation rate of transmitted light was measured under conditions that caused Mie scattering, linearity was obtained even when CRP was diluted 1 / 327,680,000 times (FIG. 8). In contrast, in the conventional measurement of color intensity of reflected light, linearity was only obtained up to a dilution rate of 1 / 40,960,000 in Comparative Example 1 (FIG. 9). At concentrations lower than 1 / 40,960,000, values ​​were obtained that were almost equivalent to those of the negative control, indicating that quantitative measurement in the low concentration range was more difficult than in Example 1. Similarly, in Comparative Example 2, linearity was only obtained up to a dilution rate of 1 / 320,000 (FIG. 9). At concentrations lower than 1 / 320,000, values ​​were obtained that were almost equivalent to those of the negative control, indicating that quantitative measurement in the low concentration range was more difficult than in Example 1. Note that the * marks in FIGS. 8 and 9 indicate the maximum dilution rate of the CRP antigen at which linearity was obtained. Furthermore, even when measuring the optical attenuation rate of transmitted light, linearity was only obtained up to a dilution rate of 1 / 640,000 under the conditions of Comparative Example 3 where Mie scattering did not occur (FIG. 10). At concentrations lower than 1 / 640,000, values ​​were almost equivalent to those of the negative control, indicating that quantitative measurement in the low concentration range was more difficult than in Example 1. The * mark in FIG. 10 indicates the maximum dilution rate of the CRP antigen at which linearity was obtained. In Comparative Example 3, instead of preparing platinum-resin composite particles under conditions that do not cause Mie scattering, gold colloid particles, which are commonly used in immunochromatography, were used. However, since the degree of change in light attenuation rate when the dilution rate is changed is determined by the relationship between particle size and the wavelength of incident light, it is thought that similar results would be obtained even if platinum-resin composite particles were used under conditions that do not cause Mie scattering. As described above, in the method of the present invention using light transmission, light is scattered in all directions mainly due to Mie scattering, which occurs depending on the particle diameter and the wavelength of the incident light according to the principles of photophysics, and therefore the attenuation of the light intensity in the straight-line direction increases exponentially with the distance of transmission. Differences in the metal material of the particles have no effect on the particle diameter and have no effect on Mie scattering. On the other hand, differences in the metal material of the particles change the color (light absorption) of the particles, which has a significant impact on conventional light reflection methods that are based on light absorption, but does not affect the method of the present invention that is based on Mie scattering. From the above, it can be concluded that measuring the light attenuation rate of transmitted light under conditions where Mie scattering occurs enables quantitative measurement in a lower concentration range than measuring the color intensity of reflected light or under conditions where Mie scattering does not occur.

[0074] [Example 2] <Measurement of optical attenuation rate at various laser light wavelengths> Mie scattering occurs due to the relationship between particle size and the wavelength of incident light. Therefore, we verified whether the reproducibility of light attenuation rate measurements could be obtained not only for the specific wavelength used in Example 1, but also when the wavelength of light was changed within the range of conditions that cause Mie scattering. Note that the particle size used was the same as in Example 1. Two types of light sources (light source 1 and light source 2) were prepared by changing the wavelength of the semiconductor laser, and immunochromatographic evaluation was carried out in the same manner as in Example 1 using each light source. Light source 1: Semiconductor laser (Civil Laser, output 10 mW, wavelength 520 nm) Light source 2: Semiconductor laser (Civil Laser, output 10 mW, wavelength 648 nm) When light source 1 is used, α is approximately 2.7, and when light source 2 is used, α is approximately 2.2, both of which are within the Mie scattering occurrence region. The results are shown in Figure 11.

[0075] <Result> The results of light attenuation rate measurements using different wavelengths, 520 nm and 648 nm, showed that quantitative and equivalent results were obtained for 520 nm and 648 nm up to the same dilution ratio as the light attenuation rate measurement using a wavelength of 450 nm (Figure 11). This confirmed that quantitative and equivalent results can be obtained even when the wavelength is changed, as long as the relationship between particle size and incident light wavelength is within the range of conditions that cause Mie scattering.

