Method for measuring target substances, and reagents thereof.

Abstract

To provide reagents and measurement methods that exhibit excellent measurement reproducibility. [Solution] A reagent for measuring a target substance contained in a sample, wherein the target substance is ferritin, and the reagent comprises a first reagent containing a buffer and a second reagent containing particles on which a ligand that specifically binds to the target substance is immobilized, wherein a first mixing step is performed to obtain a first mixed solution by mixing a sample containing 1000 ng / mL of the target substance with the first reagent in a volume ratio of sample:first reagent = 15:60, and a second mixing step is performed 280 seconds or more after mixing in the first mixing step to obtain a second mixed solution by mixing the first mixed solution and the second reagent in a volume ratio of first mixed solution:second reagent = 75:30, wherein the absorbance change of the second mixed solution becomes 0.15 or more within 50 seconds after mixing in the second mixing step.

Description

Technical Field

[0001] The present disclosure relates to a method for measuring a target substance and a reagent therefor.

Background Art

[0002] As a simple and rapid immunoassay method, immunoturbidimetry using particles can be mentioned. In this method, a dispersion of latex particles conjugated with a ligand having affinity for a target substance is mixed with a sample that may contain the target substance. At this time, since the particles cause an aggregation reaction according to the amount of the target substance contained in the sample, the target substance can be qualitatively or quantitatively determined by optically detecting this aggregation reaction as a change amount such as scattered light intensity, transmitted light intensity, absorbance, etc.

[0003] Non-Patent Documents 1 and 2 disclose a measurement reagent for ferritin by immunoturbidimetry using particles.

Prior Art Documents

Non-Patent Documents

[0004]

Non-Patent Document 1

Non-Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0005] In the reagents disclosed in Non-Patent Documents 1 and 2, there is room for improvement in measurement reproducibility. An object of the present disclosure is to provide a reagent and a measurement method excellent in measurement reproducibility.

Means for Solving the Problems

[0006] The first aspect of this disclosure is, This is a reagent for measuring target substances contained in a sample. The target substance is ferritin, The reagent comprises a first reagent containing a buffer and a second reagent containing particles on which a ligand that specifically binds to the target substance is immobilized. A first mixing step to obtain a first mixed solution by mixing the sample containing 1000 ng / mL of the target substance with the first reagent in a volume ratio of sample:first reagent = 15:60. And when a second mixing step is performed, 280 seconds or more after the mixing in the first mixing step, to obtain a second mixed solution by mixing the first mixed solution and the second reagent in a volume ratio of first mixed solution:second reagent = 75:30, This invention relates to a reagent characterized in that the change in absorbance of the second mixed solution becomes 0.15 or more within 50 seconds of mixing in the second mixing step.

[0007] Furthermore, this disclosure is, This is a reagent for measuring target substances contained in a sample. The target substance is a protein that forms a complex of hexamers or more. The reagent comprises a first reagent containing a buffer and a second reagent containing particles on which a ligand that specifically binds to the target substance is immobilized. A first mixing step to obtain a first mixed solution by mixing the sample containing 1000 ng / mL of the target substance with the first reagent in a volume ratio of sample:first reagent = 15:60. And when a second mixing step is performed, 280 seconds or more after the mixing in the first mixing step, to obtain a second mixed solution by mixing the first mixed solution and the second reagent in a volume ratio of first mixed solution:second reagent = 75:30, This invention relates to a reagent characterized in that the change in absorbance of the second mixed solution becomes 0.15 or more within 50 seconds of mixing in the second mixing step.

[0008] Furthermore, this disclosure states that This is a measurement method for measuring target substances contained in a sample. The target substance is ferritin, A first mixing step to obtain a first mixture by mixing the sample and the first reagent, A second mixing step involves mixing the first mixture with a second reagent containing particles on which a ligand that specifically binds to the target substance is immobilized to obtain a second mixture. The process includes a measurement step for measuring the absorbance of the second mixed solution, The sample contains the target substance at a concentration of 500 ng / mL or more and 1000 ng / mL or less. This invention relates to a measurement method characterized in that the change in absorbance of the second mixed solution is 0.15 or more within 50 seconds of mixing in the second mixing step.

[0009] Finally, this disclosure is This is a measurement method for measuring target substances contained in a sample. The target substance is a protein that forms a complex of hexamers or more. A first mixing step to obtain a first mixed solution by mixing the sample and the first reagent, A second mixing step to obtain a second mixture by mixing the first mixture with a second reagent containing particles on which a ligand that specifically binds to the target substance is immobilized, The process includes a measurement step for measuring the absorbance of the second mixed solution, The sample contains the target substance at a concentration of 500 ng / mL or more and 1000 ng / mL or less. This invention relates to a measurement method characterized in that the change in absorbance of the second mixed solution is 0.15 or more within 50 seconds of mixing in the second mixing step. [Effects of the Invention]

[0010] This disclosure provides reagents and measurement methods that exhibit excellent measurement reproducibility. [Modes for carrying out the invention]

[0011] Hereinafter, embodiments of the present disclosure will be described in detail, but the technical scope of the present disclosure is not limited to these embodiments. Particles immobilized with a ligand that specifically binds to a target substance are hereinafter also referred to as affinity particles, and particles before the ligand that specifically binds to the target substance is immobilized are also referred to as pre-sensitized particles.

[0012] Among target substances, there are those having multiple ligand recognition sites. For example, ferritin is a complex of 24 proteins and can have 24 antibody recognition sites. As a result of intensive studies to further improve the measurement accuracy of target substances having multiple antibody recognition sites such as ferritin, the inventors have found that it is important to control the aggregation rate of particles. Hereinafter, ferritin will be described as a specific example, but for proteins forming a complex of hexamers or more, the effects of the present disclosure can be explained in the same way.

[0013] When detecting ferritin as a target substance using an immunoturbidimetry method with particles, there is a possibility that more than two particles will aggregate per one target substance. Therefore, the aggregation patterns are diverse, and there may be cases where multi-particle aggregation of three or more occurs, or cases where multiple two-particle aggregations occur. Since the amount of change in absorbance in an immunoturbidimetry method using particles depends on the change in particle size due to aggregation, when the above-mentioned differences in aggregation states occur, even with the same amount of ferritin, different amounts of absorbance change will result. As a result, it was considered that the measured values might fluctuate even when specimens with the same amount of ferritin were measured.

[0014] Based on the above concept, as a result of detailed analysis of the aggregation behavior of particles, it was found that the faster the aggregation rate, that is, the absorbance change rate, when the specimen and affinity particles are mixed, the higher the reproducibility of the measured value.

[0015] When the particle aggregation rate is fast, the second particle quickly aggregates with the first particle that has reacted with ferritin. Since this reaction occurs simultaneously throughout the entire reaction field, it is thought that uniform two-particle aggregation is easily formed. On the other hand, when the particle aggregation rate is slow, it takes time for the second particle to aggregate with the first particle that has reacted with ferritin. As a result, the proportion of the second particle that aggregates by being drawn into other aggregates that have already formed increases, and as a result, it is thought that non-uniform aggregation is easily formed.

