Immunoassay method and immunoassay apparatus

Abstract

To provide an immunoassay method and immunoassay apparatus that can quantify the target substance in a sample even at low concentrations. [Solution] An immunoassay method comprising a container or substrate containing a sample including a target to be measured and a plurality of labeled particles carrying antibodies that can directly or indirectly bind to the target to be measured, wherein the container or substrate is irradiated with light after contact between the sample including the target to be measured and the plurality of labeled particles carrying antibodies that can directly or indirectly bind to the target to be measured, the light being irradiated into a region of the container or substrate containing the labeled particles, and the irradiated light having passed through the region of the container or substrate containing the labeled particles, wherein the wavelength of the irradiated light is 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 apparatus.

Background Art

[0002] The immunoassay method (immunometric method) is a method for detecting or quantifying trace components (antigens or antibodies) as measurement targets contained in a sample by utilizing the specific reaction between an antigen and an antibody. Since the antigen-antibody reaction has high sensitivity and reaction selectivity, it is widely used in fields such as medicine, pharmaceuticals, health foods, biotechnology, and the environment, for drugs and foods that act only on specific locations (chemical substances) in the living body, analytical devices and diagnostic agents for detecting slight changes in the living body, and the like. There are various types of immunoassay methods depending on differences in their measurement principles and the like. For example, enzyme immunoassay, radioimmunoassay, chemiluminescent immunoassay, fluorescence immunoassay, immunoradiometric assay (IRMA), immunoenzymometric assay (IEMA) in which an antigen is labeled with an enzyme, latex agglutination method, latex agglutination inhibition method, immunochromatography method, ELISA method, Western blot method, hemagglutination method, hemagglutination inhibition method, and the like are known. All of these methods are common in that they detect or quantify the target component by utilizing the specific reaction between an antigen and an antibody.

[0003] Immunoassays generally detect or quantify a target substance (antigen or antibody) by attaching a labeling substance to an antibody, antigen, or antigen-antibody complex, and measuring the intensity of the color produced by the labeling substance, the signal intensity of luminescence, fluorescence, or radiation emitted by the labeling substance, or the turbidity of aggregates formed by cross-linking of the labeling substances. Known labeling substances include particles that produce color, such as gold colloid particles, nanoparticles, and quantum dots; substances that produce signals, such as radioactive elements, enzymes, chemiluminescent substances, and fluorescent dyes; and substances that cause agglutination, such as latex and red blood cells. Among these labeling substances, colored particles are widely used, especially when measurement needs to be performed quickly, because the presence and amount of the target substance can be easily determined visually or using measuring equipment. They are mainly used in immunochromatography. Furthermore, Patent Document 1 describes an immunoassay method using a lateral flow chromatography test strip containing a resin-platinum composite as a labeling substance, in which multiple relatively small platinum particles are immobilized on resin particles. This resin-platinum composite is described as exhibiting good color development and having excellent durability and visibility. Patent Document 2 describes a chromatography measuring device that determines the concentration of a substance to be measured in the solution to be measured based on an optical signal obtained by detecting transmitted or reflected light from a chromatography test piece, which has at least three reagent-immobilized parts arranged at intervals from each other on a developing layer, and by spreading a solution to be tested on a chromatography test piece, and irradiating the chromatography test piece with light. Patent Document 3 describes an immunoassay method characterized by preparing an antibody or antigen labeled with precious metal colloid particles, adding a sample to it to react the antigen or antibody contained in the sample with the antibody or antigen to form a precious metal colloid-labeled immunocomplex, and in a cell where at least one surface is a total reflection prism, measuring light with a wavelength between visible and infrared is incident on the total reflection prism to cause total reflection, and measuring the absorption in the absorption region specific to the precious metal colloid-labeled immunocomplex among the absorption of the measurement light generated at the interface between the total reflection prism and the sample mixture containing the labeled immunocomplex. [Prior art documents] [Patent Documents]

[0004] [Patent Document 1] Patent No. 6381642 [Patent Document 2] Japanese Patent Publication No. 2006-250787 [Patent Document 3] Patent No. 3436982 [Overview of the project] [Problems that the invention aims to solve]

[0005] However, conventional immunoassay methods using colored particles all involve measuring the intensity of the color exhibited by the labeled substance (color intensity) visually or with an optical measuring device. While visually measuring color intensity has the advantage of being easy and does not require an optical measuring device, it is susceptible to individual error. There is a risk that differences in color intensity may not be visually discernible even if the concentrations of the measured substance are different, leading to the misjudgment that they are the same concentration when they are actually different. As the concentration of the substance being measured decreases, the color development becomes weaker, increasing the risk of difficulty in accurate measurement. Furthermore, with visual measurement, the test results cannot be quantified, resulting in problems with reproducibility. On the other hand, when measuring color intensity using an optical measuring device such as an absorbance meter, the absorbance method is generally used, in which a light source is shone on the colored area due to the labeling substance, and the color intensity of the reflected light is measured by a photodetector. However, there is a limit to the absorbance per labeling particle, and in particular, in the low concentration range, the number of labeling particles bound to the target of measurement is small, resulting in poor sensitivity. Thus, with conventional methods, the range of measurable concentration is limited, and it is difficult to detect or quantify the target of measurement with high sensitivity, especially in the low concentration range.

[0006] Furthermore, while Patent Document 2 mentions determining the concentration of a substance to be measured in a solution based on an optical signal obtained by detecting transmitted or reflected light from a chromatographic test piece (immunochromatographic strip), it does not describe in detail the measurement method using transmitted light, and the examples do not include the necessary components. Moreover, Patent Document 2 does not focus on the relationship between the wavelength of light to be irradiated and the particle size of the labeled particles, and makes no mention of irradiating light under conditions that cause Mie scattering. Furthermore, it does not even mention detecting or quantifying the substance to be measured even in the low-concentration range as an issue. Similarly, Patent Document 1 does not focus on the relationship between the wavelength of light to be irradiated and the particle size of the labeled particles, and makes no mention of irradiating light under conditions that cause Mie scattering. Patent Document 3 does not mention the ability to detect or quantify the target even at low concentrations as a problem; rather, it clearly states that methods using Rayleigh scattering and Mie scattering have problems with low detection sensitivity and measurement accuracy at low concentrations.

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

[0008] To solve the above problems, the inventors conducted diligent 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 targets at lower concentrations than in conventional methods by irradiating labeled particles with measurement light under conditions where Mie scattering occurs and detecting the light intensity of the transmitted light.

[0009] The gist of this invention is as follows: [1] A container or substrate comprising a sample containing a target to be measured and a plurality of labeled particles carrying antibodies that can bind directly or indirectly to the target to be measured, wherein the container or substrate is irradiated with measurement light after contact between the sample containing the target to be measured and the plurality of labeled particles carrying antibodies that can bind directly or indirectly to the target to be measured, wherein the measurement light is irradiated into the region of the container or substrate containing the labeled particles, and A step of detecting the light (transmitted light) that has passed through the region from the irradiated measurement light, Includes, An immunoassay method wherein the wavelength of the irradiated measurement light is the 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 is generated by each of the plurality of labeled particles is a wavelength that satisfies 2 < α < 10 (where α = π × particle size of labeled particle (nm) / light wavelength (nm)). [3] The immunoassay method according to [1] or [2], further comprising the step of separating the antibody supported on the labeled particle that is directly or indirectly bound to the object to be measured from the antibody supported on the labeled particle that is not bound to the object to be measured, after contact and before irradiation with the measurement light. [4] The immunoassay method according to any one of [1] to [3], further comprising the step of determining the concentration or amount of the object to be measured 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 with the measurement light includes irradiating with the measurement light into a region of the container or substrate containing the plurality of labeled particles (hereinafter referred to as the first region) and irradiating with the measurement light into a second region outside the first region. [6] The immunoassay method according to [5], comprising measuring the light intensity 1 of the measurement light transmitted through the first region and the 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) × 100 / light intensity 2 The immunoassay method according to [6], further comprising the step of determining the light attenuation rate. [8] The immunoassay method according to [7], further comprising the step of obtaining a calibration curve or approximate straight line showing the relationship between the concentration or amount of the target to be measured and the light attenuation rate, using a sample whose concentration or amount of the target to be measured is known in advance. [9] The immunoassay method according to [7], further comprising the step of determining the light attenuation rate using a sample containing a target to be measured of unknown concentration or quantity, and estimating the concentration or quantity of the target to be measured contained in the sample by comparing the light attenuation rate with a calibration curve or approximate straight line previously determined in [8].

[10] The immunoassay method according to any one of [1] to [9], wherein the antibody that can bind directly or indirectly to the target of measurement is an antibody that can specifically bind to the target of measurement.

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

[10] , wherein the light is a 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 immobilized at least partially 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 apparatus, A holding part for holding, within or on a device, a container or a substrate containing a plurality of labeled particles carrying an antibody that can be directly or indirectly bound to a measurement target; A light source disposed above the holding part and irradiating measurement light into a region containing the plurality of labeled particles of the container or the substrate held by the holding part; A light detection part disposed on the opposite side of the light source with the container or the substrate interposed therebetween, and detecting transmitted light that has been irradiated from the light source and transmitted through the region containing the labeled particles of the container or the substrate; having; The wavelength of the measurement light irradiated by the light source is a wavelength that generates Mie scattering by each of the plurality of labeled 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 wavelength that generates Mie scattering by each of the plurality of labeled particles is a wavelength satisfying 2 < α < 10 (where α = π × particle size of the labeled particle (nm) / light wavelength (nm)). The immunoassay device according to 〔18〕 or 〔19〕. 〔21〕 The immunoassay device according to any one of 〔18〕 to 〔20〕, further comprising a container or a substrate containing a plurality of labeled particles carrying an antibody that can be directly or indirectly bound to a measurement target. 〔22〕 The antibody that can be directly or indirectly bound to a measurement target is an antibody that can specifically bind to the measurement target. The immunoassay device according to any one of 〔18〕 to 〔21〕. 〔23〕 The immunoassay device according to any one of 〔18〕 to 〔22〕, further comprising a display part that displays the light intensity of the transmitted light detected by the light detection part. 〔24〕 The immunoassay device according to any one of 〔18〕 to 〔23〕, further comprising an estimation part that estimates the amount, concentration or quantity of the measurement target contained in a sample based on the light intensity of the transmitted light detected by the light detection part. [(25)] The apparatus for immunoassay is configured to irradiate measurement light onto each of a region (first region) containing the plurality of labeled particles of the container or substrate and a region (second region) outside the first region of the container or substrate. [(18)] The apparatus for immunoassay according to any one of [(18)] to [(24)]. [(26)] The apparatus for immunoassay according to [(25)], further comprising an estimation unit that estimates the concentration or amount of the measurement target 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 apparatus according to any one of [(18)] to [(26)], wherein the light source is a light source that irradiates coherent light. [(28)] The immunoassay apparatus according to [(27)], wherein the coherent measurement light is laser light. [(29)] The immunoassay apparatus according to any one of [(18)] to [(28)], wherein the light source is a light source that can irradiate measurement light within a range narrower than and within the region containing the labeled particles in the container or substrate when viewed from the light source side in the direction of the light detection unit. [(30)] The immunoassay apparatus 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 unit, measurement light is irradiated from the light source within a range narrower than and within the region containing the labeled particles in the container or substrate. [(31)] The immunoassay apparatus 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 apparatus according to any one of [(18)] to [(31)], wherein the labeled particles contain metal. [(33)] The immunoassay apparatus 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 resin particles.

