Analysis method by measurement based on polarization anisotropy, inspection kit, and inspection reagent

Abstract

To provide a reagent and a measuring method allowing specimen inspection with high sensitivity in a short time based on polarization anisotropy.SOLUTION: An analysis method is to measure a value (R) related to polarization anisotropy by using a luminescent reagent bound to a target substance, thereby determining at least one of the presence or absence of the target substance and the concentration of the target substance, and the method includes: a reaction step of mixing a sample including the target substance, the luminescent reagent, and a sensitizer and reacting the mixed solution to obtain a reaction solution; and a measuring step of measuring the R of the reaction solution. The luminescent reagent includes luminous particle matrix and a hydrophilic layer outside the luminous particle matrix, and the sensitizer includes a hydrophilic polymer.SELECTED DRAWING: Figure 1

Description

Technical field

[0001] The present invention relates to an analytical method, a test kit, and a test reagent based on polarization anisotropy. [Background technology]

[0002] In the fields of medicine and clinical testing, it is necessary to detect or quantify minute amounts of biological components from blood or a sampled organ part with high sensitivity in order to investigate the cause, presence or absence, etc. of a disease. Among the testing methods for biological components, immunoassay is widely used. Many immunoassays require a washing step called B / F (Bound / Free) separation. One of the immunoassays that does not require B / F separation is the latex agglutination method that uses antigen-antibody reactions. In the latex agglutination method, latex particles carrying an antibody or the like that specifically binds to a target substance are mixed with a liquid that can contain the target substance, and the degree of aggregation of the latex particles is measured.

[0003] In the latex aggregation method, a target substance is captured by an antibody specific to the target substance bound to latex particles, and multiple latex particles are crosslinked via the captured target substance, resulting in aggregation of the latex particles. That is, the amount of a target substance in a liquid sample such as a biological sample can be quantified by evaluating the degree of aggregation of latex particles. The degree of aggregation can be quantified by measuring and evaluating changes in the amount of light transmitted or scattered through the liquid sample.

[0004] Although the latex agglutination method allows simple and rapid detection and quantitative evaluation of antigens, which are target substances, it has the problem of a detection limit in that it cannot be detected if the amount of antigen in a liquid sample such as a biological sample is small.

[0005] In order to improve the detection sensitivity of target substances, it is required to measure the degree of aggregation with higher sensitivity. In other words, it is conceivable to replace the system that measures changes in the amount of light transmitted or scattered through a liquid sample with a detection and quantification method that utilizes more sensitive luminescence characteristics. Specifically, specimen testing methods using fluorescence depolarization methods have been proposed (Patent Documents 1 and 2).

[0006] Patent Document 1 proposes improving a fluorescence depolarization device for clinical use. Fluorescence depolarization does not require the B / F separation required by common fluorescence measurement methods. Therefore, using the fluorescence depolarization method, it is possible to easily test specimens in the same way as the latex agglutination method. Furthermore, when using the fluorescence depolarization method, it is thought that the measurement process can be performed by simply mixing a luminescent substance that specifically reacts with the target substance, and the measurement can be performed using the same testing system as the latex agglutination method. On the other hand, Patent Document 1 proposes the use of a single molecule such as fluorescein as a luminescent material, which in principle could only be applied to drugs, low-molecular antigens, and the like.

[0007] Patent Document 2 solves the problem of Patent Document 1, which is that the fluorescence depolarization method is only applicable to drugs, low-molecular antigens, and the like. That is, Patent Document 2 aims to apply the fluorescence depolarization method to polymers such as proteins, and proposes the use of a material in which a dye having long-life luminescent properties is adsorbed to latex particles as a luminescent material. In Patent Document 2, based on the principle of fluorescence depolarization, it is possible to reduce the rotational Brownian motion of a substance in a liquid due to an increase in particle size by balancing the length of the luminescence lifetime. Quantification is proposed. However, in Patent Document 2, the polarization anisotropy of the inspection particle can be stably determined by interactions between the fluorescent substances adsorbed near the particle surface, etc., because the fluorescent substance is supported on the particles after synthesis of the latex particles. is difficult. Furthermore, in Patent Document 2, in order to suppress non-specific adsorption, particles carry bovine serum albumin (BSA), a biomolecule, on their surfaces, so they have a wide particle size distribution, and BSA, a protein, causes differences between lots. There was a possibility of variation. Therefore, the concentration of the target substance is measured on the μg / mL order, and there is no significant difference in measurement sensitivity from the latex method.

[0008] Furthermore, when performing high-sensitivity measurements using the polarized fluorescence decomposition method, it is necessary to adjust the amount of fluorescent reagent to be reacted depending on the amount of the target substance. This is because, if a large amount of unreacted fluorescent reagent is contained after the reaction with the target substance, a small change in the value related to fluorescence anisotropy will be observed as a whole. On the other hand, the binding rate of the reaction between the target substance and the ligand is limited, and the agglutination reaction depends on the diffusion rate of the fluorescent reagent and antibody. That is, when the concentration of the target substance or fluorescent reagent in the reaction solution is low, the reaction takes time and it is difficult to detect the target substance with high sensitivity within a certain period of time. [Prior art documents] [Patent document]

[0009] [Patent Document 1] Special Publication No. 3-52575 [Patent Document 2] Patent No. 2893772 [Summary of the invention] [Problem to be solved by the invention]

[0010] The present invention has been made in view of such background technology, and aims to provide a reagent using particles and an analysis method that enable short-time and highly sensitive specimen testing based on polarization anisotropy. purpose. [Means to solve the problem]

[0011] In one embodiment of the present invention, at least one of the presence or absence of the target substance and the concentration of the target substance can be determined by measuring a value (R) related to polarization anisotropy using a luminescent reagent that binds to the target substance. An analysis method for determining, a reaction step of mixing and reacting a sample containing the target substance, the luminescent reagent, and the sensitizer to obtain a reaction solution; a measuring step of measuring the R of the reaction solution; has, and furthermore, The luminescent reagent comprises a luminescent particle substrate and a hydrophilic layer outside the luminescent particle substrate; the sensitizer includes a hydrophilic polymer; The present invention provides an analysis method characterized by the following.

[0012] Further, in one embodiment of the present invention, by measuring a value (R) related to polarization anisotropy using a luminescent reagent that binds to a target substance, at least one of the presence or absence of the target substance and the concentration of the target substance can be determined. A test kit used for analysis to determine one, a luminescent particle substrate, and an outer hydrophilic layer of the luminescent particle matrix; a first reagent comprising a luminescent reagent comprising; and a second reagent comprising a sensitizer with a hydrophilic polymer; Provide test kits including:

[0013] Further, in one embodiment of the present invention, by measuring a value (R) related to polarization anisotropy using a luminescent reagent that binds to a target substance, at least one of the presence or absence of the target substance and the concentration of the target substance can be determined. A test reagent used for analysis to determine one, a luminescent particle substrate, and an outer hydrophilic layer of the luminescent particle matrix; a luminescent reagent comprising; and a sensitizer with a hydrophilic polymer; We provide test reagents containing:

Effect of the invention

[0014] According to the analysis method of the present invention, changes in the anisotropy of polarized light emission can be detected with high sensitivity in response to the behavior of particle aggregation and dispersion, and furthermore, the effect of the sensitizer allows for analysis in a short time. It becomes possible. [Brief explanation of the drawing]

[0015]

Figure 1

Figure 2

[0016] Hereinafter, preferred embodiments of the present invention will be described in detail, but the scope of the present invention is not limited. The present invention provides the following analysis method as one embodiment. An analysis method for determining at least one of the presence or absence of the target substance and the concentration of the target substance by measuring a value (R) related to polarization anisotropy using a luminescent reagent that binds to the target substance, the method comprising: a reaction step of mixing and reacting a sample containing the target substance, the luminescent reagent, and the sensitizer to obtain a reaction solution; a measuring step of measuring the R of the reaction solution; has, and furthermore, The luminescent reagent comprises a luminescent particle substrate and a hydrophilic layer outside the luminescent particle substrate; the sensitizer includes a hydrophilic polymer; An analysis method characterized by:

[0017] (Values ​​related to polarization anisotropy) In this embodiment, a value related to polarization anisotropy (sometimes referred to as R) is determined as follows. In other words, it is a value indicating the relationship between the emission intensity of a polarized light component in a direction parallel to the irradiated polarized light and the emission intensity of a polarized light component in a direction perpendicular to the irradiated polarized light, for light emission generated by exciting a luminescent substance by irradiating it with polarized light. More specifically, R is a value calculated based on the luminescent intensity of a luminescent component whose vibration direction is parallel to the polarized light when a luminescent substance is excited with a certain polarized light. Furthermore, when excited with the first polarized light, the emission intensity of the emitted light component whose vibration direction is parallel to the first polarized light, and when excited with the first polarized light, the first polarized light and the vibration direction are orthogonal to each other. This is a value indicating the ratio between the difference in luminescence intensity of luminescent components and the sum of these. However, R is the emission intensity of the luminescent component whose vibration direction is orthogonal to the second polarized light when excited with the second polarized light whose vibration direction is orthogonal to the first polarized light, and whose vibration direction is orthogonal to the first polarized light. It may be corrected by the ratio of the emission intensity of the emission component whose vibration direction is parallel to the second polarization when excited by the second polarization, and other constants. Values ​​related to polarization anisotropy include values ​​called polarization anisotropy, polarization degree, and the like.

