Method for detecting a target substance and reagent for detecting a target substance

Abstract

To provide a method for detecting a target substance using silica particles, where the sensitization rate is increased without a decrease in measurement accuracy. [Solution] A method for detecting a target substance in a sample solution, comprising: a first step of preparing a liquid sample by mixing a sample solution containing at least the target substance with a dispersion of particles containing first particles having a site that specifically reacts with the target substance; and a second step of performing optical measurements of the liquid sample, wherein the liquid sample contains second particles, the ratio of the average particle size of the second particles to the average particle size of the first particles in the liquid sample is 0.03 or more and 0.24 or less, and the refractive index of the second particles is smaller than the refractive index of the first particles.

Description

Technical Field

[0001] The present invention relates to a method for detecting a target substance and a reagent for detecting a target substance.

Background Art

[0002] Currently, for detecting and measuring a target substance in a sample, immunoassay methods using antigen-antibody reactions are used in clinical tests. Among them, immunoassay methods using particles having a site that specifically reacts with a target substance such as an antibody or an antigen are known.

[0003] For example, the latex agglutination method is known. In an example of the latex agglutination method, a sample (such as serum) that may contain a target substance is reacted with a latex agglutination reagent containing latex particles (also referred to as "antibody-sensitized particles") to which an antibody against the target substance is bound. For the aggregate generated by this antigen-antibody reaction, by visually or optically detecting this aggregation, the target substance can be qualitatively and quantitatively measured.

[0004] As another example, a fluorescence polarization immunoassay method using luminescent particles has been proposed (Patent Document 1). In an example of the fluorescence polarization immunoassay method, a sample that may contain a target substance is reacted with a luminescence reagent containing luminescent particles (also referred to as "antibody-sensitized luminescent particles") to which an antibody against the target substance is bound. For the aggregate of luminescent particles generated by the antigen-antibody reaction, by detecting this aggregation by fluorescence polarization method, the target substance can be qualitatively and quantitatively measured. Detection reagents using particles such as latex particles and luminescent particles are generally used as aqueous dispersions in which the particles are dispersed in an aqueous solvent.

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Patent Document 2

[0006] While highly sensitive chemiluminescence immunoassays are used in clinical testing, they tend to have longer testing times because they require BF separation (a process to remove unreacted components). On the other hand, latex agglutination and fluorescence polarization immunoassays, which do not require BF separation, have shorter testing times and higher throughput, but their detection sensitivity may be insufficient compared to chemiluminescence immunoassays, and further improvements in detection sensitivity are needed.

[0007] Water-soluble polymers are used as sensitizers to improve the sensitivity of latex agglutination and fluorescence polarization immunoassays. For example, in Patent Document 2, sodium alginate is added to the reaction system to promote the agglutination reaction. However, while water-soluble polymers enhance sensitivity, they can also increase the viscosity of the solution and cause salting-out of the polymer, leading to a decrease in measurement accuracy. Therefore, there is still a need for technologies that can achieve high sensitivity without reducing measurement accuracy.

[0008] Furthermore, Patent Document 3 discloses a complement titer measurement reagent containing sensitized red blood cells or sensitized liposomes suspended in a buffer solution, and specifically a complement titer measurement reagent containing silica particles. According to Patent Document 3, by mixing silica particles into the complement titer measurement reagent, even if there is a long time between reagent preparation and measurement, the red blood cells in the reagent will not settle, and accurate measurement will be possible without stirring immediately before measurement.

[0009] Patent Document 3 found that the effect of mixing silica particles in complement titer measurement using micrometer-sized red blood cells was to suppress the sedimentation of sensitized red blood cells. Therefore, the effect of mixing silica particles in immunoassays using nano-sized particles and detection reagents that utilize particle aggregation remained unknown.

[0010] Furthermore, Patent Document 4 discloses a detection method using particle aggregation with latex particles immobilized with antibodies or antigens against the substance to be measured, which includes a non-specific reaction inhibitor consisting of particles immobilized with antibodies or fragments thereof that do not immunologically react with the substance to be measured, or with antigens that do not immunologically react, as well as an immunoassay using the same.

[0011] According to Patent Document 4, it is disclosed that particles bound to immunologically inactive proteins function as non-specific reaction inhibitors during latex agglutination reactions using latex particles bound to antibodies or antigens.

[0012] However, the nonspecific reaction inhibitor disclosed in Patent Document 4 has the effect of adsorbing nonspecific reaction-causing substances and does not promote particle aggregation reactions. [Means for solving the problem]

[0013] To solve the above problems, one embodiment of the present disclosure is provided. A method for detecting a target substance in a sample solution, A first step of preparing a liquid sample by mixing a sample solution containing at least a target substance with a dispersion of particles containing a first particle having a site that specifically reacts with the target substance, A second step involves performing optical measurements of the liquid sample. Includes, The liquid sample contains a second particle, The ratio of the average particle size of the second particle to the average particle size of the first particle in the liquid sample is 0.03 or more and 0.24 or less. A method for detecting a target substance in which the refractive index of the second particle is smaller than the refractive index of the first particle. Also, an embodiment of the present disclosure is A detection reagent for a target substance in a test liquid, comprising At least a first particle having a site that specifically reacts with the target substance, and A second particle different in type from the first particle, and the ratio of the average particle diameter of the second particle to the average particle diameter of the first particle is 0.03 or more and 0.24 or less, and The refractive index of the second particle is smaller than the refractive index of the first particle. A detection reagent for a target substance in which the refractive index of the second particle is smaller than the refractive index of the first particle.

Advantages of the Invention

[0014] According to the present invention, a method and a reagent for detecting a target substance with high sensitivity and high precision can be provided.

[0015] Furthermore, according to the present invention, a detection reagent with good storage stability and capable of long-term storage can also be provided.

Modes for Carrying Out the Invention

[0016] Hereinafter, a detection reagent containing at least a first particle having a site that specifically reacts with a target substance and a second particle not having a site that specifically reacts with the target substance according to an embodiment of the present disclosure, and a method for detecting a target substance in a sample using the same will be described, but the present invention is not limited thereto.

[0017] <First Embodiment> The detection reagent for a target substance according to the first embodiment is a detection reagent for a target substance in a test liquid, comprising At least a first particle having a site that specifically reacts with the target substance, and a second particle different in type from the first particle, and the ratio of the average particle diameter of the second particle to the average particle diameter of the first particle is 0.03 or more and 0.24 or less, and the refractive index of the second particle is smaller than the refractive index of the first particle.

[0018] The inventors conducted a search for a substance that promotes the aggregation reaction of the first particles having a site that specifically reacts with a target substance, aiming to establish a highly sensitive and highly accurate detection method. As a result, surprisingly, the second particles with a low refractive index and an average particle size smaller than the average particle size of the first particles were found to improve the sensitivity of the immunoassay using the first particles having a site that specifically reacts with the target substance and to achieve both the improvement of the sensitivity and the non-decrease of the measurement accuracy. In addition, it was also found that the second particles in the reagent have an effect of suppressing the natural sedimentation of the first particles having a site that specifically reacts with the target substance present in the same reagent.

[0019] The detection reagent for the target substance of the present embodiment is a detection reagent for the target substance in the specimen, and is used, for example, in the immunoassay of the target substance in the specimen using the first particles having a site that specifically reacts with the target substance. In this specification, the immunoassay method refers to an immunoassay method based on an antigen-antibody reaction, and includes immunoassay methods called, for example, latex agglutination immunoturbidimetry, latex agglutination method, fluorescence polarization immunoassay, and the like. As a specific example, a method of measuring an antigen by using particles to which an antibody is bound can be mentioned. The detection reagent for the target substance of the present embodiment preferably detects the target substance by using the latex agglutination method or the fluorescence polarization immunoassay. Hereinafter, the present invention will be described.

[0020] (Target substance in the specimen) The detection reagent of the present embodiment is used for measuring the target substance in the specimen in the detection method of the present embodiment. The target substance may be one of specific binding partners, and is one of binding partners such as ligand and acceptor, ligand and receptor, antigen and antibody. For example, it is a substance measurable by an immune reaction, and substances such as antigens and antibodies can be mentioned.

[0021] Examples of target substances include CRP (C-reactive protein), ferritin, albumin, immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulin M (IgM), immunoglobulin E (IgE), alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), prostate-specific antigen (PSA), KL-6, pepsinogen (PG), rheumatoid factor (RF), lipoproteins, insulin, infection-related antigens and antibodies against them, hormones, vitamins, and antibiotics. Nucleic acids, enzymes, enzyme substrates, viruses, and extracellular vesicles can also be targeted.

[0022] (A first particle having a site that specifically reacts with the target substance) The target substance detection reagent of this embodiment includes at least a first particle having a site that specifically reacts with the target substance, and a second particle of a different type from the first particle. The first particle that can be used in the detection reagent of this embodiment is a particle having a site that specifically reacts with the target substance.

[0023] Here, the first particle is synonymous with an insoluble carrier and refers to a particulate carrier that is substantially insoluble in water. Therefore, the first particle can also be called an insoluble carrier particle. The shape is not particularly limited as long as it can be dispersed in water and is capable of optical measurement, such as turbidity measurement or fluorescence polarization measurement. Examples of particle materials include resins such as polystyrene, natural polymers such as agarose and dextran, inorganic materials such as alumina, and metals such as iron oxide and gold colloid.

[0024] The synthesis and preparation of the particles can be carried out according to known methods. Any particles used in conventional latex agglutination reactions or fluorescence polarization immunoassays can be used, but particles that are close to perfect spheres are preferred. The particles used in fluorescence polarization immunoassays are those that emit polarized light that can be detected by this measurement, and are, for example, particles containing a light-emitting material.

[0025] Examples of suitable first particles include latex particles or luminescent particles. Here, latex particles refer to polymer particles on which antigens or antibodies against a target substance can be immobilized, and which are used in latex agglutination reactions. A preferred example of latex particles is polystyrene particles. Polystyrene particles allow for size control and can be obtained with a narrow size distribution (particle size distribution). Furthermore, methods for mass synthesis have been established, making them favorable from a cost standpoint.

[0026] Here, luminescent particles refer to luminescent particles on which antigens or antibodies against a target substance can be immobilized, such as particles used in fluorescence polarization. These particles have advantages such as being relatively easy to obtain as nano-sized particles and having their surface chemically modified according to the purpose, and are suitable carriers for use with the detection reagents of this embodiment. As luminescent particles, particles containing luminescent molecules in polystyrene particles are preferred. The luminescent molecules can be any molecules that emit fluorescence, and those that emit polarized fluorescence are particularly preferred. Rare earth complexes are particularly preferred as luminescent molecules.