[34] The immunoassay apparatus according to

[32] or

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

[35] The immunoassay apparatus according to

[32] or

[33] , wherein the metal includes platinum.

[36] An immunoassay apparatus according to any one of

[18] to

[35] , wherein, when viewed from the light source in the direction of the photodetector, the plurality of labeled particles are present in a three-dimensional overlapping arrangement within the region.

[37] The immunoassay apparatus according to any one of

[18] to

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

[38] An immunoassay apparatus according to any of

[18] to

[37] for estimating the concentration or amount of the substance to be measured contained in a sample.

[39] The apparatus described in any of

[18] to

[38] , A container or substrate containing multiple labeled particles carrying antibodies capable of directly or indirectly binding to the object to be measured, An immunoassay kit including [specific components].

[40] The apparatus described in any of

[18] to

[38] , A container or substrate capable of arranging multiple labeled particles carrying antibodies that can directly or indirectly bind to the target of measurement, An immunoassay kit including [specific components].

[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 that can directly or indirectly bind to a target for measurement, for use in the method described in any of [1] to

[17] , or in the apparatus described in any of

[18] to

[38] , or in the kit described in

[39] , or in the kit described in any of

[40] to

[41] .

[43] The container or substrate described in

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

[44] The container or substrate according to

[42] , wherein the antibody that can bind directly or indirectly to the object to be measured is an antibody that can specifically bind to the object to be measured. [Effects of the Invention]

[0010] The immunoassay method and immunoassay apparatus of the present invention can detect or quantify the target substance with high sensitivity even at low concentrations. [Brief explanation of the drawing]

[0011] [Figure 1] Figure 1 is a schematic longitudinal cross-sectional view of an immunochromatography test strip used in one embodiment of the present invention. [Figure 2] Figure 2 is a schematic diagram of the housing case used in the test strip in Figure 1, viewed from the top. [Figure 3] Figure 3 is a schematic diagram of the housing case used in the test strip in Figure 1, viewed from the bottom. [Figure 4] Figure 4 is a schematic longitudinal cross-sectional view of a conventional immunochromatography test strip. [Figure 5] Figure 5 is a schematic longitudinal cross-sectional view of an immunoassay apparatus according to one embodiment of the present invention. [Figure 6] Figure 6 is an external view (perspective view) of an immunoassay apparatus according to one embodiment of the present invention. [Figure 7] Figure 7 is a schematic longitudinal cross-sectional view of an immunoassay apparatus according to another embodiment of the present invention. [Figure 8] Figure 8 is a graph showing the relationship between the dilution rate and the optical attenuation rate of the object being measured in Example 1. [Figure 9] Figure 9 is a graph showing the relationship between the dilution ratio and color intensity of the measured samples in Comparative Examples 1 and 2. [Figure 10] Figure 10 is a graph showing the relationship between the dilution ratio and the light attenuation rate of the measured object in Example 1 and Comparative Example 3. [Figure 11]Figure 11 is a graph showing the relationship between the dilution rate and the optical attenuation rate of the object being measured when irradiated with light of various wavelengths under conditions that induce Mie scattering. [Figure 12] Figure 12 is a graph showing the relationship between the dilution rate of the object being measured and the optical attenuation rate when using labeled particles of various particle sizes under conditions that induce Mie scattering. [Modes for carrying out the invention]

[0012] <<Immunoassay Method>> A first aspect of the present invention is a container or substrate comprising a sample containing a target to be measured and a plurality of labeled particles carrying antibodies capable of directly or indirectly binding to the target to be measured, wherein the container or substrate is irradiated with measurement light after contact between the sample containing the target to be measured and the plurality of labeled particles carrying antibodies capable of directly or indirectly binding to the target to be measured, wherein the measurement light is irradiated into the region of the container or substrate containing the labeled particles. A step of detecting the light (transmitted light) that has passed through the region from the irradiated measurement light, Includes, The immunoassay method is characterized in that the wavelength of the irradiated measurement light is the wavelength that causes Mie scattering by each of the plurality of labeled particles.

[0013] The method of the first embodiment can be preferably used to detect a target substance contained in a sample and / or to quantify the target substance (to estimate its concentration or amount). In particular, it can be preferably used to quantify the target substance (to estimate its concentration or amount). The following describes each process and component.

[0014] <Step of irradiating with measurement light> A method according to the first embodiment includes a container or substrate containing a sample containing a target to be measured and a plurality of labeled particles carrying antibodies that can directly or indirectly bind to the target to be measured, the method comprising the step of irradiating the container or substrate with measurement light after contact between the sample containing the target to be measured and the plurality of labeled particles carrying antibodies that can directly or indirectly bind to the target to be measured. Here, the measurement light is irradiated into the region containing the labeled particles. In the step of irradiating with measurement light, the measurement light is irradiated into a region of the container or substrate containing multiple labeled particles. For example, in the case of (lateral flow type) immunochromatography, labeled particles that have been labeled with the target of measurement are captured on the test line. At this time, it is preferable that the width of the measurement light irradiated is 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 within the region containing the labeled particles, noise can be reduced, signal intensity can be increased, and sensitivity in the low concentration range can be improved. The measurement light may be irradiated into a first region containing multiple labeled particles of the container or substrate, and into a second region different from the first region (preferably a region not containing multiple labeled particles). The irradiation of the first and second regions with the measurement light may be simultaneous or at different timings. If irradiation is performed simultaneously, two light sources can be used to irradiate the first and second regions with measurement light at the same time. It is also possible to irradiate the first and second regions with measurement light at different timings by sequentially irradiating from two light sources. Furthermore, if the first and second regions are irradiated with measurement light at different timings, the first region may be irradiated with measurement light 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 photodetector) until the light source is positioned above the second region, thereby irradiating the second region with measurement light. Alternatively, the light source may be positioned above the first region to irradiate the first region with measurement light, and then the light source may be moved laterally (perpendicular to the direction from the light source to the photodetector) until the light source is above the second region, thereby irradiating the second region with measurement light. Furthermore, there are no particular restrictions on the order in which the measurement light is irradiated into the first and second regions; the measurement light may be irradiated into the first region first and then into the second region, or vice versa. A sliding device, described later, can be used as a means for moving the container or substrate.

[0015] (Measurement light) The measurement light is the light emitted from a light source for the purpose of immunoassay (immunological measurement). The wavelength of the irradiated measurement light is the wavelength that causes Mie scattering by each of the plurality of labeled particles. When measurement light strikes a particle, it undergoes transmission, reflection, refraction, or scattering. Of these, scattering is further divided into shielding, diffraction, Rayleigh scattering, and Mie scattering, depending on the wavelength of the measurement light and the particle size of the particle being irradiated. The particle size parameter α, expressed as α = π × particle size (nm) / light wavelength (nm), is 10 4 If <α, then light shielding occurs, and 10 < α < 10 4It is known that diffraction occurs when α < 2, Mie scattering occurs when α < 10, and Rayleigh scattering occurs when α < 2 (see, for example, "Basic Physical Properties of Powders," edited by the Japan Society of Powder Technology, published by Nikkan Kogyo Shimbun, p. 25). Note that particle size refers to the diameter of the particle. In this invention, the wavelength of the measurement light irradiated is the wavelength that causes Mie scattering by each of the multiple labeled particles. Preferably, the wavelength that causes Mie scattering by each of the multiple labeled particles is a wavelength that satisfies 2 < α < 10 (where α = π × particle size of the labeled particle (nm) / light wavelength (nm)). Based on this formula, for example, when using labeled particles having a particle size of 450 nm, it is preferable to irradiate with measurement light of a wavelength in the range of approximately 141.3 nm to approximately 706.5 nm 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 is broad and its wavelength range and phase are not aligned, so it has a divergent characteristic. Therefore, the scattering characteristics of the light in the dispersion medium are not uniform and the light penetration is poor. However, even LEDs can be used if they have the above characteristics. In contrast, coherent light (especially laser light) has properties such as wavelength and phase aligned, and the light has the characteristic of high straightness. By using coherent light (especially laser light), it becomes easier to set the relationship between particle size, which determines particle parameters, and the wavelength of the incident light.

[0016] (Measurement target) There are no particular restrictions on what can be measured; a wide variety of substances can be used as the measurement target. Furthermore, 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 the body. There are no particular restrictions on the sample, as long as it may contain the object to be measured, and various samples can be used. For example, the sample may be one that is commonly used in clinical testing, such as tissue, cells, cell extracts, or body fluids.