[0018] More specifically, for example, R can be r in the following formula (1).

number

[0019] Further, R can be r' in the following formula (2).

number

[0020] The conditions for measuring R are, for example, preferably in a liquid at a temperature of 0° C. to 50° C. and a viscosity of 0.5 mPa·s to 50 mPa·s. When the luminescent reagent is a particle containing a europium complex, the concentration of the luminescent reagent is preferably measured at 0.001 mg / ml or more and 0.1 mg / ml or less, and the excitation wavelength is preferably 500 nm or more and 700 nm or less.

[0021] (Target substance) Examples of target substances include antigens, antibodies, low molecular weight compounds, various receptors, enzymes, substrates, nucleic acids, cytokines, hormones, neurotransmitters, information transmitters, membrane proteins, and the like. Examples of antigens include allergens, bacteria, viruses, cells, cell membrane components, cancer markers, various disease markers, antibodies, blood-derived substances, food-derived substances, natural product-derived substances, and low-molecular compounds. Nucleic acids include DNA, RNA, cDNA, parts or fragments thereof, synthetic nucleic acids, primers, probes, etc. derived from bacteria, viruses, cells, etc. Examples of low-molecular compounds include cytokines, hormones, neurotransmitters, information transmitters, membrane proteins, and their receptors. Examples of antigens include CRP antigen and HBs antigen, and examples of hormones include TSH antigen. By the analysis method according to this embodiment, at least one of the presence or absence of these target substances and the concentration of the target substances can be determined. The presence or absence of the target substance can be determined by comparing the concentration of the target substance with a predetermined threshold. For example, it can be determined that the target substance is present when the concentration of the target substance is greater than or equal to a predetermined threshold, and that the target substance is absent when the concentration of the target substance is less than the predetermined threshold.

[0022] (About the reaction process) In the reaction step, a sample containing a target substance, a luminescent reagent, and a sensitizer are mixed and reacted. In the reaction step, binding of the target substance and the ligand typically occurs. The reaction solution is a solution containing a luminescent reagent, a target substance, and a sensitizer, and may also contain other additives. The reaction is carried out at a pH of 3.0 or more and a pH of 11.0 or less and a temperature of 20°C or more and 50°C or less, and the reaction time may be freely determined depending on the detected concentration of the target substance. The target substance and luminescent reagent will be described later.

[0023] (About the measurement process) In the measurement step, R of the reaction solution is measured. The measurement conditions are, for example, preferably in a liquid at a temperature of 0° C. to 50° C. and a viscosity of 0.5 mPa·s to 50 mPa·s. The concentration of the luminescent reagent is preferably measured at 0.001 mg / ml or more and 0.1 mg / ml or less, and the excitation wavelength is preferably 500 nm or more and 700 nm or less.

[0024] (sensitizer) The sensitizer according to this embodiment includes a hydrophilic polymer. The hydrophilic polymer is preferably at least one selected from the group consisting of polyvinylpyrrolidone, sodium alginate, potassium alginate, ammonium alginate, lithium alginate, alginic acid, and polyoxazoline.

[0025] Hydrophilic polymers promote the aggregation of particles through a physical phenomenon called depletion aggregation, which has the effect of increasing the reaction rate of the reaction between the target substance and the ligand. This will be explained using Figure 1. If the distance 5 between the luminescent reagents is wider than the coil diameter of the hydrophilic polymer 4, the hydrophilic polymer 4 can be present between the particles. On the other hand, when the luminescent reagents 6 approach each other due to the reaction between the target substance and the ligand, and the distance 5 between the luminescent reagents becomes narrower than the coil diameter of the hydrophilic polymer 4, the hydrophilic polymer 4 does not exist between the luminescent reagents 6. It becomes difficult. As a result, a concentration gradient occurs between the luminescent reagents and the surrounding solvent, creating a difference in osmotic pressure. This difference in osmotic pressure exerts a force in the direction of aggregating the luminescent reagents 6 together. Aggregation caused by this force is called depletion aggregation.

[0026] The conditions for causing depletion aggregation and promoting the agglutination reaction are preferably such that there is no interaction between the luminescent reagent 6 and the hydrophilic polymer 4. For example, in the case of an antigen-antibody reaction, the sensitizer is Similarly, it is preferably hydrophilic. Furthermore, a larger molecular weight of the hydrophilic polymer 4 is advantageous because depletion aggregation is more likely to occur even if the distance 5 between the luminescent reagents is wide.

[0027] The phenomenon of depletion aggregation depends on the molecular weight of the hydrophilic polymer 4, the concentration and the size of the luminescent reagent. In the case of conventional measurements based on polarization anisotropy, where the luminescent reagent is not a particle but has a size at the molecular level, the depletion aggregation phenomenon cannot occur and the reaction between the target substance and the ligand cannot be promoted. . In addition, in measurements based on polarization anisotropy, Brownian rotational motion is proportional to the viscosity of the liquid, so if the liquid viscosity of the hydrophilic polymer 4 is too high, the molecular weight is too large, or the amount added is too large, The R of the unreacted (bound) luminescent reagent becomes high.

[0028] The sensitizer is preferably hydrophilic and does not interact with the luminescent reagent, and is preferably a hydrophilic polymer. As the hydrophilic polymer, polyvinylpyrrolidone, alginate, and polyoxazoline can be suitably used. Further, for the above reasons, the weight average molecular weight of the sensitizer can be suitably used if it is about 10,000 or more and 100,000,000 or less. Particularly preferably, the molecular weight is 100,000 or more and 5,000,000 or less, more preferably 200,000 or more and 2,000,000 or less.

[0029] If the molecular weight is too small, the sensitizing effect will be reduced. If the molecular weight is too large, the viscosity of the solution will become high, and R will become high even if no reaction between the target substance and the ligand occurs, which is undesirable. The weight average molecular weight of a hydrophilic polymer is determined based on gel permeation chromatography (GPC) or viscosity measurement. The interaction between the hydrophilic polymer and the luminescent reagent is adjusted by selecting the hydrophilic polymer depending on the components of the hydrophilic layer of the luminescent reagent. In order to confirm the interaction between the sensitizer and the luminescent reagent, R can be measured by mixing only the sensitizer and the luminescent reagent. It is expected that R will increase due to the effect of increasing viscosity due to the addition of a sensitizer, but if R becomes higher than the effect of increasing viscosity, or if R is not stable, the sensitizer and luminescent reagent themselves In such cases, it is preferable to change the type of sensitizer.

[0030] The polyvinylpyrrolidone (PVP) used in this embodiment may be a homopolymer or a copolymer as long as it has a repeating unit represented by the chemical formula (I). Specific examples of the copolymer include copolymers of polyvinylpyrrolidone and polyethylene glycol, polyvinylpyrrolidone and polylactic acid, and polyvinylpyrrolidone and polyacrylic acid. [ka] (In the formula, n is an integer of 100 or more.) The molecular weight of PVP used as a sensitizer is preferably 100,000 or more and 5,000,000 or less, more preferably 200,000 or more and 2,000,000 or less. Preferred examples include, but are not limited to, PVP-K90 and PVP-130K.

[0031] The polyoxazoline used in this embodiment may be a homopolymer or a copolymer as long as it has a repeating unit represented by the chemical formula (II). Specific examples of the copolymer include copolymers of polyoxazoline and polyethylene glycol, polyoxazoline and polylactic acid, and polyoxazoline and polyacrylic acid. [ka] (In chemical formula (II), each A independently represents a hydrogen atom, a methyl group, an ethyl group, or a propyl group. m is an integer of 100 or more.)