[0027] The first particle has a site that specifically reacts with the target substance. Here, the site that specifically reacts with the target substance is preferably a site that can specifically bind to and adsorb to the target substance. Examples of sites that specifically react with the target substance include antibodies and antigens. The antibody may be a monoclonal antibody or a polyclonal antibody, and fragmented antibodies (also called antibody fragments) such as Fab, F(ab')2, F(ab'), Fv, and scFv can also be used. In addition, appropriate substances can be selected depending on the target substance. Examples include enzymes, their substrates, signaling substances such as hormones and neurotransmitters and their receptors, nucleic acids, avidin, and biotin.

[0028] The first particle has a site that specifically reacts with the target substance. This specific reaction is also called sensitization, adsorption, or immobilization. The site that specifically reacts with the target substance may be immobilized on the substrate particle by chemical bonding or by physical adsorption, but it is preferable that it be immobilized by chemical bonding. Immobilization of the site that specifically reacts with the target substance can be carried out by conventional methods, but the functional groups on the surface of the substrate particle can be reacted with primary amines, secondary amines, carboxyl groups, thiol groups, etc., which have a site that specifically reacts with the target substance.

[0029] Examples of functional groups for substrate particles that bind sites specifically reacting with the target substance include carboxyl groups, amino groups, aldehyde groups, epoxy groups, thiol groups, and maleimide groups. The particles may have only one of these functional groups, or two or more.

[0030] The presence or absence of binding and the amount of binding can be determined by using known detection methods for target substance-specific reaction sites on substrate particles. For example, if the target substance-specific reaction site is a protein, the amount of protein bound to the first particle can be determined by a protein quantification assay such as a BCA assay.

[0031] In this embodiment, the detection reagent for the target substance preferably has a first particle with an average particle size of 50 nm to 500 nm as its long axis diameter, and more preferably 100 nm to 400 nm. By setting the average particle size of the first particle to 50 nm or more, the absorbance in the visible range observed by latex agglutination measurement is within an appropriate range. Furthermore, by setting the average particle size of the particles to 50 nm or more, it becomes possible to include a large amount of luminescent material in the first particle, resulting in particles with sufficient luminescence intensity for observation by fluorescence polarization measurement.

[0032] By setting the average particle size of the first particles to 500 nm or less, the dispersion stability in solution is improved, and the absorbance in the visible range is within an appropriate range. From the viewpoint of reactivity and dispersion stability, it is preferable to use particles between 100 nm and 400 nm. The average particle size can be expressed as the z-mean particle size, which is measured by particle size measurement using dynamic light scattering. Here, the average particle size of the first particles is the average particle size of particles that have a site that specifically reacts with the target substance.

[0033] Furthermore, the concentration of the first particles can be appropriately adjusted according to the average particle size and specific gravity, the conditions of the optical measurement, and the target substance and particle material being measured. From the viewpoint of reactivity and solution turbidity, the detection reagent for the target substance of the present invention preferably has a concentration of the first particles in the detection reagent between 0.0002% by mass and 2.0% by mass. Specifically, in the latex agglutination method for measuring turbidity in the visible region, the concentration of the first particles in the reagent is preferably between 0.01% by mass and 2.0% by mass. Within this range, turbidity in the visible region can be reliably detected, and a sufficient measurement range for turbidity (absorbance) can also be ensured.

[0034] The concentration of the first particles is preferably not greater than 2.0% by mass, so as not to exceed the upper limit of turbidity (absorbance) measurement. Furthermore, when performing luminescence measurements such as fluorescence polarization using luminescent particles, the concentration of the first particles in the reagent is preferably even lower, because scattering of the first particles interferes with luminescence measurement. Specifically, the concentration of luminescent particles in the reagent is preferably between 0.0002% by mass and 0.01% by mass. Within this concentration range, the measurement of luminescence from the luminescent particles is stable.

[0035] The concentration of particles in a liquid sample is the solid content concentration of particles contained in the dispersion (hereinafter also referred to as particle dispersion), and can be measured, for example, using a heat-drying type moisture meter (such as the MX-50 heat-drying type moisture meter). In this embodiment and the examples described later, the true specific gravity (sometimes abbreviated as specific gravity) of the particles was determined by measuring the density of the particles. The true specific gravity of the particles is the relative density with respect to water, and the density of water is 1 g / cm³.3 First, the particle dispersion was freeze-dried, and its density was determined using a dry density metering method. An example of a measuring device used for dry density metering is the Shimadzu Corporation's dry automatic density meter "Accupyc II". The value obtained by dividing the determined density by the density of water was taken as the true specific gravity of the particles. As will be described later in the examples, for example, the specific gravity of polystyrene particles was 1.05, and the specific gravity of silica particles was 1.8.

[0036] (A second particle that does not have a site that specifically reacts with the target substance) The target substance detection reagent of this embodiment includes at least a first particle having a site that specifically reacts with the target substance, and a second particle of a different type from the first particle. In this embodiment, it is preferable that the second particle of the target substance detection reagent is a particle that does not have a site that specifically reacts with the target substance. In this embodiment, the component responsible for increasing the sensitivity of the target substance detection method and stabilizing the detection reagent containing the first particle having a site that specifically reacts with the target substance is the second particle that does not have a site that specifically reacts with the target substance. Unlike the first particle, the second particle does not have a site that specifically reacts with the target substance, has a smaller average particle size compared to the first particle, has a higher particle concentration compared to the first particle, and has a lower refractive index compared to the first particle.

[0037] The refractive index in this embodiment can be measured by known methods, such as the immersion method and the method using the Lorentz-Lorentz equation, and can be selected as appropriate. In the immersion method, light scattering measurements are performed by changing the refractive index of the dispersion. This method utilizes the fact that when the refractive index of the dispersion matches that of the particles, the solution appears transparent, and light scattering decreases. The refractive index at which light scattering disappears becomes the refractive index of the particles.

[0038] The refractive index can be determined using the Lorentz equation. This method first involves determining the refractive index of a dispersion of particles using a liquid refractometer. Then, the refractive index of the particles (solid content) is calculated using the Lorentz equation. The following provides a detailed explanation.

[0039] This method requires prior measurement of the solid content concentration of the particle dispersion. For example, this can be measured using a heat-drying type moisture meter (such as the MX-50). The particle density is also required, and this can be determined by freeze-drying the dispersion and using a dry-type automatic density analyzer.

[0040] First, the refractive index of the particle dispersion is measured. For liquids, an Abbe refractometer (such as the Abbemat MW multi-wavelength digital Abbe refractometer) can be used. Here, the measurement wavelength is usually 589.3 nm and the measurement temperature is 23°C. Next, the refractive index of the particles is calculated. The refractive index of a mixture depends on the volume fraction of its constituent components. The particle dispersion can be considered as a two-component system of particles and water, and the volume fraction can be calculated from the concentration and density. The refractive index of the particles can then be calculated using the Lorentz equation. Here, the specific gravity of water can be set to 1.00 and the refractive index to 1.333. As will be described later in the Examples section, as an example of the results obtained by the measurement method using the Lorentz-Lorentz equation, the refractive index of polystyrene particles was 1.59 and the refractive index of silica particles was 1.45.

[0041] In this embodiment, the fact that the second particle is a silica particle can be confirmed by known analytical methods. For example, nuclear magnetic resonance (NMR) spectroscopy and inductively coupled plasma (ICP) emission spectroscopy of silicon can be applied. By utilizing the size difference between the first and second particles, the second and first particles can be separated by centrifugation or ultrafiltration, and their respective components can be analyzed.

[0042] In this embodiment, it is preferable that the second particle is a particle that does not have a site that specifically reacts with the target substance, and the absence of a site that specifically reacts with the target substance can be confirmed by known analytical methods. For example, nuclear magnetic resonance analysis (NMR), radio frequency inductively coupled plasma (ICP) emission spectroscopy, or the second particle and the first particle can be separated and the protein quantification or immunoassay of the site that specifically reacts with the target substance can be performed on the second particle. For example, when the site that specifically reacts with the target substance is a protein, it can be confirmed that the site that specifically reacts with the target substance is not bound to the second particle by using a commercially available protein stain (such as Coomassie stain) on the second particle.

[0043] The mechanism of action of the second particle is explained below. In the target substance detection method according to this embodiment, the mechanism by which the second particle can increase the sensitivity of the detection of the target substance by the first particle having a site that specifically reacts with the target substance is thought to be the increase in apparent optical path length (increase in absorbance) due to multiple scattering and the action of depletion aggregation caused by the presence of the second particle.

[0044] Here, depletion aggregation refers to the aggregation of large particles when large particles and numerous small particles are present in a solution (in this embodiment, the small particles may be "second particles that do not have a site that specifically reacts with the target substance," and the large particles may be "first particles that have a site that specifically reacts with the target substance"). As a result, when the large particles approach each other, the small particles are expelled from the gaps between the large particles, causing osmotic pressure to act between the particles. In other words, to effectively induce depletion aggregation of large particles, it is effective to use small particles with an appropriate average particle size that have weak interactions with various media in aqueous solution.

[0045] In this embodiment, the second particle has weak interactions with salts and ions, and also weak interactions with the first particle, which has a site that specifically reacts with proteins and target substances. Furthermore, it does not significantly increase the viscosity of the solution, and because the refractive index of the second particle is low, it exhibits low scattering in aqueous solutions. For this reason, it has been discovered for the first time in this invention that it is extremely effective as an additive (sensitizer) to detection reagents that utilize optical measurement, which include the first particle having a site that specifically reacts with target substances.

[0046] In the target substance detection reagent of this embodiment, it is preferable that the second particle acts as both a sensitizer for detecting the target substance and a dispersant for the first particle. In the reagent of this embodiment, when the second particle does not have a site that specifically reacts with the target substance, the mechanism by which the first particle, which has a site that specifically reacts with the target substance, is stabilized (the second particle functions as a dispersant and stabilizer for the first particle) is thought to be due to the thickening effect of the reagent solution and the increase in the specific gravity of the reagent solution. That is, the first particle, which has a site that specifically reacts with the target substance, is prevented from spontaneously settling due to the thickening and increased specific gravity of the reagent solution. It is also thought that the second particle, which does not have a site that specifically reacts with the target substance, physically inhibits the interaction of the first particle, which has a site that specifically reacts with the target substance.