[0017] (Labeled particles carrying antibodies that can bind directly or indirectly to the target of measurement) Labeled particles carrying antibodies that can bind directly or indirectly to the target of measurement may be labeled particles carrying antibodies that can directly bind to the target of measurement, or labeled particles carrying antibodies that can indirectly bind to the target of measurement via another substance. A labeled particle is said to carry an antibody that can directly bind to the target of measurement if the labeled particle is bound to an antibody that directly binds to the target of measurement (for example, if the target of measurement is an antigen, a monoclonal antibody or polyclonal antibody that specifically binds to the antigen; if the target of measurement is an antibody (which is also an antigen), a monoclonal antibody or polyclonal antibody that binds to the antibody). A labeled particle is said to carry an antibody that can indirectly bind to the target of measurement if the labeled particle is bound to an antibody that can bind directly to a substance that binds directly to the target of measurement (for example, if the substance that binds directly to the target of measurement is a primary antibody, then a secondary antibody that binds to the primary antibody). Examples of labeled particles carrying antibodies that can directly bind to the target of measurement include, when the target of measurement is an antigen, labeled particles carrying antibodies that can (specifically) bind to that antigen. When the target of measurement is an antibody, examples include labeled particles carrying antibodies that can (specifically) bind to that antibody. The antibodies that can (specifically) bind to the target of measurement may be commercially available or manufactured in-house. Labeled particles carrying antibodies that can indirectly bind to the target of measurement via other substances can be labeled particles carrying antibodies that can bind to a substance that can bind to the target of measurement (directly or indirectly). For example, the antibody that can indirectly bind to the target of measurement via other substances may be a secondary antibody. In this case, the target of measurement can be labeled by the labeled particles when the secondary antibody carried on the labeled particle binds to an antibody (primary antibody) that can directly bind to the antigen that is the target of measurement. For examples of the target of measurement and antibodies that can bind to the target of measurement directly or indirectly, you can refer to the descriptions of direct, indirect, and sensitized methods in the section "II. Advantages and Disadvantages of Each Immunoassay Method" in the DOJINDO product catalog "First Steps in Antibody Labeling Protocols - 1" (URL) https: / / www.dojindo.co.jp / products / contents / inomu-antibody-enzyme-fluo-protein-fab-labeling-1.html. Accordingly, the method of the first embodiment includes a container or substrate comprising a target to be measured, a substance that can specifically bind to the target to be measured and is not supported on labeled particles, and a plurality of labeled particles supporting antibodies that can indirectly bind to the target to be measured (for example, a plurality of labeled particles supporting antibodies that can bind to a substance that can specifically bind to the target to be measured), wherein, by contact between these, the antibodies supported on the labeled particles may indirectly bind to the target to be measured via a substance that specifically binds to the target to be measured.

[0018] (labeled particles) The method of the present invention uses a plurality of labeled particles on which antibodies capable of directly or indirectly binding to the object to be measured are carried. Multiple labeled particles carrying antibodies capable of directly or indirectly binding to the target of measurement refer to multiple labeled particles, each of which carries an antibody capable of directly or indirectly binding to the target of measurement. Each labeled particle may carry one antibody capable of directly or indirectly binding to the target of measurement, or it may carry multiple antibodies. In the case of multiple antibodies, the multiple antibodies may be the same antibody or different antibodies. "Supporting" means that an antibody capable of directly or indirectly binding to the target to be measured is immobilized on the surface of the labeled particles, preferably the surface of the labeled particles. There are no particular restrictions on the method of immobilizing the antibody capable of directly or indirectly binding to the target to be measured on the surface of the labeled particles. For example, the antibody capable of directly or indirectly binding to the target to be measured can be immobilized on the surface of the labeled particles by passive adsorption or by covalent bonding via a crosslinking agent. For information on the passive adsorption method and the covalent bonding via a crosslinking agent, 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 particles via a linker. The particle size of the labeled particles is not particularly limited as long as Mie scattering occurs when the irradiated light strikes each of the labeled particles, and labeled particles of various particle sizes can be used. In particular, it is preferable that the labeled particles satisfy 2 < α < 10 (where α = π × particle size of labeled particle (nm) / wavelength of light (nm)). 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 in the range of approximately 286.6 nm to approximately 1433.1 nm. Also, for example, when the irradiated light is visible light (for example, wavelengths of approximately 360 nm to approximately 830 nm), the particle size of the labeled particles is preferably in the range of approximately 229.3 nm to approximately 2643.3 nm. Furthermore, in this invention, the irradiated light is not limited to visible light; for example, even if ultraviolet light or infrared light is used, it is sufficient to select a particle size within the range in which Mie scattering occurs. The particle size (or average particle size) of the labeled particles may be in the range of approximately 528.7 nm to approximately 3.2 mm when irradiated with infrared light (for example, wavelengths of approximately 830 nm to approximately 1 mm), or in 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 maintain a uniform dispersion system in the medium being measured. The average particle size can be measured using a centrifugal sedimentation particle size distribution analyzer, laser diffraction / scattering method, dynamic light scattering method, particle counter, electrolytic emission scanning electron microscope, etc. The average particle size can be determined by methods such as the area-average diameter, and is the diameter of the particle.

[0019] The labeled particles are preferably particles through which the irradiated measurement light does not pass. When light does not pass through the labeled particles, the light that passes through the region containing the particles and reaches the detector is light originating from Mie scattering, which tends to increase sensitivity. Preferred labeled particles that do not allow light to pass through include metal-containing labeled particles, as well as resin particles that do not transmit light and particles using silica. For example, resin particles include polyamines, polyamides, polypeptides, polyurethanes, polystyrenes, polyureas, polyimides, polyimidazoles, polyoxazoles, polypyrroles, and polyanilines. Furthermore, a wide range of materials can be used, such as polyamines (poly-2-vinylpyridine, poly-3-vinylpyridine, poly-4-vinylpyridine), acrylic resins, phenolic resins, epoxy resins, cellulose resins, and melamine resins. The labeled particles preferably contain metal, and more preferably contain metal on at least part or all of the surface of the labeled particles. The inclusion of metal in the labeled particles makes it easier to obtain particles in which the irradiated measurement light does not penetrate. The labeled particles may be particles composed of metal, particles on which metal is coated on the surface of non-metallic particles, or particles in which multiple metal particles are immobilized on and / or within non-metallic particles. Among these, it is preferable that the labeled particles are particles on which metal is coated on the surface of non-metallic particles, particles in which multiple metal particles are immobilized on non-metallic particles, or particles in which multiple metal particles are immobilized at least partially within non-metallic particles, more preferably particles in which multiple metal particles are immobilized on non-metallic particles or particles in which multiple metal particles are immobilized at least partially within non-metallic particles, and even more preferably metal-resin composite particles in which multiple metal particles are immobilized on resin particles or metal-resin composite particles in which multiple metal particles are immobilized at least partially within particles. Multiple metal particles being at least partially fixed within non-metal particles means that multiple metal particles are at least partially fixed within non-metal particles (preferably, some of the multiple metal particles are exposed outside the non-metal particles, while the rest are enclosed within the non-metal particles). Multiple metal particles being at least partially fixed within resin particles means that multiple metal particles are at least partially fixed within resin particles (preferably, some of the multiple metal particles are exposed outside the resin particles, while the rest are enclosed within the resin particles). Examples of metal-resin composite particles in which multiple metal particles are immobilized on resin particles, and methods for producing the same, include the metal-resin composite particles and method for producing the same described in International Publication WO2017 / 010391. Other examples of metal-resin composite particles include structures in which a resin coating is applied to the surface of metal particles, and structures in which a metal layer (which may be an aggregate of metal particles) is laminated on the surface of resin particles. Furthermore, there are no particular restrictions on the type of metal, and various metals can be used. Examples include metals such as platinum, gold, iron, iron oxide, nickel, silver, copper, or 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 contain at least one metal selected from the group consisting of platinum and gold, and even more preferably contain 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 immobilized on the surface of non-metallic particles, there are no particular restrictions on the average particle size of the multiple metal particles. 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 immobilized on non-metallic particles, there are no particular restrictions on the proportion of metal immobilized on the non-metallic particles. 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 base material) The container or substrate used in the method of the first embodiment contains a sample containing the object to be measured and a plurality of labeled particles carrying antibodies that can directly or indirectly bind to the object to be measured, and the sample containing the object to be measured and the plurality of labeled particles carrying antibodies that can directly or indirectly bind to the object to be measured are in contact. The container or substrate in which the sample containing the object to be measured and the labeled particles have been in contact is irradiated with measurement light. There are no restrictions on the type of container or substrate, and various containers or substrates can be used. However, the container or substrate must be configured such that when measurement light is irradiated from a light source onto the region containing the labeled particles (and the region not containing the labeled particles) within the container or substrate, the light can pass through the region containing the labeled particles (or each of the said region and the other regions (preferably the region not containing the 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 in at least the portion through which the measurement light is transmitted, or it may be made of a porous material, so that the measurement light can be transmitted. Examples of porous materials include porous membranes such as nitrocellulose films, woven fabrics, and nonwoven fabrics. Furthermore, when using a substrate in a form contained within a container, the container containing the substrate may have a hollow bottom portion through which light is transmitted (i.e., there is 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, test tubes, and microscope slides. Test strips and membranes may also be housed in a housing case. 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 it when light is shone from above. It is preferable that the test strip has at least a portion of its backing sheet that transmits light removed so that light can pass through when light is shone on it from above. Furthermore, if the test strip or membrane is housed in a housing case, it is preferable that at least a portion of the front and back surfaces of the housing case that transmits light is removed (opened).

[0021] The container or substrate after contact between the sample containing the target to be measured and multiple labeled particles carrying antibodies that can directly or indirectly bind to the target to be measured is the container or substrate after contact (after the antigen-antibody reaction has occurred) between the sample containing the target to be measured and multiple labeled particles carrying antibodies that can directly or indirectly bind to the target to be measured. Since the target to be measured is labeled by the labeled particles due to the antigen-antibody reaction, the presence of the target to be measured can be detected or its concentration and quantity 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 restrictions on the method of contacting a sample containing the target to be measured with multiple labeled particles carrying antibodies that can directly or indirectly bind to the target to be measured. For example, in the case of immunochromatography, multiple labeled particles carrying antibodies that can directly or indirectly bind to the target to be measured can be placed in the conjugate pad 4 of the test strip 1, and a (liquid) sample containing the target to be measured can be dropped into the sample pad 3. After dropping, the sample spreads from the sample pad 3 to the conjugate pad 4, and the sample containing the target to be measured comes into contact with the multiple labeled particles carrying antibodies that can directly or indirectly bind to the target to be measured, and the target to be measured is labeled with the labeled particles. Subsequently, the sample containing the target to be measured particles labeled with the labeled particles spreads onto the membrane 5 and passes through the test line 6. Since antibodies that can bind to the target to be measured are immobilized on the test line 6, the target to be measured labeled with the labeled particles is captured by the test line 6. This test line 6 is the region where the object to be measured, labeled with the labeled particles, is located. By irradiating this region with light, the light intensity of the transmitted light can be measured. Any other substances not captured by test line 6 are transferred to control line 7 along with the sample. Control line 7 has an immobilized antibody capable of binding to an antibody that can directly or indirectly bind to the target substance. Therefore, any labeled particles that do not bind to the target substance, despite carrying antibodies capable of directly or indirectly binding to it, are captured by control line 7. The remaining sample is then absorbed by absorption pad 8.