[0016] Based on the above considerations, this disclosure is intended to This is a reagent for measuring target substances contained in a sample. The target substance is ferritin, The reagent comprises a first reagent containing a buffer and a second reagent containing particles on which a ligand that specifically binds to the target substance is immobilized. The first mixing step involves obtaining a first mixed solution by mixing a sample containing 1000 ng / mL of the target substance with the first reagent in a volume ratio of sample:first reagent = 15:60. Furthermore, when a second mixing step is performed to obtain a second mixture by mixing the first mixture and the second reagent in a volume ratio of first mixture:second reagent = 75:30, after 280 seconds or more have elapsed since the mixing in the first mixing step, This reagent is characterized by the fact that the change in absorbance of the second mixed solution becomes 0.15 or more within 50 seconds of mixing in the second mixing step.

[0017] In this embodiment, when mixing the sample and the first reagent, it may refer to adding one to the other, or they may be added to each other. The same applies to other mixing in this specification. In this embodiment, "from mixing" means from the time when the total amounts of the two liquids are mixed, and then stirring is performed to thoroughly mix the two liquids, and the stirring is completed. The time from the time when mixing of the two liquids begins to the time when the stirring is completed is 5 seconds or less. The stirring may be performed using a stirring bar, by ultrasonic irradiation, or by tapping. For example, the statement that the change in absorbance of the second mixed solution is 0.15 or more within 50 seconds from mixing means that there are cases in which the difference in absorbance measured at two different times within 50 seconds from the time when the stirring is completed is 0.15 or more. For example, even if the difference between the absorbance measured at the time stirring is completed and the absorbance measured 50 seconds after the time stirring is completed is 0.15 or more, it is also acceptable if the difference between the absorbance measured 1 second after the time stirring is completed and the absorbance measured 42 seconds after the time stirring is completed is 0.15 or more. The same applies to the definition of "mixing" in this specification as well as to other mixing methods described herein.

[0018] If the concentration of the sample in the second mixture is less than 4.0% by volume, sufficient detection sensitivity cannot be obtained. If it is higher than 30.0% by volume, the aggregation of particles may be affected by components other than the target substance contained in the sample, which may worsen the measurement accuracy and prevent sufficient detection sensitivity from being obtained. Measurement accuracy also deteriorates if the concentration of the target substance contained in the sample is high. The concentration of the sample in the second mixture is more preferably between 7.0% by volume and 30.0% by volume, and even more preferably between 10.0% by volume and 20.0% by volume.

[0019] In the above measurement, the change in absorbance within 50 seconds after mixing the first mixture and the second reagent in a volume ratio of first mixture:second reagent = 75:30 is also called the initial absorbance change.

[0020] A change in initial absorbance of 0.15 or higher indicates a rapid aggregation rate when the antigen and affinity particles are mixed. Therefore, a change in initial absorbance of 0.15 or higher provides excellent measurement reproducibility. In particular, when the ferritin concentration is 100 ng / mL or higher, the number of antigen particles is large relative to the number of particles, making aggregation of multiple particles more likely, and thus the effect of this disclosure is greater. A change in initial absorbance of 0.20 to 0.50 is more preferable. If it exceeds 0.50, it is undesirable because it may indicate the occurrence of nonspecific aggregation other than aggregation due to the antigen-antibody reaction.

[0021] Furthermore, in the above measurement, it is preferable that the change in absorbance between 50 seconds and 250 seconds after mixing the first mixture and the second reagent in a volume ratio of first mixture:second reagent = 75:30 is 0.20 or higher. A value of 0.20 or higher means that particle aggregation proceeds appropriately even in the latter half of the agglutination reaction. Therefore, measurement results with excellent accuracy can be obtained. It is more preferable that the change in absorbance between 50 seconds and 250 seconds is between 0.25 and 0.80.

[0022] <Detection method, measurement method> The measurement method used in this disclosure is an immunoturbidimetric method using particles. This method optically detects interparticle aggregation that occurs when affinity particles of this disclosure are mixed with a sample. In this disclosure, absorbance is used as the method for detecting this optical change. There are no restrictions on the instrument used for measurement; any optical instrument capable of detecting absorbance is acceptable. In particular, it is preferable to use a general-purpose automated analyzer that allows for easy control of sample and reagent dispensing volume, mixing time, measurement wavelength, etc.

[0023] The measurement method of this embodiment is This is a measurement method for measuring target substances contained in a sample. The target substance is ferritin, The first mixing step involves obtaining a first mixture by mixing the sample and the first reagent, A second mixing step involves mixing a first mixture with a second reagent containing particles on which ligands that specifically bind to the target substance are immobilized, to obtain a second mixture. The process includes a measurement step for measuring the absorbance of the second mixed solution, The sample contains the target substance at a concentration of 500 ng / mL or more and 1000 ng / mL or less. This measurement method ensures that the change in absorbance of the second mixed solution is 0.15 or greater within 50 seconds of mixing in the second mixing step.

[0024] An example of a measurement method using an automated analyzer is described below. First, 8 μL to 25 μL of the sample is dispensed into the reaction vessel. Next, as a mixing step, 50 μL to 150 μL of the first reagent is dispensed into the same reaction vessel, stirred, and then adjusted to a predetermined temperature and kept warm for 180 to 600 seconds. The temperature at this time is preferably in the range of 20°C to 50°C.

[0025] Subsequently, as a second mixing step, 10 μL to 150 μL of the second reagent is dispensed into the same reaction vessel, stirred, and then the reaction is carried out for 180 to 600 seconds. At this time, it is preferable to start the second mixing step at least 280 seconds after the mixing in the first mixing step. By waiting 280 seconds after the mixing in the first mixing step, the sample and the first reagent are uniformly mixed, resulting in uniform particle aggregation and improved measurement reproducibility. Furthermore, it is more preferable that the reaction time for the second mixing step be between 200 seconds and 600 seconds.

[0026] Furthermore, the absorbance of the second mixture is measured, and the change in absorbance is calculated. At this time, it is preferable that the absorbance 42 seconds after mixing in the second mixing step is between 0.90 and 2.00. This is because the absorbance 42 seconds after mixing in the second mixing step represents the absorbance at the starting point before the agglutination reaction progresses, and excellent measurement reproducibility is obtained when this value falls within the above range. Additionally, it is preferable that the wavelength of absorbance measurement includes a range of 500 nm to 750 nm.

[0027] The concentration of affinity particles in the second mixture is preferably between 0.02 mass / volume% and 0.10 mass / volume%. This concentration ensures uniform particle aggregation, resulting in excellent measurement reproducibility. Furthermore, the pH of the second mixture is preferably between 5.0 and 11.0.

[0028] The viscosity of the second mixture is preferably between 0.95 mPa·s and 1.40 mPa·s. This viscosity range suppresses particle aggregation, resulting in higher measurement accuracy. Furthermore, excellent measurement accuracy can be obtained even at high concentrations of the target substance.

[0029] <Specimen and target substance> The specimens that can be used in this disclosure are not particularly limited as long as they contain the target substance, but examples include blood, serum, plasma, etc.