[0047] Here, "lacking a site that specifically reacts with the target substance" means that the substance has no site that specifically reacts with the target substance at all, or even if it has a site that specifically reacts with the target substance, it lacks the function of that site (the ability to bind to the antigen if the target substance is an antigen, or the ability to bind to the antibody if the target substance is an antibody). For example, if expressed as the amount of antigen or antibody bound to the mass of the carrier (substrate particle), this means that the amount of site that specifically reacts with the target substance is 0.1 μg or less per 1 mg of carrier.

[0048] For the second particle to exhibit the aforementioned sensitivity-enhancing effect, it is required that the second particle has extremely weak interactions with various substances in the reaction solution, including the target substance. Therefore, such a second particle is fundamentally different from the non-specific reaction inhibitor disclosed in Patent Document 4, in the sense that it is a particle that does not capture anything.

[0049] Here, the second particle, like the first particle, refers to a particulate carrier that is substantially insoluble in water. The shape is not particularly limited, as long as it disperses in water and does not interfere with optical measurements, such as turbidity measurement or fluorescence polarization measurement. Examples of particle materials include low refractive index particles such as amorphous fluoropolymers, polymethyl methacrylate, and silica particles.

[0050] In the target substance detection reagent of this embodiment, it is preferable that the refractive index of the second particle is smaller than that of the first particle. This is because a lower refractive index of the second particle reduces the scattering from the second particle, which does not interfere with turbidity measurement or fluorescence polarization measurement when detecting the target substance using the first particle. In other words, it is preferable that the first particle is a particle with a relatively high refractive index, such as polystyrene. The refractive index of polystyrene is 1.59. In the target substance detection reagent of this embodiment, it is preferable that the refractive index of the second particle is less than 1.59, more preferably 1.50 or less, and particularly preferably 1.48 or less. In other words, in the target substance detection reagent of this embodiment, it is preferable that the ratio of the refractive index of the second particle to the refractive index of the first particle is 0.92 or less.

[0051] The synthesis and preparation of the second particles can be carried out according to known methods. Preferably, the particles are nearly spherical. A suitable example of the second particles is silica particles. Silica particles have a low refractive index of 1.50 or less, can be easily synthesized as nearly spherical particles using known methods, allow for control over various average particle sizes and surface conditions, and can be produced in large quantities at low cost.

[0052] In this embodiment, it is preferable that the true specific gravity of the second particle is greater than that of the first particle in the target substance detection reagent. This is because the second particle, with its relatively higher specific gravity, contributes to increasing the specific gravity of the reagent solution, and when it coexists with the first particle, it can suppress the spontaneous sedimentation of the first particle, which has a site that specifically reacts with the target substance. If the specific gravity is too high, the second particle will settle, so the true specific gravity of the second particle is preferably between 1.1 and 3.0, and between 1.5 and 2.2.

[0053] The surface of the second particle preferably does not have sites that specifically react with the target substance, while it is preferable that it be a hydrophilic surface that does not adsorb proteins in order to suppress interactions with other substances. For example, the surface of the second particle is preferably coated with a hydrophilic polymer. The hydrophilic polymer is not particularly limited as long as it has low protein adsorption properties, but examples of hydrophilic polymers include polyvinylpyrrolidone (PVP) and polyethylene glycol.

[0054] The second particle can be coated with a hydrophilic polymer to suppress its interaction with impurities (such as proteins other than the target substance) contained in the sample. The interaction between the hydrophilic polymer used for coating and the substrate surface of the second particle may be non-covalent bonds such as hydrogen bonds, ionic bonds, or van der Waals bonds between the hydrophilic polymer and the substrate surface of the second particle, or it may be a covalent bond between the synthetic polymer and the second particle. Note that the "substrate of the second particle" described here refers to the substrate in its state before the surface is coated to obtain the second particle.

[0055] When a hydrophilic polymer is bonded to the surface of a second particle substrate by covalent bonding, for example, the functional groups of the particle substrate and the hydrophilic polymer can be utilized. Examples of functional groups include primary amines, secondary amines, carboxyl groups, thiol groups, aldehyde groups, epoxy groups, and maleimide groups. The surface of the second particle and the hydrophilic synthetic polymer present on that surface can be analyzed using known analytical methods to determine the surface composition, the presence or absence of functional groups, the presence or absence of polymer, the molecular weight of the polymer, the composition of the polymer, and the amount of polymer coating.

[0056] The second particle has a smaller average particle size compared to the first particle. The average particle size can be expressed as the z-mean particle size, which is measured, for example, by dynamic light scattering. In this embodiment, the detection reagent for the target substance preferably has an average particle size of the second particle with a major axis diameter of 5 nm to 55 nm, more preferably 10 nm to 50 nm, and particularly preferably 10 nm to 30 nm.

[0057] By setting the average particle size of the second particles to 5 nm or larger, it is possible to achieve an effective average particle size in an aqueous medium, thereby facilitating the depletion-induced agglomeration effect and improving the measurement sensitivity of the particle agglomeration reaction. Furthermore, setting the second particles to 5 nm or larger is preferable because it simplifies the preparation of the second particles, including centrifugation.

[0058] Furthermore, by setting the average particle size of the second particle to 55 nm or less, scattering in the visible range can be suppressed. Setting the second particle size to 55 nm or less is preferable because it is considered that the scattering component of the second particle itself will have little effect on the optical measurement signal (absorbance and luminescence).

[0059] In this embodiment, the size ratio of the first particles to the second particles is important. Specifically, in the target substance detection reagent of this embodiment, the ratio of the average particle size of the second particles to the average particle size of the first particles is between 0.03 and 0.24. It is believed that by having a ratio of 0.03 or more, the second particles exhibit an effective size in the aqueous medium, as described above, allowing the depletion agglutination effect to be realized and improving the measurement sensitivity of the particle agglutination reaction.

[0060] On the other hand, if the ratio of the average particle size of the second particle to the average particle size of the first particle becomes too large, for example, if the second particle is the same size as the first particle (ratio of average particle size is 1), depletion aggregation will no longer occur, and the aggregation reaction of the first particle may be inhibited. Also, if the ratio of the average particle size of the second particle to the average particle size of the first particle becomes too large, light scattering from the second particle may significantly affect the optical measurement of the first particle.

[0061] As demonstrated in the examples described later, when the ratio of the average particle size of the second particle to the average particle size of the first particle is 0.24 or less, the depletion and aggregation of the first particle is more likely to occur as described above, and the effect of light scattering from the second particle can be reduced. Therefore, in the reagent of this embodiment, it is preferable to adjust the ratio of the average particle size of the second particle to the average particle size of the first particle to 0.03 or more and 0.24 or less.

[0062] Furthermore, from the viewpoint of further reducing scattering by the second particle, it is preferable that the second particle is small, so the ratio of average particle sizes is preferably 0.03 or more and 0.12 or less, and from the viewpoint of achieving both high sensitivity due to the second particle and maintenance of measurement accuracy (good reproducibility), it is more preferable that the ratio of average particle sizes is 0.04 or more and 0.10 or less.

[0063] In this embodiment, the concentration of the second particle is an important parameter of the target substance detection reagent, and it is preferable that the ratio of the concentration of the second particle (by mass, by mass%) to the concentration of the first particle (by mass, by mass%) in the detection reagent is 1 or more and 840 or less. This means that the concentration of the second particle is at least equal to or greater than that of the first particle, and if the ratio of the concentration of the second particle to the concentration of the first particle is 1 or more, there may be a large amount of the second particle compared to the first particle, so the depletion and aggregation effect of the first particle by the second particle can be effectively expressed, and more sensitive detection can be achieved.

[0064] On the other hand, if the amount of second particles is excessively high compared to the amount of first particles, the viscosity of the solution will increase, which may lead to problems such as the generation of bubbles during dispensing or mixing of the solution, and variations in the amount of solution dispensed. In addition, light scattering by the second particles may interfere with optical measurements. Since these problems affect measurement accuracy, it is preferable to keep the ratio of the concentration of second particles to the concentration of first particles to 840 or less. From the viewpoint of achieving both high sensitivity due to the second particle and maintaining measurement accuracy, a concentration ratio of 10 to 600 is more preferable, and 20 to 250 is particularly preferable.

[0065] The concentration of the second particles can be appropriately adjusted according to their average particle size and specific gravity, the optical measurement conditions, the target substance being measured, and the material of the particles. In this embodiment, from the viewpoint of reactivity and solution turbidity, it is preferable that the concentration of the second particles in the detection reagent is 0.08% by mass or more and 22.0% by mass or less. The effect of the second particles is clearly observed when the amount of the second particles in the reagent is 0.08% by mass or more, and the effect of the second particles can be improved in a dose-dependent manner as the amount of the second particles in the reagent is increased.

[0066] The upper limit of the concentration of the second particles in the reagent is preferably not to exceed 22.0% by mass. Within this range, as described above, it does not affect the measurement accuracy and has a high effect in increasing sensitivity. From the viewpoint of achieving both increased sensitivity with the second particles and maintenance of measurement accuracy (reproducibility), it is even more preferable that the concentration of the second particles be 0.5 to 20%, and more preferably 1.0 to 15.0%.

[0067] A preferred form of the second particle is PVP-coated silica particles (hereinafter referred to as PVP-coated silica particles). PVP is preferred because it exhibits strong hydrogen bonding interactions with silica particles, preventing the PVP from detaching from the PVP-coated silica particles during reagent storage or particle aggregation reactions. Silica particles can be synthesized by conventional methods, and commercially available products can also be used. For example, PVP-coated silica particles can be obtained by synthesizing silica particle nanoparticles using the Stöber method or the sol-gel method, and then coating their surface with polyvinylpyrrolidone (PVP), a hydrophilic polymer.

[0068] Furthermore, PVP-coated silica particles can be obtained by coating commercially available silica particle nanoparticles, Sicastar® (manufactured by micromod), with polyvinylpyrrolidone (PVP), a hydrophilic polymer. A suitable example of PVP-coated silica particles is Percoll® (manufactured by Cytiva), which has an average particle size of 20 nm and is coated with polyvinylpyrrolidone. Percoll® is a solution for creating density gradients in a solution and is commercially available.

[0069] (Latex agglutination reagent) As an example of a target substance detection reagent in this embodiment, a latex agglutination reagent is provided. An example of the latex agglutination reagent of this embodiment is a latex agglutination reagent containing at least latex particles having a site that specifically reacts with a target substance and second particles (preferably without a site that specifically reacts with the target substance). Here, the second particles function as a sensitizer. The latex particles and second particles of this embodiment are dispersed in an aqueous solvent. Examples of aqueous solvents include purified water, phosphate buffer, glycine buffer, Good's buffer, Tris buffer, ammonia buffer, and various other buffers such as phosphate buffer and phosphate-buffered saline. In particular, phosphate buffer, Good's buffer, and phosphate-buffered saline (sometimes abbreviated as PBS) are preferred.