[0032] The alginic acid or alginate used in this embodiment is more preferably an alginate, and sodium alginate, potassium alginate, ammonium alginate, lithium alginate, etc. can be suitably used.

[0033] Further, it is preferable that the concentration at the time of measurement of R after the reaction between the test reagent and the target substance is 0.01 w / v% or more and 2.0 w / v% or less. If the concentration is within this range, the viscosity of the solution will be low and the solution will be easy to handle. If the concentration of the sensitizer is too low, it will be difficult to obtain a sufficient sensitizing effect. If the concentration of the sensitizer is too high, the reaction solution will thicken and R will increase. Specifically, the viscosity of the solution is preferably 0.5 mPa·s or more and 15.0 mPa·s or less. With this viscosity range, even if the R of the luminescent reagent increases due to the effect of thickening, it will not become saturated and can be measured. Furthermore, if the viscosity of the liquid exceeds 15.0 mPa·s, there is a high possibility that air bubbles will be mixed into the sample during the mixing of reagents during the measurement process. Therefore, also from the viewpoint of reagent handling, the viscosity of the liquid is preferably 15.0 mPa·s or less. Further, the concentration can be set depending on the target substance to be measured and the type of insoluble carrier.

[0034] (luminescent reagent) In this embodiment, a luminescent reagent binds a target substance and also has a luminescent particle substrate and a hydrophilic layer outside the luminescent particle substrate (ie, on the surface of the luminescent reagent). The luminescent particle substrate contains a luminescent molecule, and the luminescent molecule is preferably a molecule that emits light when excited by light irradiation, and one that emits luminescence due to a chemical reaction such as luminol is not preferred. Luminescence includes phosphorescence and fluorescence, preferably phosphorescence. More preferably, in this embodiment the luminescent particle substrate comprises a europium complex. The hydrophilic layer on the surface of the luminescent reagent preferably includes a hydrophilic polymer.

[0035] The luminescent reagent preferably has a ligand that binds to the target substance. A ligand is a compound that specifically binds to a specific target substance. Any ligand can be used as long as it shows affinity for a specific substance. Examples of combinations of a ligand and a target substance or a combination of a target substance and a ligand include the following. That is, examples include antigens and antibodies, low-molecular compounds and their receptors, enzymes and substrates, and complementary nucleic acids. Furthermore, antibodies and their specific allergens, bacteria, viruses, cells, cell membrane constituents, cancer markers, various disease markers, antibodies, blood-derived substances, food-derived substances, natural product-derived substances, all kinds of low-molecular compounds, etc. can be mentioned. Further examples include receptors and specific low molecular weight compounds, cytokines, hormones, neurotransmitters, information transmitters, membrane proteins, and the like. Further examples include DNA, RNA, cDNA derived from bacteria, viruses, cells, etc., parts or fragments thereof, synthetic nucleic acids, primers, probes, etc., and nucleic acids complementary thereto. In addition to the above, any combination known to have affinity can be used as a combination of a target substance and a ligand. The ligand in this embodiment typically includes any one of an antibody, an antigen, and a nucleic acid.

[0036] The luminescent reagent is shown in FIG. 1 as luminescent reagent 6 and has a particle matrix 1 containing a europium complex 3, a hydrophilic layer 2 on the outside of the particle matrix, and a ligand (not shown) that binds to the target substance. The luminescent reagent in FIG. 1 is in the form of particles, and the diameter of the particles is 25 nm or more and 500 nm or less.

[0037] The luminescent reagent used in this embodiment preferably has a small particle size distribution and the surfaces of the particles are coated with hydrophilic properties. Europium complex 3 is present inside the particle.

[0038] The diameter of the particles can be determined by dynamic light scattering. When particles dispersed in a solution are irradiated with a laser beam and the scattered light is observed with a photon detector, the intensity distribution due to the interference of the scattered light is constantly changing because the particles are constantly moving their positions due to Brownian motion. It's shaking. Dynamic light scattering is a measurement method that observes this Brownian motion as fluctuations in the intensity of scattered light. The fluctuation of scattered light with respect to time is expressed by an autocorrelation function, and the translational diffusion coefficient is determined. By determining the Stokes diameter from the determined diffusion coefficient, the size of the particles dispersed in the solution can be derived.

[0039] From the viewpoint of maintaining particle uniformity and monodispersity, it is desirable that no luminescent reagent be applied to the surface of the particles. However, in order to use it in the analysis method according to this embodiment, it is necessary to prevent non-specific adsorption of substances other than the target onto the particles, so in order to keep the surface of the luminescent reagent hydrophilic, a hydrophilic layer is added to the surface. have

[0040] As a hydrophilic layer on the surface, a method of supporting BSA on the surface of particles is commonly used to maintain hydrophilicity, but this method may cause lot variations. Therefore, the luminescent reagent preferably includes a hydrophilic layer containing a hydrophilic polymer. The concentration of the luminescent reagent in the reaction solution is preferably 0.000001% by mass or more and 1% by mass or less, more preferably 0.00001% by mass or more and 0.001% by mass or less.

[0041] The luminescent reagent used in this embodiment can emit long-lived phosphorescence by containing a europium complex. When the luminescent reagent used in this embodiment is in the form of particles, preferably the average particle diameter is 25 nm or more and 500 nm or less, more preferably 50 nm or more and 300 nm or less. It is as follows. When the average particle diameter exceeds 500 nm, the polarization anisotropy before aggregation becomes high, and the difference from the polarization anisotropy after the aggregation reaction becomes small. Furthermore, if the average particle diameter is less than 25 nm, the change in size before and after aggregation will be small, making it difficult to capture the change in R by depolarizing phosphorescent emission.

[0042] By reducing the particle size distribution of the luminescent reagent and introducing a europium complex as a luminescent molecule, it is possible to detect changes in polarized luminescence characteristics even if there is a slight change in the dispersion state of particles in the liquid. . Specifically, even if the concentration of the target substance in the solution is on the order of nanograms to picograms per mL, when the luminescent reagent aggregates through the target substance, the change in the rotational Brownian motion of the luminescent reagent can be measured by polarization anisotropy. This can be seen as a change in

[0043] Polarized light emission means that in a light emitting material whose transition moment (transition dipole moment) is anisotropic, if excitation light is polarized along the transition moment, the emitted light will also be polarized along the transition moment. say. Europium complexes exhibit fluorescence emission based on energy transfer from the ligand to the central metal ion, so the transition moment is complicated, but the red emission at around 610 nm is derived from the electronic transition from the lowest excited state 5D0 to 7F2. emits polarized light.

[0044] The principle of polarization anisotropy is to measure the deviation of the transition moment due to the rotational movement of the luminescent material during the time when polarized light emission is occurring. The rotational motion of the luminescent material can be expressed by equation (3). Q=3Vη / kT (3) here, Q: Material rotational relaxation time V: Volume of material η: Solvent viscosity k: Boltzmann constant T: Absolute temperature It is. The rotational relaxation time of a material is the time required for molecules to rotate through an angle θ (68.5°) where cos θ=1 / e.

[0045] From equation (3), it can be seen that the rotational relaxation time of a luminescent material is proportional to the volume of the material, that is, when the luminescent material is in the form of particles, to the cube of the particle size. On the other hand, the relationship between the luminescence lifetime of a luminescent material and the degree of polarization, which is a value related to polarization anisotropy, can be expressed by equation (4). p0 / p=1+A(τ / Q)...(4) here, p0: degree of polarization when the material is at rest (Q=∞) p: degree of polarization A:Constant τ: Luminous lifetime of material Q: Rotational relaxation time It is.

[0046] From equations (3) and (4), the degree of polarization is influenced by the luminescent material's luminescent lifetime and rotational relaxation time, that is, the volume (particle size) of the luminescent material, that is, the balance between the luminescent material's particle size and luminescent lifetime. It can be seen that this has an influence.

[0047] When determining the degree of polarization of a luminescent material expressed by equation (4) through experiments, polarized light may be incident on the luminescent material and luminescence detected in a direction 90 degrees from the traveling direction and vibration direction of the excitation light. At this time, the detection light may be detected separately into polarization components parallel to and perpendicular to the polarization of the incident light, and the polarization anisotropy shown in equation (5), for example, may be used as a value related to the polarization anisotropy. R(t)=(I∥(t)−GI⊥(t)) / (I∥(t)+2GI⊥(t))···(5) here, R(t): polarization anisotropy at time t I∥(t): Emission intensity of the emission component parallel to the excitation light at time t I⊥(t): Emission intensity of the emission component perpendicular to the excitation light at time t G: Correction value, ratio of I⊥ / I∥ measured with excitation light whose vibration direction is 90 degrees different from the excitation light used for sample measurement It is.