[0022] (The region where the object to be measured, labeled with labeled particles, is located) After contact between a sample containing the target to be measured and a plurality of labeled particles carrying antibodies capable of directly or indirectly binding to the target to be measured, the labeled particles that have labeled the target to be measured are present in or move within the region where the target to be measured, labeled by the labeled particles, is located. Preferably, the region where the target to be measured, labeled by the labeled particles, is located has thickness in the direction from the light source to the detector. The greater the thickness of the region, the more labeled particles are present across that thickness, meaning that light strikes more labeled particles before reaching the photodetector. The attenuation of light increases exponentially with respect to the thickness (depending on the frequency of strikes by labeled particles), so by adjusting the thickness, it becomes easier to measure the concentration or quantity of the substance being measured with high sensitivity. For example, in immunochromatography, the substance being measured, 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 test line membrane. In the case of ELISA, the substance being measured, labeled with labeled particles, is present in the liquid in each well of the 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 substance being measured, labeled with labeled particles, is present). The region where the substance being measured, labeled with labeled particles, is present is the region that transmits light irradiated from the light source to the detector, and can therefore be called the medium. Similarly, the thickness of the region can be called the thickness of the medium. In this case, there are no particular restrictions on the type of medium, and various mediums can be used. Examples include the membranes of test strips used in immunochromatography, the liquids (developing solvent, buffer, water, organic solvent) contained in the wells of well plates used in ELISA, liquids, gels and thin layers (silica particle gel, ceramic particles, resin particles, cellulose particles), woven fabrics, nonwoven fabrics, and paper. For reference, when light travels through the thickness of a homogeneous, semi-transparent medium, the attenuation is exponential and can be expressed by the Lambert-Beer law shown below. JPEG2026089266000002.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-propagated position (unit: [W / nm]), and α(λ) is the spectral absorption coefficient of the medium.

[0023] There are no particular restrictions on the thickness of the area or medium in which the object to be measured exists; the most suitable option should be used based on the type of object to be measured and its expected concentration.

[0024] <Separation process> The method of the first embodiment may further include a step of separating the antibodies supported on the labeled particles that are directly or indirectly bound to the target for measurement from the antibodies supported on the labeled particles that are not bound to the target for measurement, after contact between the sample containing the target for measurement and a plurality of labeled particles on which antibodies capable of directly or indirectly binding to the target for measurement are carried, and before irradiation with measurement light. By including a separation step, the region containing the labeled particles irradiated with the measurement light is more likely to contain only the antibodies supported on the labeled particles that are directly or indirectly bound to the target of measurement, thereby further improving sensitivity. The separation step may be configured, as in a lateral flow immunochromatographic assay, to capture only the antibodies supported on labeled particles that are directly or indirectly bound to the target of measurement in a predetermined area while the sample or labeled particles flow through the test strip, and to not capture antibodies supported on labeled particles that are not bound to the target of measurement in the same area, thereby (automatically) separating antibodies supported on labeled particles that are directly or indirectly bound to the target of measurement from antibodies supported on labeled particles that are not bound to the target of measurement. Alternatively, the two can be separated manually by providing an area at the bottom of the container to capture antibodies supported on labeled particles that are directly or indirectly bound to the target of measurement, and discarding the liquid containing antibodies supported on labeled particles that are not bound to the target of measurement.

[0025] <Process for detecting light> The method according to the first embodiment includes the step of detecting transmitted light that has passed through a region of the measurement light that contains labeled particles on the container or substrate. There are no particular limitations on the means for detecting the transmitted light; for example, a photodiode, photomultiplier tube, photoconductive element, or imaging element may be used to detect the transmitted light. Furthermore, in the step of irradiating with measurement light, if the measurement light is irradiated into a first region containing multiple labeled particles of the container or substrate, and into a second region outside the first region, the step of detecting transmitted light will not only detect the transmitted light that has passed through the first region, but also detect the transmitted light that has passed through the second region. In this specification, the light irradiated from the light source toward the container or substrate is referred to as the measurement light, and the light that passes through the container or substrate is referred to as the transmitted light.

[0026] <Steps for detecting the target to be measured / estimating the concentration or amount of the target to be measured> The method according to the first aspect of the present invention may include a step of detecting a target to be measured from the light intensity of the detected light, and may further include a step of estimating the concentration or amount of the target to be measured from the light intensity of the detected light. Note that the light intensity is, The values ​​obtained from an illuminance meter using a photodiode (e.g., lux) are used.

[0027] Furthermore, when estimating the concentration or amount of the substance to be measured, light may be irradiated into a first region containing multiple labeled particles of a container or substrate, and into a second region different from the first region (preferably a region not containing multiple labeled particles), and the light intensity 1 of the light transmitted through the first region and the light intensity 2 of the light transmitted through the second region may be measured. In addition, the method of the first embodiment of the present invention may further include a step of determining the light attenuation rate by the following formula. Light attenuation rate = (light intensity 2 - light intensity 1) × 100 / light intensity 2 The optical attenuation rate is an index that indicates how much the light intensity of transmitted light decreases when light is irradiated into a first region containing a measurement target labeled with multiple labeled particles, compared to the light intensity of transmitted light when light is irradiated into a second region. It allows us to understand the degree of attenuation of transmitted light caused by Mie scattering.

[0028] <Process for determining the calibration curve> The method of the first embodiment may further include a step of determining a calibration curve showing the relationship between the concentration or amount of the object to be measured and the light attenuation rate, using a sample whose concentration or amount of the object to be measured is known in advance. The calibration curve is preferably linear, and more preferably an approximate straight line. For example, the calibration curve or approximate straight line may have the vertical axis (y) as the light attenuation rate (%) and the horizontal axis (x) as the dilution rate (%), with both axes being logarithmic. 10The values ​​obtained above can be plotted as a log-log graph of the scale, and a calibration curve or approximate line can be obtained using the least squares method and the regression equation (y = ax + b) of simple linear regression analysis. By obtaining a reference line, the optical attenuation rate can be calculated by performing the method of the first embodiment on a sample containing a target whose concentration or quantity is unknown, and the concentration or quantity of the target can be estimated by comparing the calculated optical attenuation rate with the calibration curve.

[0029] <Steps for estimating the concentration or quantity of the object to be measured> The method of the first embodiment may further include a step of estimating the concentration or amount of the substance to be measured contained in the sample of unknown concentration or amount by comparing the optical attenuation rate obtained using a sample of unknown concentration or amount with a calibration curve that has been determined in advance. A calibration curve plots the concentration or amount (dilution ratio) of the substance being measured on the horizontal axis and the light attenuation rate on the vertical axis. Therefore, by comparing the light attenuation rate obtained using a sample with an unknown concentration or amount of the substance being measured with the calibration curve, the concentration or amount of the substance being measured in the calibration curve corresponding to the light attenuation rate obtained using the sample with an unknown concentration or amount can be determined. The determined concentration or amount can then be estimated to be the concentration or amount of the substance being measured in the sample.

[0030] <<Immunoassay device>> An apparatus according to a second aspect of the present invention is an immunoassay apparatus, A container or substrate containing a holding part for holding a plurality of labeled particles carrying antibodies that can bind directly or indirectly to the object to be measured, A light source positioned above the holding portion for irradiating light into the region of the container or substrate held by the holding portion that contains the plurality of labeled particles, A photodetector is positioned on the opposite side of the container or substrate from the light source and detects light that has been irradiated from the light source and has passed through the region of the container or substrate containing the labeled particles. It has, The wavelength of the light emitted by the light source is such that Mie scattering occurs in each of the plurality of labeled particles. This is the immunoassay apparatus mentioned above. The apparatus of the second embodiment can preferably be used to detect the substance to be measured contained in a sample, or to estimate the concentration or amount of the substance to be measured. Furthermore, the apparatus of the second embodiment can be suitably used in the method of the first embodiment. Therefore, the apparatus of the second embodiment is preferably an apparatus for use in the method of the first embodiment. The object to be measured, the antibody that can bind directly or indirectly to the object to be measured, the labeled particles, etc., may be those listed in the first embodiment.

[0031] (light source) The apparatus of the second embodiment has a light source positioned above the holding portion, and the light source is for irradiating light into a region of a container or substrate held by the holding portion that contains a plurality of labeled 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 × particle size (nm) of the labeled particle / wavelength of light (nm)). Furthermore, while there are no particular restrictions on the type of light source, it is preferable that the light source has coherent characteristics and generates light that can withstand exponential light attenuation due to Mie scattering, and more preferably that it is laser light. However, other types of measurement light can also be used as long as they possess the above properties. Furthermore, it is preferable that the light source is capable of irradiating light to the same area as or a narrower area 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 it is more preferable that the light source is capable of irradiating light to a narrower area than the area containing the labeled particles in the container or substrate. Furthermore, the apparatus of the second embodiment is preferably configured to irradiate light into a first region of the container or substrate containing the plurality of labeled particles, and into a second region of the container or substrate that does not contain the plurality of labeled particles. The irradiation of light into the first region and the second region may be simultaneous or at different timings. When irradiation is performed simultaneously, two light sources can be provided to irradiate light into the first region and the second region at the same time. 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 timings, the light source may be positioned above the first region to irradiate light into 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 to irradiate light into the second region. Alternatively, the light source may be positioned above the first region to irradiate the first region with light, and then the light source 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 to irradiate the second region with light. Furthermore, there are no particular restrictions on the order in which light is irradiated into the first and second regions; the first region may be irradiated first and then the second region, or the second region may be irradiated first and then the first region. As a means of moving the container or substrate, a sliding device described later can be mentioned.

[0032] (Slit section) The apparatus of the second embodiment may have a slit between the light source and the container or substrate. This makes it easier to irradiate a narrower area with light than the area containing the labeled particles in the container or substrate when viewed from the light source side toward the photodetector. The slit is made by providing a small slit hole in a plate-shaped member, and the light from the light source passes through the slit hole, allowing light to irradiate a narrower area. There are no particular restrictions on the shape of the slit hole; it may be circular, square, rectangular, triangular, or any other shape, and an appropriate shape of slit hole should be selected according to the shape of the area containing the labeled particles. Preferably, the maximum width of the slit hole is smaller than the width of the light irradiated from the light source into the slit. Furthermore, the apparatus of the second embodiment 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 photodetector to detect only the light irradiated into the area containing the labeled particles. There are no particular restrictions on the shape of the slit hole; it may be circular, square, rectangular, triangular, or any other shape, and a slit hole of an appropriate shape can be selected according to the shape of the area containing the labeled particles. Preferably, the maximum width of the slit hole is smaller than the width of the light irradiated from the light source into the area containing the labeled particles and transmitted through the slit.