[0070] An example of a one-component latex agglutination reagent of this embodiment is shown. The second particle is added to phosphate-buffered saline (sometimes abbreviated as PBS). In the case where the target substance is an antigen, polystyrene particles sensitized with an antibody that specifically binds to the target substance (sometimes abbreviated as antibody-sensitized latex particles) can be added to this solution to create an example of an embodiment of the latex agglutination reagent of this embodiment. A sample such as serum or plasma (sometimes abbreviated as sample) is added to this one-component latex agglutination reagent, and the turbidity is measured with a spectrophotometer.

[0071] If the sample does not contain the target substance, the antibody-sensitized latex particles remain dispersed, and no change in turbidity over time is observed. On the other hand, if the sample contains the target substance, the antibody-sensitized latex particles agglutinate due to the antigen-antibody reaction with the target substance. This agglutination causes a change in turbidity over time; that is, the turbidity increases.

[0072] By measuring turbidity using a standard solution of the target substance with a known concentration, a calibration curve (a graph showing the relationship between turbidity and the concentration of the target substance) can be used to quantify the target substance contained in the sample. The latex agglutination reagent of this embodiment may contain various additives. Generally, it may contain pH buffers, proteins such as albumin and globulin, amino acids, surfactants, preservatives, etc.

[0073] The target substance detection reagent of this embodiment includes a first dispersion containing at least first particles and a second dispersion containing second particles, which are different from the first dispersion. Each dispersion may be independent, or it may contain a dispersion containing at least the first and second particles. Even in the former case, these particle dispersions are mixed during optical measurement. The detection reagent consists of at least two solutions, and one consisting of two reagents is called a two-component latex agglutination reagent.

[0074] The types of solutions include not only the sample diluent, the first dispersion, and the second dispersion, but also standard solutions (solutions containing known concentrations of the target substance) for obtaining calibration curves, and controls for confirming measurement accuracy. A reagent set consisting of multiple reagents is also called a reagent kit.

[0075] A particularly preferred example of the latex agglutination reagent of this embodiment is a two-component latex agglutination reagent comprising a first reagent (sometimes called R1 solution) for diluting the sample, which contains a second particle, and a second reagent (sometimes called R2 solution) which contains the first particle.

[0076] Here, the R1 solution is sometimes called the sample diluent. In latex agglutination assays using this reagent, for example, after adding the sample to a sample diluent containing silica particles, the detection of the target substance (optical measurement) can be made more sensitive by mixing this solution with the R2 solution, which contains antibody-sensitized latex particles.

[0077] (Luminescent reagent) An example of a target substance detection reagent in this embodiment is a luminescent reagent containing at least a luminescent particle (first particle) having a site that specifically reacts with the target substance, and a second particle that does not have a site that specifically reacts with the target substance. An example of the luminescent reagent of this embodiment is shown. The second particle is added to PBS. By adding luminescent particles sensitized with an antibody against the target substance (sometimes abbreviated as antibody-sensitized luminescent particles) to this solution, the luminescent reagent of this embodiment can be obtained. The sample is added to this one-solution type luminescent reagent, and the degree of polarization (or polarization anisotropy) of the emission from the antibody-sensitized luminescent particles (first particle) is measured using a fluorescence polarization analyzer.

[0078] If the sample does not contain the target substance, the antibody-sensitized luminescent particles remain dispersed, and no change in the polarization of their luminescence over time is observed. On the other hand, if the sample contains the target substance, the antibody-sensitized luminescent particles aggregate due to the antigen-antibody reaction with the target substance. This aggregation reduces the motility of the antibody-sensitized luminescent particles.

[0079] In other words, the degree of polarization of the luminescence of antibody-sensitized luminescent particles increases. By measuring the degree of polarization using a standard solution of a target substance of known concentration, a calibration curve (a graph showing the relationship between the degree of polarization and the concentration of the target substance) can be obtained, making it possible to quantify the target substance contained in the sample from the increase in the degree of polarization. Various additives may be included in the luminescent reagent of this embodiment.

[0080] Generally, the reagent may contain pH buffers, proteins such as albumin and globulin, amino acids, surfactants, preservatives, etc. The luminescence reagent of this embodiment may be a reagent kit composed of multiple reagents, similar to the latex agglutination reagent described above. A particularly preferred example of the luminescence reagent of this embodiment is a luminescence reagent composed of a sample diluent containing silica particles and an R2 solution containing antibody-sensitized luminescent particles.

[0081] <Second Embodiment> The second embodiment is a method for detecting a target substance in a sample solution, comprising: a first step of preparing a liquid sample by mixing a sample solution containing at least the target substance with a dispersion of particles containing first particles having a site that specifically reacts with the target substance; and a second step of performing optical measurements of the liquid sample, wherein the liquid sample contains second particles, the ratio of the average particle size of the second particles to the average particle size of the first particles in the liquid sample is 0.03 or more and 0.24 or less, and the refractive index of the second particles is smaller than the refractive index of the first particles. Here, "in the liquid sample" is used interchangeably with "in the reaction solution." Some items described in the first embodiment may be omitted from the explanation in the second embodiment.

[0082] <First step> The method for detecting a target substance according to this embodiment is a method for detecting a target substance in a sample solution, and includes a first step of preparing a liquid sample by mixing a sample solution containing at least the target substance with a dispersion of particles containing first particles having a site that specifically reacts with the target substance. The first step is to have first particles having a site that specifically reacts with the target substance, preferably specifically binding with the target substance, and the first particles aggregate depending on the amount of target substance. In this embodiment, the presence of second particles in the liquid sample in this step promotes the aggregation of the first particles.

[0083] In the target substance detection method of this embodiment, the sizes of the first and second particles used, i.e., the average particle size, are appropriately selected from viewpoints such as the effect of increasing the measurement sensitivity and whether or not there is interference with measurement signals such as absorbance, depending on the target substance, the detection method (turbidity or luminescence), and the type of particles. However, as explained in the first embodiment, it is important that the average particle size of the second particles is smaller than that of the first particles. In the target substance detection method of this embodiment, the ratio of the average particle size of the second particles to the average particle size of the first particles in the liquid sample is 0.03 or more and 0.24 or less.

[0084] As mentioned above, within this range, the second particle exhibits an effective size in the liquid sample, enabling the depletion agglomeration effect to manifest and improving the measurement sensitivity of the particle agglomeration reaction. On the other hand, if the ratio of the average particle size of the second particle to the average particle size of the first particle becomes too large, for example, if the second particle becomes the same size as the first particle (ratio of average particle size is 1), depletion agglomeration will no longer occur, and the agglomeration reaction of the first particle will be inhibited.

[0085] Furthermore, light scattering from the second particle significantly affects the optical measurement of the first particle, for example, reducing the measurable range of absorbance, which is a major disadvantage. As mentioned above, from the viewpoint of reducing scattering by the second particle, it is preferable that the second particle is small in size, so the ratio of average particle sizes is preferably between 0.03 and 0.12, and from the viewpoint of achieving both high sensitivity due to the second particle and maintaining measurement accuracy, it is more preferable that the ratio of average particle sizes is between 0.04 and 0.10.

[0086] Furthermore, in the target substance detection method of this embodiment, the refractive index of the second particle is smaller than that of the first particle. This is because the low refractive index of the second particle reduces the scattering from the second particle, so as not to interfere with turbidity measurement or fluorescence polarization measurement. In the target substance detection method of this embodiment, it is preferable that the ratio of the refractive index of the second particle to the refractive index of the first particle is 0.92 or less. Furthermore, in the target substance detection method of this embodiment, it is preferable that the refractive index of the second particle is less than 1.59, more preferably 1.50 or less, and particularly preferable 1.48 or less. By making the second particle a low refractive index particle, the particle aggregation reaction of the first particle and its optical measurement are less likely to be affected.

[0087] Furthermore, in the target substance detection method of this embodiment, it is preferable that the true specific gravity of the second particle is greater than the true specific gravity of the first particle. This is particularly preferable when the first and second particles are stored together for a long period of time. By adding the second particle to the reagent, the specific gravity of the solvent increases, resulting in a decrease in the natural sedimentation rate of the first particle, thereby improving the dispersion stability of the first particle. If the specific gravity is too high, the second particle will settle, so the true specific gravity of the second particle is preferably between 1.1 and 3.0, and more preferably between 1.5 and 2.2. As mentioned above, silica particles are preferred as the second particle. This is because they satisfy the preferred range of refractive index and specific gravity, and can be produced in large quantities inexpensively and easily.

[0088] Furthermore, in the target substance detection method of this embodiment, it is preferable that the second particle does not have a site that specifically reacts with the target substance. This is to avoid inhibiting the aggregation reaction of the first particle. In this embodiment, it is important to minimize the interaction with substances in the reaction solution as much as possible.

[0089] In this embodiment of the target substance detection method, it is preferable that the average particle size of the second particles is 5 nm to 55 nm, and the average particle size of the first particles is 50 nm to 500 nm. Within this range and combination, the second particles can cause depletion and aggregation of the first particles, achieving high sensitivity. Furthermore, the particle aggregation reaction of the first particles is less likely to be affected and its optical measurement is less affected, without narrowing the measurement range or deteriorating the measurement accuracy.

[0090] In the detection method of this embodiment, the concentrations of the first and second particles used are appropriately selected from viewpoints such as the effect of increasing the measurement sensitivity and whether or not there is interference with measurement signals such as absorbance, depending on the target substance, the detection method (turbidity or luminescence), and the type of particles. However, it is important that the concentration of the second particles is equal to or greater than that of the first particles when detected. In the target substance detection method of the present invention, it is preferable that the concentration of the first particles in the sample solution is 0.0001% by mass or less, and that the ratio of the concentration of the second particles (by mass) to the concentration of the first particles (by mass) in the sample solution is 1 or more and 840 or less.

[0091] Under these conditions, there is a large proportion of second particles relative to the first particles, which allows the second particles to deplete and aggregate the first particles, resulting in increased sensitivity. On the other hand, if there is an excessive amount of second particles relative to the first particles, the viscosity of the solution increases, leading to problems such as the generation of bubbles during solution dispensing and mixing, and variations in the amount of solution dispensed. Furthermore, light scattering by the second particles can interfere with optical measurements.

[0092] Since these issues affect measurement accuracy, it is preferable that the ratio of the concentration of the second particle to the concentration of the first particle be 840 or less. From the viewpoint of achieving both high sensitivity due to the second particle and maintaining measurement accuracy, it is even more preferable that the concentration ratio be between 10 and 600, and particularly preferable that it be between 20 and 250.