[0076] [Example 3] <Measurement of light attenuation rate for various particle sizes> As mentioned above, Mie scattering occurs due to the relationship between particle size and the wavelength of incident light. Therefore, in Example 3, we verified whether the reproducibility of light attenuation rate measurements could be obtained not only for the specific particle size used in Example 1, but also when the particle size was changed within the Mie scattering region. The same light wavelength as in Example 1 was used. (1) Synthesis of labeled particles Labeled particles 1 (platinum-resin composite particles, average particle size 454 nm) were prepared according to the procedure of Example 1. Labeled particles 2 (platinum-resin composite particles, average particle size 382 nm) were synthesized according to the following procedure. When labeled particle 1 is used, α is approximately 3.2, and when labeled particle 2 is used, α is approximately 2.7, both of which are within the Mie scattering generation region. Aliquat 336 (Aldrich) (3.00 g) and polyethylene glycol methyl ether methacrylate (PEGMA, 10.00 g) were dissolved in 300 g of purified water, followed by the addition of 2-vinylpyridine (2-VP, 49.50 g) and divinylbenzene (DVB, 0.50 g). The mixture was stirred under a nitrogen stream at 30°C for 50 minutes, then at 60°C for 30 minutes. After stirring, 2,2-azobis(2-methylpropionamidine) dihydrochloride (AIBA, 0.50 g) dissolved in 18.00 g of purified water was added dropwise and stirred at 60°C for 3.5 hours to obtain resin particles with an average particle size of 370 nm. The resulting mixture was precipitated by centrifugation (9000 rpm, 45 minutes). The supernatant was removed, and impurities were removed by dialysis. The concentration was then adjusted to obtain a 10 wt% resin particle dispersion. 245 ml of pure water was added to the above resin particle dispersion (90 ml), followed by 400 mM chloroplatinic acid aqueous solution (90 ml). The mixture was stirred at 30°C for 3 hours and then left at room temperature for 24 hours. The resin particles were then precipitated by centrifugation (3100 rpm, 30 minutes), and the supernatant was removed to remove excess chloroplatinic acid. The concentration was then adjusted to prepare a 5 wt% platinum ion-adsorbing resin particle dispersion. Next, the 5 wt% platinum ion-adsorbing resin particle dispersion (55 ml) was added to 3825 ml of pure water, and while stirring at 3°C, 132 mM dimethylamine borane aqueous solution (110 ml) was added dropwise, followed by stirring at 3°C ​​for 1 hour. Stirring was then continued at 25°C for 3 hours to obtain a platinum-resin composite with an average particle size of 382 nm. The platinum-resin composite was precipitated by centrifugation (3100 rpm, 60 minutes), the supernatant was removed, and then purified by dialysis to remove impurities. The concentration was then adjusted to obtain a 1 wt% platinum-resin composite dispersion. The formed platinum particles had an average particle size of 5 nm, and the platinum loading was 38.5 wt%.

[0077] (2) Binding of labeled particles to antibodies After mixing 25 μg of anti-CRP antibody with 0.45 mL of 50 mM HEPES buffer (pH 7), 0.05 mL of 1 wt% of any of the platinum-resin composite particle dispersions (solvent: water) prepared in (1) above was added, and the mixture was stirred end-over-end at room temperature for 1 hour to obtain labeled antibody dispersion A containing anti-CRP antibody labeled with platinum-resin composite particles. Other than that, immunochromatographic evaluation was carried out in the same manner as in Example 1. The results are shown in FIG.

[0078] <Result> As a result of measuring the light attenuation rate using platinum-resin composite particles with different particle sizes of 382 nm and 454 nm, quantitative results were obtained for both particle sizes down to the low concentration range (Figure 12). This confirmed that, within the range of conditions in which the relationship between particle size and the wavelength of incident light causes Mie scattering, quantitative and equivalent results can be obtained even when the particle size is changed. The following aspects of the present invention are also preferred. [1] A step of irradiating a measurement light onto a container or substrate containing a sample containing an analyte and a plurality of labeled particles carrying antibodies capable of directly or indirectly binding to the analyte after the sample containing the analyte and the plurality of labeled particles carrying antibodies capable of directly or indirectly binding to the analyte have come into contact with each other, wherein the measurement light is irradiated onto an area of ​​the container or substrate containing the labeled particles; and a step of detecting light (transmitted light) that has passed through the region out of the irradiated measurement light; Including, An immunoassay method, wherein the wavelength of the irradiated measurement light is a wavelength that causes Mie scattering by each of the plurality of labeled particles. [2] The immunoassay method according to [1], wherein the wavelength at which Mie scattering occurs by each of the plurality of labeled particles satisfies 2<α<10 (where α=π×particle diameter (nm) of labeled particle / wavelength of light (nm)). [3] The immunoassay method according to [1], further comprising a step of separating, after the contact and before the irradiation of the measurement light, the antibodies carried by the labeled particles that are directly or indirectly bound to the object to be measured from the antibodies carried by the labeled particles that are not bound to the object to be measured. [4] The immunoassay method according to [1], further comprising a step of determining the concentration or amount of the analyte from the light intensity of the detected transmitted light. [5] The immunoassay method according to [1], wherein the step of irradiating the measurement light includes irradiating the measurement light into a region of the container or substrate containing the plurality of labeled particles (hereinafter referred to as a first region), and irradiating the measurement light into a second region outside the first region. [6] The immunoassay method according to [5], comprising measuring a light intensity 1 of the measurement light transmitted through the first region and a light intensity 2 of the measurement light transmitted through the second region. [7] The following formula: Light attenuation rate = (light intensity 2 - light intensity 1) x 100 / light intensity 2 The immunoassay method according to [6], further comprising a step of determining the light attenuation rate by the following method. [8] The immunoassay method according to [7], further comprising the step of obtaining a calibration curve or an approximate straight line showing the relationship between the concentration or amount of the target substance and the light attenuation rate using a sample in which the concentration or amount of the target substance is known in advance. [9] The immunoassay method according to [7], further comprising the step of determining a light attenuation rate using a sample containing an analyte whose concentration or amount is unknown, and comparing the light attenuation rate with the calibration curve or approximate line obtained in advance in [8], thereby estimating the concentration or amount of the analyte contained in the sample.