[0048] In other words, if the particle size and luminescence lifetime are within appropriate ranges, changes in the size of the luminescent material due to reactions with target substances, etc. can be sensitively read as changes in polarization anisotropy. That is, r(t) of non-agglomerated luminescent material is observed to be low, and r(t) of aggregated luminescent material is observed to be high. This is the principle of polarization anisotropy. Note that the value related to polarization anisotropy may be corrected using G and 2G, or may be a value excluding G and 2G.

[0049] (particle matrix 1) FIG. 1 shows an example of a particle-shaped (spherical) luminescent reagent 6, and the luminescent reagent 6 includes a particle substrate 1. Although FIG. 1 shows an example in which both the luminescent reagent 6 and the particle substrate 1 are spherical, the shapes of the luminescent reagent 6 and the particle substrate 1 of this embodiment are not limited. The particle matrix 1 is not particularly specified as long as it is a material that can stably incorporate the europium complex, but it is preferably a polymer containing a styrene unit and an organic silane unit. Polymers obtained by polymerizing compositions containing the above-mentioned components are preferably used. By containing styrene as a main component in the composition, it is possible to produce particles with a very uniform particle size distribution by the emulsion polymerization method described below, and by using a polymer containing an organic silane unit, Silanol groups (Si-OH) are generated in the polymer in an aqueous solvent, forming siloxane bonds (Si-O-Si) with each other on the particle substrate surface, and through this, a hydrophilic layer and a ligand, which will be described later, can be added. can. The particles according to this embodiment preferably have a ligand-binding functional group capable of binding a ligand on the outside of the particle matrix.

[0050] (Hydrophilic layer 2) The hydrophilic layer 2 can be comprised of a hydrophilic polymer or hydrophilic molecules on the outside of the particle matrix 1. A hydrophilic polymer or a hydrophilic molecule is a polymer or molecule containing a hydrophilic group, and specific examples of the hydrophilic group include molecules and polymers having a hydroxyl group, ether, pyrrolidone, betaine structure, etc. Specific examples of hydrophilic polymers include polyethylene glycol, polyvinylpyrrolidone, sulfobetaine polymers, phosphobetaine polymers, and polyglycidyl methacrylic acid with ring-opened glycidyl groups and modified hydroxyl groups at the end of the molecule. It can be the main component of the hydrophilic layer 2. Alternatively, the hydrophilic layer 2 may be formed by directly applying a monomolecule having a hydrophilic group to the surface of the particle substrate 1 using a silane coupling agent or the like. There is no limit to the thickness of the hydrophilic layer 2, but it does not need to be thicker than the thickness that can exhibit hydrophilicity. If the hydrophilic layer 2 is too thick, it will resemble a hydrogel, and the thickness of the hydrophilic layer may become unstable due to hydration due to the influence of ions in the solvent. The thickness of the hydrophilic layer 2 is preferably 1 nm or more and 15 nm or less.

[0051] (Europium complex 3) Europium complex 3 has the characteristics that the wavelength and intensity of the emitted light are not easily affected by the surrounding environment, and the emitted light has a long life. Europium complex 3 is composed of europium element and a ligand. Considering the luminescence lifetime, visible emission wavelength range, etc., the luminescent dye is preferably a europium complex. Europium generally has a luminescence lifetime of 0.1ms or more and 1.0ms or less. It is necessary to appropriately adjust this luminescence lifetime and the rotational relaxation time obtained from equation (1). In the case of europium in an aqueous dispersion, if the diameter of the luminescent reagent is 50 nm or more and 300 nm or less, R changes significantly before and after aggregation.

[0052] At least one of the ligands constituting the europium complex 3 is a ligand having a light-concentrating function. The light condensing function is an action that excites the central metal of the complex through energy transfer by excitation at a specific wavelength. Further, it is preferable that a ligand such as β-diketone exists in the ligand constituting the europium complex 3 to prevent coordination of water molecules. Ligands such as β-diketones coordinated to europium ions suppress the deactivation process due to energy transfer to solvent molecules, etc., resulting in strong luminescence.

[0053] Europium complex 3 may be a polynuclear complex. Further, as a specific example of a europium complex, [Tris(2-thenoyltrifluoroacetone)(Bis(triphenylphosphineoxide))europium(III)] ), [Tris(2-thenoyltrifluoroacetone)(triphenylphosphineoxide)(dibenzylsulfoxide)europium(III)], Examples include [Tris(2-thenoyltrifluoroacetone)(phenanthroline)europium(III)].

[0054] When the Brownian rotational motion of the europium complex 3 in the medium is considered to be stopped, it is desirable that the polarization anisotropy expressed by formula (3) is 0.08 or more. The state in which the Brownian rotational motion can be considered to have stopped is a state in which the rotational relaxation time of the particles is sufficiently longer than the emission lifetime of the europium complex 3.

[0055] It is preferable that a large amount of europium complex 3 is incorporated into the particle matrix 1 because the luminescence intensity per particle becomes stronger. On the other hand, when the europium complex 3 aggregates in the particle matrix 1, the interaction between the ligands affects the excitation efficiency of the europium complex 3, making it difficult to measure polarization anisotropy while maintaining reproducibility. It becomes difficult. Whether europium complex 3 exhibits non-agglomerative luminescence behavior in particle matrix 1 can be determined from the excitation spectrum of the sample.

[0056] Particles with strong luminescence not only enable highly sensitive measurements, but also maintain luminescence even when the particle size is reduced, making it possible to accelerate biochemical reaction rates. The smaller the particle size, the larger the diffusion coefficient of Brownian motion in the liquid, making it possible to detect reactions in a shorter time.

[0057] (Production method of luminescent reagent) Next, an example of a method for manufacturing the luminescent reagent used in this embodiment will be described. A method for producing a luminescent reagent includes preparing an emulsion by mixing at least a radically polymerizable monomer containing styrene and a radically polymerizable organic silane, a radical polymerization initiator, a polarized luminescent europium complex, and a hydrophilic polymer with an aqueous medium. (first step).

[0058] Furthermore, the method for producing a luminescent reagent includes a step (second step) of heating the emulsion and polymerizing a radically polymerizable monomer.

[0059] The method for producing a luminescent reagent can include a step (third step) of providing a ligand-binding functional group to the surface of the luminescent reagent, which will be described later. Here, the ligand-binding functional group is a functional group that can bind a ligand, and specifically includes a carboxy group, an amino group, a thiol group, an epoxy group, a maleimide group, a succinimidyl group, or an alkoxysilyl group (silicon alkoxide structure) can be used.

[0060] (radical polymerizable monomer) The luminescent reagent is produced by polymerizing radically polymerizable monomers, and the radically polymerizable monomers include at least styrene and radically polymerizable organic silane. The radically polymerizable monomer can further contain at least one monomer selected from the group consisting of acrylate monomers and methacrylate monomers. Examples of the monomer include butadiene, vinyl acetate, vinyl chloride, acrylonitrile, methyl methacrylate, methacrylonitrile, methyl acrylate, and mixtures thereof. That is, one or more of these monomers can be used in addition to styrene and radically polymerizable organic silane. Furthermore, a monomer having two or more double bonds in one molecule, such as divinylbenzene, may be used as a crosslinking agent.

[0061] Since the radically polymerizable monomer contains a radically polymerizable organic silane, a siloxane bond is imparted to the particle substrate 1. Examples of radically polymerizable organic silanes include vinyltrimethoxysilane, vinyltriethoxysilane, p-styryltrimethoxysilane, 3-methacryloxypropylmethyldimethoxysilane, 3-methacryloxypropyltrimethoxysilane, and 3-methacryloxypropylmethyl. Mention may be made of diethoxysilane, 3-methacryloxypropyltriethoxysilane, 3-acryloxypropyltrimethoxysilane, or combinations thereof. By using a radically polymerizable organic silane, an inorganic oxide skeleton is formed within the particle matrix 1, which plays a role in improving the physical and chemical stability of the luminescent reagent. Furthermore, by using a radically polymerizable organic silane, the affinity between the particle substrate 1 and the hydrophilic layer 2 and the ligand-binding functional group increases.