[0093] The concentration of the second particles can be appropriately adjusted according to their average particle size and specific gravity, the optical measurement conditions, the target substance being measured, and the material of the particles. In this embodiment, from the viewpoint of reactivity and solution turbidity, it is preferable that the concentration of the second particles in the liquid sample is 0.04% by mass or more and 11.0% by mass or less. The effect is clearly observed when the amount of the second particles in the liquid sample is 0.04% by mass or more, and the effect of the second particles can be improved in a dose-dependent manner as the amount in the liquid sample is increased.

[0094] The upper limit of the concentration of the second particles in the liquid sample is preferably not more than 11.0% by mass of the total liquid sample. Within this range, as described above, the effect of increased sensitivity can be obtained without affecting the measurement accuracy. From the viewpoint of achieving both increased sensitivity due to the second particles and maintenance of measurement accuracy, it is even more preferable that the concentration of the second particles in the liquid sample be between 0.3% by mass and 10.0% by mass, and particularly preferable that it be between 0.5% by mass and 7.5% by mass.

[0095] In this embodiment, the method for detecting a target substance preferably involves a second particle acting as both a sensitizer for detecting the target substance and a dispersant for the first particle. This allows for a detection method using a detection reagent that has good storage stability and can be stored for a long period of time.

[0096] <Second step> The method for detecting a target substance according to this embodiment includes a second step of performing optical measurement of a liquid sample. The method for detecting a target substance in a sample according to this embodiment includes a step of performing optical measurement of a liquid sample prepared according to the above. The target substance is detected by examining the degree of particle aggregation through optical measurement of the liquid sample. Detection of the target substance can be qualitative or quantitative. This detection may be performed by visually observing aggregation formation, but from the viewpoint of detection reproducibility and throughput, it is preferable to measure the degree of particle aggregation using an optical measuring device.

[0097] In the method for detecting a target substance in a sample according to this embodiment, for example, a relationship between the concentration of the target substance and the degree of particle aggregation (calibration curve) can be determined in advance using a target substance solution of known concentration. The concentration of the target substance can be determined from the degree of particle aggregation obtained by measuring the target substance. Here, the degree of particle aggregation can be measured optically by absorbance or fluorescence polarization.

[0098] (Example of detecting a target substance using immunoassay) The detection method of this embodiment will be described in detail below, using immunoassay as an example. Here, immunoassay refers to an immunoassay based on an antigen-antibody reaction, and includes, for example, immunoassays called latex agglutination immunoturbidimetry, latex agglutination, and fluorescence polarization immunoassay. In the detection method of the target substance of this embodiment, it is preferable to use optical measurement, either latex agglutination or fluorescence polarization immunoassay.

[0099] (Latex agglutination method using latex particles) In the latex agglutination method of this embodiment, a sample containing the target substance is added to a latex agglutination reagent comprising latex particles to which an antibody against the target substance is bound (antibody-sensitized latex particles) and second particles that do not have a site that specifically reacts with the target substance. The target substance can be qualitatively and quantitatively measured by visually or optically detecting the aggregates of latex particles generated by the antigen-antibody reaction. From the viewpoint of sensitivity and avoiding absorption from impurities contained in the sample, the detection wavelength is preferably in the visible region, and wavelengths between 400 nm and 800 nm are preferred.

[0100] As a preferred example, a liquid sample is prepared by mixing a sample containing a target substance (antigen) with a dispersion of particles including antibody-sensitized latex particles and silica particles. Next, the absorbance of the liquid sample is measured. At this time, the ratio of the average particle size of the silica particles to the average particle size of the antibody-sensitized latex particles is between 0.03 and 0.24, and the refractive index of the silica particles is smaller than the refractive index of the antibody-sensitized latex particles.

[0101] Under these conditions, highly sensitive latex agglutination measurement becomes possible. The reason these conditions are preferable is as described above: the depletion agglutination effect by silica particles is efficiently expressed, and the silica particles do not optically affect the absorbance changes originating from antibody-sensitized latex particles.

[0102] The ratio of the concentration of silica particles to the concentration of antibody-sensitized latex particles in the liquid sample is particularly preferably between 1 and 230. The latex agglutination reaction is preferably carried out with a silica particle concentration in the range of 0.04% by mass to 11.0% by mass. The amount of sample, the amount of the first reagent, and the amount of the second reagent can be appropriately set to satisfy this range. During the latex agglutination reaction, the viscosity of each solution constituting the reagent is low, making the solutions easy to handle and improving the accuracy of the measurement.

[0103] In the detection of target substances by latex agglutination, the concentration of antibody-sensitized latex particles in the liquid sample is particularly preferred to be, for example, between 0.005% by mass and 1.9% by mass. Within this range, changes in the absorbance of antibody-sensitized latex particles can be detected with high accuracy.

[0104] The reaction temperature for latex agglutination is generally in the range of 4°C to 50°C. Since low temperatures reduce the reactivity of the antigen-antibody, and high temperatures reduce the stability of the antigen-antibody immune complex, a temperature of 10°C to 40°C is preferable, and 30°C to 40°C is more preferable. The reaction time for latex agglutination is generally in the range of 0 minutes to 60 minutes, preferably 1 minute to 30 minutes.

[0105] (Fluorescence-polarized immunoassay using luminescent particles) In the fluorescence polarization immunoassay method of this embodiment, a sample potentially containing a target substance is added to a luminescent reagent comprising luminescent particles conjugated with an antibody against the target substance (antibody-sensitized luminescent particles) and second particles that do not have a site that specifically reacts with the target substance. The aggregates generated by the antigen-antibody reaction are detected by fluorescence polarization, thereby allowing for qualitative and quantitative measurement of the target substance. From the viewpoint of sensitivity and avoiding absorption due to impurities in the sample, the detection wavelength of the emission is preferably in the visible region, and wavelengths between 400 nm and 800 nm are preferred.

[0106] Fluorescence polarization is used to analyze the mobility of fluorescent molecules in solution. The principle of fluorescence polarization will be explained using the example of the luminescent particles in this embodiment. When luminescent particles in a liquid (the particles contain luminescent molecules that exhibit fluorescence polarization) are excited by plane-polarized light, they emit polarized fluorescence in the same plane. However, if the luminescent particles rotate due to Brownian motion while excited, they emit fluorescence in a plane different from the excitation plane, and the fluorescence polarization is eliminated. In other words, the degree of fluorescence polarization represents the degree of rotational motion of the luminescent particles from the time of excitation until fluorescence is emitted.

[0107] When luminescent particles in a liquid are dispersed individually in the solution, they exhibit a low degree of polarization due to their Brownian motion, which causes them to rotate vigorously. On the other hand, when luminescent particles aggregate, Brownian motion in the solution decreases, and the degree of polarization increases. Therefore, in fluorescence polarization, the change in degree of polarization is used as an indicator to analyze the mobility of luminescent particles in the solution. The degree of polarization can be measured using milli P (hereinafter abbreviated as mp), which indicates the change in plane polarization. In the fluorescence polarization immunoassay method in this embodiment, the aggregation reaction of antibody-sensitized luminescent particles, which occurs depending on the amount of antigen, can be evaluated using the degree of polarization mp.

[0108] The preferred method for detecting target substances by fluorescence polarization immunoassay is the same as the latex agglutination method using latex particles described above. However, due to the high sensitivity of fluorescence measurement, the concentration of antibody-sensitized luminescent particles is preferably in the range of 0.0001% by mass or more and 0.009% by mass or less. Within this range, changes in fluorescence intensity and fluorescence polarization degree of antibody-sensitized luminescent particles can be detected with high accuracy. Although fluorescence polarization is also called fluorescence depolarization, in this specification, fluorescence polarization and fluorescence depolarization are synonymous.

[0109] (Method for storing a first particle having a site that specifically reacts with a target substance) This embodiment includes a method for storing first particles having a site that specifically reacts with a target substance in a reagent containing second particles that do not have a site that specifically reacts with a target substance. In this specification, "storage" means storing at a predetermined temperature for a predetermined period of time. A preferred storage method is to store the reagent of the present invention described above, which comprises first particles having a site that specifically reacts with a target substance and second particles that do not have a site that specifically reacts with a target substance.

[0110] There are no particular restrictions on the storage temperature, but for example, it is between 0°C and 50°C, more preferably between 0°C and 20°C, and most preferably between 0°C and 10°C. The effect of the second particles can be obtained within this range. There are no particular restrictions on the storage period, but for example, it can be 2 days, 7 days, 14 days, 1 month, 3 months, 6 months, or 1 year, and should be selected according to the storage temperature and how the reagent is used. As the amount of the second particles increases, the effect of the second particles in suppressing the natural sedimentation of the first particles becomes dose-dependent, thus stably maintaining the detection performance of the target substance by the first particles.

[0111] (others) In this embodiment, the method for detecting a target substance preferably involves the first particle being a particle containing polystyrene (which may be polystyrene particles), and the second particle being a particle containing silica (which may be silica particles). Although the method for detecting the target substance in this embodiment appears to be a process in which the first and second steps are performed at separate times as described above, the first and second steps may be performed simultaneously. [Examples]

[0112] The following describes some embodiments (hereinafter referred to as "these embodiments") for the purpose of explaining the present invention, but the present invention is not limited thereto. <Example 1: Preparation of latex agglutination reagent for detecting CRP> A latex agglutination reagent was prepared consisting of a first particle, a latex particle having a site that specifically reacts with the target substance, and a second particle, a silica particle that does not have a site that specifically reacts with the target substance. Percoll® was used as the silica particle. Percoll® is a silica particle whose surface is coated with PVP, and it was confirmed to have an average particle size (z-average particle size) of 20 nm when analyzed by dynamic light scattering. The silica particle concentration of Percoll® was 23% by mass.

[0113] Antibody-sensitized latex particles were prepared as follows, according to a known method. Polystyrene latex particles (with a z-average particle size of 213.5 nm; manufactured by Fujikura Chemical Co., Ltd.) were used as the particles. A dispersion of latex particles was prepared by adding 50 μL of the polystyrene latex particle solution with a particle concentration of 10% and 950 μL of 10 mM HEPES buffer (pH 7.0) to a microtube. Water-soluble carbodiimide (WSC) was dissolved in HEPES buffer (pH 7.0) (WSC concentration 1 mg / mL). 300 μL of the WSC solution was added to the latex dispersion (1 mL). The microtube was stirred in a tube mixer at room temperature for 30 minutes.