[0033] (holding part) The apparatus of the second embodiment has a holding part for holding a container or substrate containing a plurality of labeled particles carrying antibodies that can directly or indirectly bind to the object to be measured. There are no particular restrictions on the shape of the holding part as long as it can hold the container or substrate. For example, the immunoassay apparatus may have a holding part 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 unidirectional extension of the concave shape. Having a holding part with a concave shape extending in one direction makes it easier to slide the container or substrate in either the front-to-back direction or the left-to-right direction. Alternatively, the immunoassay apparatus may have a holding part with a mechanism for clamping and fixing the container or substrate. In this case, it is preferable that the holding part has the function of moving the container or substrate in the front-to-back direction and / or the left-to-right direction.

[0034] (Container or base material) The apparatus of the second embodiment may further include a container or substrate containing a plurality of labeled particles on which antibodies capable of directly or indirectly binding to the object to be measured are supported. Furthermore, the apparatus of the second embodiment may further include a container or a substrate. Alternatively, the container or substrate may be prepared separately from the apparatus, and after the sample containing the target to be measured and a plurality of labeled particles carrying antibodies that can directly or indirectly bind to the target to be measured are brought into contact within the container or substrate, it may be held in a holding section within the apparatus. For example, in the case of the ELISA method, the plurality of labeled particles carrying antibodies that can directly or indirectly bind to the target to be measured are not included in the container beforehand but are added at the time of the test. Therefore, the apparatus of the second embodiment may further include the container or substrate in a form that does not include the plurality of labeled particles carrying antibodies that can directly or indirectly bind to the target to be measured. Examples of containers or substrates include those listed in the first embodiment. When a container or substrate is irradiated with light from a light source, it is preferable that, when viewed from the light source towards the photodetector, multiple labeled particles are present in an overlapping manner within a region containing multiple labeled particles. The overlapping of multiple labeled particles means that within the region of the container or substrate, multiple labeled particles are densely present in all three directions: the X-axis direction (left-right direction), the Y-axis direction (front-back direction), and the Z-axis direction (thickness direction), and when viewed from the light source towards the photodetector, the multiple labeled particles appear to overlap each other.

[0035] (Light detection unit) The apparatus of the second embodiment has a photodetector positioned on the opposite side of the container or substrate from the light source, which detects light irradiated from the light source and transmitted through the area of ​​the container or substrate containing the labeled particles. The photodetector may be composed of, for example, a photodiode. Furthermore, when the light source irradiates light into a first region containing multiple labeled particles of a container or substrate, and into a second region different from the first region (preferably a region that does not contain multiple labeled particles), the light detection unit not only detects the light transmitted through the first region, but also detects the light transmitted through the second region.

[0036] (Display) The apparatus of the second embodiment may further include a display unit that displays the light intensity of light detected by the photodetector. The display unit is connected to the photodetector by wire or wireless, and displays the light intensity detected by the photodetector, the light attenuation rate calculated from the light intensity, and / or the concentration or amount of the object to be measured estimated from the light intensity or light attenuation rate.

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

[0038] (Slide section) The apparatus of the second embodiment may further have a sliding part for sliding a container or substrate. The sliding part may slide the container or substrate away from the sliding part, or it may slide the container or substrate towards the sliding part, or it may have both mechanisms. Furthermore, the sliding part may be capable of sliding the container or substrate only in the left-right direction, or it 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 both the left-right and front-back directions, for example, even when there are multiple wells in both the front-back and left-right directions, such as in a 96-well plate, the well plate can be moved by the sliding part, and the light intensity of the object to be measured present in 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 apparatus according to the second aspect and a container or substrate containing a plurality of labeled particles on which antibodies capable of directly or indirectly binding to a target for measurement are carried. The objects to be measured, antibodies that can directly or indirectly bind to the objects to be measured, labeled particles, containers, or substrates can be those described in the first or second embodiment. The kit of the third embodiment can be preferably used in the method of the first embodiment.

[0040] <<Immunoassay Kit 2>> A fourth embodiment of the present invention is an immunoassay kit comprising the apparatus of the second embodiment and a container or substrate on which a plurality of labeled particles carrying antibodies capable of directly or indirectly binding to a target for measurement can be placed. The kit of the fourth embodiment preferably further comprises a plurality of labeled particles carrying antibodies that can bind directly or indirectly to the object to be measured. The objects to be measured, antibodies that can directly or indirectly bind to the objects to be measured, labeled particles, containers, or substrates can be those described in the first or second embodiment. The kit of the fourth embodiment can be preferably used in the method of the first embodiment.

[0041] <<Containers or substrates for immunoassays>> A container or substrate according to a fifth aspect of the present invention is a container or substrate containing a plurality of labeled particles carrying antibodies that can directly or indirectly bind to a target for measurement, for use in the method of the first aspect, or in the apparatus of the second aspect, or in the kit of the third aspect, or in the kit of the fourth aspect. The objects to be measured, antibodies that can directly or indirectly bind to the objects to be measured, labeled particles, containers, or substrates can be those described in the first or second embodiment.

[0042] <Application to various immunoassay methods> The methods, apparatus, kits, and containers or substrates of the present invention can be applied to various types of immunoassay methods. Examples of immunoassay methods to which the methods of the present invention can be applied include, for example, enzyme immunoassay, radioimmunoassay, chemiluminescence immunoassay, fluorescence immunoassay, immunoradiometric assay (IRMA), immunoenzymometric assay (IEMA) that labels antigens with enzymes, latex agglutination assay, latex agglutination inhibition assay, immunochromatography assay, ELISA assay, Western blotting assay, hemagglutination assay, hemagglutination inhibition assay, and the like.

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

[0044] When the test solution containing the substance to be measured is dropped onto the sample pad, a substance-gold colloid-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 the first capture antibody-substance-gold colloid-labeled complex, and a colored line appears on the test line. Gold colloid-labeled antibodies that do not form a complex with the substance to be measured are captured by the second capture antibody on the control line, and a colored line appears on the control line, indicating that the test was performed correctly. Conventional methods detect and quantify the substance to be measured by visually inspecting or using an optical measuring device to measure the color intensity of the colored line on the test line. To replace this measurement method with the method of the present invention, instead of measuring the color intensity of the colored line on the test line, the test line is irradiated with light such as laser light under conditions that cause Mie scattering, and the intensity of the light transmitted through the test strip (membrane) is measured. The amount or concentration of the substance to be measured is then estimated from this light intensity. This makes it possible to quantify the substance to be measured down to lower concentration ranges than with conventional methods.

[0045] (Enzyme immunoassay) ELISA is a widely known enzyme immunoassay method. In ELISA, the target substance (the protein or antibody of interest) is immobilized in each well of a well plate, and if the target substance is a protein, an enzyme-labeled antibody that specifically binds to that protein is added. Subsequently, a substrate that reacts with the enzyme is added, causing an enzymatic reaction, and the target substance is detected or quantified based on the resulting enzyme activity. By replacing the labeling enzyme with the labeled particles used in the present invention, it is possible to detect or quantify the target substance using the method of the present invention. In this case, the addition of a substrate that reacts with the enzyme becomes unnecessary. In addition to enzyme immunoassay, other methods such as radioimmunoassay, chemiluminescence immunoassay, and fluorescence immunoassay are known, differing in that radioimmunoassay labels the antigen with a radioisotope, chemiluminescence immunoassay labels the antigen with a chemiluminescent substance, and fluorescence immunoassay labels the antigen with a fluorescent dye, but the measurement principle is the same. To replace these measurement methods with the method of the present invention, the above-mentioned labeling substance can be replaced with the labeling particles used in the present invention, and the measurement can be performed using the method of the present invention. Furthermore, by installing a membrane or the like that matches the bottom shape of the wells of the well plate and fixing the object to be measured to each membrane, the region where the object to be measured, labeled with the labeling particles, is present will have thickness in the direction from the light source to the detector, enabling more sensitive measurement.

[0046] (Immunometric assay method) Immunometric assays involve reacting an antigen (or antibody) with an excess amount of labeled antibody (or labeled antigen) to form a complex, and then removing the unreacted labeled antibody (or labeled antigen). The greater the amount of the formed complex (the greater the amount of target substance), the greater the signal intensity of the labeled substance. By determining the signal intensity when a sample containing the target substance is added, the amount and concentration of the target substance can be estimated. In this case, by pre-determining the relationship between the signal intensity and an antigen (or antibody) of known concentration as a calibration curve, the amount and concentration of the target substance in the sample can be estimated by comparing the signal intensity when a sample containing the target substance is added with the calibration curve. Depending on the type of labeling, variations exist, such as immunoradiometric assays that label antibodies (or antigens) with radioisotopes, and immunoenzymometric assays that label antibodies (or antigens) with enzymes. In the case of enzyme labeling, a substrate is added to induce 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 antibodies (or antigens) labeled with the above-mentioned radioisotopes or enzymes can be replaced with antibodies (or antigens) labeled with the labeled particles used in the present invention, and the measurement can be performed using the method of the present invention.

[0047] (Sandwich assay method) The sandwich assay method is known as a variation of the immunoradiometric assay method or the immunoenzymometric assay method, or as a variation of the ELISA method. Immunochromatography is also a method that utilizes the sandwich assay method. In the sandwich assay method, an antibody or antigen is immobilized in a well, and a sample containing the target to be measured (antigen or antibody) is added and reacted. Then, a labeled antibody or labeled antigen that recognizes and binds to a different target site than the immobilized antibody or antigen is added, and the target to be measured is sandwiched between the antibody (or antigen) and the labeled antibody (or labeled antigen). Unreacted target to be measured or labeled antibody (or labeled antigen) is removed. The more the amount of target to be measured, the stronger the signal intensity of the label becomes because the labeled antibody or labeled antigen binds to the target to the target. At this time, by first determining the relationship between the signal intensity and the target to be measured at a known concentration as a calibration curve, it is possible to compare the signal intensity when a sample containing a target to be measured at an unknown concentration or amount with the pre-determined calibration curve and estimate the concentration or amount of the target to be measured in the sample. To replace these measurement methods with the method of the present invention, the labeled antibody (or labeled antigen) described above can be changed to an antibody (or antigen) labeled with the labeled particles used in the present invention, and the measurement can be performed using the method of the present invention. It is also possible to change the labeled substances used in conventional immunoassays, such as latex particles used in latex agglutination methods and latex agglutination inhibition methods, to the labeled particles used in the present invention, and perform the measurement using the method of the present invention. Furthermore, by installing a membrane that matches the shape of the bottom surface of the well plate and fixing the object to be measured to each membrane rather than each well, the region where the object to be measured, labeled with the labeled particles, is located will have thickness in the direction from the light source to the detector, enabling more sensitive measurements.