[0062] Furthermore, since the radically polymerizable monomer contains a radically polymerizable organic silane, silanol groups are imparted to the surface of the particle substrate 1. Silanol groups and hydrophilic polymers, such as PVP, form hydrogen bonds. As a result, the hydrophilic polymer such as PVP is more firmly adsorbed onto the surface of the particle matrix 1.

[0063] (radical polymerization initiator) As the radical polymerization initiator, a wide variety of azo compounds, organic peroxides, etc. can be used. Specifically, 2,2'-azobis(isobutyronitrile), 2,2'-azobis(2,4-dimethylvaleronitrile), 2,2'-azobis(2-methylbutyronitrile), 4 ,4'-azobis(4-cyanovaleric acid), 2,2'-azobis(2-methylpropionamidine) dihydrochloride, dimethyl 2,2'-azobis(2-methylpropionate), tert-butyl hydroperoxide , benzoyl peroxide, ammonium persulfate (APS), sodium persulfate (NPS), potassium persulfate (KPS), and the like.

[0064] (hydrophilic polymer) The luminescent reagent can include a hydrophilic polymer as a hydrophilic layer. The hydrophilic polymer preferably suppresses non-specific adsorption. Examples of hydrophilic polymers include hydrophilic polymers containing units having ether, betaine, pyrrolidone rings, and the like. The hydrophilic layer is included in the synthesized luminescent reagent and is preferably present mainly on the particle surface outside the particle matrix. In this specification, a polymer having a pyrrolidone ring may be abbreviated as "PVP". By adding PVP during the synthesis of a luminescent reagent, it is possible to impart nonspecific adsorption suppression ability and ligand binding ability to the luminescent reagent at the same time. Since PVP introduced during synthesis has higher hydrophilicity than radically polymerizable monomers, it is present at the interface between the solvent and the particle substrate during polymerization during synthesis. The particle matrix 1 adsorbs PVP on the outside by partially involving PVP during polymerization and by physical / chemical adsorption such as interaction between the pyrrolidone ring and styrene (radical polymerizable monomer).

[0065] The molecular weight of PVP is preferably 10,000 or more and 100,000 or less, more preferably 40,000 or more and 70,000 or less. If the molecular weight is less than 10,000, the surface of the luminescent reagent will have weak hydrophilicity and non-specific adsorption will likely occur. When the molecular weight is greater than 100,000, the hydrophilic layer becomes too thick and gels, making it difficult to handle.

[0066] In addition to PVP, a hydrophilic polymer may be added as a protective colloid during particle matrix synthesis.

[0067] Further, the luminescent reagent preferably satisfies A2-A1≦0.1. A1 and A2 are defined as follows. That is, the absorbance of a mixture of 60 μL of a buffer containing 16 μL of human serum diluted 15 times and 30 μL of a 0.1% by weight luminescent reagent dispersion immediately after the addition is defined as A1, and the absorbance at 37°C after the addition is defined as A1. The absorbance after standing for 5 minutes is defined as A2. Absorbance is measured using a 10 mm optical path and a wavelength of 572 nm. Particles with A2-A1 of 0.1 or less are preferable because non-specific adsorption of impurities in serum is small.

[0068] (aqueous medium) The aqueous medium (aqueous solution) used in the above-described method for producing a luminescent reagent preferably contains water in an amount of 80% by weight or more and 100% by weight or less. The aqueous solvent is preferably water or an organic solvent soluble in water, and examples include a solution of methanol, ethanol, isopropyl alcohol, or acetone mixed in water. If the organic solvent other than water is contained in an amount exceeding 20% ​​by weight, there is a risk that the polymerizable monomer will be dissolved during particle production.

[0069] Further, the pH of the aqueous medium is preferably adjusted in advance to 6 or more and 9 or less. If the pH is less than 6 or greater than 9, the alkoxide groups and silanol groups of the radically polymerizable organosilane will undergo polycondensation or react with other functional groups before forming a polymer, resulting in agglomeration of the resulting particles. There is a risk. In this embodiment, the alkoxide is not intentionally subjected to condensation polymerization before polymerization.

[0070] The pH adjustment described above is preferably carried out using a pH buffer, but may also be adjusted using an acid or a base.

[0071] In addition, surfactants, antifoaming agents, salts, thickeners, etc. may be added to the aqueous medium at a rate of 10% or less.

[0072] When producing a luminescent reagent, it is preferable to first dissolve PVP in an aqueous medium whose pH is adjusted to 6 or more and 9 or less. The content of PVP is preferably 0.01% by weight or more and 10% by weight or less, more preferably 0.03% by weight or more and 5% by weight or less based on the aqueous medium. If it is less than 0.01% by weight, the amount of adsorption to the particle matrix will be small, making it difficult to exhibit its effect. Moreover, if the amount is more than 10% by weight, the viscosity of the aqueous medium increases, and there is a possibility that sufficient stirring cannot be performed.

[0073] Subsequently, a radically polymerizable monomer containing styrene (A) and radically polymerizable organic silane (B) is added to the aqueous medium to form an emulsion. The weight ratio of styrene (A) and radically polymerizable organic silane (B) is from 6:4 to 100:1. Furthermore, a europium complex is mixed into the prepared emulsion. At this time, if the solubility of the europium complex is low, a water-insoluble organic solvent may be added. The weight ratio of the europium complex to the radically polymerizable monomer is 1:1000 to 1:10.

[0074] If the weight ratio of styrene (A) and radically polymerizable organic silane (B) is less than 6:4, the specific gravity of the entire particles may increase, and sedimentation of the particles may become significant. Furthermore, in order to increase the adhesion between PVP and the luminescent particles, it is desirable that the weight ratio of styrene (A) and radically polymerizable organic silane (B) be 100:1 or more.

[0075] The weight ratio of the weight of the aqueous medium to the total amount of radically polymerizable monomers is preferably from 5:5 to 9.5:0.5. If the weight ratio of the weight of the aqueous medium to the total amount of radically polymerizable monomers is less than 5:5, there is a possibility that the agglomeration of the generated particles will become significant. Furthermore, if the weight ratio of the weight of the aqueous medium to the total amount of the radically polymerizable monomer is greater than 9.5:0.5, there will be no problem in the generation of particles, but there is a risk that the amount of generation will decrease.

[0076] The radical polymerization initiator is used after being dissolved in water, a buffer, or the like. The radical polymerization initiator can be used in an amount of 0.5% by mass or more and 10% by mass or less based on the total weight of styrene (A) and radically polymerizable organic silane (B) in the emulsion.

[0077] In the step of heating the emulsion, the entire emulsion may be heated uniformly. The heating temperature can be arbitrarily set between 50°C and 80°C, and the heating time between 2 hours and 24 hours. By heating the emulsion, the radically polymerizable monomer is polymerized.

[0078] The luminescent reagent can have a ligand-binding functional group on its surface. The ligand-binding functional group is not particularly limited as long as it is a functional group capable of binding an antibody, antigen, enzyme, etc., but examples thereof include a carboxy group, an amino group, a thiol group, an epoxy group, a maleimide group, a succinimidyl group, a silicon alkoxide group, etc. or can contain these functional groups. For example, by mixing a silane coupling agent having a ligand-binding functional group with synthesized particles, it is possible to impart a functional group to the particle surface. Specifically, a carboxy group can be imparted to the particle surface by preparing an aqueous solution of a silane coupling agent having a carboxyl group and mixing it with the synthesized particle dispersion. At this time, a dispersant such as Tween 20 may be added to the reaction solution. The reaction temperature can be set arbitrarily between 0°C and 80°C, and the reaction time can be set arbitrarily between 1 hour and 24 hours. In order to suppress the rapid condensation reaction of the silane coupling agent, it is preferable to set the temperature at a room temperature of about 25° C. or lower and the reaction time to be 3 hours or more and 14 hours or less. Depending on the ligand-binding functional group, an acid or alkali catalyst may be added to promote the reaction on the particle surface.

[0079] By binding a luminescent reagent with a ligand such as various antibodies, it can be used as a particle for specimen testing. What is necessary is to select an optimal method for binding the target antibody or the like using the functional groups present in the hydrophilic layer 2.

[0080] (Introduction of ligand) For the chemical reaction for chemically bonding the ligand-binding functional group and the ligand, conventionally known methods can be applied to the extent that the object of the present invention can be achieved. Further, when the ligand is bonded to an amide bond, a catalyst such as 1-[3-(dimethylaminopropyl)-3-ethylcarbodiimide] can be used as appropriate.