[0114] Next, 0.3 mg of anti-human CRP polyclonal antibody (manufactured by Oriental Yeast Co., Ltd.) was added to a microcentrifuge tube and stirred at room temperature for 1 hour. The mixture was centrifuged at 20000 G for 10 minutes at 4°C, the supernatant was removed, and the mixture was washed twice with HEPES buffer containing albumin and then redispersed with HEPES buffer. The resulting antibody-sensitized polystyrene particles are hereafter referred to as antibody-sensitized latex particles LA1. Dynamic light scattering analysis of antibody-sensitized latex particles LA1 confirmed that they had an average particle size (z-mean particle size) of 238.3 nm.

[0115] Percoll®, which has a silica particle concentration of 23% by mass, and antibody-sensitized latex particles LA1 were mixed in HEPES buffer to prepare reagent S1, which has an antibody-sensitized latex particle concentration of 0.025% by mass and a silica particle concentration of 0.092% by mass (Table 1).

[0116] Table 1 shows the average particle size of reagent S1 (z-average particle size by dynamic light scattering method, with the average particle size of the first particle being d1 and the average particle size of the second particle being d2), the ratio of the concentration of the second particle to the concentration of the first particle (c2 / c1), the ratio of the z-average particle size of the second particle to the z-average particle size of the first particle (d2 / d1), the material of the particles, the refractive index of the particles, the specific gravity of the particles, and the surface coating of the particles. In Table 1, c1, c2, c1', and c2' are rounded to the third decimal place, while c2 / c1 and c2' / c1' are calculated by calculating c2' / c1' without rounding the calculated c1, c2, c1', and c2', and then rounding the calculated value to the third decimal place.

[0117] The refractive index of the particles was determined using the Lorentz equation. The refractive index of the polystyrene particles was 1.59, and the refractive index of the silica particles was 1.45. Therefore, although not shown in Table 1, the refractive index ratio of the second particle (made of silica) to the first particle (made of polystyrene) is 0.92. Using the dry density measurement method, the specific gravity of polystyrene particles was found to be 1.05, and the specific gravity of silica particles was found to be 1.8.

[0118] The stability of the first particle of reagent S1 was evaluated. For the stability evaluation, the rate of change in absorbance on day 14 was used as the indicator. Specifically, the absorbance (measurement wavelength 572 nm, optical path length 1 cm) was measured immediately after preparation of the reagent and after 14 days of refrigerated storage, and the ratio (i.e., rate of change) of the absorbance after 14 days of refrigerated storage to the absorbance immediately after preparation was determined.

[0119] If the antibody-sensitized latex particles LA1 are stably dispersed after 14 days of refrigerated storage, the absorbance will not change, resulting in a 100% change rate. However, if the antibody-sensitized latex particles LA1 are unstable, such as by spontaneous sedimentation, the absorbance will decrease over time, and the ratio will also decrease. The stability of reagent S1 was found to be 90.3%, which is higher than the 85% of comparative reagent C1 described later (i.e., it can be said that the measurement accuracy is higher in the long term). Furthermore, from this perspective, the second particle can be used as both a sensitizer and a dispersant.

[0120] The measurable range of reagent S1 was evaluated. A larger measurable range is preferable for a reagent's performance, as it allows for measurement of the target substance's concentration over a wide range. In the measurable range evaluation, an equal volume of HEPES buffer was added to reagent S1 immediately after preparation, and the absorbance of the solution (measurement wavelength 572 nm, optical path length 1 cm) was measured. In spectroscopic measurements, the upper limit of absorbance measurement is approximately 2.0, and 2.0 was set as the upper limit of measurement here.

[0121] Therefore, the measurable range was expressed as the upper limit absorbance of 2.0 minus the absorbance of reagent S1. For example, if the absorbance of reagent S1 was 0.5, the measurable range of reagent S1 was 1.5 (2.0 minus 0.5). As will be described later, this was equivalent to comparative reagent C1 which does not contain silica particles, and no effect of silica particles on the measurable range was observed.

[0122] Another aspect of evaluating the measurable range is the inhibition of absorbance changes of antibody-sensitized latex particles LA1 by silica particles. In other words, it is antibody-sensitized latex particles LA1 that react with the target substance and change its absorbance, and ideally, it is better if the absorbance originating from silica particles is small. This is because it is impossible to distinguish between the absorbance originating from antibody-sensitized latex particles LA1 in the reagent and the absorbance originating from silica particles.

[0123] The increase in absorbance derived from antibody-sensitized latex particles LA1 due to the addition of silica particles is preferably within 20%. If the increase exceeds this, as mentioned above, the measurable range will narrow, and there is a high possibility that the change in absorbance derived from antibody-sensitized latex particles LA1 will not be accurately observed.

[0124] <Example 2: Preparation of latex agglutination reagent> Reagent S2 was prepared in the same manner as in Example 1, except that the silica particle concentration was 0.69% by mass. Table 1 shows the parameters for reagent S2. Evaluation in the same manner as in Example 1 revealed that reagent S2 had a stability of 92.8%, which is higher than the 85% stability of comparative reagent C1, which will be discussed later. The measurable range for reagent S2 was 1.5. No influence on the measurable range was observed due to silica particles.

[0125] <Example 3: Preparation of latex agglutination reagent> Reagent S3 was prepared in the same manner as in Example 1, except that the silica particle concentration was 1.38% by mass. Table 1 shows the parameters for reagent S3. Evaluation in the same manner as in Example 1 revealed that reagent S3 had a stability of 96.8%, which is higher than the 85% stability of comparative reagent C1, which will be discussed later. The measurable range of reagent S3 was 1.5. No influence on the measurable range was observed due to silica particles.

[0126] <Example 4: Preparation of latex agglutination reagent> Reagent S4 was prepared in the same manner as in Example 1, except that the silica particle concentration was 5.52% by mass. Table 1 shows the parameters for reagent S4. Evaluation in the same manner as in Example 1 revealed that reagent S4 had a stability of 98.3%, which is higher than the 85% stability of comparative reagent C1, which will be discussed later. The measurable range of reagent S4 was 1.5. No influence on the measurable range was observed due to silica particles.

[0127] <Example 5: Preparation of latex agglutination reagent> Reagent S5 was prepared in the same manner as in Example 1, except that the silica particle concentration was 11.5% by mass. Table 1 shows the parameters for reagent S5. Evaluation in the same manner as in Example 1 revealed that reagent S5 had a stability of 99.7%, which is higher than the 85% stability of comparative reagent C1, which will be discussed later. The measurable range of reagent S5 was 1.5. No influence on the measurable range was observed due to silica particles.

[0128] <Example 6: Preparation of latex agglutination reagent> Reagent S6 was prepared in the same manner as in Example 1, except that the silica particle concentration was 16.1% by mass. Table 1 shows the parameters for reagent S6. Evaluation in the same manner as in Example 1 revealed that reagent S6 had a stability of 99.1%, which is higher than the 85% stability of comparative reagent C1, which will be discussed later. The measurable range of reagent S6 was 1.4. There was almost no influence on the measurable range from silica particles.

[0129] <Example 7: Preparation of latex agglutination reagent> Reagent S7 was prepared in the same manner as in Example 1, except that the silica particle concentration was 20.7% by mass. Table 1 shows the parameters for reagent S7. Evaluation in the same manner as in Example 1 revealed that reagent S7 had a stability of 99.2%, which is higher than the 85% stability of comparative reagent C1, which will be discussed later. The measurable range of reagent S7 was 1.4. There was almost no influence on the measurable range from silica particles.

[0130] <Example 8: Preparation of latex agglutination reagent> Commercially available silica nanoparticles, Sicastar® (manufactured by micromod, refractive index 1.45), were coated with polyvinylpyrrolidone (PVP), a hydrophilic polymer. First, an equal amount of polyvinylpyrrolidone (PVP-K30: manufactured by Tokyo Chemical Industry Co., Ltd.) aqueous solution (PVP concentration 1%) was added to an aqueous solution of Sicastar® with an average particle size (z-average particle size) of 10 nm (particle concentration 2.5%).

[0131] Subsequently, the PVP-coated Sicastar® was washed with water by ultrafiltration to obtain PVP-coated Sicastar®. Reagent S8 was prepared in the same manner as in Example 1, except that these PVP-coated silica particles were used and the concentration of the PVP-coated silica particles was 5.52% by mass (Table 1).

[0132] Table 1 shows the parameters for reagent S8. Evaluation in the same manner as in Example 1 revealed that reagent S8 had a stability of 92.1%, which is higher than the 85% stability of comparative reagent C1, which will be discussed later. The measurable range of reagent S8 was 1.5. No influence on the measurable range was observed due to silica particles.

[0133] <Example 9: Preparation of latex agglutination reagent> Reagent S9 was prepared in the same manner as in Example 8, except that the average particle size of the silica particles was 30 nm. The parameters for reagent S9 are shown in Table 1. As a result of evaluation in the same manner as in Example 1, the stability of reagent S9 was found to be 96.7%, which is higher than the 85% stability of comparative reagent C1, which will be described later. The measurable range of reagent S9 was 1.5. No influence on the measurable range was observed due to silica particles.

[0134] <Example 10: Preparation of latex agglutination reagent> Reagent S10 was prepared in the same manner as in Example 8, except that the average particle size of the silica particles was 50 nm. The parameters for reagent S10 are shown in Table 1. As a result of evaluation in the same manner as in Example 1, the stability of reagent S10 was found to be 95.5%, which is higher than the 85% stability of comparative reagent C1, which will be described later. The measurable range of reagent S10 was 1.4. There was almost no influence on the measurable range from silica particles.

[0135] <Comparative Example 1> Comparative reagent C1 was prepared in the same manner as in Example 1, except that it did not contain silica particles (the second particle concentration was 0%). The parameters for comparative reagent C1 are shown in Table 1. As a result of evaluation in the same manner as in Example 1, the stability of comparative reagent C1 was found to be 85.0%, which is lower than that of the reagents in the examples of the present invention. This is thought to be because the antibody-sensitized latex particles LA1 spontaneously settle over time. The measurable range of comparative reagent C1 was 1.5.

[0136] <Comparative Example 2> Comparative reagent C2 was prepared in the same manner as in Example 8, except that the average particle size of the silica particles was 70 nm. The parameters for comparative reagent C2 are shown in Table 1. As a result of evaluation in the same manner as in Example 1, the measurable range of comparative reagent C2 was 1.2. Compared to the reagent in the example, the absorbance of the silica particles was higher, and the measurable range was narrower. In addition, comparative reagent C2 could not distinguish between the absorbance derived from antibody-sensitized latex particles LA1 and the absorbance derived from silica particles. Therefore, the stability of comparative reagent C2, which is indicated by the change in absorbance of antibody-sensitized latex particles LA1, could not be measured and was not evaluated.

