Target substance testing kit and target substance detection method

Abstract

Provided are a target substance detection method and a target substance testing kit for detecting a target substance with high sensitivity on the basis of a change in fluorescence anisotropy. This target substance testing kit for detecting a target substance in a specimen liquid using a value related to fluorescence anisotropy is characterized by including: first particles having a first site specifically binding to the target substance and a light-emitting component; a first compound having a second site specifically binding to a site different from the site specifically binding to the first site in the target substance, and a third site different from the second site; and second particles that have a fourth site specifically binding to the third site and that have an average particle diameter larger than that of the first particles.

Description

Target substance testing kit and method for detecting target substances

[0001] This disclosure relates to a test kit for target substances and a method for detecting target substances.

[0002] In the fields of medicine and clinical testing, the highly sensitive detection of trace amounts of biological components from blood or collected organ tissue is necessary for investigating the causes of disease. Among the methods for detecting biological components, immunoassays, which utilize antigen-antibody reactions and use particles to which capture substances such as antibodies and antigens are bound, are well known.

[0003] For example, a fluorescence polarization immunoassay method using luminescent particles has been proposed (Patent Document 1). In one example of a fluorescence polarization immunoassay method, a sample that may contain a target substance is reacted with a luminescent reagent containing luminescent particles to which antibodies against the target substance are bound (also called "antibody-sensitized luminescent particles"). The aggregates of luminescent particles generated by the antigen-antibody reaction are then detected by fluorescence polarization, allowing for qualitative and quantitative measurement of the target substance. Fluorescence polarization measures the mobility, i.e., size, of the luminescent particles used as probes. To make fluorescence polarization highly sensitive, it is necessary to significantly change the mobility, i.e., the size, of the luminescent particles.

[0004] Patent Document 1 discloses a method for detecting and measuring a target substance by increasing the particle size through the aggregation reaction of antibody-sensitized luminescent particles. In Patent Document 1, for example, when two 100 nm luminescent particles aggregate via a single molecule, the approximate size becomes about 200 nm, and the mobility of the aggregate of luminescent particles that has reached about 200 nm becomes the limit of the signal change. Therefore, when there are few objects to be measured, a size increase of more than 200 nm cannot be expected, making it difficult to capture changes in fluorescence polarization with high sensitivity.

[0005] Patent Document 2 discloses a fluorescence polarization method that includes the step of preparing a first particle containing a rare earth complex that specifically binds to the target substance, and a second particle that has a larger average particle size than the first particle and also specifically binds to the target substance, in order to increase the sensitivity of the fluorescence polarization method. Subsequently, the sample solution is mixed with the first particle and the second particle to obtain a mixture, and at least one of the presence or absence and concentration of the target substance is determined from the fluorescence anisotropy of the obtained mixture. However, in this method, the reactivity of the antibody immobilized on the second particle (larger particle) to the target substance is greatly reduced, making it difficult to capture changes in fluorescence polarization with sufficiently high sensitivity.

[0006] Furthermore, Patent Document 3 discloses a chemiluminescence assay using two types of particles, in which these particles are linked using biotin and avidin. Specifically, one particle is bound to an antibody, and this antibody-bound particle captures a target substance, which is then reacted with a biotinylated antibody. Finally, the other avidin-bound particle is reacted to perform a close-proximity homogeneous chemiluminescence assay. Because biotin and avidin are highly reactive, the reaction between particles can be promoted.

[0007] Japanese Patent Publication No. 2022-187791, Japanese Patent Publication No. 2022-187939, Japanese Patent Publication No. 09-505888

[0008] However, the method described in the above-mentioned patent document was insufficiently sensitive as a method for detecting target substances based on changes in fluorescence anisotropy.

[0009] A first aspect of this embodiment is a method for detecting a target substance in a sample solution, comprising the steps of: obtaining a first mixture having a first complex comprising a sample solution that may contain the target substance; a first particle having a first site that specifically binds to the target substance and a luminescent component; and a first compound having a second site that specifically binds to a site different from the site that specifically binds to the first site of the target substance and a third site different from the second site; obtaining a second mixture having a second complex comprising a second particle having a fourth site that specifically binds to the third site and having a larger average particle size than the first particle, and the first complex; and obtaining a value relating to the fluorescence anisotropy of the second mixture.

[0010] Furthermore, a second aspect of this embodiment is a target substance test kit for detecting a target substance in a sample solution using a value relating to fluorescence anisotropy, comprising: a first particle having a first site that specifically binds to the target substance and a luminescent component; a first compound having a second site that specifically binds to a site in the target substance different from the site that specifically binds to the first site, and a third site different from the second site; and a second particle having a fourth site that specifically binds to the third site and having an average particle size larger than the first particle.

[0011] According to this disclosure, target substances in a sample solution can be detected with high sensitivity using values ​​related to fluorescence anisotropy. In particular, the reaction can be more sensitively captured from the change in fluorescence anisotropy detected when particles aggregate in the sample solution. Furthermore, by using a first compound that specifically binds to the target substance and also binds to a second particle, the reaction rate of binding between the first particle and the larger second particle is increased, enabling even more sensitive measurement.

[0012] This figure illustrates each step of the target substance detection method according to the embodiment of this disclosure. This is a schematic diagram of the target substance detection according to the embodiment of this disclosure. This figure illustrates the flow of the steps in the target substance detection method according to the embodiment of this disclosure.

[0013] Preferred embodiments of this disclosure will be described in detail below, but this will not limit the scope of this disclosure.

[0014] This disclosure provides, as one embodiment, a method for detecting a target substance in a sample solution, as shown in Figure 1, comprising the following steps: (1) A step of obtaining a first mixture (S1001) having a sample solution that may contain a target substance, a first particle having a first site that specifically binds to the target substance and a luminescent component, and a first compound having a second site that specifically binds to a site different from the site that specifically binds to the first site of the target substance, and a third site different from the second site. (2) A step of obtaining a second mixture (S1002) having a second complex comprising a second particle having a fourth site that specifically binds to the third site and having a larger average particle size than the first particle, and the first complex. (3) A step of obtaining a value relating to the fluorescence anisotropy of the second mixture (S1003).

[0015] Fluorescence anisotropy is an index that indicates the tendency of the emission emitted by an object irradiated with polarized light to retain the component of the irradiated polarized light orientation. Values ​​related to fluorescence anisotropy in this disclosure include, for example, fluorescence polarization degree and fluorescence anisotropy. Specifically, fluorescence anisotropy is calculated by <r> in the following equation (1). (However, <r>...fluorescence anisotropy I ＶＶ ...The emission intensity of the emission component whose vibration direction is parallel to the first polarization when excited by the first polarization I ＶＨ ...The emission intensity of the emission component whose vibration direction is perpendicular to the first polarization when excited by the first polarization I ＨＶ ...When excited with a second polarization whose vibration direction is perpendicular to the first polarization, the emission intensity of the emission component whose vibration direction is perpendicular to the second polarization is I. ＨＨ ...The emission intensity G of the emission component whose vibration direction is parallel to the second polarization when excited with a second polarization whose vibration direction is perpendicular to the first polarization (this is a correction value).

[0016] By placing a polarizer on the incident side of the light, the target is excited with a first polarization. By placing a polarizer on the detection side parallel to the incident polarizer and measuring the intensity of the emitted light, the intensity of the emitted light component whose vibration direction is parallel to the first polarization, i.e., I ＶＶThis can be measured. On the other hand, if a polarizer is set on the detection side in a direction perpendicular to the incident polarizer, and the intensity of the light emission is measured, the light emission intensity of the light emission component whose vibration direction is perpendicular to the first polarization, i.e., I ＶＨ It can measure [something].

[0017] A polarizer is placed on the incident side of the light, perpendicular to the direction from which the first polarization was obtained, that is, the target is excited with the second polarization. A polarizer is placed on the detection side, parallel to the incident polarizer, and the intensity of the emission is measured. The emission intensity of the emission component whose vibration direction is parallel to the second polarization, i.e., I ＨＶ This can be measured. On the other hand, if a polarizer is set on the detection side in a direction perpendicular to the incident polarizer, and the intensity of the light emission is measured, the light emission intensity of the light emission component whose vibration direction is perpendicular to the second polarization, i.e., I ＨＨ It can measure [something].

[0018] In this disclosure, "specifically binding" means having the ability to specifically bind, as long as it possesses the property of specific interaction, the form is not specified, but it is particularly preferable that it has the ability to specifically bind. Examples of mechanisms that give rise to binding include electrostatic interaction, van der Waals interaction, and hydrogen bonding. The binding sites and mechanisms of the first particle and the first compound may be the same or different.

[0019] Any substance that interacts with any other substance can be a target substance. Examples of target substances include antigens, antibodies, small molecules, various receptors, enzymes, substrates, nucleic acids, cytokines, hormones, neurotransmitters, signaling molecules, and membrane proteins. Antigens include allergens, bacteria, viruses, cells, cell membrane components, cancer markers, various disease markers, antibodies, blood-derived substances, food-derived substances, natural product-derived substances, and all small molecules. Nucleic acids include DNA, RNA, cDNA derived from bacteria, viruses, cells, etc., parts or fragments thereof, synthetic nucleic acids, primers, probes, etc. Small molecules include cytokines, hormones, neurotransmitters, signaling molecules, membrane proteins, etc., and their receptors, etc.

[0020] The first particle is a particle that binds to the target substance and is not particularly limited as long as it is luminescent. That is, it is acceptable as long as it has the property of exhibiting luminescence when irradiated with an excitation wavelength. Preferably, the first particle has a luminescent rare earth complex as a luminescent component. Examples of rare earth complexes include those of europium, terbium, neodymium, erbium, yttrium, lanthanum, cerium, samarium, gadolinium, dysprosium, thulium, ytterbium, and scandium. Since fluorescence anisotropy depends on the change in rotational motion of the luminescent material during the luminescence time, it is preferable to use a rare earth complex with a long luminescence lifetime as the luminescent material. Here, in this embodiment and disclosure, fluorescence anisotropy can also be referred to as polarization anisotropy.