[0081] The luminescent reagent used in this embodiment can be preferably applied to immunolatex agglutination assays that are widely used in fields such as clinical testing and biochemical research.

[0082] (Test reagent) As a further embodiment of the present invention, A test reagent used in an analysis to determine at least one of the presence or absence of the target substance and the concentration of the target substance by measuring a value (R) related to polarization anisotropy using a luminescent reagent that binds to the target substance. And, a luminescent particle substrate, and an outer hydrophilic layer of the luminescent particle matrix; a luminescent reagent comprising; and a sensitizer with a hydrophilic polymer; We provide test reagents containing: In the test reagent of this embodiment, preferably, the hydrophilic polymer is at least one selected from the group consisting of polyvinylpyrrolidone, sodium alginate, potassium alginate, ammonium alginate, lithium alginate, alginic acid, and polyoxazoline, and also has a molecular weight is 200,000 or more and 2,000,000 or less. Preferably, the luminescent molecule is a europium complex. Preferably, the luminescent reagent has a ligand that binds to the target substance. Preferably, R0≧0.001, where R0 is the R measured for a luminescent reagent that has not reacted with the target substance. The test reagent in this embodiment is used for analysis of a target substance in a sample in in vitro diagnosis.

[0083] The test reagent can include a luminescent reagent, a sensitizer, and further a dispersion medium. The amount of the luminescent reagent according to the present embodiment contained in the test reagent is preferably 0.000001% by mass or more and 20% by mass or less, more preferably 0.0001% by mass or more and 1% by mass or less. The amount of the hydrophilic polymer sensitizer according to the present embodiment contained in the test reagent according to the present embodiment is preferably 0.01 w / v% or more and 2.0 w / v% or less. The test reagent according to the present embodiment may contain a third substance such as an additive or a blocking agent in addition to the luminescent reagent according to the present embodiment, within a range that can achieve the object of the present invention. A combination of two or more types of third substances such as additives and blocking agents may be included. Examples of the dispersion medium used in this embodiment include various buffers such as phosphate buffer, glycine buffer, Good's buffer, Tris buffer, and ammonia buffer. The dispersion medium included is not limited to these. The test reagent in this embodiment may be a test reagent in which each component exists independently. By mixing test reagents in which each component exists independently at the time of measurement, a test reagent containing the components necessary for specimen testing can be prepared.

[0084] When the test reagent in this embodiment is used to detect an antigen or antibody in a specimen, an antibody or an antigen can be used as the ligand.

[0085] (test kit) As a further embodiment of the present invention, A test kit used for analysis that determines at least one of the presence or absence of the target substance and the concentration of the target substance by measuring a value (R) related to polarization anisotropy using a luminescent reagent that binds to the target substance. And, a luminescent particle substrate, and an outer hydrophilic layer of the luminescent particle matrix; a first reagent comprising a luminescent reagent comprising; and a second reagent comprising a sensitizer with a hydrophilic polymer; Provide test kits including:

[0086] In the test reagent of this embodiment, preferably, the hydrophilic polymer is at least one selected from the group consisting of polyvinylpyrrolidone, sodium alginate, potassium alginate, ammonium alginate, lithium alginate, alginic acid, and polyoxazoline, and also has a molecular weight is 200,000 or more and 2,000,000 or less. Preferably, the luminescent molecule is a europium complex. Preferably, the luminescent reagent has a ligand that binds to the target substance. Preferably, R0≧0.001, where R0 is the value of R measured for a luminescent reagent that has not reacted with the target substance. The test kit in this embodiment is used for analysis of a target substance in a sample in in vitro diagnosis.

[0087] The first reagent and the second reagent can each have a dispersion medium. The test reagent according to the present embodiment may contain a third substance such as an additive or a blocking agent in addition to the luminescent reagent according to the present embodiment, within a range that can achieve the object of the present invention. A combination of two or more types of third substances such as additives and blocking agents may be included. Examples of the dispersion medium used in this embodiment include various buffers such as phosphate buffer, glycine buffer, Good's buffer, Tris buffer, and ammonia buffer. The dispersion medium included is not limited to these.

[0088] The first reagent and the second reagent are mixed with a sample containing the target substance, and the value (R) related to polarization anisotropy is measured to determine at least one of the presence or absence of the target substance and the concentration of the target substance. Used for analysis to determine one side. Regardless of the order of mixing, the second reagent of this kit may be mixed with the first reagent, mixed with a sample containing the target substance, or mixed with the target substance. The sample containing the sample and the first reagent may be mixed together, and then the second reagent may be mixed therein.

[0089] In the mixed solution, the concentration of the first reagent is preferably adjusted so that the amount of the luminescent reagent is preferably 0.000001% by mass or more and 20% by mass or less, and preferably 0.0001% by mass or more and 1% by mass or less. . Further, the concentration of the second reagent is preferably adjusted so that the amount of the sensitizer in the mixed solution is 0.01 w / v% or more and 2.0 w / v% or less.

[0090] The test kit can further include a container containing the first reagent or the second reagent, and a casing containing them. The first reagent and the second reagent may each be diluted as appropriate, and the test kit may further include a positive control, a negative control, a diluent, and the like. As a medium for the positive control and negative control, a solvent may be used in addition to serum and physiological saline that do not contain a measurable target substance.

Example

[0091] Hereinafter, the present invention will be specifically explained with reference to Examples. However, the present invention is not limited to such embodiments.

[0092] (1) Preparation of luminescent particles Solvent A was prepared by dissolving polyvinylpyrrolidone (PVP-K30: manufactured by Tokyo Kasei Kogyo Co., Ltd.) in a pH 7 MES (2-morpholinoethanesulfonic acid) buffer (manufactured by Kishida Chemical Co., Ltd.). The europium complex is [Tris(2-thenoyltrifluoroacetone)(Bis(triphenylphosphineoxide))europium(III)] (Central Techno Co., Ltd.) Manufactured by the company, hereinafter referred to as “Eu(TTA)” 3 (TPPO) 2 ”), styrene monomer (manufactured by Kishida Chemical Co., Ltd.), and 3-methacryloxypropyltrimethoxysilane (manufactured by Tokyo Kasei Kogyo Co., Ltd., hereinafter abbreviated as “MPS”) to prepare reaction solution B. Reaction solution B was added to a four-necked flask containing solvent A, and stirred with a mechanical stirrer set at 300 rpm. After stirring for 15 minutes under nitrogen flow conditions, the temperature of the prepared oil bath was set to 70°C, and nitrogen flow was further performed for 15 minutes. After heating and stirring the mixture, an aqueous solution containing potassium persulfate (hereinafter referred to as "KPS") (manufactured by Aldrich) was added to the reaction solution, and emulsion polymerization was carried out for 20 hours. After the polymerization reaction, the resulting suspension was ultrafiltered with about 4 L of ion-exchanged water using an ultrafiltration membrane with a molecular weight cut off of 100K, and the product was washed to obtain a dispersion of luminescent particles.

[0093] A dispersion of luminescent particles obtained by emulsion polymerization was separated and added to an aqueous solution containing 1% by mass of Tween 20 (manufactured by Kishida Chemical Co., Ltd.) dissolved therein. After stirring for 10 minutes, a silane coupling agent, X12-1135 (manufactured by Shin-Etsu Chemical Co., Ltd.) was added and stirred overnight. After stirring, the dispersion was centrifuged, the supernatant was removed, and the precipitate was redispersed with pure water. Centrifugation and redispersion were performed three times or more to wash the product. The washed precipitate was redispersed in pure water. As described above, a ligand-binding functional group was introduced into particles 1 to 8. The mass ratio of the charged particles, pure water, and X12-1135 was 1:300:2.

[0094] (Preparation of luminescent reagent with anti-CRP antibody) 0.25 mL of a 1.2 wt% particle dispersion corresponding to the synthesized luminescent particles was collected, and the solvent was replaced with 1.6 mL of a pH 6.0 MES buffer. 0.5 wt% of 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide and sodium N-hydroxysulfosuccinimide were added to the particle MES buffer solution, and the mixture was reacted at 25°C for 1 hour. After the reaction, the dispersion was washed with a pH 5.0 MES buffer, 100 μg / mL of anti-CRP antibody was added, and the anti-CRP antibody was allowed to bind to the particles at 25° C. for 2 hours. After binding, the particles were washed with pH 8 Tris buffer. After the reaction, the particles were washed with a phosphate buffer to obtain an anti-CRP antibody-modified luminescent reagent (also referred to as affinity particles) at a concentration of 0.3 wt%.