[0137] <Comparative Example 3> Comparative reagent C3 was prepared in the same manner as in Example 8, except that the average particle size of the silica particles was 100 nm. The parameters for comparative reagent C3 are shown in Table 1. As a result of evaluation in the same manner as in Example 1, the measurable range of comparative reagent C3 was 0.1. Compared to the reagent in the example, the absorbance of the silica particles was higher, and the measurable range was narrower. In addition, comparative reagent C3 could not distinguish between the absorbance derived from antibody-sensitized latex particles LA1 and the absorbance derived from silica particles. Therefore, the stability of comparative reagent C3, which is indicated by the change in absorbance of antibody-sensitized latex particles LA1, could not be measured and was therefore left unevaluated.

[0138] <Example 11: Latex agglutination assay> The sensitivity of the latex agglutination reagent in this example was evaluated using C-reactive protein (hereinafter abbreviated as CRP). In the assay, 1 μL of a CRP solution with a CRP concentration of 0.5 mg / dL was added to 50 μL of phosphate-buffered saline (reagent 1). This was placed in a spectrophotometer measurement cell (optical path length 10 mm) and held at 37 degrees Celsius for 5 minutes.

[0139] Next, 50 μL of reagent S1, which is the second reagent in this embodiment, was added to 51 μL of the first reagent described above and thoroughly mixed. After this, the absorbance at 572 nm was measured using a spectrophotometer (represented as the absorbance at 0 minutes). After 5 minutes, the absorbance at 572 nm was measured again (represented as the absorbance at 5 minutes). In Table 1, c2' / c1' was calculated, with c1' being the concentration of the first particles and c2' being the concentration of the first particles in the mixture (a mixture of 50 μL of reagent S1 and 51 μL of the first reagent) for which the absorbance was measured.

[0140] The difference in absorbance between the two obtained points ((absorbance at 5 minutes) - (absorbance at 0 minutes)) is calculated and multiplied by 10,000 to express the absorbance change (ΔOD × 10000). If particle aggregation occurs due to the reaction between CRP and antibody-antigen, the absorbance change will be high. A higher absorbance change indicates higher sensitivity. The absorbance change was calculated as the average of three measurements.

[0141] The sensitization rate of reagent S1 in Example 1 for CRP measurement was evaluated according to the measurement method described above. The sensitization rate for CRP measurement was determined by setting the absorbance change in Comparative Example 4, described later, to 100%. Following the measurement method described above, the coefficient of variation (CV) of the change in absorbance for the CRP measurement of reagent S1 in Example 1 was determined. A smaller coefficient of variation indicates higher measurement accuracy (i.e., higher reproducibility).

[0142] In the measurement method described above, the change in absorbance of reagent S1 was also examined, following the same measurement method as described above, except that the CRP concentration was set to 0, i.e., 1 μL of phosphate-buffered saline was added to 50 μL of reagent 1. This was a test to determine whether or not nonspecific agglutination occurred in the latex agglutination reaction. If the change in absorbance was close to zero (within 100), it was determined that nonspecific agglutination had not occurred, and if the change in absorbance exceeded 100, it was determined that nonspecific agglutination had occurred.

[0143] The results are shown in Table 1. As shown in Table 1, in reagent S1, which was a mixture of antibody-sensitized latex particles LA1 and silica particles (concentration of silica particles at absorbance measurement was 0.046%), the sensitization rate for CRP measurement was 121%. This means that the sensitivity of CRP measurement was improved compared to Comparative Example 4, which does not contain silica particles, as described later, confirming that the addition of silica particles to the reagent has the effect of improving sensitivity. The coefficient of variation for CRP measurement was 8.2%, which was equivalent to that of Comparative Example 4. No change in the coefficient of variation was observed due to the addition of silica particles to the reagent. Non-specific agglutination did not occur in reagent S1.

[0144] <Example 12: Latex Agglutination Assay> In the same manner as in Example 11, the sensitization rate, coefficient of variation, and non-specific aggregation of CRP were evaluated for reagent S2, which had a silica particle concentration of 0.342% during absorbance measurement (Table 1). The sensitization rate of reagent S2 for CRP measurement was 179%. Compared to Comparative Example 4, which does not contain silica particles and will be described later, the sensitivity of CRP measurement was improved. The coefficient of variation of CRP measurement was 7.8%, which was equivalent to that of Comparative Example 4. No non-specific aggregation occurred with reagent S2.

[0145] <Example 13: Latex Agglutination Assay> In the same manner as in Example 11, the sensitization rate, coefficient of variation, and non-specific aggregation of CRP were evaluated for reagent S3, which had a silica particle concentration of 0.683% during absorbance measurement (Table 1). The sensitization rate of reagent S3 for CRP measurement was 242%. Compared to Comparative Example 4, which does not contain silica particles and will be described later, the sensitivity of CRP measurement was improved. The coefficient of variation of CRP measurement was 8.6%, which was equivalent to that of Comparative Example 4. No non-specific aggregation occurred with reagent S2.

[0146] <Example 14: Latex Agglutination Assay> In the same manner as in Example 11, the sensitization rate, coefficient of variation, and non-specific aggregation of reagent S4, which had a silica particle concentration of 2.733% during absorbance measurement, were evaluated (Table 1). The sensitization rate of reagent S4 for CRP measurement was 263%. Compared to Comparative Example 4, which does not contain silica particles and will be described later, the sensitivity of CRP measurement was improved. The coefficient of variation of CRP measurement was 7.9%, which was equivalent to that of Comparative Example 4. No non-specific aggregation occurred with reagent S4.

[0147] <Example 15: Latex Agglutination Assay> In the same manner as in Example 11, the sensitization rate, coefficient of variation, and non-specific aggregation of reagent S5, which had a silica particle concentration of 5.693% during absorbance measurement, were evaluated (Table 1). The sensitization rate of reagent S5 for CRP measurement was 256%. Compared to Comparative Example 4, which does not contain silica particles and will be described later, the sensitivity of CRP measurement was improved. The coefficient of variation of CRP measurement was 9.7%, which was equivalent to that of Comparative Example 4. No non-specific aggregation occurred with reagent S5.

[0148] <Example 16: Latex Agglutination Assay> In the same manner as in Example 11, the sensitization rate, coefficient of variation, and non-specific aggregation of reagent S6, which had a silica particle concentration of 7.97% during absorbance measurement, were evaluated (Table 1). The sensitization rate of reagent S6 for CRP measurement was 285%. Compared to Comparative Example 4, which does not contain silica particles and will be described later, the sensitivity of CRP measurement was improved. The coefficient of variation of CRP measurement was 12.0%, which is larger than that of Comparative Example 4, but was within the acceptable range (20% or less). No non-specific aggregation occurred with reagent S6.

[0149] <Example 17: Latex Agglutination Assay> In the same manner as in Example 11, the sensitization rate, coefficient of variation, and non-specific aggregation of reagent S7, which had a silica particle concentration of 10.248% during absorbance measurement, were evaluated (Table 1). The sensitization rate of reagent S7 for CRP measurement was 279%. Compared to Comparative Example 4, which does not contain silica particles and will be described later, the sensitivity of CRP measurement was improved. The coefficient of variation of CRP measurement was 18.8%, which is larger than that of Comparative Example 4, but was within the acceptable range (20% or less). No non-specific aggregation occurred with reagent S7.

[0150] <Example 18: Latex Agglutination Assay> In the same manner as in Example 11, the sensitization rate, coefficient of variation, and non-specific aggregation of CRP were evaluated for reagent S8, which had a silica particle concentration of 2.733% during absorbance measurement and an average silica particle size of 10 nm (Table 1). The sensitization rate of reagent S8 for CRP measurement was 181%. Compared to Comparative Example 4, which does not contain silica particles and will be described later, the sensitivity of CRP measurement was improved. The coefficient of variation of CRP measurement was 11.3%, which is larger than that of Comparative Example 4, but was within the acceptable range (20% or less). No non-specific aggregation occurred with reagent S8.

[0151] <Example 19: Latex Agglutination Assay> In the same manner as in Example 11, the sensitization rate, coefficient of variation, and non-specific aggregation of CRP were evaluated for reagent S9, which had a silica particle concentration of 2.733% during absorbance measurement and an average silica particle size of 30 nm (Table 1). The sensitization rate of reagent S9 for CRP measurement was 136%. Compared to Comparative Example 4, which does not contain silica particles and will be described later, the sensitivity of CRP measurement was improved. The coefficient of variation of CRP measurement was 12.7%, which is larger than that of Comparative Example 4, but was within the acceptable range (20% or less). No non-specific aggregation occurred with reagent S9.

[0152] <Example 20: Latex Agglutination Assay> In the same manner as in Example 11, the sensitization rate, coefficient of variation, and non-specific aggregation of CRP were evaluated for reagent S10, which had a silica particle concentration of 2.733% during absorbance measurement and an average particle size of 50 nm (Table 1). The sensitization rate of reagent S10 for CRP measurement was 112%. Compared to Comparative Example 4, which does not contain silica particles and will be described later, the sensitivity of CRP measurement was improved. The coefficient of variation of CRP measurement was 15.9%, which is larger than that of Comparative Example 4, but was within the acceptable range (20% or less). No non-specific aggregation occurred with reagent S10.

[0153] <Comparative Example 4> In the same manner as in Example 11, the sensitization rate, coefficient of variation, and non-specific aggregation of CRP were evaluated for comparative reagent C1, which did not contain silica particles (silica particle concentration was 0%) during absorbance measurement (Table 1). The sensitization rate of comparative reagent C1 for CRP measurement was set to 100%. The coefficient of variation of the CRP measurement was 8.2%. No non-specific aggregation occurred with comparative reagent C1.

[0154] <Comparative Example 5> In the same manner as in Example 11, the sensitization rate, coefficient of variation, and non-specific aggregation of CRP were evaluated for comparative reagent C2, which had a silica particle concentration of 2.733% during absorbance measurement and an average particle size of 70 nm (Table 1). The sensitization rate of comparative reagent C2 for CRP measurement was 81%, which was lower than that of Comparative Example 4, which did not contain silica particles. The coefficient of variation for CRP measurement was 22.4%, which was higher. Non-specific aggregation was observed in comparative reagent C2. These results are thought to be due to the large average particle size of the silica particles as the second particle. In other words, the addition of large silica particles is thought to inhibit the aggregation reaction of the first particle, and the absorbance originating from the silica particles reduces the measurement accuracy.