[0021] An example of an embodiment will be described using Figure 2. The second particle 11 is not particularly limited as long as it has a larger average particle size than the first particle 1, does not specifically bind to the target substance 6, and specifically binds to the first compound 8. As will be described later, because the second particle 11 has a larger average particle size than the first particle 1, the rotational motion of the first particle 1 is significantly suppressed in the first particle-target substance-first compound-second particle complex formed via the target substance 6, resulting in a large increase in fluorescence anisotropy.

[0022] In the target substance detection method of this embodiment, in a mixture obtained by mixing the sample solution and the reagent, the first particle 1 and the first compound 8 form a first complex 12 via the target substance 6. Furthermore, the first complex 12 specifically binds to the second particle 11 to form a second complex 13. That is, the first particle 1 and the second particle 11 form the second complex 13 via the target substance 6 and the first compound 8. It is desirable that the fluorescence anisotropy of the first particle 1 is relatively low not only before mixing with the sample solution but also before mixing with the second particle 11, i.e., the measured fluorescence anisotropy is low and the fluorescence polarization is depolarized.

[0023] On the other hand, when the first particle 1 and the second particle 11 aggregate via the target substance 6 and the first compound 8, it is desirable that the fluorescence anisotropy is relatively high, that is, that some (or all) of the depolarization of fluorescence is canceled out.

[0024] The first particle 1 and the second particle 11 form a second complex 13 in the dispersion medium via the target substance 6 and the first compound 8. At this time, aggregation of the first particles 1 and the second particles 11 may also occur. The fluorescence anisotropy exhibited by the second complex 13 in the dispersion medium is preferably higher than the fluorescence anisotropy exhibited by the unaggregated first particles 1 in the dispersion medium. Preferably, it increases by 0.004 or more, more preferably by 0.01 or more, and even more preferably by 0.02 or more.

[0025] The first particle 1 exhibits fluorescence anisotropy in the dispersion medium preferably of 0.2 or less, more preferably of 0.15 or less, and even more preferably of 0.12 or less. The second composite 13 exhibits fluorescence anisotropy in the dispersion medium preferably of 0.05 or more, more preferably of 0.09 or more, and even more preferably of 0.13 or more.

[0026] Furthermore, the first particle 1 and the second particle 11 used in the measurement preferably together constitute 0.000001% to 1% by mass in the dispersion medium, and their mass ratio is preferably in the range of 1:9 to 9:1.

[0027] When measuring fluorescence anisotropy, a polarizer such as a polarizing filter can be placed on the incident light side to irradiate the area with polarized excitation light (first polarization). If the polarizing filter is placed perpendicular to this, a second polarization whose vibration direction is perpendicular to the first polarization can be irradiated. If a polarizer is placed on the detection side parallel to the incident polarizer, the emission intensity of the emission component whose vibration direction is parallel to the excitation side can be measured. If the detection side is placed perpendicular to the incident polarizer, the emission intensity of the emission component whose vibration direction is perpendicular to the excitation light can be measured. The emission intensity can be measured using a spectrophotometer or the like.

[0028] The measurement is preferably performed in a dispersion solution at a temperature in the range of 0°C to 100°C. More preferably, the measurement is performed at a temperature in the range of 4°C to 50°C. The solution is preferably an aqueous solvent, such as a buffer solution, physiological saline, or water.

[0029] The measurement conditions can be set as appropriate by those skilled in the art, and in doing so, they may refer to, but are not limited to, the examples described later in this specification.

[0030] In order to produce a sufficient change in fluorescence anisotropy before and after particle aggregation, it is desirable that the first particle 1 is not too large. On the other hand, if the first particle 1 is too small, problems such as insufficient luminescence may occur, or manufacturing problems may arise. The average particle size of the first particle 1 is preferably 10 nm or more and 1 μm or less. That is, the first particle 1 is preferably 1 μm (micron) or less, more preferably 500 nm or less, even more preferably 400 nm or less, and even more preferably 250 nm or less. The average particle size of the first particle 1 is preferably 10 nm or more, more preferably 20 nm or more, and even more preferably 50 nm or more. Therefore, for example, it is preferable that the average particle size of the first particle 1 be 20 nm or more and 400 nm or less. Note that the average particle size in this disclosure is the number-average particle size, and the average particle size can be measured by dynamic light scattering.

[0031] The first particle 1 preferably has a small particle size distribution, and preferably has a polydispersity index (hereinafter also referred to as PDI) of 0.1 or less. The first particle 1 may also contain a particle substrate 2 consisting of polystyrene and a polymer having siloxane bonds. Furthermore, the first particle 1 may have a layer (hydrophilic layer 3) on its surface containing a hydrophilic polymer that includes either an ether structure, a betaine structure, or a pyrrolidone ring. By including a hydrophilic polymer on the particle surface, nonspecific adsorption is suppressed. In this way, by suppressing nonspecific adsorption with a hydrophilic polymer rather than with proteins, the particle size distribution is suppressed, which is advantageous for measurements based on fluorescence anisotropy.

[0032] Preferably, the second particle 11 has a larger average particle size than the first particle 1, does not bind to the target substance 6, and has affinity for the first compound 8. Having affinity is used synonymously with specifically binding. Preferably, the second particle 11 has an average particle size of 100 nm or more and 5 μm or less. That is, the lower limit of the average particle size of the second particle 11 is 100 nm or more, more preferably 150 nm or more, and even more preferably 1 μm or more, in order to sufficiently increase the fluorescence anisotropy of the composite. The upper limit is preferably 5 μm or less, and more preferably 3 μm or less, from the viewpoint of particle dispersion stability, etc. Therefore, for example, it is preferable that the second particle 11 is 1 μm or more and 5 μm or less.

[0033] In this disclosure, it is preferable that the average particle size of the second particle 11 is at least twice and up to 500 times the average particle size of the first particle 1. Furthermore, for both the first particle 1 and the second particle 11, it is preferable that their average particle sizes be small from the viewpoint of increasing reactivity in solution, while for sufficiently increasing the fluorescence anisotropy of the composite, it is preferable that the second particle 11 be large. Therefore, it is even more preferable that the average particle size of the second particle 11 be at least twice and up to 50 times the average particle size of the first particle 1.

[0034] Furthermore, the second particle 11 preferably has a small particle size distribution, and preferably a PDI of 0.1 or less. The second particle 11 may include a second particle carrier 10 made of polystyrene and a polymer having siloxane bonds. The second particle 11 may also have a layer on its surface containing a hydrophilic polymer that includes either an ether structure, a betaine structure, or a pyrrolidone ring. Including a hydrophilic polymer on the particle surface makes it easier to suppress nonspecific adsorption. In this way, suppressing nonspecific adsorption with a hydrophilic polymer instead of proteins, etc., suppresses the particle size distribution, which is advantageous for measurements based on fluorescence anisotropy.

[0035] Furthermore, the second particle 11 is preferably an agarose particle, a dextran particle, a polystyrene particle, a silica particle, or a magnetic particle.

[0036] The second particle 11 may also contain a luminescent component, but it is preferable that the excitation wavelength of the first particle 1 is different from the excitation wavelength of the second particle 11, the second particle 11 does not have a maximum absorption wavelength in the excitation wavelength region of the first particle 1, or the emission wavelength of the first particle 1 is different from the emission wavelength of the second particle 11. Alternatively, the excitation wavelength of the first particle 1 may be different from the excitation wavelength of the second particle 11, and the emission wavelength of the first particle 1 may be different from the emission wavelength of the second particle 11. That is, the second particle 11 can emit light at an excitation wavelength different from the excitation wavelength of the first particle 1, and can also emit light different from the emission wavelength of the first particle 1. By providing the second particle 11 with luminescence characteristics different from those of the first particle 1, multi-dimensional measurement such as excitation at multiple wavelengths or observation of luminescence at multiple wavelengths becomes possible.

[0037] On the other hand, it is desirable that the second particle 11 is not excited at a wavelength suitable for exciting the first particle 1. This is because when the excitation wavelength of the first particle 1 is different from the excitation wavelength of the second particle 11, or when the second particle 11 does not have a maximum absorption wavelength in the excitation wavelength region of the first particle 1, it is possible to sufficiently excite the first particle 1 with the excitation light and to obtain emission only from the first particle 1. Specifically, when the luminescent component contained in the first particle 1 is a europium complex, which is one of the rare earth complexes 4, it is preferable that the second particle 11 does not have a maximum absorption wavelength in the wavelength region of 300 nm or more and 450 nm or less, which is the range of the maximum absorption wavelengths of many europium complexes.

[0038] Also, as described above, it is preferable that the second particle 11 does not produce luminescence overlapping with that of the first particle 1. Specifically, when the luminescent component is a europium complex, it is preferable that the second particle 11 does not have a maximum emission wavelength in the wavelength region of 550 nm or more and 650 nm or less. This is because the maximum emission wavelength of the europium complex is often included in the above wavelength region.

[0039] The first particle 1 and the first compound 8 can have a ligand as the first site 5A or the second site 5B in order to have affinity for the target substance 6. Any ligand that shows affinity for a specific substance can be used. Examples of ligand-target substance 6 or target substance 6-ligand combinations include the following: antigens and antibodies, small molecules and their receptors, enzymes and substrates, and complementary nucleic acids. Furthermore, antibodies and substances specific to them, such as allergens, bacteria, viruses, cells, cell membrane components, cancer markers, various disease markers, antibodies, blood-derived substances, food-derived substances, natural product-derived substances, and all kinds of small molecules. Furthermore, receptors and substances specific to them, such as small molecules, cytokines, hormones, neurotransmitters, signaling molecules, and membrane proteins. Furthermore, DNA, RNA, cDNA, parts or fragments thereof, synthetic nucleic acids, primers, probes, etc. derived from bacteria, viruses, cells, etc., and nucleic acids that are complementary to them can be included. In addition to the above, any combination of target substance 6 and ligand known to exhibit affinity can be used. Typical examples of ligands include antigens, antibodies, and nucleic acids.