[0030] The target substance in this disclosure is a protein with a molecular weight of 150 kDa or more and 500 kDa or less, or a protein forming a complex of hexamers or more. More preferably, it is a protein with a molecular weight of 300 kDa or more and 600 kDa or less. The ferritin exemplified in this disclosure has a molecular weight of 445 kDa, and because of its large molecular weight, it reacts easily with antibodies on the particle surface, making it easier to obtain the effects of this disclosure. In this embodiment, it is preferable that the sample contains the target substance in a concentration of 500 ng / mL or more and 1000 ng / mL or less.

[0031] <First reagent and second reagent> The reagents disclosed herein include a first reagent containing a buffer and a second reagent containing affinity particles. The first reagent is used to dilute the sample and prevent nonspecific reactions of the sample. For this reason, the first reagent may contain buffers, sugars, surfactants, sensitizers, and nonspecific reaction inhibitors as described later.

[0032] The surface tension of the first reagent is preferably between 20 mN / m and 50 mN / m. This range ensures uniform mixing of affinity particles and the sample, resulting in excellent measurement reproducibility.

[0033] The electrical conductivity of the first reagent is preferably 0.5 mS / cm to 70.0 mS / cm, and more preferably 10.0 mS / cm to 70.0 mS / cm. This range maintains the electrostatic repulsion of affinity particles, resulting in more uniform interaction between affinity particles, particularly with anionic sample components, and thus superior measurement reproducibility. The electrical conductivity of the first reagent is more preferably 0.5 mS / cm to 65.0 mS / cm.

[0034] The pH of the first and second reagents is preferably between 5.0 and 11.0. Being within this range allows for uniform dispersion of affinity particles and uniform mixing of the samples. Therefore, better measurement reproducibility can be obtained. The pH values ​​of the first and second reagents may be different.

[0035] The first and second reagents preferably contain a buffer. The type of buffer is not particularly limited and any substance that provides buffering capacity is acceptable. For example, MES, Bis-Tris, ADA, PIPES, ACES, MOPSO, BES, MOPS, TES, HEPES, TAPSO, POPSO, HEPSO, EPPS, tricine, bicine, TAPS, CHES, and CAPS are preferably used as acetic acid, citrate, phosphoric acid, Tris, glycine, boric acid, and Good's buffers. One type of buffer may be used alone, or two or more types may be used in combination. Furthermore, the buffers used in the first and second reagents may be the same or different.

[0036] The first and second reagents preferably further contain sugars or sugar alcohols. The inclusion of sugars promotes hydration of the surface of affinity particles and sample components, reducing the interaction between affinity particles and sample components, and improving measurement reproducibility. Examples of such sugars and sugar alcohols include, but are not limited to, monosaccharides such as glucose and fructose, disaccharides such as sucrose, lactose, maltose, cellobiose, and trehalose, or oligosaccharides such as maltotriose and dextran, and sugar alcohols such as erythritol, mannitol, sorbitol, and xylitol. One type of sugar or sugar alcohol may be used alone, or two or more types may be used in combination.

[0037] The first and second reagents preferably further contain surfactants. Known nonionic surfactants, anionic surfactants, cationic surfactants, and amphoteric surfactants can be used. Among these, it is preferable to contain one or more of sorbitan fatty acid esters, polyoxyethylene alkyl ethers, and polyoxyethylene phenyl ethers. These surfactants have high hydrophilicity and a high solubilizing and stabilizing effect on sample components. Therefore, the interaction between affinity particles and sample components is reduced, resulting in excellent measurement reproducibility. The concentration of the surfactant in the second mixture is preferably 0.001% by mass or more and 0.200% by mass or less.

[0038] The first reagent may further contain a sensitizer. Examples of sensitizers include water-soluble polymers such as polyethylene glycol, carboxymethylcellulose, methylcellulose, polyvinylpyrrolidone, polyvinyl alcohol, polyacrylic acid, and polyglycosylethyl methacrylate.

[0039] In addition, the first and second reagents of this disclosure may contain chelating agents such as EDTA, CyDTA, DTPA, EGTA, NTA, and NTP; proteins such as bovine serum albumin, casein, and gelatin; protein degradation products; amino acids; animal serum, antibodies, antibody fragments; nonspecific reaction inhibitors such as reducing agents; stabilizers such as proteins and preservatives; and inorganic salts such as sodium chloride, potassium chloride, and calcium chloride.

[0040] <Affinity particles and pre-sensitized particles> The zeta potential of the affinity particles in this disclosure is preferably between -50mV and -15mV. A zeta potential of -50mV and -15mV ensures an appropriate balance between the electrostatic repulsive and attractive forces of the affinity particles. This stabilizes the dispersion of the affinity particles, resulting in superior measurement reproducibility. More preferably, the zeta potential of the affinity particles is between -40mV and -15mV.

[0041] The volume-average particle size of the affinity particles in this disclosure is preferably between 200 nm and 500 nm. Having the volume-average particle size within this range ensures an appropriate particle number concentration. Therefore, the effect of the sample components on the affinity particles becomes more uniform, resulting in excellent measurement reproducibility. A volume-average particle size of 250 nm to 400 nm is more preferable.

[0042] Furthermore, the affinity particles of this disclosure are affinity particles obtained by mixing two types of affinity particles with different volume-average particle sizes, and it is preferable that the difference in volume-average particle sizes between the two types of affinity particles is 50 nm or more and 250 nm or less. By mixing two types of affinity particles with different volume-average particle sizes, excellent measurement accuracy can be obtained for a wider range of target substance concentrations. In this case, because the particle size difference is within the above range, the effect of the sample components on the affinity particles is less likely to be biased, and thus excellent measurement accuracy can be obtained for a wide range of target substance concentrations.

[0043] Furthermore, the refractive index of the affinity particles in this disclosure is preferably 1.600 or more and 1.800 or less. Moreover, it is more preferable that the affinity particles be a mixture of two types of affinity particles with different refractive indices, which provides a better measurement range.

[0044] Furthermore, the affinity particles of this disclosure preferably have a protein content of 200 μg or less per 1 mg of particle relative to the pre-sensitized particles. Being within this range means that the amount of albumin and other stabilizers in the affinity particles is very small. Therefore, excellent sensitivity can be obtained.

[0045] Conventionally known pre-sensitized particles can be used in this disclosure. For example, polystyrene, styrene-butadiene copolymer, styrene-styrene sulfonate copolymer, styrene-glycidyl methacrylate copolymer, etc. can be used. In particular, it is preferable that the pre-sensitized particles are particles having a polymer containing repeating units represented by the following formula (1). [ka] (R1 represents a methyl group or a hydrogen atom. R2 represents a group having one of the following: an epoxy group, a hydroxyl group, or a carboxyl group.)