[0048] (One embodiment when applied to immunochromatography) Hereinafter, with reference to Figures 1 to 6, an embodiment of the present invention will be described in which the method of the present invention is applied to immunochromatography, one of the immunoassay methods. Figure 1 shows a schematic longitudinal cross-sectional view of the test strip 1 used in this embodiment. The test strip 1 is a lateral flow type test strip and has a structure that consists 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. On the backing sheet, the components are arranged in the following order: sample pad 3, conjugate pad 4, membrane 5, and absorbent pad 8. A hole is provided in the housing case 9 above the sample pad 3, and a (liquid) sample that may contain the target to be measured is dropped onto the sample pad 3 through this hole. The conjugate pad 4 contains multiple labeled particles carrying antibodies that can directly or indirectly bind to the target to be measured. When the sample is dropped onto the sample pad 3, the sample solution spreads to the conjugate pad 4, and the multiple labeled particles carrying antibodies that can directly or indirectly bind to the target to be measured bind to the target to be measured in the sample, forming a complex. The sample solution spreads onto the membrane, and the sample solution containing the complex moves to the test line 6. A substance (such as an antibody) that specifically binds to the target to be measured is placed on the test line 6, and the target to be measured (complex) labeled with the labeled particles is captured on the test line 6. On the other hand, the labeled particles that are not bound to the target to be measured pass through the test line with the sample solution and move to the control line 7. Control line 7 contains an antibody (such as an IgG antibody) that can bind directly or indirectly to the target substance, and labeled particles that are not bound to the target substance are captured here. The sample solution ultimately spreads and is absorbed by the absorption pad 7. Figure 5 is a schematic longitudinal cross-sectional view of an immunoassay apparatus of one embodiment that can be used in this embodiment, showing the case when light 12 from a light source 10 is irradiated onto an immunochromatographic slip 1 held in a holding part (not shown) of the immunoassay apparatus. Figure 6 is an external view (perspective view) of an immunoassay apparatus of one embodiment that can be used in this embodiment, showing that the immunochromatographic slip 1 is housed in a holding part that is formed by a concave shape extending in one direction. In Figures 5 and 6, a slit portion 11 is placed between the light source and the test strip 1, but the slit portion 11 is not required. Also, the slit portion 11 may be placed between the test strip 1 and the photodetector 13, rather than between the light source and the test strip 1, or it may be placed both between the light source 10 and the test strip 1 and between the test strip 1 and the photodetector 13. Furthermore, in Figure 6, the cover next to the slit portion 11 is shown open to make the location of the slit portion 11 clearer, but this cover is closed during use to prevent light from entering from the outside. Unlike conventional test strips (Figure 4), the test strip 1 used in this invention lacks a backing sheet 2 and housing case 9 on a portion of the back surface of the membrane 5 (see Figures 1, 3, and 5). As a result, light irradiated onto the membrane 5 and test line 6 reaches the light detection unit 13 on the opposite side of the light source 10 via the test strip 1, and the intensity of the received light can be measured. The measured light intensity and other information can be displayed on the display unit 14. In this case, the light 12 emitted from the light source 10 may be emitted not only on the test line 6, but also on the membrane 5 other than the test line 6 (regions that do not contain labeled particles). When irradiating the test line 6 and the membrane 5 with light, the test strip 1 may be slid by hand to sequentially irradiate each part with light, or the test strip 1 may be slid laterally using the slide device 16. In this embodiment, the housing case 9 containing the test strip is placed on a base connected to the slide device 16. In this case, the base has a cavity similar to the housing case 9, and transmitted light reaches the light detection unit 13 on the opposite side of the light source 10, and the light intensity of the transmitted light that reaches it can be measured. Furthermore, a handle 17 for the slide device is provided, and by rotating the handle 17, the base of the slide device 16 connected to the handle 17 is slid to the left by a lead screw mechanism. Alternatively, the slide device 16 may be equipped with a member for connecting the test strip 1 (e.g., a hook, magnet, spring, etc.). In this case, by rotating the handle 17, the screw inside the slide 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 slide device 16 to slide to the right or left. Light 12 transmitted through the region containing the labeled particles of the test line 6 is detected by the photodetector 13. The intensity of the detected light is determined by the estimation unit. The light intensity may be displayed on the display unit 14. The estimation unit and the display unit may be integrated. Similarly, light 12 transmitted through the membrane 5 is detected by the photodetector 13. The intensity of the detected light is determined by the estimation unit. The light intensity may be displayed on the display unit 14. The estimation unit and the display unit may be integrated. 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 the membrane 5 and / or the control line 7. The calculated light attenuation rate may be displayed on the display unit 14. In addition to the above measurements, it is preferable to determine a calibration curve (or approximate straight line) showing the relationship between the concentration or amount of the target to be measured and the light attenuation rate, using a sample whose concentration or amount of the target to be measured is known in advance. By pre-determining a calibration curve, the concentration or amount of the target substance in the sample can be determined by comparing the optical attenuation rate with the calibration curve when measuring with a sample containing a target substance of unknown concentration or amount. This allows for accurate estimation of the concentration or amount of the target substance in the sample. The estimated concentration or amount of the target substance in the sample may be displayed on the display unit 14.

[0049] (One embodiment when applied to the ELISA method) Next, with reference to Figure 7, an embodiment of the present invention will be described in which the method of the present invention is applied to ELISA, another immunoassay method. Figure 7 is a schematic longitudinal cross-sectional view of a well plate 18 held in the holding section (not shown) of the immunoassay apparatus used in this embodiment, when light 12 is irradiated from the light source 10. The well plate 18 comprises one or more wells, preferably more than one well (for example, 96 wells). Each well is preferably made of a transparent material so that when light 12 is shone into the well, the light can pass through to the bottom of the well. In the test sample well 20, where immobilization antibody is adsorbed onto the surface of the well, a sample containing the target to be measured is added. After the reaction is complete, the excess liquid is discarded and the well is washed to allow the target to bind to the immobilization antibody. Next, a sample containing multiple labeled particles carrying antibodies that can directly or indirectly bind to the target to be measured is added and reacted. Excess standard particles are washed away, and the sample is used as a test sample. Alternatively, a membrane or the like, matched to the shape of the bottom surface of the well, is placed on the bottom, immobilization antibody is adsorbed onto the membrane or the like, a sample containing the target to be measured is added, and after the reaction is complete, the excess liquid is discarded. The well with the membrane or the like is washed, allowing the target to bind to the immobilization antibody. Next, a sample containing multiple labeled particles carrying antibodies that can directly or indirectly bind to the target to be measured is added and reacted. Excess standard particles are washed away. The region where the target to be measured, labeled with the labeled particles, is present will have thickness in the direction from the light source to the detector, allowing for more sensitive measurement. For control sample well 19, use a well on which the immobilized antibody has been adsorbed onto the surface. Alternatively, if using a test sample with a membrane or the like placed on the bottom of the well, place the membrane or the like to match the shape of the bottom of the well, and use a test sample on which the immobilized antibody has been adsorbed onto the membrane. Light 12 is shone from the light source 10 into the test sample well 20. The shone light passes through the test sample well and is detected by the light detection unit 13 located on the opposite side of the test sample well 20 from the light source 10. This allows the intensity of the light that reaches the well to be measured. The measured light intensity and other information may be displayed on the 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 on the opposite side of the light source 10, and the light intensity of the transmitted light can be measured. Furthermore, a handle 17 for the slide device is provided, and by rotating the handle 17, the base of the slide device 16 connected to the handle 17 is slid to the left by a lead screw mechanism. Alternatively, the slide device 16 may be equipped with a member (e.g., a hook, magnet, spring, etc.) that connects the well plate 18. In this case, by rotating the handle 17, a screw inside the slide device 16 connected to the handle 17 rotates and extends to the right or contracts to the left, thereby allowing the well plate 18 connected to the slide device 16 to slide to the right or left. Light 12 transmitted through the area of ​​the test sample well 20 is detected by the photodetector 13. The intensity of the detected light is determined by the estimation unit. The light intensity may be displayed on the display unit 14. The estimation unit and the display unit may be integrated. Similarly, light 12 transmitted through the control sample well 19 is detected by the photodetector 13. The intensity of the detected light is determined by the estimation unit. The light intensity may be displayed on the display unit 14. The estimation unit and the display unit may be integrated. Next, the light attenuation rate is calculated from the light intensity transmitted through the test sample well 20 and the light intensity transmitted through the control sample well 19. The calculated light attenuation rate may be displayed on the display unit 14. In addition to the above measurements, it is preferable to determine a calibration curve (or approximate straight line) showing the relationship between the concentration or amount of the target to be measured and the light attenuation rate, using a sample whose concentration or amount of the target to be measured is known in advance. By pre-determining a calibration curve, the concentration or amount of the target substance in the sample can be determined by comparing the optical attenuation rate with the calibration curve when measuring with a sample containing a target substance of unknown concentration or amount. This allows for accurate estimation of the concentration or amount of the target substance in the sample. The estimated concentration or amount of the target substance in the sample may be displayed on the display unit 14.

[0050] The above describes in detail embodiments of applying the method of the first embodiment (and the apparatus of the second embodiment) to immunochromatography and ELISA as representative examples of immunoassay methods. However, the method, apparatus, kit, and container or substrate of the present invention are not limited to application to these two immunoassay methods, but can be applied to a variety of other immunoassay methods.