[0040] The first site 5A or the second site 5B is a ligand that can specifically bind to the target substance 6 and can be bound to the first particle 1 or the first compound 8 via a functional group. Since the first compound 8 is preferably small in size, it may be a molecule in which the ligand constituting the second site 5B has a third site 7 introduced into it.

[0041] In this embodiment, particles that bind to the target substance 6 may be referred to as affinity particles, particles that exhibit luminescence may be referred to as luminescent particles, and particles with a relatively large average particle size may be referred to as large particle size particles. Particles that have luminescence and bind to the target substance 6 may be referred to as luminescent affinity particles. Further, particles that have a relatively large average particle size and bind to the first compound 8 may be referred to as large particle size affinity particles. In the present disclosure, the first particle 1 corresponds to the luminescent affinity particle, and the second particle 11 corresponds to the large particle size affinity particle.

[0042] In the first particle 1, by reducing the particle size distribution of the particles and introducing the luminescent rare earth complex 4 into the particles, a fine change in the dispersion state of the particles in the test liquid can be captured as a change in the polarized luminescence characteristics. That is, through the first compound 8, the target substance 6 is sandwiched, and particles having affinity aggregate (a sandwich-type reaction occurs), thereby causing a change in the rotational Brownian motion of the particles. This can be captured as a change in fluorescence anisotropy. According to this method, it is possible to detect the target substance 6 at a level of nanograms to picograms per 1 mL of the solution.

[0043] The rare earth complex 4 can generate polarized light of long-lived phosphorescence. Fluorescence anisotropy is related to the anisotropy of the transition moment (transition dipole moment) of the luminescent dye. In the case of a luminescent dye having anisotropy in the transition moment, when polarized light along the transition moment is used as the excitation light, the emitted light is also polarized light along the transition moment. In the case of the rare earth complex 4, when a ligand having anisotropy is excited, it exhibits fluorescence emission based on energy transfer from the ligand to the central metal ion, so polarized light is emitted by exciting the ligand with polarized light.

[0044] The principle of fluorescence polarization decay measures the deviation of the transition moment due to the rotational motion of the luminescent material within the time when polarized luminescence occurs. The rotational motion of the luminescent material can be expressed by Equation (2).Q = 3Vη / kT... (2) Q: Rotational relaxation time of the material V: Volume of the material η: Viscosity of the solvent k: Boltzmann constant T: Absolute temperature [[ID=]]

[0045] The rotational relaxation time of a material is the time required for the molecule to rotate by an angle θ (68.5°) such that cosθ = 1 / e.

[0046] From equation (2), it can be seen that the rotational relaxation time of the luminescent material is proportional to the volume of the material, that is, the cube of the particle size of the luminescent material. On the other hand, the relationship between the luminescence lifetime of the material and the degree of polarization in fluorescence depolarization can be expressed by equation (3). p0 / p = 1 + A(τ / Q) ... (3) p0: degree of polarization when the material is stationary (Q = ∞) p: degree of polarization A: constant τ: luminescence lifetime of the material Q: rotational relaxation time

[0047] From equations (2) and (3), it is clear that in order to measure a large change in polarization degree, the relationship between the luminescence lifetime of the luminescent material and the rotational relaxation time, i.e., the volume (particle size) of the luminescent material, is important, and the larger the particle size of the luminescent material, the longer the luminescence lifetime needs to be.

[0048] To experimentally determine the degree of polarization of the emission shown in equation (3), polarized light should be incident on the sample, and the emission should be detected at a 90-degree angle to the direction of propagation and vibration of the excitation light. At this time, the detected light should be separated into polarization components parallel and perpendicular to the polarization of the incident light and evaluated according to equation (4). r(t) = (I∥(t) - GI⊥(t)) / (I∥(t) + 2GI⊥(t)) ... (4) r(t): Fluorescence anisotropy at time t I∥(t): Emission intensity of the emission component parallel to the excitation light at time t I⊥(t): Emission intensity of the emission component perpendicular to the excitation light at time t G: Correction value, ratio of I⊥ / I∥ measured with excitation light whose vibration direction is 90 degrees different from the excitation light used for sample measurement

[0049] In other words, within the appropriate particle (or aggregate) size and luminescence lifetime range, it is possible to sensitively read changes in the size of the luminescent material due to antigen-antibody reactions, etc., as a value of fluorescence anisotropy. Note that fluorescence anisotropy is the value corrected for G and 2G, and the degree of polarization is the value obtained by removing G and 2G from equation (4). In actual measurements, a correction value for G is necessary, so fluorescence anisotropy is determined.

[0050] In terms of material design, it is desirable for the first particle 1 to be small, and for its particle size to increase to the point where the fluorescence anisotropy saturates upon binding with the target substance 6. However, when performing a sandwich-type antigen-antibody reaction using only one type of particle, the particle size formed after the reaction is limited. If the size of the first particle 1 is increased to increase the fluorescence anisotropy, the anisotropy before binding increases, and ultimately, the change in fluorescence anisotropy cannot be significantly increased.

[0051] Therefore, a technique has been proposed to increase the difference in fluorescence anisotropy before and after the reaction by reacting the first particle 1 with a second particle 11 having a larger average particle size than the first particle 1 in a sandwich-type manner. However, the second particle 11, with its larger average particle size, has reduced reactivity in solution due to its large size. This disclosure is characterized by using the first compound 8 as a linker to increase the reactivity of the large-sized second particle 11.

[0052] Preferably, the first compound 8 acts as a linker that specifically binds to the target substance 6 and also binds to the second particle 11. For example, it may be a molecule having an antibody that binds to the target substance 6 and biotin for binding to the second particle 11 (also called a biotinylated antibody). In this disclosure, in order to obtain a large change in fluorescence anisotropy dependent on the target substance 6, the second particle 11, which is larger in size than the luminescent particle, is bound to the first compound 8. Therefore, the first compound 8 is preferably a molecule that is relatively smaller than the first particle 1, in other words, it is desirable that it does not change the rotational motion of the luminescent particle, which is the first particle 1. In this view, the size of the first compound 8 may be between 1 nm and 50 nm, and is particularly preferably between 1 nm and 20 nm. A small size of the first compound 8 is preferable because it increases the rate of the binding reaction to the target substance 6 captured by the first particle 1.

[0053] (Steps of the method for detecting target substances by fluorescence polarization measurement) As described above, the detection method of this embodiment includes the following steps, which will be explained in detail with reference to Figure 3. (1) A step of obtaining a first mixture having a sample solution that may contain the target substance 6, a first particle 1 having a first site 5A that specifically binds to the target substance 6, a first particle carrier 14 containing a particle substrate and a luminescent component, and a first compound 8 having a second site 5B that specifically binds to a site different from the site that specifically binds to the first site 5A of the target substance 6, and a third site 7 different from the second site 5B. (2) A step of obtaining a second mixture having a second compound 13 having a second particle 11 having a fourth site 9 that specifically binds to the third site 7 and containing a second particle carrier 10, and the first compound 12. (3) A step of obtaining a value related to the fluorescence anisotropy of the second mixture.

[0054] First, a first particle 1 containing a rare earth complex 4 is prepared, which specifically binds to the target substance 6 via a first site 5A, and a first compound 8 is prepared, which specifically binds to the target substance 6 via a second site 5B and also binds to the second particle 11.

[0055] Next, the sample solution (which may contain the target substance 6), the first particle 1, and the first compound 8 are mixed to obtain the first mixture. If the target substance 6 is present in the obtained first mixture, then a first complex 12 containing the first particle, the target substance, and the first compound is present. In this step, mixing is preferably carried out in the pH range of 3.0 to 11.0. The mixing temperature is in the range of 0°C to 100°C, or 4°C to 50°C, or even 20°C to 50°C, and the mixing time is in the range of 1 second to 2 hours, or 1 minute to 60 minutes. Furthermore, in the method according to this embodiment, the concentration of the first particle 1 is preferably 0.000001% by mass (corresponding to 0.00001 mg / mL) to 1% by mass (corresponding to 10 mg / mL) in total amount in the reaction system, and more preferably 0.00001% by mass (corresponding to 0.0001 mg / mL) to 0.001% by mass (corresponding to 0.01 mg / mL).

[0056] The detection method according to this embodiment is characterized by detecting the agglutination reaction resulting from the mixing of the first particle 1, the first compound 8, the target substance 6, and the second particle 11 using fluorescence polarization. Specifically, the detection method includes the steps of: mixing a test reagent and a sample solution to obtain a mixture; irradiating the mixture with polarized light; and separating and detecting the polarization component of the emission of affinity particles in the mixture. Therefore, fluorescence polarization measurement of the first mixture may also be performed. This measurement allows for confirmation of the blank level of fluorescence anisotropy.

[0057] In the next step, after the steps described above, the first mixture and the second particles 11 are mixed to obtain a second mixture. At this time, the obtained second mixture contains a second complex 13 comprising the first particles, target substance, first compound, and second particles, and the fluorescence anisotropy of the first particles 1 (luminescent particles) changes depending on the amount of target substance 6. The mixing conditions (pH, temperature, time) are preferably the same as those in the steps described above, and the concentration of the second particles 11 is also the same as the preferred range for the first particles 1. However, since the average particle size of the second particles 11 is larger than that of the first particles 1, it is preferable that the particle concentration of the second particles 11 is relatively higher than that of the first particles 1. Therefore, in the method according to this embodiment, the concentration of the second particles 11 is particularly preferably between 0.001% by mass (corresponding to 0.01 mg / mL) and 0.1% by mass (corresponding to 1 mg / mL) in the reaction system.