[0155] <Comparative Example 6> In the same manner as in Example 11, the sensitization rate, coefficient of variation, and non-specific aggregation of CRP were evaluated for comparative reagent C3, which had a silica particle concentration of 2.733% during absorbance measurement and an average particle size of 100 nm (Table 1). The sensitization rate for CRP measurement of comparative reagent C3 was 55%, which was lower than that of Comparative Example 4, which did not contain silica particles. The coefficient of variation for CRP measurement was 28.7%, which was higher. Non-specific aggregation was observed in comparative reagent C2. These results are thought to be due to the large average particle size of the silica particles as the second particle. In other words, the addition of large silica particles is thought to inhibit the aggregation reaction of the first particle, and the absorbance originating from the silica particles reduces the measurement accuracy.

[0156] [Table 1-1]

[0157] [Table 1-2]

[0158] This embodiment includes the following methods and configurations. (Method 1) A method for detecting a target substance in a sample solution, A first step involves preparing a liquid sample by mixing a sample solution containing at least a target substance with a dispersion of particles containing a first particle having a site that specifically reacts with the target substance. A second step involves performing optical measurements of the liquid sample. Includes, The liquid sample contains a second particle, The ratio of the average particle size of the second particle to the average particle size of the first particle in the liquid sample is 0.03 or more and 0.24 or less. A method for detecting a target substance, wherein the refractive index of the second particle is smaller than the refractive index of the first particle. (Method 2) The method for detecting a target substance according to Method 1, wherein the ratio of the refractive index of the second particle to the refractive index of the first particle is 0.92 or less. (Method 3) The method for detecting a target substance according to method 1 or 2, wherein the refractive index of the second particle is 1.50 or less. (Method 4) A method for detecting a target substance according to any one of methods 1 to 3, wherein the true specific gravity of the second particle is greater than the true specific gravity of the first particle. (Method 5) A method for detecting a target substance according to any one of methods 1 to 4, wherein the average particle size of the second particle is 5 nm or more and 55 nm or less. (Method 6) A method for detecting a target substance according to any one of methods 1 to 5, wherein the concentration of the second particle is 0.04% by mass or more and 11.0% by mass or less. (Method 7) A method for detecting a target substance according to any one of methods 1 to 6, wherein the first particle is a polystyrene-containing particle and the second particle is a silica particle. (Method 8) A method for detecting a target substance according to any one of methods 1 to 7, wherein the second particle is a particle that does not have a site that specifically reacts with the target substance. (Method 9) A method for detecting a target substance according to any one of methods 1 to 8, wherein the average particle size of the first particles is 50 nm or more and 500 nm or less. (Method 10) A method for detecting a target substance according to any one of methods 1 to 9, wherein the ratio of the concentration (by mass) of the second particle to the concentration (by mass) of the first particle in the sample solution is 1 or more and 840 or less. (Method 11) A method for detecting a target substance according to any one of methods 1 to 10, wherein the concentration of the first particles in the sample solution is 0.0001% by mass or less (1.0% by mass or less). (Method 12) A method for detecting a target substance according to any one of methods 1 to 11, wherein the optical measurement uses a latex agglutination method or a fluorescence polarization immunoassay. (Method 13) A method for detecting a target substance according to any one of methods 1 to 12, wherein the first step and the second step are performed simultaneously. (Method 14) A method for detecting a target substance according to any one of methods 1 to 13, wherein the second particle is a sensitizer for detecting the target substance and a dispersant for the first particle. (Composition 1) A reagent for detecting a target substance in a sample solution, A first particle having at least a site that specifically reacts with the target substance, The present invention includes a second particle of a different type from the first particle, The ratio of the average particle size of the second particle to the average particle size of the first particle is 0.03 or more and 0.24 or less. A detection reagent for a target substance, wherein the refractive index of the second particle is smaller than that of the first particle. (Configuration 2) A target substance detection reagent according to configuration 1, wherein the ratio of the refractive index of the second particle to the refractive index of the first particle is 0.92 or less. (Composition 3) A detection reagent for a target substance according to configuration 1 or 2, wherein the refractive index of the second particle is 1.50 or less. (Composition 4) A detection reagent for a target substance according to any one of configurations 1 to 3, wherein the true specific gravity of the second particle is greater than the true specific gravity of the first particle. (Composition 5) A detection reagent for a target substance according to any one of configurations 1 to 4, wherein the average particle size of the second particle is 5 nm or more and 55 nm or less. (Composition 6) A detection reagent for a target substance according to any one of configurations 1 to 5, wherein the concentration of the second particle in the detection reagent is 0.08% by mass or more and 22.0% by mass or less. (Composition 7) A target substance detection reagent according to any one of configurations 1 to 6, wherein the second particle is a silica particle. (Composition 8) A target substance detection reagent according to any one of configurations 1 to 7, wherein the second particle is a particle that does not have a site that specifically reacts with the target substance. (Composition 9) A detection reagent for a target substance according to any one of configurations 1 to 8, wherein the average particle size of the first particles is 50 nm or more and 500 nm or less. (Composition 10) A detection reagent for a target substance according to any one of configurations 1 to 9, wherein the ratio of the concentration (by mass) of the second particle to the concentration (by mass) of the first particle in the detection reagent is 1 or more and 840 or less. (Composition 11) A detection reagent for a target substance according to any one of configurations 1 to 10, wherein the concentration of the first particles in the detection reagent is 0.0002% by mass or more and 2.0% by mass or less. (Composition 12) A target substance detection reagent according to any one of components 1 to 11, which detects the target substance using latex agglutination or fluorescence polarization immunoassay. (Composition 13) The target substance detection reagent according to any one of configurations 1 to 12, wherein the target substance detection reagent comprises a first dispersion in which at least the first particles are dispersed, and a second dispersion different from the first dispersion in which the second particles are dispersed. (Composition 14) A target substance detection reagent according to any one of configurations 1 to 13, wherein the target substance detection reagent comprises a dispersion containing at least the first particles and the second particles. (Composition 15) A target substance detection reagent according to any one of configurations 1 to 14, wherein the second particle is a sensitizer for the detection of the target substance and a dispersant for the first particle.

Claims

1. A method for detecting a target substance in a sample solution, A first step involves preparing a liquid sample by mixing a sample solution containing at least a target substance with a dispersion of particles containing a first particle having a site that specifically reacts with the target substance. A second step involves performing optical measurements of the liquid sample. Includes, The liquid sample contains a second particle, The ratio of the average particle size of the second particle to the average particle size of the first particle in the liquid sample is 0.03 or more and 0.24 or less. A method for detecting a target substance, wherein the refractive index of the second particle is smaller than the refractive index of the first particle.

2. The method for detecting a target substance according to claim 1, wherein the ratio of the refractive index of the second particle to the refractive index of the first particle is 0.92 or less.

3. The method for detecting a target substance according to claim 1, wherein the refractive index of the second particle is 1.50 or less.

4. The method for detecting a target substance according to claim 1, wherein the true specific gravity of the second particle is greater than the true specific gravity of the first particle.

5. The method for detecting a target substance according to claim 1, wherein the average particle size of the second particle is 5 nm or more and 55 nm or less.

6. The method for detecting a target substance according to claim 1, wherein the concentration of the second particles is 0.04% by mass or more and 11.0% by mass or less.

7. The method for detecting a target substance according to claim 1, wherein the first particle is a particle containing polystyrene, and the second particle is a silica particle.

8. The method for detecting a target substance according to claim 1, wherein the second particle is a particle that does not have a site that specifically reacts with the target substance.

9. The method for detecting a target substance according to claim 1, wherein the average particle size of the first particles is 50 nm or more and 500 nm or less.

10. The method for detecting a target substance according to claim 1, wherein the ratio of the concentration (by mass) of the second particle to the concentration (by mass) of the first particle in the sample solution is 1 or more and 840 or less.

11. The method for detecting a target substance according to claim 1, wherein the concentration of the first particles in the sample solution is 0.0001% by mass or less (1.0% by mass or less).

12. The method for detecting a target substance according to claim 1, wherein the optical measurement uses a latex agglutination method or a fluorescence polarization immunoassay.

13. The method for detecting a target substance according to claim 1, wherein the first step and the second step are performed simultaneously.

14. The method for detecting a target substance according to claim 1, wherein the second particle is a sensitizer for detecting the target substance and a dispersant for the first particle.

15. A reagent for detecting a target substance in a sample solution, At least, a first particle having a site that specifically reacts with the target substance, The present invention includes a second particle of a different type from the first particle, The ratio of the average particle size of the second particle to the average particle size of the first particle is 0.03 or more and 0.24 or less. A detection reagent for a target substance, wherein the refractive index of the second particle is smaller than that of the first particle.

16. The target substance detection reagent according to claim 15, wherein the ratio of the refractive index of the second particle to the refractive index of the first particle is 0.92 or less.

17. The target substance detection reagent according to claim 15, wherein the refractive index of the second particle is 1.50 or less.

18. The target substance detection reagent according to claim 15, wherein the true specific gravity of the second particle is greater than the true specific gravity of the first particle.

19. The target substance detection reagent according to claim 15, wherein the average particle size of the second particle is 5 nm or more and 55 nm or less.

20. The detection reagent for a target substance according to claim 15, wherein the concentration of the second particles in the detection reagent is 0.08% by mass or more and 22.0% by mass or less.

21. The target substance detection reagent according to claim 15, wherein the second particle is a silica particle.

22. The target substance detection reagent according to claim 15, wherein the second particle is a particle that does not have a site that specifically reacts with the target substance.

23. The target substance detection reagent according to claim 15, wherein the average particle size of the first particles is 50 nm or more and 500 nm or less.

24. The detection reagent for a target substance according to claim 15, wherein the ratio of the concentration (by mass) of the second particle to the concentration (by mass) of the first particle in the detection reagent is 1 or more and 840 or less.

25. The detection reagent for a target substance according to claim 15, wherein the concentration of the first particles in the detection reagent is 0.0002% by mass or more and 2.0% by mass or less.

26. A target substance detection reagent according to claim 15, wherein the target substance is detected using a latex agglutination method or a fluorescence polarization immunoassay.

27. The target substance detection reagent according to claim 15, wherein the target substance detection reagent comprises a first dispersion in which at least the first particles are dispersed, and a second dispersion different from the first dispersion in which the second particles are dispersed.

28. The target substance detection reagent according to claim 15, wherein the target substance detection reagent comprises a dispersion containing at least the first particles and the second particles.

29. The target substance detection reagent according to claim 15, wherein the second particle is a sensitizer for the detection of the target substance and a dispersant for the first particle.

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