[0051] <Effects> This invention utilizes the principle that light scattering increases when the wavelength of light used as measurement light in an immunoassay and the particle size of labeled particles carrying the target substance are combined under conditions that cause Mie scattering. When the measurement light passes through a region containing the target substance, it strikes the labeled particles carrying the target substance, and the amount of light scattering increases exponentially due to repeated scattering and transmission. As a result, the transmitted light is exponentially attenuated, and it has been found that the attenuation of light intensity can be measured even at low concentrations of labeled particles carrying the target substance. The method of the present invention irradiates labeled particles with light under conditions that induce Mie scattering and detects the transmitted light, thereby enabling quantitative measurement of the target substance down to lower concentrations than conventional methods. The present invention is a novel measurement method that utilizes a unique measurement principle that enables quantitative measurement of optical attenuation in a dispersed medium. This principle involves using coherent laser light, preferably with consistent properties such as wavelength and phase, as the input wavelength, and taking advantage of the characteristic that the straight-line propagation of light in the Mie scattering region increases depending on the combination of particle sizes of the labeled particles. This method differs from existing measurement principles that utilize reflected light. A key feature of this measurement method is that it not only achieves a sensitivity that cannot be measured by reflection methods, but also possesses the reproducibility that is important for quantitative measurement. This measurement method is preferably used in measurement systems in which labeled particles, which have been labeled with the target of measurement by an antigen-antibody reaction, exist with thickness not only in the two-dimensional direction but also in the optical axis direction within the measurement area (or medium). 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 applying the method of the first embodiment to immunochromatography is a completely different method from conventional immunochromatography using light reflection, and has the characteristics of rapid and specific testing, as well as high sensitivity in the low concentration range of the target substance. With the present invention, linear results can be obtained down to even lower concentration ranges of the target substance, enabling rapid quantitative testing in immunochromatography. Conventional and commonly used optical reflection methods measure reflected light obtained as a result of attenuation due to two-dimensional (planar) absorption and scattering by particles present on the optical axis relative to incident light. In contrast, the present invention solves the problem by applying the relationship between particle size and the wavelength of incident light, and the optical effect of labeled particles, to measure the attenuation of detected light relative to incident light in three dimensions due to the Mie scattering effect by all particles present on the optical vertical axis in the test line zone, rather than using an absorption method that measures reflected light from particles near the surface of the test line in two dimensions. As described above, the method and apparatus of the present invention can be applied to various immunoassay methods and are not limited to immunochromatography or ELISA. [Examples]

[0052] The following are some test examples relating to the present invention, but these are not intended to limit the present invention.

[0053] [Example 1] <Measurement of optical attenuation rate of transmitted light under Mie scattering conditions> As described below, each material used in Example 1 was prepared, and the optical attenuation rate of transmitted light was determined when the C-reactive protein (CRP), which was the target of measurement, was diluted to various concentrations. The relationship between the dilution rate and the optical attenuation rate was then investigated. In Example 1, when using a wavelength of 450 nm and a labeled particle size of 454 nm, α = approximately 3.2, and the wavelength of the measured light is the 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 pure water. Then, 2-vinylpyridine (2-VP, 49.50 g) and divinylbenzene (DVB, 0.50 g) were added, and the mixture was stirred under a nitrogen stream at 30°C for 50 minutes, followed by stirring at 60°C for 30 minutes. After stirring, 2,2-azobis(2-methylpropionamidine) dihydrochloride (AIBA, 0.50 g) dissolved in 18.00 g of pure water was added dropwise, and the mixture was stirred at 60°C for 3.5 hours to obtain resin particles with an average particle size of 430 nm. The mixture was precipitated by centrifugation (9000 rpm, 45 minutes), the supernatant was removed, and impurities were removed by dialysis. Subsequently, the concentration was adjusted to obtain a 10 wt% resin particle dispersion. After adding 245 ml of pure water to the above resin particle dispersion (90 ml), 90 ml of 400 mM chloroplatinic acid aqueous solution was added, and the mixture was stirred at 30°C for 3 hours, then left at room temperature for 24 hours. Subsequently, the resin particles were precipitated by centrifugation (3100 rpm, 30 minutes), and excess chloroplatinic acid was removed by removing the supernatant. After that, the concentration was adjusted to prepare a 5 wt% platinum ion adsorbed resin particle dispersion. Next, 55 ml of the 5 wt% platinum ion adsorbent resin particle dispersion was added to 3825 ml of pure water, and 110 ml of a 132 mM aqueous dimethylamine borane solution was added dropwise while stirring at 3°C. The mixture was then stirred at 3°C ​​for 1 hour. After that, the mixture was stirred 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 impurities were removed by dialysis. Subsequently, the concentration was adjusted to obtain a 1 wt% platinum-resin composite dispersion. The average particle size of the formed platinum particles was 5 nm, and the platinum load was 37.7 wt%. <Measurement of average particle size of metal particles> A substrate was prepared by dropping a platinum-resin composite particle dispersion onto a metallic mesh with a carbon support film. Images were observed using a field emission scanning electron microscope (FE-SEM; Hitachi High-Technologies Corporation, SU-9000). The area-average diameter of 100 arbitrary metal particles was measured from these images and determined as the average particle size.

[0054] <Measurement of average particle size of resin particles and platinum-resin composite particles> The measurements were performed using a centrifugal sedimentation particle size distribution analyzer (LUMiSizer610, manufactured by LUM GmbH). The measurements were taken 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 inverted and stirred at room temperature for 1 hour to obtain labeled antibody dispersion A containing anti-CRP antibody labeled with platinum-resin composite particles.

[0056] (3) Block 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 settled sediment, and after ultrasonic dispersion, the mixture was inverted and stirred at room temperature for 1 hour to obtain labeled antibody dispersion B.

[0057] (4) Washing 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 settled sediment, and sonication was performed. This procedure was repeated three times to obtain labeled antibody dispersion C.

[0058] (5) Fabrication of conjugate pads Labeled antibody dispersion C was centrifuged at 3000 rpm for 5 minutes, and the supernatant was removed. Then, a 5 mM Tris aqueous solution (pH 8.5) containing 5 wt% sucrose and 2.5 wt% BSA was added to the settled sediment, and ultrasonic dispersion was performed to obtain labeled antibody dispersion D. Labeled antibody dispersion D was uniformly impregnated into a glass fiber nonwoven fabric, and then dried at 50°C for 1 hour to prepare a conjugate pad 4. At this time, the concentration of labeled antibody dispersion D and the amount applied to the glass fiber nonwoven fabric were adjusted so that the content of platinum-resin composite particles in conjugate pad 4 was 3 μg per test of evaluation by the immunochromatographic method described later.

[0059] (6) Preparation of test strips As shown in Figure 1, a nitrocellulose membrane 5 with a width of 25 mm and a thickness of 100 μm was coated with a 1 mg / mL anti-CRP antibody solution to draw a test line 6. A control line 7 was also drawn downstream of test line 6 by coating it with a 0.5 mg / mL anti-mouse IgG antibody solution. After drying the nitrocellulose membrane 5 at 50°C for 1 hour, a backing sheet 2 (partially removed to allow light transmission), a conjugate pad 4, a sample pad 3 (glass fiber nonwoven fabric), and an absorbent pad 8 (cotton nonwoven fabric) were laminated together, 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 a test strip 1, which was then placed in a housing case 9. As shown in Figures 2 and 3, the housing case 9 has openings 21 not only on the top surface of the housing case but also on the bottom surface of the housing case, so that the light transmitted through the membrane can reach the photodetector without being obstructed. (7) Preparation of the 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 and used as the developing solution.

[0060] (8) Construction of an immunoassay device (immunochromatograph) As shown in Figures 5 and 6, the configuration of the immunochromatograph leader consists 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 stand 15, a slide device 16, a handle for the slide device 17, and a light attenuation rate measurement result display unit 14. The housing for the light source 10 and the slit 11 holder, the housing for the slide base 15, the housing for the slide device 16, and the handle 17 for the slide device were manufactured using a 3D printer (FRASHFORGE, product name Adventurer5M Pro). Specifically, the light source 10 uses a semiconductor laser (manufactured by CivilLaser, output 10mW, wavelength 450nm) with a beam shaped into a line. The slit 11 has an opening of 2mm in the direction of the long side of the test line 6 and 0.3mm in the direction of the short side, parallel to the test line 6 of the immunochromatographic slip (for example, if the long side is 3.5mm and the short side is 1mm). The laser light emitted from the light source 10 is adjusted to illuminate only the test line 6 on the membrane 5 of the test strip 1, and is fixed in place by a holding unit. The holding unit is structured so that the laser light 12 emitted from the light source 10 passes through the slit 11, then through the membrane 5 of the immunochromatographic slip 1, and reaches the photodetector 13. The slide stand 15 is designed to move while holding the housing case 9 containing the immunochromatographic slip 1. Two bar-shaped protrusions are provided along the long side of the upper side of the slide stand 15's housing, and the housing case 9 containing the test strip 1 is held between the two protrusions, allowing it to 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 that is irradiated from the light source 10 and passes through the slit 11 directly below it. The holding unit 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. The measurement result display unit 14 uses an illuminance meter (manufactured by Zhangzhou WeiHua Electronic, product name LX-1010B). As shown in Figures 5 and 6, the sliding device 16 was designed using a lead screw mechanism to allow it to slide precisely in the long-side direction while holding the housing case 9. The sliding device 16 was designed to be moved by turning the sliding device handle 17.

[0061] (9) Measurement and evaluation by immunochromatography Positive and negative control (antigen-free) sample solutions were prepared by diluting 3.12 mg / mL of CRP antigen using a developing solution as shown in Tables 1 and 2. 50 μL of the sample solution was dropped onto sample pad 3 of the test strip. The concentrations of CRP antigen (pg / mL) at each dilution ratio are shown in Tables 1 and 2.

[0062] [Table 1]

[0063] [Table 2]

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

[0065] [Comparative Example 1] <Measuring color intensity using the conventional method of reflected light (Part 1)>

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

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

[0068] [Comparative Example 2] <Measuring color intensity using the conventional method of 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 performed. That is, as in Comparative Example 1, the color intensity was measured using reflected light, a conventional method, rather than transmitted light, and the relationship between the dilution ratio and the color intensity was investigated.

[0070] Specifically, the 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, a 5 mM Tris aqueous solution (pH 9.5) was used as the binding buffer, and a 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 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 under conditions that did not cause Mie scattering (specifically, conditions that caused Rayleigh scattering), similar to Example 1, and the relationship between the dilution ratio and the color intensity was investigated.