[0058] Finally, the target substance 6 can be detected (at least one of the presence or absence and concentration of the target substance can be determined) by measuring the fluorescence anisotropy of the obtained second mixture. In this disclosure, the "value related to fluorescence anisotropy" may be not only the absolute value of the fluorescence anisotropy of the second mixture, but also the change in the difference value between the fluorescence anisotropy of the first mixture and the second mixture.

[0059] By optically detecting the agglutination reaction that occurs in the mixture, the target substance 6 in the sample can be detected, and its concentration can also be measured. For example, by obtaining a calibration curve (a graph showing the relationship between fluorescence anisotropy and the concentration of target substance 6) using a standard solution of target substance 6 with a known concentration, it becomes possible to quantify the target substance 6 contained in the sample solution from the fluorescence anisotropy.

[0060] (Detailed description of the particles) An example of the particles according to this embodiment will be described in more detail with reference to Figure 2.

[0061] (First Particle) The first particle 1 (luminescent affinity particle) in this embodiment has a small particle size distribution and the surface of the particle is hydrophilically coated. A rare earth complex 4, which is a luminescent component, is present inside the particle.

[0062] Figure 2 shows an example where the particles are spherical, but is not particularly limited. The diameter (average particle size) of the first particle 1 is preferably 10 nm or more, or 20 nm or more, more preferably 25 nm or more, and also preferably 1 μm or less, more preferably 400 nm or less. In this embodiment, it is particularly preferable that the average particle size of the first particle 1 is 20 nm or more and 400 nm or less. In addition, it is preferable that the particle size distribution is more uniform. For example, it can be suitably used if the pdi measured by dynamic light scattering is 0.1 or less.

[0063] PDI can be determined by irradiating particles dispersed in a solution with laser light and observing the scattered light with a photon detector. Since particles are constantly moving due to Brownian motion, the intensity distribution due to the interference of scattered light is also constantly fluctuating. Dynamic light scattering is a particle measurement method that observes the Brownian motion as fluctuations in scattered light intensity. The fluctuation of scattered light with respect to time is represented by an autocorrelation function, and the translational diffusion coefficient is determined. From the determined diffusion coefficient, the Stokes diameter can be found, and the particle size dispersed in the solution can be derived. Furthermore, by performing cumulant analysis on the autocorrelation function, the cumulant diameter and PDI can be determined. PDI indicates the degree of polydispersity of the particle size distribution, and the smaller this value, the more uniform the particle size distribution is. Generally, if PDI is 0.1 or less, the particles are considered to be monodisperse in the solvent.

[0064] The first particle 1 according to this embodiment preferably has a pdi of 0.1 or less. In order to stably maintain a pdi of 0.1 or less, it is desirable not to apply a nonspecific adsorption inhibitor to the surface of the particle. However, for the purposes of this disclosure, it is necessary to prevent substances other than the target substance 6 from being nonspecifically adsorbed onto the particle, and therefore, it is preferable to have a coating to keep the surface of the particle hydrophilic.

[0065] On the other hand, with BSA loading, which is commonly performed for the above purposes, it is difficult to stably maintain a pdi of 0.1 or less. Furthermore, lot-to-lot variability can occur with BSA loading. Therefore, a coating using a hydrophilic polymer is necessary to ensure that the pdi relative to particle size is 0.1 or less.

[0066] In this embodiment, if the average particle size of the first particle 1 is larger than 1 μm, the fluorescence anisotropy before aggregation increases, and the difference with the fluorescence anisotropy after aggregation becomes smaller. Also, if the average particle size is less than 10 nm, the volume per particle decreases, the amount of rare earth complex 4 that can be contained decreases, and it may become impossible to obtain the emission intensity necessary for measurement.

[0067] (Particle Substrate) Particle substrate 2 refers to the core portion of the first particle 1. Particle substrate 2 is preferably spherical or nearly spherical in shape. The material of particle substrate 2 is not particularly specified as long as it can stably contain the rare earth complex 4. As particle substrate 2, polystyrene particles polymerized with styrene monomer as the main component, or a polymer containing siloxane bonds in addition to polystyrene particles can be preferably used. Polystyrene particles can be produced as particles with a very uniform particle size distribution by the emulsion polymerization method described later. Furthermore, silanol present in the polymer containing siloxane bonds can be used to impart a hydrophilic coating or ligand, as described later.

[0068] (Hydrophilic Layer) The hydrophilic layer 3 is formed on the outside of the particle substrate 2 and is preferably composed of a hydrophilic polymer such as a molecule or polymer containing a hydrophilic group. A molecule containing a hydrophilic group means a molecule or polymer having a hydroxyl group, an ether structure, a pyrrolidone ring, a betaine structure, etc. Specifically, the hydrophilic layer 3 can be mainly composed of polyethylene glycol, polyvinylpyrrolidone, sulfobetaine, phosphobetaine polymers, or polyglycidyl methacrylic acid, which has a glycidyl group opened and a hydroxyl group modified at the end of the molecule. The hydrophilic layer 3 can be formed by imparting a single molecule having a hydrophilic group to the surface of the first particle 1 using a silane coupling agent, or it may be formed simultaneously during the synthesis of the particle substrate 2, as described later. There is no limit to the thickness of the hydrophilic layer 3; it is sufficient as long as it exhibits hydrophilicity. However, if the hydrophilic layer 3 is too thick, it may become hydrogel-like, and hydration due to the influence of ions in the solvent may change the thickness of the hydrophilic layer 3, potentially leading to instability. The thickness of the hydrophilic layer 3 is preferably between 1 nm and 50 nm.

[0069] (Rare Earth Complex) Due to the characteristics that the wavelength and intensity of the emission are less affected by the surroundings and the emission has a long lifetime, it is preferable to use a luminescent rare earth complex 4 as the luminescent component in this disclosure. The rare earth complex 4 is composed of a rare earth element and a ligand. The rare earth element is selected from europium, terbium, neodymium, erbium, yttrium, lanthanum, cerium, samarium, gadolinium, dysprosium, thulium, ytterbium, scandium, etc. Considering the emission lifetime and the visible emission wavelength range, europium and terbium are particularly preferred. For example, europium generally has an emission lifetime of 0.1 to 1.0 ms. It is preferable that the emission lifetime and the rotational relaxation time obtained from formula (2) are appropriately adjusted. In the case of europium in an aqueous dispersion, if the first particle 1 has a diameter of approximately 50 nm to 300 nm, the fluorescence anisotropy represented by equation (4) changes significantly before and after aggregate formation.

[0070] Regarding the ligands constituting the rare-earth complex 4, at least one of them must be a ligand with a light-harvesting function. The light-harvesting function is the action of being excited at a specific wavelength and exciting the central metal of the complex by energy transfer. Furthermore, the ligand with the light-harvesting function is preferably a molecule with anisotropic transition moment, for example, phenanthroline is suitably used, but this is not limited to this. In addition, it is preferable that the ligands constituting the rare-earth complex 4 include ligands such as β-diketones to prevent coordination of water molecules. Ligands such as β-diketones that coordinate to the rare-earth ions suppress the deactivation process due to energy transfer to solvent molecules, etc., and strong fluorescence emission is obtained.

[0071] The rare-earth complex 4 may be a multinuclear complex as long as it exhibits anisotropy in its transition moment. It is desirable that the fluorescence anisotropy be 0.1 or greater when the Brownian rotation of the rare-earth complex 4 is considered to have stopped in the medium. The state in which the Brownian rotation is considered to have stopped means that the rotational relaxation time of the particles is sufficiently longer than the luminescence lifetime of the rare-earth complex 4.

[0072] (First and Second Sites) In this embodiment, the first site 5A and the second site 5B are ligands that specifically bind to the target substance 6 or a part thereof. Preferably, the first site 5A and the second site 5B have selective or specific affinity for the target substance 6. Examples of combinations of the target substance 6 with the first site 5A or the second site 5B, or the first site 5A or the second site 5B with the target substance 6 include, but are not limited to, antigens and antibodies, enzyme proteins and their substrates, signaling substances such as hormones and neurotransmitters and their receptors, nucleic acids and nucleic acids. Typical nucleic acids include deoxyribonucleic acid. In Figure 2, the first site 5A is contained in the first particle 1, and the second site 5B is contained in the first compound 8.

[0073] The first site 5A and the second site 5B can be bound to the particle substrate 2 or to the hydrophilic layer 3 via binding functional groups present in the hydrophilic layer 3, or they can be incorporated as ligands during the preparation of the particle substrate 2 or the formation of the hydrophilic layer 3.

[0074] Furthermore, the first site 5A and the second site 5B can also be similar to known ligands used in latex agglutination. Ligands that cause agglutination in latex agglutination are likely to be effectively used in this embodiment as well. Typical examples of the first site 5A and the second site 5B in this embodiment include antibodies, antigens, and nucleic acids.

[0075] (Target Substance) In Figure 2, the target substance 6 is the object to be measured, and it is preferable that the first site 5A and the second site 5B are substances that show affinity to and specifically bind (may be called adsorption or reaction) to the target substance 6 via different sites of the target substance 6. The target substance 6 can be any substance as long as the corresponding ligand is available. Examples of target substance 6 include the following: antibodies, antigens, nucleic acids, small molecule compounds, various receptors, enzymes, and substrates. Furthermore, all substances such as allergens, cytokines, hormones, bacteria, viruses, DNA, RNA, cDNA, cells, cell membrane components, cancer markers, various disease markers, antibodies, blood-derived substances, food-derived substances, and natural product-derived substances can also be cited. In this embodiment, it is preferable that the target substance 6 includes at least one of antibodies, antigens, and nucleic acids.