[10] The immunoassay method according to [1], wherein the light is coherent measurement light.

[11] The immunoassay method according to

[10] , wherein the coherent measurement light is laser light.

[12] The immunoassay method according to [1], wherein the labeled particles contain a metal.

[13] An immunoassay device, a holder for holding, in or on the device, a container or substrate containing a plurality of labeled particles carrying antibodies capable of directly or indirectly binding to the object to be measured; a light source disposed above the holder for irradiating a measurement light into an area of ​​the container or substrate held by the holder, the area including the plurality of labeled particles; a light detection unit that is disposed on the opposite side of the container or the substrate from the light source and detects transmitted light that has been irradiated from the light source and transmitted through an area of ​​the container or the substrate that contains the labeled particles; and the wavelength of the measurement light emitted by the light source is a wavelength that causes Mie scattering by each of the plurality of label particles; The immunoassay device.

[14] The immunoassay device according to

[13] , for use in the method according to [1].

[15] The immunoassay device according to

[13] , wherein the wavelength at which Mie scattering occurs by each of the plurality of labeled particles satisfies 2<α<10 (where α=π×particle diameter (nm) of labeled particle / wavelength of light (nm)).

[16] The immunoassay device according to

[13] , further comprising a container or substrate containing a plurality of labeled particles carrying antibodies capable of directly or indirectly binding to the object to be measured.

[17] The immunoassay device is configured to irradiate a measurement light onto a region (first region) of the container or substrate containing the plurality of labeled particles and a region (second region) of the container or substrate outside the first region. The immunoassay device according to

[13] .

[18] The device according to

[13] , a container or substrate containing a plurality of labeled particles carrying antibodies capable of directly or indirectly binding to the object to be measured; A kit for immunoassay comprising:

[19] The device according to

[13] , a container or substrate capable of arranging a plurality of labeled particles carrying antibodies capable of directly or indirectly binding to the object to be measured; A kit for immunoassay comprising:

[20] The immunoassay kit according to

[19] , further comprising a plurality of labeled particles carrying antibodies capable of binding directly or indirectly to the object to be measured.

[21] A container or substrate containing a plurality of labeled particles carrying antibodies capable of directly or indirectly binding to the object to be measured, for use in the method according to [1], for use in the device according to

[13] , for use in the kit according to

[18] , or for use in the kit according to

[19] . [Industrial Applicability]

[0079] According to the present invention, even low concentrations of an analyte in a sample can be quantified, and therefore the present invention is extremely useful industrially. [Explanation of symbols]

[0080] 1...Test strip 2...Backing sheet 3...Sample pad 4...Conjugate pad 5...Membrane 6...Test line 7...Control line 8...Absorbent pad 9...Housing case 10…Light source 11...Slit 12...Light (laser light) 13...Light detection unit 14...Display section 15...Slide 16...Slide device 17...Slide device handle 18...well plate 19...Control sample well 20...Test sample wells 21...Opening

Claims

1. a step of irradiating a measurement light onto a container or substrate containing a sample containing an analyte and a plurality of labeled particles carrying antibodies capable of directly or indirectly binding to the analyte after the sample containing the analyte has been brought into contact with the plurality of labeled particles carrying antibodies capable of directly or indirectly binding to the analyte, wherein the measurement light is irradiated onto an area of ​​the container or substrate containing the labeled particles; and detecting light (transmitted light) that has passed through the region out of the irradiated measurement light; Including, The wavelength of the irradiated measurement light is a wavelength that causes Mie scattering by each of the plurality of label particles; and The step of irradiating the measurement light includes irradiating the measurement light into a region (hereinafter referred to as a first region) of the container or the substrate containing the plurality of labeled particles, and irradiating the measurement light into a second region outside the first region. Immunoassay methods.