[0046] Having a polymer containing repeating units represented by formula (1) in the pre-sensitization particles facilitates hydration of the affinity particle surface, and its low ionicity suppresses the influence of hydrophobic ionic components. As a result, higher measurement accuracy is obtained. Formula (1) is more preferably the structure represented by formula (1-A). [ka] (At least one of R3 and R4 represents a hydroxyl group, and the other represents a hydroxyl group or a group represented by formula (1-B).) [ka] (R5 represents a single bond or a methylene group. R6, R7, and R8 are selected from a hydrogen atom, a methyl group, a hydroxyl group, a carboxyl group, a hydroxymethyl group, or a carboxymethyl group, and include at least one hydroxyl group or a carboxyl group. Y1 represents a sulfur atom or an imino group. *1 indicates the bond position with the structure shown in formula (1-A).)

[0047] Examples of specific structures of equation (1-A) are shown below in (1-A-1) to (1-A-12), but are not limited to these. [ka]

[0048] One method for giving particles these structures is to use glycidyl methacrylate. Specifically, particles having the structure of formula (1) can be obtained by using glycidyl methacrylate. Furthermore, particles having the structure of formula (1-A) can be obtained by bonding a compound having a mercapto group or a compound having an amino group to the epoxy group of glycidyl methacrylate.

[0049] Furthermore, it is preferable that the pre-sensitized particles have nitrogen-containing functional groups, and more preferably that they contain a compound having an amidine group represented by formula (2). The amidine group suppresses the electrostatic repulsion force of the particles and increases the aggregation rate, so uniform two-particle aggregation is easily achieved, and excellent measurement reproducibility can be obtained. [ka]

[0050] One method for imparting amidine groups to particles is to use an initiator containing amidine groups during particle manufacturing. Examples of initiators containing amidine groups include V-50 (2,2'-azobis(2-methylpropionamidine) dihydrochloride).

[0051] Furthermore, it is preferable that the pre-sensitization particles are particles having a polymer containing repeating units represented by the following formula (3). [ka] (R9 represents a hydrogen atom or a methyl group. 10 The group (3) represents a substituted or unsubstituted phenyl group or naphthyl group. Substituents include methyl and ethyl groups. Formula (3) may have two or more structures.

[0052] The structures shown in formulas (1), (1-A), (2), and (3) may be present in the pre-sensitized particles or in the affinity particles. Furthermore, it is preferable that the affinity particles have formula (4) or formula (5), or both formula (4) and formula (5). [ka] (R 11 (This indicates a functional group having one or more hydroxyl groups.) [ka] (R 12 This indicates a functional group having at least one carboxyl group.

[0053] By having the structure shown in formula (4) or formula (5), the hydrophilicity of the particles can be increased. Furthermore, since a state that does not increase the electrostatic repulsion force can be maintained, excellent measurement reproducibility and accuracy can be obtained.

[0054] One method for giving particles the structure shown in formula (4) is to use an amino alcohol as a reaction stopper used in the production of affinity particles. Another method for giving particles the structure shown in formula (5) is to use an amino acid as a reaction stopper used in the production of affinity particles.

[0055] <Ligands that specifically bind to proteins> A ligand is a compound that specifically binds to a receptor on a particular target substance. The binding site of a ligand to the target substance is fixed, and it has a selective or specific high affinity. Examples include, but are not limited to, antigens and antibodies, enzyme proteins and their substrates, signaling substances such as hormones and neurotransmitters and their receptors, nucleic acids, avidin and biotin, etc., as long as the objectives of this disclosure can be achieved. Specifically, examples of ligands include antigens, antibodies, antigen-binding fragments (e.g., Fab, F(ab')2, F(ab'), Fv, scFv, etc.), naturally occurring nucleic acids, artificial nucleic acids, aptamers, peptide aptamers, oligopeptides, enzymes, coenzymes, etc.

[0056] In this embodiment, the ligand is preferably an antibody or antigen, and more preferably an antibody with an isoelectric point between 5.0 and 8.0. Affinity particles using antibodies with an isoelectric point in this range have a reduced charge on the surface of the affinity particles due to the influence of the antibody. As a result, the interaction between the affinity particles and the cationic components contained in the sample is reduced, resulting in excellent measurement accuracy. The isoelectric point can be measured by isoelectric focusing electrophoresis.

[0057] In this disclosure, the method for immobilizing ligands on pre-sensitized particles can be any known method, and the ligands can be immobilized by physically or chemically binding them to the pre-sensitized particles. For example, ligands can be immobilized on pre-sensitized particles by covalent bonds, hydrogen bonds, ionic bonds, electrostatic attraction, or van der Waals forces. Examples of chemical binding methods include methods utilizing carbodiimide-mediated reactions or NHS ester activation reactions, or methods in which avidin is bound to a carboxyl group and then a biotin-modified ligand is bound to it.

[0058] The amount of ligand per 1.0 mg of pre-sensitized particles is preferably 1.0 μg or more and 150.0 μg or less, and more preferably 2.0 μg or more and 100.0 μg or less. Furthermore, the affinity particles in this disclosure are affinity particles obtained by mixing two types of affinity particles, and it is even more preferable that the amount of ligand in the two types of affinity particles is different.

[0059] Furthermore, the amount of ligand in the second mixture is preferably 0.01 mg / mL or more and 2.00 mg / mL or less.

[0060] An example of a method for measuring physical properties in this disclosure is described below.

[0061] <Method for measuring viscosity> Viscosity was measured using RE-85L (Toki Sangyo Co., Ltd.). 1.1 mL of the sample was placed in the measuring container, and the measurement was performed using a cone rotor at a shear rate of 100 rpm. The measurement temperature was set to 25°C by connecting a circulating constant temperature bath.

[0062] <Method for measuring surface tension> Surface tension is measured using an automated surface tension meter, DyneMaster (Kyowa Interface Science Co., Ltd.). The measurement is performed using the plate method with a platinum plate at a temperature of 25°C.

[0063] <Method for measuring electrical conductivity> Electrical conductivity is measured using an electrical conductivity meter AS710 (AS ONE Corporation). Measurements are taken under conditions of 23°C and 50% humidity.

[0064] <Method for measuring the zeta potential of particles> The zeta potential of the particles is measured using a zetasizing device, Nano-ZS (Malvern Panalytical). In this disclosure, the zeta potential is measured with the particles dispersed in a 0.01N potassium hydroxide aqueous solution at pH 7.8 at a concentration of 0.003 mass / volume%. The measurement conditions are 25°C, latex (n≈1.59) is selected as the refractive index of the particles, and pure water is selected as the solvent. Ten measurements are taken, and the average value of the ten measurements is adopted as the zeta potential.

[0065] <Method for measuring the volume-average particle size> The volume-average particle size is measured using a Zetasizer Nano-ZS (Malvern Panalytical). In this disclosure, the volume-average particle size is measured with the particles dispersed in ion-exchanged water at a concentration of 0.003 mass / volume%. The measurement conditions are 25°C, latex (n≈1.59) is selected as the refractive index of the particles, and pure water is selected as the solvent. Ten measurements are taken, and the average of the ten measurements is adopted as the volume-average particle size.