[0076] The target substance 6 binds to the first site 5A of the first particle 1 and to the second site 5B of the first compound 8. This forms the first complex 12 (first particle - target substance - first compound) via the target substance 6. Note that Figure 2 schematically shows one example each of the first compound 8, the target substance 6, and the first particle 1. In reality, the first particle 1 has numerous ligands, binds to numerous target substances 6, and through these, further binds to numerous first particles 1 and / or the first compound 8 to form the first complex 12.

[0077] (First compound) The first compound 8 according to this embodiment has a second site 5B that can bind to the target substance 6 and can bind to the second particle 11. The first compound 8 and the second particle 11 are bound together by the binding of a third site 7 of the first compound 8 and a fourth site 9 of the second particle 11.

[0078] The third site 7 and the fourth site 9 are not particularly limited as long as their binding constants are smaller than the binding constant between the target substance 6 and the second site 5B. For example, it is preferable that one of the third site 7 and the fourth site 9 is a site containing biotin and the other is a site containing avidin, and it is more preferable that the third site 7 is a site containing biotin and the fourth site 9 is a site containing avidin. In that case, the first compound 8 may have multiple biotin molecules as the third site 7, but preferably 1 to 8, and more preferably 1 to 4. This is because a large number of third sites 7 may reduce the affinity of the second site 5B.

[0079] (Second particle 11) The second particle 11 (large particle size affinity particle) according to this embodiment preferably consists of at least the second particle carrier 10 and the fourth moiety 9. It can have the same properties as the first particle 1, except that it does not contain the second moiety 5B and the rare earth complex 4, and its particle size is larger than that of the first particle 1.

[0080] The larger the second particle 11, the greater the effect of causing a change in fluorescence anisotropy due to aggregate formation. On the other hand, if the particle size is too large, the dispersion stability in the sample solution will decrease, and the particles may settle. Also, if the particles are too large, the light scattering per particle will become stronger, and the polarization may be eliminated. Therefore, the average particle size of the second particle 11 is preferably 100 nm or more and 5 μm or less, more preferably 1 μm or more and 5 μm or less.

[0081] It is preferable that the second particle 11 does not emit or absorb light to an extent that would hinder the measurement in the wavelength range for measuring fluorescence anisotropy or the wavelength range for exciting the rare-earth complex 4 that exhibits fluorescence anisotropy.

[0082] By using a solution in which the first particle 1, the first compound 8, and the second particle 11 according to this embodiment are dispersed, the anisotropy of polarized light emission can be detected with high sensitivity in relation to the aggregation and dispersion behavior of the particles. Therefore, the aqueous solvent in which the first particle 1, the first compound 8, and the second particle 11 are dispersed can be used as a highly sensitive test reagent using fluorescence polarization. A buffer solution can also be used as the aqueous solvent. Furthermore, surfactants, preservatives, sensitizers, etc., may be added to the aqueous solvent to increase the stability of the dispersion.

[0083] (Example of a method for producing the first particle 1) Next, an example of a method for producing the first particle 1 according to this embodiment will be described. In this embodiment, the luminescent particles are produced by mixing a radical polymerizable monomer, a radical polymerization initiator, a rare earth complex 4, and a hydrophilic polymer with an aqueous medium to prepare an emulsion (first step). Furthermore, the emulsion is heated to polymerize the monomer (second step). The luminescent particles according to this embodiment are luminescent particles obtained by copolymerizing a compound having a double bond that is radically polymerizable.

[0084] Furthermore, the process may optionally include a step (third step) of applying a functional group for binding the first site 5A to the surface of the luminescent particle. Here, the functional group to which the first site 5A can be bound can be any of the following: a carboxyl group, an amino group, a thiol group, an epoxy group, a maleimide group, a succinimidyl group, or a silicon alkoxide group.

[0085] (1. Radical Polymerizable Monomers) Radical polymerizable monomers may include styrene monomers. Furthermore, monomers selected from the group consisting of acrylate monomers and methacrylate monomers may also be included. Examples of such monomers include butadiene, vinyl acetate, vinyl chloride, acrylonitrile, methyl methacrylate, methacrylonitrile, methyl acrylate, etc. In addition to styrene monomers, at least one selected from the above group of monomers can be used. That is, these monomers can be used individually or in combination. Also, monomers having two or more double bonds in a single molecule, such as divinylbenzene, may be used as a crosslinking agent.

[0086] Furthermore, radical polymerizable monomers containing organosilanes may be used to incorporate siloxane bonds into the luminescent particles. Examples of organosilane compounds include vinyltrimethoxysilane, vinyltriethoxysilane, p-styryltrimethoxysilane, 3-methacryloxypropylmethyldimethoxysilane, 3-methacryloxypropyltrimethoxysilane, 3-methacryloxypropylmethyldiethoxysilane, 3-methacryloxypropyltriethoxysilane, and 3-acryloxypropyltrimethoxysilane.

[0087] At least one selected from this group of monomers can be used. That is, these organosilane compounds may be used individually or in combination of several types. Radical polymerizable monomers containing organosilanes form an inorganic oxide backbone within the synthesized luminescent particles, improving the physical and chemical stability of the luminescent particles. Furthermore, radical polymerizable monomers containing organosilanes enhance the affinity between the unit having a functional group capable of binding a ligand to the hydrophilic polymer on the surface of the luminescent particles and the backbone material of the polymer nanoparticles containing styrene.

[0088] When incorporating siloxane bonds into luminescent particles, it is preferable that the proportion of units having siloxane bonds to styrene monomer be 40% by mass or less. Furthermore, by using a radical polymerizable monomer containing an organic silane, silanol groups can be imparted to the surface of the luminescent particles. Hydrogen bonding between the silanol groups and a hydrophilic polymer, such as polyvinylpyrrolidone, allows the polyvinylpyrrolidone to adsorb more strongly onto the particle surface.

[0089] (2. Radical Polymerization Initiators) Radical polymerization initiators can be broadly selected from azo compounds, organic peroxides, etc. Specifically, examples of radical polymerization initiators include 2,2'-azobis(isobutyronitrile), 2,2'-azobis(2,4-dimethylvaleronitrile), 2,2'-azobis(2-methylbutyronitrile), 4,4'-azobis(4-cyanovaleric acid), 2,2'-azobis(2-methylpropionamidine) dihydrochloride, 2,2'-azobis(2-methylpropionic acid) dimethyl, tert-butyl hydroperoxide, benzoyl peroxide, ammonium persulfate (APS), sodium persulfate (NPS), potassium persulfate (KPS), etc.

[0090] (3. Hydrophilic Polymers) Examples of hydrophilic polymers for suppressing nonspecific adsorption include hydrophilic polymers containing units having ether structures, betaine structures, pyrrolidone rings, etc. Hydrophilic polymers are contained in the synthesized luminescent particles and can mainly exist on the particle surface. As one preferred example, the case in which a polymer having a pyrrolidone ring (hereinafter sometimes abbreviated as PVP) is used will be described.

[0091] PVP can be added from the time of particle synthesis, and by adding PVP, it is possible to simultaneously impart non-specific adsorption suppression ability and ligand binding ability to the particles. Since PVP is more hydrophilic than radical polymerizable monomers, during synthesis, PVP is present at the interface between the solvent and the particles being formed. PVP is adsorbed onto the surface of the particles by physico- and chemi-adsorption, such as by partially incorporating the PVP polymer chain during polymerization or by interactions between the pyrrolidone ring and styrene (radical polymerizable monomer).

[0092] The molecular weight of PVP is preferably between 10,000 and 100,000, and more preferably between 40,000 and 70,000. If the molecular weight is less than 10,000, the hydrophilicity of the surface of the luminescent particles is weak, making nonspecific adsorption more likely. If the molecular weight is greater than 100,000, the hydrophilic layer 3 on the surface of the luminescent particles becomes too thick, causing the luminescent particles to gel and become difficult to handle. In addition to PVP during the synthesis of luminescent particles, or separately from PVP, an additional hydrophilic polymer may be added as a protective colloid.

[0093] In an example of luminescent particles prepared as described above, 30 μL of a dispersion of 0.1% by mass of particles was added to 60 μL of buffer mixed with 16 μL of serum, and the mixture was left at 37°C for 5 minutes. The difference in the absorption spectrum at a wavelength of 572 nm with a 10 mm optical path before and after this addition was 0.1 or less. In other words, nonspecific adsorption of impurities in the serum was sufficiently small.

[0094] (4. Aqueous Medium) The aqueous solution (aqueous medium) used in the above synthesis preferably contains 80% by mass or more and 100% by mass or less water. Examples of aqueous solutions include water and water-soluble organic solvents, such as methanol, ethanol, isopropyl alcohol, and acetone, mixed with water. If the content of organic solvents other than water exceeds 20% by mass, dissolution of polymerizable monomers may occur during the production of luminescent particles.

[0095] Furthermore, when using a radical polymerizable monomer containing an organosilane, it is preferable that the pH of the aqueous solution (aqueous medium) is pre-adjusted to between 6 and 9. If the pH is less than 6 or greater than 9, the alkoxide and silanol groups in the organosilane compound may undergo condensation polymerization or react with other functional groups before the formation of luminescent particles, potentially causing the resulting particles to aggregate. In this embodiment, it is desirable not to intentionally perform condensation polymerization of the alkoxide before particle formation.

[0096] The pH adjustment described above is preferably done using a pH buffer, but it may also be done with an acid or a base. In addition, surfactants, defoamers, salts, thickeners, etc. may be added to the aqueous medium in a proportion of 10% by mass or less.