2. The immunoassay method of claim 1, wherein the wavelength at which Mie scattering occurs by each of the plurality of labeled particles satisfies 2 < α < 10 (where α = π × labeled particle diameter (nm) / light wavelength (nm)).

3. The immunoassay method of claim 1, further comprising a step of separating, after the contact and before the irradiation of the measurement light, the antibodies carried by the labeled particles that are directly or indirectly bound to the object to be measured from the antibodies carried by the labeled particles that are not bound to the object to be measured.

4. The immunoassay method according to claim 1, further comprising the step of determining the concentration or amount of the analyte from the intensity of the detected transmitted light.

5. 2. The immunoassay method according to claim 1, further comprising measuring a light intensity 1 of the measurement light transmitted through the first region and a light intensity 2 of the measurement light transmitted through the second region.

6. The following formula: Light attenuation rate = (light intensity 2 - light intensity 1) x 100 / light intensity 2 The immunoassay method of claim 5, further comprising the step of determining the light attenuation rate by:

7. 7. The immunoassay method according to claim 6, further comprising the step of obtaining a calibration curve or an approximate straight line showing the relationship between the concentration or amount of the analyte and the light attenuation rate using a sample whose concentration or amount of the analyte is known in advance.

8. The immunoassay method according to claim 6, further comprising a step of determining a light attenuation rate using a sample containing an analyte whose concentration or amount is unknown, and comparing the light attenuation rate with a calibration curve or an approximate straight line obtained in advance in claim 7, thereby estimating the concentration or amount of the analyte contained in the sample.

9. The immunoassay method according to claim 1 , wherein the light is coherent measurement light.

10. The immunoassay method according to claim 9 , wherein the coherent measurement light is laser light.

11. The immunoassay method of claim 1 , wherein the label particles comprise a metal.

12. 1. An immunoassay device, comprising: a holder for holding, in or on the device, a container or substrate containing a plurality of labeled particles carrying antibodies capable of directly or indirectly binding to the object to be measured; a light source disposed above the holder for irradiating a measurement light into an area of ​​the container or substrate held by the holder, the area including the plurality of labeled particles; a light detection unit that is disposed on the opposite side of the container or the substrate from the light source and detects transmitted light that has been irradiated from the light source and transmitted through an area of ​​the container or the substrate that contains the labeled particles; and the wavelength of the measurement light emitted by the light source is a wavelength that causes Mie scattering by each of the plurality of label particles; The immunoassay device, The immunoassay device is configured to irradiate a measurement light onto a region (first region) of the container or substrate containing the plurality of labeled particles and a region (second region) of the container or substrate outside the first region. The immunoassay device.

13. 13. An immunoassay device according to claim 12 for use in the method of claim 1.

14. The immunoassay device of claim 12, wherein the wavelength at which Mie scattering occurs by each of the plurality of labeled particles satisfies 2 < α < 10 (where α = π × labeled particle diameter (nm) / light wavelength (nm)).

15. The immunoassay device according to claim 12, further comprising a container or substrate containing a plurality of labeled particles carrying antibodies capable of directly or indirectly binding to the analyte.

16. 13. An apparatus according to claim 12; a container or substrate containing a plurality of labeled particles carrying antibodies capable of directly or indirectly binding to the object to be measured; A kit for immunoassay comprising:

17. 13. An apparatus according to claim 12; a container or substrate capable of arranging a plurality of labeled particles carrying antibodies capable of directly or indirectly binding to the object to be measured; A kit for immunoassay comprising:

18. 18. The immunoassay kit according to claim 17, further comprising a plurality of labeled particles carrying antibodies capable of directly or indirectly binding to the analyte.

19. A container or substrate containing a plurality of labeled particles carrying antibodies capable of directly or indirectly binding to the object to be measured, for use in the method of claim 1, for use in the device of claim 12, for use in the kit of claim 16, or for use in the kit of claim 17.

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