[0066] Furthermore, the difference in volume-average particle size when two or more particles with different particle sizes are mixed can be determined by observation using a scanning electron microscope or similar device. Specifically, more than 300 particles are photographed at a magnification of 50,000x, and the particle sizes of these 300 particles are measured using a known image processing method. The volume of each particle is determined from the measured particle sizes, and a volume distribution is created. The particle size that causes a peak in the created volume distribution is taken as the volume-average particle size of each particle, and the difference can be calculated to determine the difference in particle sizes between two types of particles.

[0067] <Method for measuring the refractive index of particles> The refractive index of particles can be measured using Abbemat (manufactured by Anton Paar). In the following examples, the refractive index was specifically measured when the particle dispersion was dispersed to a concentration of 5% by mass. The measurement conditions selected were 25°C and a measurement wavelength of 589.3 nm. The particle refractive index was calculated from the Lorentz-Lorentz equation using the measured refractive index value, the specific gravity of the dispersion medium, the refractive index, and the specific gravity of the particles.

[0068] <Method for measuring the amount of antibody against particles (antibody sensitization amount of affinity particles)> This disclosure describes the method for measuring the antibody sensitization level of affinity particles. The antibody sensitization level of affinity particles was determined by protein quantification. Here, the antibody sensitization level of particles refers to the amount of antibody bound to or adsorbed per 1 mg of particle.

[0069] First, mix 7 mL of solution A and 140 μL of solution B from the Protein Assay BCA Kit (Fujifilm Wako Pure Chemical Corporation) to prepare a solution called solution AB. Next, add 25 μL of affinity particle dispersion (particle concentration 0.1 mass / volume%) to 200 μL of solution AB and incubate at 60°C for 30 minutes. Then, centrifuge the solution at 20400 × g at 4°C for 5 minutes, and pipette 200 μL of the supernatant into a 96-well microplate. Add 200 μL of standard samples, prepared by mixing antibody in 10 mM HEPES buffer at arbitrary concentrations (5 points in the concentration range from 0 μg / mL to 200 μg / mL), to a separate microplate well. Measure the absorbance at 562 nm using a microplate reader and calculate the antibody amount from the calibration curve of the standard samples. The antibody amount per particle (μg / mg) is determined by dividing the calculated antibody amount by the particle mass. [Examples]

[0070] The present disclosure will be described in detail below with reference to examples, but the present disclosure is not limited to these examples.

[0071] (Synthesis of particle 1) The synthesis of particle 1 involves the following steps 1 to 3.

[0072] (Step 1) 71.75 g of styrene (St: Kishida Chemical Co., Ltd.), 1.30 g of divinylbenzene (DVB: Kishida Chemical Co., Ltd.), and 1190.67 g of deionized water were weighed into a 2 L four-neck separable flask to form a mixture. This mixture was kept at 70°C while stirring at 200 rpm, and the inside of the four-neck separable flask was deoxygenated by flowing nitrogen at a flow rate of 200 mL / min. Next, a solution prepared separately by dissolving 3.11 g of V-50 (Fujifilm Wako Pure Chemical Corporation) in 50 g of deionized water was added to the mixture, and a polymerization reaction was carried out for 48 hours to obtain a copolymer particle dispersion of St and DVB.

[0073] (Step 2) Next, 148.29 g of the above dispersion diluted with deionized water to a solid content concentration of 2.0% by mass was weighed into another four-necked separable flask. Then, 0.39 g of glycidyl methacrylate (GMA: Kishida Chemical Co., Ltd.) was added and the mixture was kept at 70°C while stirring at 100 rpm, and the inside of the four-necked separable flask was deoxygenated by flowing nitrogen at a flow rate of 200 mL / min. Then, a solution prepared separately by dissolving 0.018 g of V-50 in 1 g of deionized water was added to the mixture, and stirring was continued for 17 hours to obtain a dispersion containing St / DVB / GMA composite particles.

[0074] (Step 3) Finally, an aqueous solution containing mercaptosuccinic acid (MSA: Fujifilm Wako Pure Chemical Industries, Ltd.) and 3-mercapto-1,2-propanediol (MPD: Fujifilm Wako Pure Chemical Industries, Ltd.), which had been prepared in advance, was added to the dispersion containing St / DVB / GMA composite particles. At this time, the aqueous solution was prepared so that the ratio of 3-mercapto-1,2-propanediol to mercaptosuccinic acid was 6:4 (mol fraction), and the total number of moles of MSA and MPD was equal to the number of moles of glycidyl methacrylate. Next, triethylamine (Kishida Chemical Co., Ltd.) was added to adjust the pH to 10. Then, the dispersion was heated to 70°C while stirring at 200 rpm, and maintained in this state for 18 hours to obtain a dispersion of particle 1. Subsequently, particle 1 was separated from the dispersion using a centrifuge, and then the particle 1 was redispersed in ion-exchanged water. This process was repeated eight times to purify particle 1, and finally a particle 1 dispersion with a solid content of 5.0% by mass was obtained. The volume-average particle size of the obtained particle 1 was 400 nm.

[0075] (Synthesis of particle 2 to particle 6) Particles 2 through 4 were synthesized using the same experimental procedure as for particle 1, except that the amounts of styrene, DVB, V-50, and stirring speed used in step 1, the amount of GMA in step 2, and the molar fractions of MSA and MPD in step 3 were changed as shown in Table 1. These particles have a structure on their surface that is derived from GMA, as shown in formula (1). Furthermore, they have a structure that is derived from V-50, as shown in formula (2), which is a nitrogen-containing compound. In addition, JSR's polystyrene particles IMMUTEX P0307 and IMMUTEX P0322 were prepared as particles 5 and 6. The physical properties of the obtained particles 1 through 6 are summarized in Table 1.

[0076] In each of particles 1 to 6, the core particle contains a styrene-divinylbenzene copolymer and has a polymer on its surface that includes a structural unit represented by formula (1). More specifically, the polymer in formula (1) has a methyl group as R1 and a structural unit represented by the following formulas (31), (32), (33), or (34) as R2. [ka] [ka] [ka] [ka] (* indicates the bonding position with the structure shown in equation (1).)

[0077] [Table 1]

[0078] (Production of affinity particle 1) For the dispersion of particle 1, 300 μL of the dispersion (3 mg as particle solids), diluted with deionized water to a solid content concentration of 1.0 mass / volume%, was placed in a 1.5 mL microtube. 90 μL of a 5.0 mass% aqueous solution of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 90 μL of a 5.0 mass% aqueous solution of N-hydroxysulfosuccinimide sodium were added, and the mixture was stirred at room temperature for 30 minutes to obtain an activated particle dispersion containing carboxyl groups (activated particle dispersion).

[0079] After centrifugal washing, 270 μL of pH 5.5 phosphate buffer-physiological saline (PBS) was added, and the particles with activated carboxyl groups were dispersed using ultrasound.

[0080] To this, 24 μL of a 5.0 mg / mL dispersion of mouse monoclonal anti-ferritin antibody (isoelectric point 7.1) (0.12 mg of antibody) was added as a ligand, and the mixture was stirred at room temperature for 3 hours to sensitize the particles with the antibody. Subsequently, 1.0 mL of a pH 8.0 1 mol / L trishydroxymethylaminomethane (Tris) solution was added as a reaction stopper, and the mixture was left to stand overnight at 4°C to obtain antibody-sensitized test particles. After centrifugation washing of these test particles, 500 μL of PBS was added to obtain affinity particle 1.