[0095] (Preparation of luminescent reagent with anti-TSH antibody) 0.25 mL of a 1.2 wt% particle dispersion corresponding to the synthesized luminescent particles was collected, and the solvent was replaced with 1.6 mL of a pH 6.0 MES buffer. 0.5 wt% of 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide and sodium N-hydroxysulfosuccinimide were added to the particle MES buffer solution, and the mixture was reacted at 25°C for 1 hour. After the reaction, the dispersion was washed with a pH 5.0 MES buffer, 100 μg / mL of anti-TSH antibody was added, and the anti-TSH antibody was allowed to bind to the particles at 25° C. for 2 hours. After binding, the particles were washed with pH 8 Tris buffer. After the reaction, the particles were washed with a phosphate buffer to obtain an anti-TSH antibody-modified luminescent reagent (also referred to as affinity particles) at a concentration of 1.0 wt%. The anti-TSH antibodies used were monoclonal antibodies, and two types of anti-TSH antibodies were modified into luminescent particles in order to cause at least two particles to react with the TSH antigen, which was the analyte.

[0096] The binding of the antibody to the particles was confirmed by measuring the amount of decrease in the antibody concentration in the buffer solution to which the antibody was added using a BCA assay.

[0097] (Preparation of luminescent reagent solution) The obtained luminescent reagent was diluted with a pH 7.4 phosphate (PBS) buffer to a concentration of 0.1 mg / mL to prepare a luminescent reagent solution.

[0098] (Preparation of diluted solution) Mix 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer and PBS buffer at a volume ratio of 1:1, add a sensitizer as appropriate, and mix with the diluted solution. did. The type and amount of the sensitizer added will be shown in Examples below.

[0099] Reaction between target substance and ligand (antigen-antibody reaction) A mixture of HEPES buffer and CRP antigen was prepared and heated to 37°C. Preparation: A luminescent reagent solution was added thereto, stirred quickly, and the polarization anisotropy of the mixed solution was observed. In Examples 1, 2, 3, and 4 and Comparative Example, the luminescent reagent was used at 0.01 mg / mL, and the CRP antigen concentration was used at 200 pM. In Example 5, the luminescence reagent concentration was 0.0025 mg / mL, and the CRP antigen concentration was 1 pM. In Example 6, the luminescence reagent concentration was 0.04 mg / mL, and the TSH antigen concentration was 100 pM. The observation temperature was 37°C. Polarization anisotropy will be described later.

[0100] (Example 1) Sodium alginate 80~120 (manufactured by Fuji Film Wako Pure Chemical Industries, Ltd.) was added to the final concentration of the sensitizer at a final concentration of 0.1 w / v%, an antigen-antibody reaction was carried out, and fluorescence polarization was measured. The concentration was evaluated.

[0101] (Example 2) CRP antigen concentration was evaluated in the same manner as in Example 1, except that 0.2 w / v% of PVP-K90 (manufactured by Tokyo Kasei Kogyo Co., Ltd., molecular weight 360,000) was used as a sensitizer.

[0102] (Example 3) CRP antigen concentration was evaluated in the same manner as in Example 1, except that 0.4 w / v% of PVP-K90 (manufactured by Tokyo Kasei Kogyo Co., Ltd., molecular weight 360,000) was used as a sensitizer.

[0103] (Example 4) CRP antigen concentration was evaluated in the same manner as in Example 1, except that 0.1 w / v% of PVP1300K (manufactured by Merck & Co., Ltd., molecular weight 1,300,000) was used as a sensitizer.

[0104] (Example 5) At the final concentration, 0.2 w / v% of sodium alginate 80~120 (manufactured by Fuji Film Wako Pure Chemical Industries, Ltd.) and polyethylene glycol (hereinafter abbreviated as PEG) (manufactured by Fuji Film Wako Pure Chemical Industries, Ltd., molecular weight 500,000) were added as sensitizers at the final concentration. The CRP antigen concentration was evaluated based on the results of adding 0.2 w / v% of antigen-antibody reaction and measuring fluorescence polarization.

[0105] (Example 6) Add poly(2-ethyl-2-oxazoline) (manufactured by Sigma-Aldrich, molecular weight 500,000) to the sensitizer at a final concentration of 2.0 w / v%, perform antigen-antibody reaction, and measure fluorescence polarization. Based on the results, TSH antigen concentration was evaluated.

[0106] (Comparative example 1) CRP antigen concentration was evaluated in the same manner as in Example 1 except that no sensitizer was used.

[0107] (Product evaluation) The products in Examples and Comparative Examples were evaluated as follows. The shape of the product was evaluated using an electron microscope (S5500, manufactured by Hitachi High Technologies).

[0108] The average particle size of the product was evaluated using dynamic light scattering (Zetasizer Nano S manufactured by Malvern).

[0109] The concentration of the suspension in which the product was dispersed was evaluated using a gravimetric analyzer (Thermoplus TG8120 manufactured by Rigaku).

[0110] The following equipment was used to measure R. Prepare an LED light source with excitation light of 340 nm, insert a polarizing filter (manufactured by Sigma Koki Co., Ltd., NSPFU-30C) and a short pass filter (manufactured by Edmont Optics, Inc., 84-706) into the optical path, and irradiate a 1 cm square quartz cell. I set up an optical system that would allow me to do so. A polarizing filter (manufactured by Thorlab, PIVISC050) and a bandpass filter (manufactured by Thorlab, FB610-10) were set in a direction 90° to the incident light. Luminescence is I VV and I VH In order to measure in two directions simultaneously, we prepared two sets with different polarizer configurations for the incident light and 90° directions. To detect polarized light, spectroscopic measurements were performed using QEPro manufactured by Ocean Optics. A temperature control was set on the sample holder so that measurements could be taken at 37°C. The polarization anisotropy r was measured with the LED light source fixed at an output of 12 mW and an integration time of 3 seconds. The measurement interval was 15 seconds. From the emission spectrum of the obtained polarized light emission, the emission intensity in the wavelength range of 600 nm to 630 nm was applied to equation (1) and was determined as R. R was measured for 2400 seconds and the value of R was plotted against time. R in Examples and Comparative Examples was compared by subtracting R (R0) immediately after the reaction, ie, immediately after mixing the luminescent reagent and CRP antigen, from R (R300) 300 seconds after the reaction to calculate ΔR300-R0. Alternatively, ΔR900-R0 was calculated by subtracting R(R0) immediately after mixing the CRP antigen from R(R900) 900 seconds after the reaction.

[0111] Evaluation of non-specific aggregation inhibition of the product was performed as follows. 60 μl of a human serum solution diluted 15 times with a buffer solution was added to the luminescent reagent dispersion (3 mg / mL), and the mixture was incubated at 37° C. for 5 minutes. Absorbance at 527 nm was measured before and after incubation, and the amount of change in absorbance before and after incubation was measured three times. Table 2 shows the average value of the three measurements. When the amount of change in the value of absorbance x 10000 was less than 1000, it was evaluated that non-specific aggregation was suppressed, and when it was 1000 or more, it was evaluated that non-specific aggregation was occurring.

[0112] (Performance evaluation) The particle size of the synthesized luminescent reagent was approximately 100 nm, and it emitted strong red light with excitation light of 340 nm. As a result of the non-specific aggregation suppression evaluation, the change in absorbance was below the specified value (the amount of change in the value of absorbance x 10000 was 1000 or less), confirming that the particles were capable of suppressing non-specific adsorption. The results of Example 1 and Comparative Example 1 are shown in FIG.

[0113] FIG. 2 is a diagram in which reaction time is plotted on the horizontal axis and R (polarization anisotropy r) is plotted on the vertical axis. It can be seen that in Example 1 plotted with circles in FIG. 2, R was 0.065 immediately after the reaction, and rapidly increased to 0.1 after 1000 seconds. On the other hand, in Comparative Example 1 plotted with a cross in FIG. 2, R immediately after the reaction is 0.056, but R after 1000 seconds is about 0.064, and although R increases with reaction time, the difference is small. Further, when the sample of Example 1 was left overnight and then remeasured, R was about 0.015. This value is the same as R of the luminescent reagent in the gel, indicating that the polarization anisotropy is saturated. As can be seen from FIG. 2, R in Example 1 increased to 0.113 after 2400 seconds, indicating that the reaction was promoted until R was sufficiently saturated in about 40 minutes of reaction.