[0097] In the production of particles according to this embodiment, it is preferable to first dissolve PVP in an aqueous medium whose pH is adjusted to 6 to 9. The amount of PVP added to the aqueous medium is 0.01% to 10% by mass, preferably 0.03% to 5% by mass. If the amount is 0.01% by mass or less, the amount adsorbed to the polymer fine particles will be small and the effect will not be exhibited. If the amount is 10% by mass or more, the viscosity of the aqueous medium will increase, and sufficient stirring may not be possible.

[0098] Next, the radical polymerizable monomer (A) is added to the aqueous medium to form an emulsion. At this time, in addition to (A), a radical polymerizable monomer (B) containing an organosilane may also be added to the aqueous medium to form an emulsion. The mass ratio of (A) to (B) is preferably 6:4 to 10:0. Furthermore, the luminescent rare earth complex 4 is mixed into the prepared monomer solution. At this time, if the solubility of the rare earth complex 4 is low, an insoluble organic solvent may be added. The mass ratio of the rare earth complex 4 to the monomer is preferably 1:1000 to 1:10.

[0099] If (A) is 60% by mass or less, the specific gravity of the entire particle will increase, and particle sedimentation may become significant. Also, (B) is not necessary, but it is preferable to add (B) in order to improve the adhesion between PVP and luminescent particles. The mass ratio of the total amount of the aqueous medium, (A), and (B) is preferably 5:5 to 9.5:0.5. If the ratio of the aqueous medium is 50% by mass or less, nonspecific aggregation of the generated particles may become a problem. Also, even if it is 95% by mass or more, there is no problem in the generation of luminescent particles, but the amount generated may be small.

[0100] The radical polymerization initiator is used after being dissolved in water, a buffer, etc. The amount of radical polymerization initiator relative to the total mass of (A) and (B) can be used between 0.5% by mass and 10% by mass.

[0101] The process of heating the emulsion described above only needs to be performed so that the entire emulsion is heated uniformly. The heating temperature can be arbitrarily set between 50°C and 80°C, and the heating time between 2 hours and 24 hours. By heating the emulsion, the monomers are polymerized.

[0102] The functional group that can bind to the first site 5A is preferably a functional group that can bind to proteins, nucleic acids, peptides, amino acids, amino groups, etc. Examples of such functional groups include carboxyl groups, amino groups, thiol groups, epoxy groups, maleimide groups, succinimidyl groups, and silicon alkoxide groups. It is possible to impart the functional group to the surface of the luminescent particles by mixing a silane coupling agent having a functional group that can bind to the first site 5A with the synthesized luminescent particles. Specifically, for example, by preparing an aqueous solution of a silane coupling agent having a carboxyl group and mixing it with a dispersion of synthesized luminescent particles, a carboxyl group can be imparted to the particle surface. At this time, a dispersant such as polysorbate 20 may be added to the reaction solution. The reaction temperature can be arbitrarily set between 0°C and 80°C, and the reaction time between 1 hour and 24 hours. In order to suppress the rapid condensation reaction of the silane coupling agent, it is preferable to set the reaction time to 3 to 14 hours at or below room temperature of about 25°C. Depending on the functional group that can bond to the first site 5A, an acid or alkali catalyst can be added to accelerate the reaction on the particle surface.

[0103] Affinity for the target substance 6 can be conferred to particles synthesized by emulsion polymerization or the like by attaching a first site 5A, such as various antibodies. The method for attaching the first site 5A is not particularly limited; the optimal method for attaching the desired antibody can be selected using functional groups present in the hydrophilic layer 3 or particle substrate 2.

[0104] (Affinity Particles) In this embodiment, the first site 5A is a ligand that specifically binds to a particular target substance 6. The site to which the first site 5A binds to the target substance 6 is fixed and has a selective or specific high affinity. For example, typical examples of combinations of target substance 6 and the first site 5A, or the first site 5A and target substance 6, include the following: antigens and antibodies, enzyme proteins and their substrates, signaling substances such as hormones and neurotransmitters and their receptors, nucleic acids, etc. However, the first site 5A in this embodiment is not limited to these. Examples of nucleic acids include deoxyribonucleic acid. The affinity particles in this embodiment have a selective or specific affinity for the target substance 6. Typical examples of the first site 5A in this embodiment include antibodies, antigens, and nucleic acids.

[0105] The method for the chemical reaction that chemically bonds the reactive functional group of the particles according to this embodiment to the first site 5A can be a conventionally known method to the extent that the objectives of this disclosure can be achieved. Furthermore, when forming an amide bond with the first site 5A, a catalyst such as 1-[3-(dimethylaminopropyl)-3-ethylcarbodiimide] can be used as appropriate.

[0106] In this embodiment, when the affinity particle uses an antibody (antigen) as the first site 5A and an antigen (antibody) as the target substance 6, it can be preferably applied to the immunolatex agglutination assay, which is widely used in fields such as clinical testing and biochemical research.

[0107] (Method for producing the second particle) The second particle 11, i.e., a large particle, can be obtained in the same manner as the synthesis method for the first particle 1 described above, except for the step of introducing the rare earth complex 4. However, the second particle 11 may contain a different fluorescent dye than the rare earth complex 4 contained in the first particle 1. Alternatively, commercially available particles that do not contain the rare earth complex 4 can be obtained and used as the second particle 11. Examples of the second particle 11 include particles selected from the group consisting of agarose particles, dextran particles, polystyrene particles, silica particles, and magnetic particles. These particles are readily available commercially.

[0108] Unlike the first particle 1, the fourth portion 9 is immobilized on the surface of the second particle carrier 10. The immobilization of the fourth portion 9 on the second particle carrier 10 is the same as the method for synthesizing the affinity particles described above.

[0109] (Method for producing the first compound) The first compound 8 can be synthesized by binding the second site 5B and the third site 7. The binding of the third site 7 to the second site 5B can be done using known methods. For example, when producing a biotinylated antibody that binds to a target substance 6 as the first compound 8, a site having biotin can be prepared as the third site 7, and the carboxyl group of biotin can be condensed with the amino group contained in the antibody, which is the second site 5B, to synthesize the first compound 8.

[0110] (Test Kit) This disclosure provides, as one embodiment, the following test kit for detecting a target substance.

[0111] According to this disclosure, a target substance test kit for detecting a target substance in a sample solution using a value relating to fluorescence anisotropy, comprising: a first particle having a first site that specifically binds to the target substance and a luminescent component; a first compound having a second site that specifically binds to a site in the target substance different from the site that specifically binds to the first site, and a third site different from the second site; and a second particle having a fourth site that specifically binds to the third site and having an average particle size larger than that of the first particle.

[0112] The test kit according to this embodiment comprises a first particle 1, a first compound 8, and a second particle 11. Furthermore, the test kit may include a dispersion medium. In addition, the first particle 1, the first compound 8, and the second particle 11 may be dispersed in the dispersion medium in the test kit. Moreover, the test kit may include a reagent containing the first particle 1, the first compound 8, the second particle 11, and the dispersion medium.

[0113] The first particle 1 preferably has a rare earth complex 4 as a luminescent component, and the rare earth complex 4 is more preferably a europium complex. The average particle size of the first particle 1 is preferably 1 μm (micron) or less, more preferably 500 nm or less, even more preferably 400 nm or less, and even more preferably 250 nm or less. The average particle size of the first particle 1 is preferably 10 nm or more, more preferably 20 nm or more, and even more preferably 50 nm or more. Therefore, for example, it is preferable that the average particle size of the first particle 1 be 20 nm or more and 400 nm or less.

[0114] The average particle size of the second particle 11 is 100 nm or more, more preferably 150 nm, and even more preferably 1 μm. The upper limit is preferably 5 μm or less, and more preferably 3 μm or less, from the viewpoint of particle dispersion stability, etc. Therefore, for example, it is preferable that the second particle 11 be 1 μm or more and 5 μm or less. Also, it is preferable that the average particle size of the second particle 11 is 2 times or more and 500 times or less than the average particle size of the first particle 1. The second particle 11 preferably contains any of the group consisting of agarose particles, dextran particles, polystyrene particles, silica particles, and magnetic particles. It is preferable that the second particle 11 does not have a maximum absorption wavelength in the wavelength region of 300 nm to 450 nm. Also, it is preferable that it does not have a maximum emission wavelength in the wavelength region of 550 nm to 650 nm.

[0115] It is preferable that one of the third site 7 and the fourth site 9 is a site containing biotin and the other is a site containing avidin, and it is more preferable that the third site 7 is a site containing biotin and the fourth site 9 is a site containing avidin.

[0116] A suitable target substance 6 for the test kit of this embodiment is a substance comprising at least one selected from the group consisting of antibodies, antigens, and nucleic acids.

[0117] While there is no particular designation for the form of the test kit, one preferred test kit of this disclosure is a test kit comprising a first reagent containing at least a first particle 1 and a first compound 8, and a second reagent containing a second particle 11, wherein each reagent is independent. Another preferred test kit of this disclosure is a test kit comprising a first reagent containing the first particle 1, a second reagent containing the first compound 8, and a third reagent containing the second particle 11, wherein each reagent is independent. Yet another preferred test kit of this disclosure is a test kit comprising a first reagent containing the first particle 1 and the first compound 8, and a second reagent containing the second particle 11, wherein each reagent is independent.

[0118] The amount of affinity particles according to this embodiment contained in the reagent in this embodiment is preferably 0.000001% by mass or more and 20% by mass or less, and more preferably 0.0001% by mass or more and 1% by mass or less. The reagent according to this embodiment may contain, in addition to the affinity particles according to this embodiment, third substances such as solvents and blocking agents, to the extent that the objectives of this disclosure can be achieved. Two or more types of third substances such as solvents and blocking agents may be included in combination. Examples of solvents used in this embodiment include various buffers such as phosphate buffer, glycine buffer, Good's buffer, Tris buffer, and ammonia buffer, but the solvents contained in the reagent in this embodiment are not limited to these.