[0072] Specifically, the 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, a 5 mM Tris aqueous solution (pH 9.5) was used as the binding buffer, and a 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, which measured the optical attenuation rate of transmitted light under conditions that cause Mie scattering, linearity was obtained even when CRP was diluted to 1 / 327,680,000 times (Figure 8). In contrast, with conventional measurement of reflected light color intensity, linearity was only obtained down to a dilution ratio of 1 / 40,960,000 in Comparative Example 1 (Figure 9). At concentrations lower than 1 / 40,960,000, the values ​​were almost the same as 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 down to a dilution ratio of 1 / 320,000 (Figure 9). At concentrations lower than 1 / 320,000, the values ​​were almost the same as those of the negative control, indicating that quantitative measurement in the low concentration range was more difficult than in Example 1. Note that the asterisks (*) in Figures 8 and 9 indicate the maximum dilution ratio of the CRP antigen at which linearity was obtained. Furthermore, even when measuring the optical attenuation rate of transmitted light, under the conditions in Comparative Example 3 where Mie scattering does not occur, linearity could only be obtained down to a dilution of 1 / 640,000 (Figure 10). At concentrations lower than 1 / 640,000, the values ​​were almost the same as those of the negative control, indicating that quantitative measurement in the low-concentration range was more difficult than in Example 1. The asterisk (*) in Figure 10 indicates the maximum dilution 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 commonly used in immunochromatography were used. However, since the degree of change in the optical attenuation rate when the dilution ratio is changed is determined by the relationship between the particle size and the wavelength of the incident light, it is expected 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, the light transmission method of the present invention primarily involves Mie scattering, which occurs under the conditions of particle diameter and incident light wavelength according to the principles of photophysics. Because light is scattered in all directions, the attenuation of light in the straight-line direction increases exponentially with the distance transmitted. Differences in the metallic material of the particles have no effect on the particle diameter and therefore do not affect Mie scattering at all. On the other hand, differences in the metallic material of the particles cause changes in the particle color (light absorption), which greatly affects conventional light reflection methods that primarily rely on absorption, but does not affect the Mie scattering method of the present invention. From the above, we can conclude that measuring the optical attenuation rate of transmitted light under conditions where Mie scattering occurs allows for quantitative measurement in a lower concentration range compared to measuring the color intensity of reflected light or measuring under conditions where Mie scattering does not occur.

[0074] [Example 2] <Measurement of optical attenuation rate at various laser wavelengths> Mie scattering is caused by the relationship between particle size and the wavelength of incident light. Therefore, we investigated whether the reproducibility of the optical attenuation rate measurement could be obtained not only when the wavelength of light was changed within the range of conditions under which Mie scattering occurs, but also when the wavelength of light was changed, not just for the specific wavelength used in Example 1. The particle size used was the same as in Example 1. Two types of light sources (light source 1 and light source 2) with different semiconductor laser wavelengths were prepared, and immunochromatographic evaluation was performed using each light source in the same manner as in Example 1. Light source 1: Semiconductor laser (manufactured by CivilLaser, output 10mW, wavelength 520nm) Light source 2: Semiconductor laser (manufactured by CivilLaser, output 10mW, wavelength 648nm) Furthermore, when using light source 1, α = approximately 2.7, and when using light source 2, α = approximately 2.2, both of which are within the Mie scattering region. The results are shown in Figure 11.

[0075] <Result> Measurements of optical attenuation using different wavelengths of 520 nm and 648 nm yielded quantitative and equivalent results up to the same dilution ratio as the optical attenuation measurement using 450 nm (Figure 11). This confirms that, within the range of conditions that cause Mie scattering, 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 that range.

[0076] [Example 3] <Measurement of optical attenuation rate for various particle sizes> As described above, Mie scattering is caused by the relationship between particle size and the wavelength of incident light. Therefore, in Example 3, we verified whether the reproducibility of the optical attenuation rate measurement could be obtained not only with the specific particle size used in Example 1, but also when the particle size was changed within the range of the Mie scattering region. The same wavelength of light 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. Furthermore, when labeled particle 1 is used, α = approximately 3.2, and when labeled particle 2 is used, α = 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 pure water. Then, 2-vinylpyridine (2-VP, 49.50 g) and divinylbenzene (DVB, 0.50 g) were added, and the mixture was stirred under a nitrogen stream at 30°C for 50 minutes, followed by stirring at 60°C for 30 minutes. After stirring, 2,2-azobis(2-methylpropionamidine) dihydrochloride (AIBA, 0.50 g) dissolved in 18.00 g of pure water was added dropwise, and the mixture was stirred at 60°C for 3.5 hours to obtain resin particles with an average particle size of 370 nm. The mixture was precipitated by centrifugation (9000 rpm, 45 minutes), the supernatant was removed, and impurities were removed by dialysis. Subsequently, the concentration was adjusted to obtain a 10 wt% resin particle dispersion. After adding 245 ml of pure water to the above resin particle dispersion (90 ml), 90 ml of 400 mM chloroplatinic acid aqueous solution was added, and the mixture was stirred at 30°C for 3 hours, then left at room temperature for 24 hours. Subsequently, the resin particles were precipitated by centrifugation (3100 rpm, 30 minutes), and excess chloroplatinic acid was removed by removing the supernatant. After that, the concentration was adjusted to prepare a 5 wt% platinum ion adsorbed resin particle dispersion. Next, 55 ml of the 5 wt% platinum ion adsorbent resin particle dispersion was added to 3825 ml of pure water, and 110 ml of 132 mM dimethylamine borane aqueous solution was added dropwise while stirring at 3°C. The mixture was then stirred at 3°C ​​for 1 hour. After that, the mixture was stirred 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 impurities were removed by dialysis. Subsequently, the concentration was adjusted to obtain a 1 wt% platinum-resin composite dispersion. The average particle size of the formed platinum particles was 5 nm, and the platinum load 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 one of the 1 wt% platinum-resin composite particle dispersions (solvent: water) prepared in (1) above was added, and the mixture was inverted and stirred at room temperature for 1 hour to obtain labeled antibody dispersion A containing anti-CRP antibody labeled with platinum-resin composite particles. Otherwise, the immunochromatographic evaluation was performed in the same manner as in Example 1. The results are shown in Figure 12.

[0078] <Result> Measurements of the optical attenuation rate using platinum-resin composite particles with different particle sizes, 382 nm and 454 nm, yielded quantitative results down to low concentrations for both particle sizes (Figure 12). This confirms that, within the range where the relationship between particle size and incident light wavelength is within the conditions for Mie scattering, equivalent quantitative results can be obtained even when the particle size is changed. [Industrial applicability]

[0079] According to the present invention, the substance to be measured in a sample can be quantified even at low concentrations. Therefore, the present invention is extremely useful in industry. [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 pads 9…Housing Case 10…Light source 11…Slit 12…Light (laser light) 13…Light detection unit 14...Display section 15…Sliding base 16…Slide device 17...Handle for sliding device 18... Well plate 19…Control sample well 20…Test sample wells 21…Opening

Claims

1. A container or substrate comprising a sample containing the target to be measured and a plurality of labeled particles carrying antibodies capable of directly or indirectly binding to the target to be measured, wherein the container or substrate is irradiated with measurement light after contact between the sample containing the target to be measured and the plurality of labeled particles carrying antibodies capable of directly or indirectly binding to the target to be measured, wherein the measurement light is irradiated into the region of the container or substrate containing the labeled particles, and A step of detecting the light (transmitted light) that has passed through the region from the irradiated measurement light, Includes, An immunoassay method wherein the wavelength of the irradiated measurement light is the wavelength that causes Mie scattering by each of the plurality of labeled particles.

2. The immunoassay method according to claim 1, wherein the wavelength at which Mie scattering is generated by each of the plurality of labeled particles is a wavelength that satisfies 2 < α < 10 (where α = π × particle size of labeled particle (nm) / light wavelength (nm)).

3. The immunoassay method according to claim 1, further comprising the step of separating the antibody supported on the labeled particle that is directly or indirectly bound to the target to be measured from the antibody supported on the labeled particle that is not bound to the target to be measured, after contact and before irradiation with the measurement light.

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

5. The immunoassay method according to claim 1, wherein the step of irradiating with the measurement light includes irradiating with the measurement light into a region of the container or substrate containing the plurality of labeled particles (hereinafter referred to as the first region) and irradiating with the measurement light into a second region outside the first region.

6. The immunoassay method according to claim 5, comprising measuring the light intensity 1 of the measurement light transmitted through the first region and the 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) × 100 / light intensity 2 The immunoassay method according to claim 6, further comprising the step of determining the light attenuation rate.

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

9. The immunoassay method according to claim 7, further comprising the steps of determining the light attenuation rate using a sample containing a target to be measured of unknown concentration or quantity, and estimating the concentration or quantity of the target to be measured contained in the sample by comparing the light attenuation rate with a calibration curve or approximate straight line determined in advance in claim 8.

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

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

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

13. An immunoassay device, A holding unit for holding a container or substrate containing multiple labeled particles carrying antibodies capable of directly or indirectly binding to the object to be measured, inside or on the device; A light source positioned above the holding portion for irradiating measurement light into the region of the container or substrate held by the holding portion that contains the plurality of labeled particles, A photodetector is positioned on the opposite side of the container or substrate from the light source and detects transmitted light that has been irradiated from the light source and passed through the region of the container or substrate containing the labeled particles. It has, The wavelength of the measurement light emitted by the light source is the wavelength that causes Mie scattering by each of the plurality of labeled particles. The aforementioned immunoassay apparatus.

14. An immunoassay apparatus according to claim 13, for use in the method described in claim 1.

15. The immunoassay apparatus according to claim 13, wherein the wavelength at which Mie scattering is generated by each of the plurality of labeled particles is a wavelength that satisfies 2 < α < 10 (where α = π × particle size of labeled particle (nm) / light wavelength (nm)).

16. The immunoassay apparatus according to claim 13, further comprising a container or substrate containing a plurality of labeled particles on which antibodies capable of directly or indirectly binding to a target for measurement are carried.

17. The immunoassay apparatus is configured to irradiate measurement light into the region of the container or substrate containing the plurality of labeled particles (first region) and into the region of the container or substrate outside the first region (second region). The immunoassay apparatus according to claim 13.

18. The apparatus according to claim 13, A container or substrate containing multiple labeled particles carrying antibodies capable of directly or indirectly binding to the object to be measured, An immunoassay kit including [specific components].

19. The apparatus according to claim 13, A container or substrate capable of arranging multiple labeled particles carrying antibodies that can directly or indirectly bind to the target of measurement, An immunoassay kit including [specific components].

20. The immunoassay kit according to claim 19, further comprising a plurality of labeled particles carrying antibodies capable of directly or indirectly binding to a target for measurement.

21. A container or substrate containing a plurality of labeled particles carrying antibodies capable of directly or indirectly binding to a target for measurement, for use in the method described in claim 1, or in the apparatus described in claim 13, or in the kit described in claim 18, or in the kit described in claim 19.

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