[0081] Furthermore, since the reaction is stopped when the amino groups contained in the reaction stopper agent bind to the activated carboxyl groups on the particle surface, it can be said that the surface of each affinity particle has a structure derived from the reaction stopper agent.

[0082] (Production of affinity particle 10 from affinity particle 2) Affinity particle 10 was produced from affinity particle 2 using the same experimental procedure as for affinity particle 1, except that the particle type, antibody type, amount of antibody dispersion added, and type and concentration of reaction stopper were changed as shown in Table 2.

[0083] [Table 2]

[0084] (Preparation of Reagent 1) Second reagent 1 was prepared using the prepared affinity particles 1 and 4. Specifically, affinity particles 1 and 4 were centrifuged and redispersed in 500 μL of a buffer (HEPES buffer) containing 10 mM HEPES, 0.01% by mass polyoxyethylene nonylphenyl ether (Triton X-100: Kishida Chemical Co., Ltd.), and 10% by mass sucrose (viscosity modifier) ​​dissolved in deionized water. Then, the particles were mixed and diluted with HEPES buffer so that affinity particle 1 was 0.08% by mass / volume, affinity particle 2 was 0.08% by mass / volume, and the total was 0.16% by mass / volume, to obtain second reagent 1.

[0085] (Preparation of Reagent 2 to Reagent 11) Second reagent 11 was prepared from second reagent 2 using the same experimental procedure as for second reagent 1, except that the type of affinity particles, the concentration of affinity particles, and the composition of the viscosity modifier added to the HEPES buffer were changed as shown in Table 3. The physical properties of the obtained second reagents 1 to 11 are summarized in Table 3.

[0086] [Table 3]

[0087] (Preparation of the first reagent) As shown in Table 4, the first reagent was prepared by mixing each material in predetermined amounts with deionized water. The physical properties of the obtained first reagent are shown in Table 4.

[0088] [Table 4]

[0089] (Measurement of change in absorbance) A discrete-type automated clinical chemistry analyzer (TBA-120FR: Canon Medical Systems Corporation) was used as the measurement device. A sample containing 1000 ng / mL of the target substance, ferritin, was used, and the assay parameters were as follows: sample: 15 μL, first reagent 1: 60 μL, second reagent: 30 μL, measurement wavelength: 572 nm. The optical path length of this instrument is 5 mm, but the absorbance obtained as the measurement value is converted to the value for an optical path length of 1 cm.

[0090] In the obtained reaction curve, the change in absorbance from immediately after dispensing the second reagent to 50 seconds later, and the change in absorbance between 50 seconds and 250 seconds later were calculated. Since the photometric points of the instrument were not set at 50 seconds and 250 seconds, an approximate formula was calculated from the graph of the obtained reaction curve to calculate the absorbance at 50 seconds and 250 seconds later.

[0091] (Creating a calibration curve) First, standard solutions with ferritin concentrations of 0, 100, 250, 500, and 1000 ng / mL were prepared. These standard solutions were then measured using the following assay parameters: sample: 15 μL, first reagent 1: 60 μL, second reagent: 30 μL, and measurement wavelength: 572 nm.

[0092] The difference between the absorbance OD(19) at photometric point 19 and the absorbance OD(33) at photometric point 33, OD(33)-OD(19), was calculated as the absorbance change ΔOD in the obtained reaction curve. Next, a calibration curve showing the relationship between each ferritin concentration and ΔOD was created. These experiments were performed for each second reagent used in each example.

[0093] (Method for evaluating measurement reproducibility) Using similar measuring devices and assay parameters, the difference between the absorbance OD(19) at photometric point 19 and the absorbance OD(33) at photometric point 33, OD(33)-OD(19), was calculated as the absorbance change ΔOD, and the ferritin concentration was quantified using a pre-prepared calibration curve.

[0094] Three samples were used: Sample A containing 970 ng / mL of ferritin, Sample B containing 550 ng / mL of ferritin, and Sample C containing 106 ng / mL of ferritin. Each sample was measured five times. The coefficient of variation (CV) of the obtained ferritin concentrations was calculated. The obtained CVs were evaluated according to the following criteria. A: CV is less than 0.8 B:CV is between 0.8 and less than 1.0 C:CV is between 1.0 and 1.5 D:CV is between 1.5 and less than 2.0 E:CV is between 2.0 and less than 3.0 F:CV is 3.0 or higher

[0095] (Method for evaluating the accuracy of measurements) Using the same measurement method as for evaluating measurement reproducibility, the average ferritin concentration was calculated from five measurements of sample A containing 970 ng / mL, sample B containing 550 ng / mL, and sample C containing 106 ng / mL. The average ferritin concentration obtained was evaluated according to the following criteria. A: The difference between the obtained average value and the ferritin concentration mentioned above is less than 3%. B: The difference between the obtained average value and the ferritin concentration mentioned above is 3% or more but less than 5%. C: The difference between the obtained average value and the ferritin concentration mentioned above is 5% or more but less than 10%. D: The difference between the obtained average value and the ferritin concentration mentioned above is 10% or more but less than 15%. E: The difference between the obtained average value and the ferritin concentration mentioned above is 15% or more.

[0096] (Examples 1 to 8, and Comparative Examples 1 to 3) We evaluated reagents 1 through 11 of the second reagent. The obtained physical properties and evaluation results are summarized in Tables 5 and 6.

[0097] [Table 5]

[0098] [Table 6] These results demonstrate that reagents meeting the requirements of this disclosure provide excellent measurement reproducibility and accuracy across a wide range of ferritin concentrations.

[0099] Ferritin was used as an example of a target substance that can be used in this disclosure. Proteins like ferritin, which are composed of multiple associated subunits, may have multiple sites that can be recognized by antibodies. Such proteins may aggregate with more than two particles for each target substance. Therefore, the aggregation patterns are diverse, and it is thought that there may be cases of multi-particle aggregation of three or more particles, or multiple aggregations of two particles. Since the change in absorbance in immunoturbidimetry using particles depends on the change in particle size due to aggregation, if differences in the aggregation state occur as described above, it is possible that the change in absorbance will differ even with the same amount of target substance. As a result, even if the same amount of target substance is measured, there is a possibility that the measured values ​​will fluctuate. From this perspective, it can be said that the measurement method of this disclosure targets substances that have multiple reaction sites with respect to ligands that specifically bind to the target substance.