[0119] The test kit may also include the reagents described above and a housing that encloses the reagents.

[0120] Furthermore, the kit according to this embodiment may contain a sensitizer that promotes particle aggregation via the target substance 6. Examples of sensitizers include polyvinyl alcohol, polyvinylpyrrolidone, and polyalginic acid, but this disclosure is not limited to these. The kit according to this embodiment may also include a positive control, a negative control, a serum diluent, etc. As the medium for the positive control and negative control, serum or saline solution that does not contain the target substance 6 may be used, or a solvent may be used. The kit according to this embodiment can be used in the method for detecting the target substance 6 according to this embodiment in the same way as a kit used for detecting the target substance 6 in a sample by conventional in vitro diagnostics. Furthermore, the concentration of the target substance 6 can also be measured by conventionally known methods, and it is particularly suitable for use in detecting the target substance 6 in a sample by the latex agglutination method.

[0121] The following examples illustrate a detection method targeting human TSH (thyroid-stimulating hormone).

[0122] (1) Preparation of luminescent affinity particles (first particles) that bind to the target substance First, particles (particle substrate 2) were prepared by mixing a polymer of styrene monomer and 3-methacrylateoxypropyltrimethoxysilane with a europium complex (rare earth complex 4), and the particle surface was made hydrophilic with Tween 20 (hydrophilic layer 3). Subsequently, luminescent affinity particles (first particle 1) were prepared by binding an anti-TSH antibody (first site 5A) to the particle surface. Details of the preparation example are shown below.

[0123] Solvent A was prepared by dissolving polyvinylpyrrolidone (PVP-K30: manufactured by Tokyo Chemical Industry Co., Ltd.) in MES (2-morpholinoethanesulfonic acid) buffer solution at pH 7 (manufactured by Kishida Chemical Co., Ltd.).

[0124] The europium complex is [tris(2-thenoyltrifluoroacetone)bis(triphenylphosphine oxide)europium(III)] (manufactured by Central Techno Co., Ltd., hereinafter referred to as "Eu(TTA)").３ (TPPO) ２ (abbreviated as "」), styrene monomer (manufactured by Kishida Chemical Co., Ltd.), and 3-methacryloxypropyltrimethoxysilane (manufactured by Tokyo Chemical Industry Co., Ltd., hereinafter abbreviated as "MPS") were mixed to prepare reaction solution B. Reaction solution B was added to a four-neck flask containing solvent A, and a mechanical stirrer was set to 300 rpm for stirring. After stirring for 15 minutes under nitrogen flow conditions, the temperature of the prepared oil bath was set to 70 °C, and nitrogen flow was carried out for another 15 minutes. After heating and stirring the mixture, an aqueous solution in which potassium persulfate (hereinafter abbreviated as "KPS") (manufactured by Aldrich) was dissolved was added to the reaction solution, and emulsion polymerization was carried out for 20 hours. After the polymerization reaction, the obtained suspension was subjected to ultrafiltration with an ultrafiltration membrane having a fractional molecular weight of 100K using about 4 L of ion-exchanged water to wash the product, and a dispersion of luminescent particles was obtained.

[0125] A dispersion of luminescent particles obtained by emulsion polymerization was separated and added to an aqueous solution in which 1% by mass of Tween 20 (manufactured by Kishida Chemical Co., Ltd.) was dissolved. After stirring for 10 minutes, a silane coupling agent, X12-1135 (manufactured by Shin-Etsu Chemical Co., Ltd.) was added and stirred overnight. After stirring, the dispersion was centrifuged, the supernatant was removed, and the precipitate was redispersed in pure water. The operations of centrifugation and redispersion were performed three or more times to wash the product. The washed precipitate was redispersed in pure water. As described above, ligand-binding functional groups were introduced into the particles. The mass ratio of the charged particles, pure water, and X12-1135 was 1:300:2. The concentration of the luminescent particles was evaluated using a thermogravimetric analyzer (Thermo Plus TG8120 manufactured by Rigaku).

[0126] 0.25 mL of a 1.2% by mass particle dispersion, corresponding to the synthesized luminescent particles, was taken and the solvent was replaced with 1.6 mL of pH 6.0 MES buffer. 0.5% by mass of 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide and N-hydroxysulfosuccinimide sodium were added to the particle MES buffer and reacted at 25°C for 1 hour. After the reaction, the dispersion was washed with pH 5.0 MES buffer, and 100 μg / mL of anti-TSH antibody was added. The anti-TSH antibody was bound to the particles at 25°C for 2 hours. After binding, the particles were washed with pH 8 Tris buffer. After the reaction, the particles were washed with phosphate buffer to obtain a 0.1% by mass anti-TSH antibody-modified luminescent reagent solution. At this time, the anti-TSH antibody was tested using clone number 3F10 manufactured by Mikli Immunology Laboratory Co., Ltd.

[0127] The binding of antibodies to the particles was confirmed by measuring the decrease in antibody concentration in the antibody-added buffer using a BCA assay. The size of the luminescent particles was 120 nm. The Pdi was 0.05 (dynamic light scattering method, Malvern Zetasizer Nano S).

[0128] (2) Preparation of the first compound that binds to the target substance (Preparation of biotinylated anti-TSH antibody) Next, an example of the preparation of a biotinylated anti-TSH antibody (first compound 8) is shown, in which biotin (third site 7) is labeled onto the anti-TSH antibody (second site 5B).

[0129] Biotin was labeled onto anti-TSH antibodies according to the protocol of the biotin labeling kit (LK03 Biotin Labeling Kit-NH2: Dojin Chemical Laboratories). After labeling, the biotinylated anti-TSH antibodies were stored in phosphate buffer solution. The binding of biotin to the antibodies was confirmed using HABA (4'-hydroxyazobenzene-2-carboxyl acid). The biotinylated anti-TSH antibodies contained approximately 4 biotin molecules per antibody molecule. The anti-TSH antibodies used for this study were clone number 9G11 manufactured by Mikli Immunology Laboratories, Inc.

[0130] (3) Preparation of the second particle solution Next, a magnetic particle (second particle 11) to which streptavidin (fourth site 9) is bound is shown as an example.

[0131] As the second particle, Dynabeads MyOne Streptavidin C1 (manufactured by Thermo Fisher) was used. This is a 1 μm particle with a hydrophilic surface, based on carboxyl-activated beads, and has streptavidin bound to its surface. It can bind to 2500 pmol of biotin per 1 mg of particle. The particle size was 1.0 μm, and the particle concentration was 10 mg / mL. The second particle has streptavidin on its surface and can bind to the biotin of the first compound.

[0132] (4) Fluorescence-polarized immunoassay of TSH (evaluation of Δr) (Example 1) Fluorescence-polarized immunoassay of TSH (manufactured by Sigma-Aldrich) was performed in the following steps.

[0133] First, luminescent affinity particles were added to a PBS buffer solution at pH 7.4 at a concentration of 0.01 mg / mL. Biotinylated anti-TSH antibody was also added at a concentration of 0.01 mg / mL. Furthermore, a solution of human TSH (Aldrich) dissolved in PBS buffer was mixed in, and the mixture was heated at 37°C for 10 minutes to perform an antigen-antibody reaction, resulting in the first mixture (reaction step 1). The fluorescence anisotropy <r> of the first mixture was measured. The fluorescence anisotropy measured here is defined as r0.

[0134] Next, the second particle solution was added to this solution to form the second mixture (reaction step 2). Ten minutes after addition, the fluorescence anisotropy <r> of the second mixture was measured. The fluorescence anisotropy measured here is denoted as r10. The value obtained by subtracting r0 from r10 is denoted as Δr.

[0135] The fluorescence anisotropy <r> was measured using apparatus 1. Apparatus 1 has the following configuration.

[0136] An LED light source with excitation light of 340 nm was prepared, and a polarizing filter (Sigma Optical Co., Ltd., NSPFU-30C) and a short-pass filter (Edmond Optics, Ltd., 84-706) were inserted into the optical path to set up an optical system capable of irradiating a 1 cm quartz square cell. A polarizing filter (Sawlab, Ltd., PIVISC050) and a band-pass filter (Sawlab, Ltd., FB610-10) were set at a 90° angle to the incident light. Emission was I ＶＶ and I ＶＨ To measure both directions simultaneously, two sets were prepared, each with a different polarizer configuration for the incident light and the 90° direction. For polarization detection, a QE-PRO from Ocean Optics was used for spectroscopic measurement. The sample holder was temperature-controlled to allow measurement at 37°C. For the measurement of fluorescence anisotropy <r>, the LED light source was fixed at an output of 12 mW, and the integration time was set to 1 second. The measurement interval was 3 seconds. From the emission spectrum of the obtained polarized emission, the emission intensity in the wavelength range of 600 nm to 630 nm was applied to equation (4) to determine the fluorescence anisotropy <r>.

[0137] In Example 1, the TSH concentration was adjusted to 200 pM and reaction step 1 was carried out. Then, the second particle concentration was adjusted to 0.5 mg / mL and reaction step 2 was carried out. Immediately after the start of reaction step 2, the fluorescence anisotropy <r> of the mixture was measured and the measurement was carried out for 10 minutes.

[0138] (Example 2) The same procedure as in Example 1 was followed, except that the TSH antigen concentration was adjusted to 50 pM.

[0139] (Comparative Example 1) The same procedure as in Example 1 was followed, except that the TSH antigen concentration was adjusted to 0 pM. That is, the change in fluorescence anisotropy when TSH was not present was investigated.

[0140] (Comparative Example 2) The same procedure as in Example 1 was followed, except that the final concentration of the second particle was adjusted to 0 mg / mL. That is, the change in fluorescence anisotropy when the second particle was not included was investigated.

[0141] (Comparative Example 3) The same procedure as in Example 1 was followed, except that the final concentration of the biotinylated anti-TSH antibody was adjusted to 0 mg / mL. Specifically, the change in fluorescence anisotropy when the biotinylated anti-TSH antibody was not included was examined.