[0114] Table 1 shows the results of Examples 1 to 6 and Comparative Example 1. In all Examples and Comparative Examples, PVP-K30 is present as a hydrophilic polymer on the surface of the luminescent reagent. Comparing the initial value of polarization anisotropy, R0, of all Examples and Comparative Example 1, it is higher in Example than 0.05676 in Comparative Example 1. This reflects the increase in liquid viscosity due to the addition of the sensitizer. However, the increase in R0 in the example is small compared to the saturation value of R (approximately 0.150), so it is within a range that can be fully evaluated. Further, the viscosity of the liquid of Example 1 was measured and found to be 2.3 mPa·s. From Table 1, when comparing ΔR300-R0, which is obtained by subtracting R0 from the polarization anisotropy value R300 after 300 seconds, the results are 0.0143, 0.0087, 0.0073, 0.007, and 0.00138 in the order of Examples 1, 2, 3, and Comparative Example 1. The effect of the sensitizer in the example could be confirmed. Furthermore, from Table 1, when comparing ΔR900-R0, which is obtained by subtracting R0 from the polarization anisotropy value R900 after 900 seconds, Examples 1, 2, 3 and Comparative Example 1 are 0.0377, 0.0225, 0.00136, 0.0198, and 0.00594 in that order. Therefore, it was confirmed that the difference between the example and the comparative example was even wider. In particular, when sodium alginate was used as the sensitizer in Example 1, the value of ΔR300-R0 was more than twice as high as ΔR900-R0 of the comparative example, and the sensitizer reduced the reaction time during measurement by a third. This shows that it is possible to reduce the number to 1 or less. Example 5 shows that by using multiple hydrophilic polymers as sensitizers, CRP antigen at a concentration of only 1 pM can be sufficiently detected within 300 seconds (ΔR300-R0=0.00601). Example 6 shows that the sensitizer is effective in TSH antigen-antibody reactions other than CRP. Furthermore, it has been shown that using polyoxazoline as a hydrophilic polymer is also effective (ΔR300-R0=0.0046). Further, when the study was conducted by subtracting polyoxazoline from the study in Example 6, almost no change in polarization anisotropy could be observed.

[0115]

table 1

[0116] From the above, it was shown that the CRP antigen, which is a target substance, could be measured with high sensitivity and in a short time by the analysis method according to the present embodiment.

[0117] Using the analysis method according to this embodiment, it becomes possible to measure a target substance in a short time and with high sensitivity. It is believed that by using the analysis method according to this embodiment, it is possible to realize a device that performs measurements with high sensitivity for applications such as specimen testing where a large number of tests are performed in a short period of time.

[0118] The disclosure of this embodiment includes the following methods and configurations. (Method 1) An analysis method for determining at least one of the presence or absence of the target substance and the concentration of the target substance by measuring a value (R) related to polarization anisotropy using a luminescent reagent that binds to the target substance, the method comprising: a reaction step of mixing and reacting a sample containing the target substance, the luminescent reagent, and the sensitizer to obtain a reaction solution; a measuring step of measuring the R of the reaction solution; has, and furthermore, The luminescent reagent comprises a luminescent particle substrate and a hydrophilic layer outside the luminescent particle substrate; the sensitizer includes a hydrophilic polymer; An analysis method characterized by: (Method 2) The analysis method according to method 1, wherein the hydrophilic polymer is at least one selected from the group consisting of polyvinylpyrrolidone, sodium alginate, potassium alginate, ammonium alginate, lithium alginate, alginic acid, and polyoxazoline. (Method 3) The analysis method according to method 1 or method 2, wherein the hydrophilic polymer has a molecular weight of 200,000 or more and 2,000,000 or less. (Method 4) The analysis method according to any one of methods 1 to 3, wherein the luminescent particle substrate contains a europium complex. (Method 5) The analysis method described in Methods 1 to 4, wherein the luminescent reagent has a ligand that binds to the target substance. (Method 6) The analysis method according to any one of methods 1 to 5, characterized in that R0≧0.001, where R0 is the R measured for the luminescent reagent that has not reacted with the target substance. (Method 7) 6. The analysis method according to any one of methods 1 to 6, wherein the R is defined by r in the following formula (1).

number

[0119] 1 particle substrate 2 hydrophilic layers 3 europium complex 4 Hydrophilic polymer 5 Distance between luminescent reagents 6 luminescence reagent

Claims

1. By measuring the value related to polarization anisotropy (R) using a luminescent reagent that has a ligand that binds to the target substance, An analytical method for determining at least one of the presence or absence of the target substance and the concentration of the target substance, A reaction step to obtain a reaction solution by mixing a sample containing the target substance, the luminescent reagent, and a sensitizer to react the target substance with the ligand, and A measurement step for measuring the R of the reaction solution, It has, and further, The luminescent reagent comprises a luminescent particle substrate and a hydrophilic layer outside the luminescent particle substrate. The sensitizer comprises a hydrophilic polymer. An analysis method characterized by the following.

2. The analytical method according to claim 1, characterized in that the hydrophilic polymer is at least one selected from the group consisting of polyvinylpyrrolidone, sodium alginate, potassium alginate, ammonium alginate, lithium alginate, alginic acid, and polyoxazoline.

3. The analytical method according to claim 1, characterized in that the hydrophilic polymer has a molecular weight of 200,000 or more and 2,000,000 or less.

4. The analytical method according to claim 1, characterized in that the luminescent particle substrate contains a europium complex.

5. The analytical method according to claim 1, characterized in that when R measured with respect to the luminescent reagent that has not reacted with the target substance is defined as R0, R0 ≥ 0.

001.

6. The analysis method according to claim 1, characterized in that the aforementioned R is determined by r in the following formula (1). [Math 1] (In formula (1), IVV... The emission intensity of the emission component whose vibration direction is parallel to the first polarization when excited by the first polarization. IVH... The emission intensity of the emission component whose vibration direction is perpendicular to the first polarization when excited by the first polarization. IHV... The emission intensity of the emission component whose vibration direction is perpendicular to the second polarization when excited with a second polarization whose vibration direction is perpendicular to the first polarization. IHH... The emission intensity of the emission component whose vibration direction is parallel to the second polarization when excited by a second polarization whose vibration direction is perpendicular to the first polarization. G... Correction value (That is the case.)

7. By measuring the value related to polarization anisotropy (R) using a luminescent reagent that has a ligand that binds to the target substance, A test kit used for analysis to determine at least one of the presence or absence of the target substance and the concentration of the target substance, Luminescent particle substrate, The hydrophilic layer on the outside of the luminescent particle substrate, and ligand that binds to the target substance A first reagent containing a luminescent reagent, and A second reagent containing a sensitizer having a hydrophilic polymer, A test kit that includes [the following].

8. The test kit according to claim 7, characterized in that the hydrophilic polymer is at least one selected from the group consisting of polyvinylpyrrolidone, sodium alginate, potassium alginate, ammonium alginate, lithium alginate, alginic acid, and polyoxazoline.

9. The test kit according to claim 7, characterized in that the luminescent particle substrate contains a europium complex.

10. The test kit according to claim 8, wherein R is measured by mixing the first reagent and the second reagent with a sample containing the target substance, and the amount of the sensitizer in the mixed solution is 0.01 w / v% or more and 2.0 w / v% or less.

11. The test kit according to claim 7, wherein R is measured by mixing the first reagent and the second reagent with a sample containing the target substance, and the amount of the luminescent reagent in the mixed solution is 0.000001% by mass or more and 20% by mass or less.

12. By using a luminescent reagent that binds to the target substance, and measuring the value related to polarization anisotropy (R), A test reagent used in an analysis to determine at least one of the presence or absence of the target substance and the concentration of the target substance, Luminescent particle substrate, The hydrophilic layer on the outside of the luminescent particle substrate, and ligand that binds to the target substance Luminescent reagents containing A sensitizer having a hydrophilic polymer, and dispersion medium Test reagents containing [specific components / materials].

13. The test reagent according to claim 12, characterized in that the hydrophilic polymer is at least one selected from the group consisting of polyvinylpyrrolidone, sodium alginate, potassium alginate, ammonium alginate, lithium alginate, alginic acid, and polyoxazoline.

14. The test reagent according to claim 12, characterized in that the luminescent particle substrate contains a europium complex.

15. The test reagent according to claim 12, characterized in that the hydrophilic polymer has a molecular weight of 200,000 or more and 2,000,000 or less.

16. The test reagent according to claim 12, characterized in that the hydrophilic polymer does not interact with the luminescent reagent.

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