[0100] This embodiment includes the following configurations and methods. (Composition 1) This is a reagent for measuring target substances contained in a sample. The target substance is ferritin, The reagent comprises a first reagent containing a buffer and a second reagent containing particles on which a ligand that specifically binds to the target substance is immobilized. A first mixing step to obtain a first mixed solution by mixing the sample containing 1000 ng / mL of the target substance with the first reagent in a volume ratio of sample:first reagent = 15:60. And when a second mixing step is performed, 280 seconds or more after the mixing in the first mixing step, to obtain a second mixed solution by mixing the first mixed solution and the second reagent in a volume ratio of first mixed solution:second reagent = 75:30, A reagent characterized in that the amount of change in absorbance of the second mixed solution becomes 0.15 or more within 50 seconds from the mixing in the second mixing step. (Configuration 2) The reagent according to configuration 1, characterized in that the sample is selected from the group consisting of blood, serum, and plasma containing the target substance. (Composition 3) The reagent according to configuration 1 or 2, characterized in that, after mixing the first mixture and the second reagent in a volume ratio of first mixture:second reagent = 75:30, the change in absorbance of the second mixture becomes 0.20 or more within 50 seconds. (Composition 4) The reagent according to any one of configurations 1 to 3, characterized in that, after mixing the first mixture and the second reagent in a volume ratio of first mixture:second reagent = 75:30, the change in absorbance of the second mixture between 50 seconds and 250 seconds is 0.20 or more. (Composition 5) The reagent according to any one of configurations 1 to 4, characterized in that the surface tension of the first reagent is 20 mN / m or more and 50 mN / m or less. (Composition 6) The reagent according to any one of configurations 1 to 5, characterized in that the first reagent and the second reagent contain at least one of sugars and sugar alcohols. (Composition 7) The reagent according to any one of configurations 1 to 6, characterized in that the first reagent and the second reagent contain one or more surfactants selected from the group consisting of sorbitan fatty acid esters, polyoxyethylene alkyl ethers, and polyoxyethylene phenyl ethers. (Composition 8) The reagent according to any one of configurations 1 to 7, characterized in that the electrical conductivity of the first reagent is 0.5 mS / cm or more and 70.0 mS / cm or less. (Composition 9) The reagent according to any one of configurations 1 to 8, characterized in that the zeta potential of the particles is -50mV or more and -15mV or less. (Composition 10) The reagent according to any one of configurations 1 to 9, characterized in that the particles contain a polymer having the structure shown in the following formula (1). [ka] (R1 represents a methyl group or a hydrogen atom. R2 represents a group having one of the following: an epoxy group, a hydroxyl group, or a carboxyl group.) (Composition 11) The reagent according to configuration 10, characterized in that formula (1) has the structure shown by formula (1-A) below. [ka] (At least one of R3 and R4 represents a hydroxyl group, and the other represents a hydroxyl group or a group represented by formula (1-B).) [ka] (R5 represents a single bond or a methylene group. R6, R7, and R8 are selected from a hydrogen atom, a methyl group, a hydroxyl group, a carboxyl group, a hydroxymethyl group, or a carboxymethyl group, and include at least one hydroxyl group or a carboxyl group. Y1 represents a sulfur atom or an imino group. *1 indicates the bond position with the structure shown in formula (1-A).) (Composition 12) The reagent according to any one of configurations 1 to 11, characterized in that the particles have nitrogen-containing functional groups. (Composition 13) The reagent according to any one of configurations 1 to 12, characterized in that the particles contain a compound having an amidine group represented by the following formula (2). [ka] (Composition 14) The reagent according to any one of configurations 1 to 13, characterized in that the particles have a polymer containing repeating units represented by the following formula (3). [ka] (R9 represents a hydrogen atom or a methyl group. 10 The group (3) represents a substituted or unsubstituted phenyl group or naphthyl group. Substituents include methyl and ethyl groups. Formula (3) may have two or more structures. (Composition 15) The reagent according to any one of configurations 1 to 14, characterized in that the particles have a structure represented by the following formula (4). [ka] (R 11 (This indicates a functional group having one or more hydroxyl groups.) (Composition 16) The reagent according to any one of configurations 1 to 15, characterized in that the particles have a structure represented by the following formula (5). [ka] (R 12 This indicates a functional group having at least one carboxyl group. (Composition 17) The reagent according to any one of configurations 1 to 16, characterized in that the volume-average particle size of the particles is 200 nm or more and 500 nm or less. (Composition 18) The reagent according to any one of configurations 1 to 17, characterized in that the particles are a mixture of two types of particles with different volume-average particle sizes, and the difference in volume-average particle sizes between the two types of particles is 250 nm or less. (Composition 19) A reagent according to any one of configurations 1 to 18, characterized in that the absorbance measurement wavelength includes 500 nm to 750 nm. (Composition 20) This is a reagent for measuring the target substance contained in a sample. The target substance is a protein that forms a complex of hexamers or more. The reagent comprises a first reagent containing a buffer and a second reagent containing particles on which a ligand that specifically binds to the target substance is immobilized. A first mixing step to obtain a first mixed solution by mixing the sample containing 1000 ng / mL of the target substance with the first reagent in a volume ratio of sample:first reagent = 15:60. And when a second mixing step is performed, 280 seconds or more after the mixing in the first mixing step, to obtain a second mixed solution by mixing the first mixed solution and the second reagent in a volume ratio of first mixed solution:second reagent = 75:30, A reagent characterized in that the amount of change in absorbance of the second mixed solution becomes 0.15 or more within 50 seconds from the mixing in the second mixing step. (Composition 21) The reagent according to configuration 20, characterized in that the target substance is a protein with a molecular weight of 300 kDa or more and 600 kDa or less. (Composition 22) The reagent according to configuration 20 or 21, characterized in that the ligand is an antibody having an isoelectric point of 5.0 to 8.0. (Method 23) This is a measurement method for measuring target substances contained in a sample. The target substance is ferritin, A first mixing step to obtain a first mixed solution by mixing the sample and the first reagent, A second mixing step to obtain a second mixture by mixing the first mixture with a second reagent containing particles on which a ligand that specifically binds to the target substance is immobilized, The process includes a measurement step for measuring the absorbance of the second mixed solution, The sample contains the target substance at a concentration of 500 ng / mL or more and 1000 ng / mL or less. A measurement method using a reagent according to any one of configurations 1 to 22, wherein the change in absorbance of the second mixed solution within 50 seconds from the mixing in the second mixing step is 0.15 or more. (Method 24) The measurement method according to method 23, characterized in that the concentration of the sample in the second mixed solution is 7.0% by volume or more and 30.0% by volume or less. (Method 25) This is a measurement method for measuring target substances contained in a sample. The target substance is a protein that forms a complex of hexamers or more. A first mixing step to obtain a first mixed solution by mixing the sample and the first reagent, A second mixing step to obtain a second mixture by mixing the first mixture with a second reagent containing particles on which a ligand that specifically binds to the target substance is immobilized, The process includes a measurement step for measuring the absorbance of the second mixed solution, The sample contains the target substance at a concentration of 500 ng / mL or more and 1000 ng / mL or less. A measurement method using a reagent according to any one of configurations 1 to 22, wherein the change in absorbance of the second mixed solution within 50 seconds from the mixing in the second mixing step is 0.15 or more. (Method 26) The measurement method according to method 25, characterized in that the concentration of the sample in the second mixed solution is 7.0% by volume or more and 30.0% by volume or less.