[0142] (Comparative Example 4) The same procedure as in Example 1 was followed, except that the final concentration of the biotinylated anti-TSH antibody was adjusted to 0 mg / mL, and a different ligand-bound latex particle (particle size 0.5 μm) was added as the second particle to bring the final concentration to 0.5 mg / mL. Specifically, the change in fluorescence anisotropy was investigated when using latex particles that did not contain the biotinylated anti-TSH antibody and had a larger particle size than the first particle.

[0143] (Measurement results of Δr) The results for Examples 1-2 and Comparative Examples 1-4 are shown in Table 1. In Table 1, Δr is the change in the fluorescence anisotropy <r> value during a 10-minute fluorescence anisotropy measurement period. In Examples 1 and 2, the values ​​of Δr were 0.053 and 0.026, respectively, indicating that Δr changed in a TSH concentration-dependent manner. On the other hand, in Comparative Example 1, the value of Δr was 0.006, and virtually no change in r was observed. From these results, it was found that when TSH is absent, the r of the luminescent particles does not change, and when TSH is present, r increases. The fluorescence anisotropy value increases depending on the TSH concentration, and it was shown that by using this relationship as a calibration curve, the TSH concentration can be measured for sample solutions with an unknown TSH concentration.

[0144] In Comparative Example 2, TSH was present, but the second particle (1-micron particle) was not mixed in, and the value of Δr was 0.003, indicating virtually no change in r. These results show that the second particle is necessary for highly sensitive detection of TSH.

[0145] In Comparative Example 3, TSH was present, but the biotinylated anti-TSH antibody (first compound 8) was not mixed in. The value of Δr was 0.005, indicating virtually no change in r. These results indicate that a biotinylated anti-TSH antibody is necessary for highly sensitive detection of TSH.

[0146] In Comparative Example 4, TSH was present, but the system did not include a mixture of the second particle (1 micron particle) and the biotinylated anti-TSH antibody. Instead, ligand-bound latex particles were used. In this case, the ligand bound to the particle was an anti-TSH antibody (clone number: 9G11) that recognizes and binds to the target substance, TSH. That is, in this reaction, particle aggregation is expected to occur among the first particle, the target substance, and the ligand-bound latex particles. As a result, the value of Δr was 0.003, and virtually no change in r was observed. From the results of Example 1 and Comparative Example 4, it was shown that in this embodiment, which utilizes the high reaction rate of biotin-avidin to bind the second large particle to the first luminescent particle, it was possible to significantly increase the fluorescence anisotropy compared to the conventional fluorescence polarization measurement method. From the above, it was shown that the detection method according to this disclosure has extremely high detection sensitivity, and that highly sensitive measurement is possible with the test kit of this disclosure, or with luminescent affinity particles, the first compound, or large particle size particles.

[0147] (Comparative Example 5) As the second particle, a different streptavidin-bound magnetic particle (average particle size 0.1 μm, Therma Max, manufactured by SEGNOS Corporation) from that used in Example 1 was used. The procedure was the same as in Example 1, except that the final concentration was increased to 0.1 mg / mL and the temperature for measuring the fluorescence anisotropy <r> of reaction step 2 and the second mixture was kept at room temperature. In other words, the change in fluorescence anisotropy when using a second particle that has almost the same average particle size as the first particle and specifically binds to the first compound was investigated.

[0148] (Comparative Example 6) The same procedure as in Comparative Example 5 was followed, except that the TSH antigen concentration was adjusted to 50 pM.

[0149] The results are shown in Table 1. In Comparative Example 5, the size of the second particles is almost the same as that of the first particles. That is, in this reaction, it is expected that the increase in fluorescence anisotropy obtained using second particles, which have a larger average particle size than the first particles and specifically bind to the first compound, as seen in Example 1, will be smaller. As a result, the value of Δr is 0.011, and in Comparative Example 5, as in Example 1, it is possible to bind the second particles to the first luminescent particles by utilizing the high reaction rate of biotin-avidin, but it is thought that the increase in fluorescence anisotropy could not be made large because the second particles were small.

[0150] In Comparative Example 6, the TSH antigen concentration was set to 50 pM, but the value of Δr was 0.005, indicating virtually no change in r. Since no increase in r was observed, as seen in Example 2 where the TSH antigen concentration was the same, it was found that a second particle with a larger average particle size than the first particle and that specifically binds to the first compound is necessary for highly sensitive detection of TSH.

[0151]

[0152] This disclosure is not limited to the embodiments described above, and various modifications and alterations are possible without departing from the spirit and scope of this disclosure. Accordingly, the following claims are attached to make the scope of this disclosure public.

[0153] This application claims priority based on Japanese Patent Application No. 2025-016901, filed on 4 February 2025, and Japanese Patent Application No. 2026-011488, filed on 27 January 2026, and all of the contents of those applications are incorporated herein by reference.

[0154] 1. First particle (luminescent affinity particle) 2. Particle substrate 3. Hydrophilic layer 4. Rare earth complex 5A. First site 5B. Second site 6. Target substance 7. Third site 8. First compound 9. Fourth site 10. Second particle carrier 11. Second particle 12. First complex (complex of first particle - target substance - first compound) 13. Second complex (complex of first particle - target substance - first compound - second particle) 14. First particle carrier

Claims

1. A target substance test kit for detecting a target substance in a sample solution using a value relating to fluorescence anisotropy, comprising: a first particle having a first site that specifically binds to the target substance and a luminescent component; a first compound having a second site that specifically binds to a site in the target substance different from the site that specifically binds to the first site, and a third site different from the second site; and a second particle having a fourth site that specifically binds to the third site and having an average particle size larger than the first particle.

2. The target substance testing kit according to claim 1, wherein the luminescent component comprises a rare earth complex.

3. The target substance testing kit according to claim 2, wherein the rare earth complex is a europium complex.

4. The target substance testing kit according to claim 3, wherein the second particle does not have a maximum absorption wavelength in the wavelength range of 300 nm to 450 nm.

5. The target substance testing kit according to any one of claims 3 or 4, wherein the second particle does not have a maximum emission wavelength in the wavelength region of 550 nm to 650 nm.

6. The target substance testing kit according to any one of claims 1 to 5, wherein the average particle size of the first particles is 20 nm or more and 400 nm or less.

7. The target substance testing kit according to any one of claims 1 to 6, wherein the average particle size of the second particle is 1 μm or more and 5 μm or less.

8. A target substance testing kit according to any one of claims 1 to 7, wherein the average particle size of the second particles is 2 times or more and 500 times or less the average particle size of the first particles.

9. The target substance testing kit according to any one of claims 1 to 8, wherein the testing kit for the target substance comprises a first reagent containing the first particles, a second reagent containing the first compound, and a third reagent containing the second particles.

10. A target substance testing kit according to any one of claims 1 to 8, comprising a first reagent containing the first particles and the first compound, and a second reagent containing the second particles.

11. A target substance testing kit according to any one of claims 1 to 10, wherein one of the third and fourth sites is a site containing biotin and the other is a site containing avidin.

12. A test kit for a target substance according to any one of claims 1 to 11, wherein the target substance comprises at least one selected from the group consisting of antibodies, antigens, and nucleic acids.

13. The target substance testing kit according to any one of claims 1 to 12, wherein the second particle is selected from the group consisting of agarose particles, dextran particles, polystyrene particles, silica particles, and magnetic particles.

14. A method for detecting a target substance in a sample solution, comprising the steps of: obtaining a first mixture having a first complex comprising a sample solution that may contain the target substance; a first particle having a first site that specifically binds to the target substance and a luminescent component; and a first compound having a second site that specifically binds to a site different from the site that specifically binds to the first site of the target substance and a third site different from the second site; obtaining a second mixture having a second complex comprising a second particle having a fourth site that specifically binds to the third site and having a larger average particle size than the first particle, and the first complex; and obtaining a value relating to the fluorescence anisotropy of the second mixture.

15. The method for detecting a target substance according to claim 14, wherein the binding constant between the third site and the fourth site is smaller than the binding constant between the target substance and the second site.

16. The method for detecting a target substance according to claim 14 or 15, wherein the average particle size of the first particles is 20 nm or more and 400 nm or less.

17. The method for detecting a target substance according to any one of claims 14 to 16, wherein the average particle size of the second particle is 1 μm or more and 5 μm or less.

18. A method for detecting a target substance according to any one of claims 14 to 17, wherein the average particle size of the second particles is 2 times or more and 500 times or less the average particle size of the first particles.

19. A method for detecting a target substance according to any one of claims 14 to 18, wherein the third site is a site having biotin and the fourth site is a site having avidin.

20. A method for detecting a target substance according to any one of claims 14 to 19, wherein the target substance comprises at least one selected from the group consisting of antibodies, antigens, and nucleic acids.

21. The method for detecting a target substance according to any one of claims 14 to 20, wherein the second particle is selected from the group consisting of agarose particles, dextran particles, polystyrene particles, silica particles, and magnetic particles.

22. The method for detecting a target substance according to any one of claims 14 to 21, wherein the first particle comprises polystyrene, a particle substrate containing a polymer having siloxane bonds, and a hydrophilic polymer present on the surface of the particle substrate.

23. The method for detecting a target substance according to any one of claims 14 to 22, wherein the luminescent component comprises a rare earth complex.

24. The method for detecting a target substance according to claim 23, wherein the rare earth complex is a europium complex.

25. The method for detecting a target substance according to claim 24, wherein the second particle does not have a maximum absorption wavelength in the wavelength region of 300 nm to 450 nm.

26. The method for detecting a target substance according to claim 24 or 25, wherein the second particle does not have a maximum emission wavelength in the wavelength region of 550 nm to 650 nm.

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