Claims

1. This is a reagent for measuring target substances contained in a sample. The target substance is ferritin, The reagent comprises a first reagent containing a buffer and a second reagent containing particles on which a ligand that specifically binds to the target substance is immobilized. A first mixing step to obtain a first mixed solution by mixing the sample containing the target substance at a volume ratio of 1000 ng / mL with the first reagent at a volume ratio of sample:first reagent = 15:

60. And when a second mixing step is performed 280 seconds or more after the mixing in the first mixing step, to obtain a second mixed solution by mixing the first mixed solution and the second reagent in a volume ratio of first mixed solution:second reagent = 75:30, A reagent characterized in that the change in absorbance of the second mixed solution becomes 0.15 or more within 50 seconds from the mixing in the second mixing step.

2. The reagent according to claim 1, characterized in that the sample is selected from the group consisting of blood, serum, and plasma containing the target substance.

3. The reagent according to claim 1, characterized in that, after mixing the first mixture and the second reagent in a volume ratio of first mixture:second reagent = 75:30, the change in absorbance of the second mixture becomes 0.20 or more within 50 seconds.

4. The reagent according to claim 1, characterized in that, after mixing the first mixture and the second reagent in a volume ratio of first mixture:second reagent = 75:30, the change in absorbance of the second mixture between 50 seconds and 250 seconds is 0.20 or more.

5. The reagent according to claim 1, characterized in that the surface tension of the first reagent is 20 mN / m or more and 50 mN / m or less.

6. The reagent according to claim 1, characterized in that the first reagent and the second reagent each contain at least one of sugars and sugar alcohols.

7. The reagent according to claim 6, characterized in that the first reagent and the second reagent each contain one or more surfactants selected from the group consisting of sorbitan fatty acid esters, polyoxyethylene alkyl ethers, and polyoxyethylene phenyl ethers.

8. The reagent according to claim 1, characterized in that the electrical conductivity of the first reagent is 0.5 mS / cm or more and 70.0 mS / cm or less.

9. The reagent according to claim 1, characterized in that the zeta potential of the particles is -50 mV or more and -15 mV or less.

10. The reagent according to claim 1, characterized in that the particles contain a polymer having a structure represented by the following formula (1). 【Chemistry 1】 (R １ R represents a methyl group or a hydrogen atom. ２ (This indicates a group having either an epoxy group, a hydroxyl group, or a carboxyl group.)

11. The reagent according to claim 10, characterized in that the formula (1) has the structure shown by the following formula (1-A). 【Chemistry 2】 (R ３ , R ４ At least one of the elements represents a hydroxyl group, and the other represents a hydroxyl group or a group represented by formula (1-B). 【Transformation 3】 (R ５ represents a single bond or a methylene group. R ６ , R ７ , R ８ is selected from any of a hydrogen atom, a methyl group, a hydroxy group, a carboxy group, a hydroxymethyl group, or a carboxymethyl group, and at least one of them contains a hydroxy group or a carboxy group. Y １ represents a sulfur atom or an imino group. * １ indicates the bonding position with the structure represented by the formula (1-A). )

12. The reagent according to claim 1, characterized in that the particles have nitrogen-containing functional groups.

13. The reagent according to claim 1, characterized in that the particles contain a compound having an amidine group represented by the following formula (2). 【Chemistry 4】

14. The reagent according to claim 1, characterized in that the particles have a polymer containing repeating units represented by the following formula (3). 【Transformation 5】 (R ９ R represents a hydrogen atom or a methyl group. １０ The group (3) represents a substituted or unsubstituted phenyl group or naphthyl group. Substituents include methyl and ethyl groups. Formula (3) may have two or more structures.

15. The reagent according to claim 1, characterized in that the particles have a structure represented by the following formula (4). 【Transformation 6】 (R １１ (This indicates a functional group having one or more hydroxyl groups.)

16. The reagent according to claim 1, characterized in that the particles have a structure represented by the following formula (5). 【Transformation 7】 (R １２ This indicates a functional group having at least a carboxyl group.

17. The reagent according to claim 1, characterized in that the volume-average particle size of the particles is 200 nm or more and 500 nm or less.

18. The reagent according to claim 1, characterized in that the particles are a mixture of two types of particles with different volume-average particle sizes, and the difference in volume-average particle sizes between the two types of particles is 250 nm or less.

19. The reagent according to claim 1, characterized in that the absorbance measurement wavelength includes 500 nm to 750 nm.

20. This is a reagent for measuring the target substance contained in a sample. The target substance is a protein that forms a complex of hexamers or more. The reagent comprises a first reagent containing a buffer and a second reagent containing particles on which a ligand that specifically binds to the target substance is immobilized. A first mixing step to obtain a first mixed solution by mixing the sample containing the target substance at a volume ratio of 1000 ng / mL with the first reagent at a volume ratio of sample:first reagent = 15:

60. And when a second mixing step is performed 280 seconds or more after the mixing in the first mixing step, to obtain a second mixed solution by mixing the first mixed solution and the second reagent in a volume ratio of first mixed solution:second reagent = 75:30, A reagent characterized in that the change in absorbance of the second mixed solution becomes 0.15 or more within 50 seconds from the mixing in the second mixing step.

21. The reagent according to claim 20, characterized in that the target substance is a protein with a molecular weight of 300 kDa or more and 600 kDa or less.

22. The reagent according to claim 20, characterized in that the ligand is an antibody having an isoelectric point of 5.0 or higher and 8.0 or lower.

23. This is a measurement method for measuring target substances contained in a sample. The target substance is ferritin, A first mixing step to obtain a first mixed solution by mixing the sample and the first reagent, A second mixing step to obtain a second mixture by mixing the first mixture with a second reagent containing particles on which a ligand that specifically binds to the target substance is immobilized, The process includes a measurement step for measuring the absorbance of the second mixed solution, The sample contains the target substance at a concentration of 500 ng / mL or more and 1000 ng / mL or less. A measurement method using the reagent according to any one of claims 1 to 22, wherein the change in absorbance of the second mixed solution is 0.15 or more within 50 seconds from the mixing in the second mixing step.

24. The measurement method according to claim 23, characterized in that the concentration of the sample in the second mixed solution is 7.0% by volume or more and 30.0% by volume or less.

25. This is a measurement method for measuring target substances contained in a sample. The target substance is a protein that forms a complex of hexamers or more. A first mixing step to obtain a first mixed solution by mixing the sample and the first reagent, A second mixing step to obtain a second mixture by mixing the first mixture with a second reagent containing particles on which a ligand that specifically binds to the target substance is immobilized, The process includes a measurement step for measuring the absorbance of the second mixed solution, The sample contains the target substance at a concentration of 500 ng / mL or more and 1000 ng / mL or less. A measurement method using the reagent according to any one of claims 1 to 22, wherein the change in absorbance of the second mixed solution is 0.15 or more within 50 seconds from the mixing in the second mixing step.

26. The measurement method according to claim 25, characterized in that the concentration of the sample in the second mixed solution is 7.0% by volume or more and 30.0% by volume or less.

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