Method for measuring target substances, and reagents thereof.

Abstract

To provide a measurement method and reagents that improve the detection limit and offer high reproducibility at high concentrations. [Solution] A measurement method for measuring a target substance contained in a sample, wherein the target substance is a protein forming a complex of hexamers or more, comprising: a first mixing step of mixing the sample with a first reagent to obtain a first mixed solution; a second mixing step of mixing the first mixed solution with a second reagent containing particles on which ligands that specifically bind to the target substance are immobilized to obtain a second mixed solution; and a measurement step of measuring the absorbance of the second mixed solution, wherein the absorbance at a time of 35 seconds to 45 seconds after mixing in the second mixing step is 1.40 or higher, the magnitude of the change in absorbance with respect to concentration in the low concentration region of the target substance is large, and the magnitude of the change in absorbance with respect to concentration in the high concentration region is kept within a specified range.

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, an immunoturbidimetry method using particles can be mentioned. In this method, a dispersion of particles bound with a ligand having an 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, by using a device that optically detects this aggregation reaction as a change amount such as scattered light intensity, transmitted light intensity, absorbance, etc., the target substance can be qualitatively or quantitatively determined.

[0003] In an immunological measurement method by immunoturbidimetry using particles, a device for measuring the absorbance of a reaction solution is used. Since there is a maximum measurable absorbance value in this device, it is known that absorbance above this maximum value cannot be measured (Patent Document 1, Patent Document 2). On the other hand, specific absorbance ranges and conditions that can be stably measured have not been clearly defined.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0005] The immunoturbidimetry method using particles is a simple and highly rapid evaluation method, but the study for measuring a low-concentration target substance has not been sufficiently advanced. As a method for measuring a low-concentration target substance, it is common to use a chemiluminescent enzyme immunoassay method.

[0006] Chemiluminescent enzyme immunoassay is a method in which an antigen is reacted with an immobilized antibody, an enzyme-labeled antibody is used in a secondary reaction with the antigen, and a chemiluminescent substrate is added to measure the luminescence intensity. While chemiluminescent enzyme immunoassay can measure low concentrations of target substances, it is a more complex procedure than particle-based immunoturbidimetry, resulting in longer measurement times and higher costs. Therefore, there is a need to improve the detection sensitivity of particle-based immunoturbidimetry for even lower concentrations of target substances. [Means for solving the problem]

[0007] 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 involves mixing the sample with the first reagent to obtain a first mixed solution, 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 absorbance at a time of 35 seconds to 45 seconds after the mixing in the second mixing step is 1.40 or higher. The change in absorbance of sample A containing the target substance at Ang / mL is expressed as ΔODA. Let ΔODB(B>A) be the change in absorbance of sample B containing the target substance at Bng / mL. When (ΔODB-ΔODA) / (BA)=X, This invention relates to a measurement method in which, within the range where both A and B are between 0 and 100, there exists an X that is 0.00100 or greater, and within the range where both A and B are between 1000 and 1500, there exists an X that is 0.00003 or greater and 0.00010 or less.

[0008] 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. The reagent is obtained in a first mixing step, which involves mixing the sample and the first reagent in a volume ratio of sample:first reagent = 15:60 to obtain a first mixed solution. When the second mixing step involves mixing the first mixture and the second reagent in a volume ratio of first mixture:second reagent = 75:30 to obtain a second mixture, When using the sample containing 0 ng / mL of the target substance The absorbance of the second mixed liquid at a time of 35 seconds to 45 seconds after the mixing in the second mixing step is 1.40 or higher. When using the sample containing 0 ng / mL of the target substance In the second mixing step, the change in absorbance from 40 seconds to 300 seconds after mixing is defined as ΔOD(0). When using the sample containing 10 ng / mL of the target substance The change in absorbance from 40 seconds to 300 seconds after mixing in the second mixing step is defined as ΔOD(10). When using the sample containing 90 ng / mL of the target substance The change in absorbance between 40 seconds and 300 seconds after mixing in the second mixing step is defined as ΔOD(90). When using the sample containing 100 ng / mL of the target substance The change in absorbance from 40 seconds to 300 seconds after mixing in the second mixing step is defined as ΔOD(100). When using the sample containing 1000 ng / mL of the target substance The change in absorbance from 40 seconds to 300 seconds after mixing in the second mixing step is defined as ΔOD(1000). When using the sample containing 1100 ng / mL of the target substance The change in absorbance between 40 seconds and 300 seconds after mixing in the second mixing step is defined as ΔOD(1100). When using the sample containing 1400 ng / mL of the target substance The change in absorbance from 40 seconds to 300 seconds after the mixing in the second mixing step is defined as ΔOD(1400). When using the sample containing 1500 ng / mL of the target substance When the change in absorbance from 40 seconds to 300 seconds after the mixing in the second mixing step is defined as ΔOD(1500), (ΔOD(10) - ΔOD(0)) / (10 - 0) or (ΔOD(100) - ΔOD(90)) / (100 - 90) has a value of 0.00100 or more, (ΔOD(1100) - ΔOD(1000)) / (1100 - 1000) or (ΔOD(1500) - ΔOD(1400)) / (1500 - 1400) has a value of 0.00003 or more and 0.00010 or less. The present invention relates to a reagent characterized by this. [Advantages of the Invention]

[0009] According to the present disclosure, in the immunoturbidimetry using particles, a measurement method with improved detection limit and high reproducibility even at high concentrations, and a reagent thereof can be provided, with a protein of hexamer or more as the target substance. [Modes for Carrying Out the Invention]

[0010] 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 to which a ligand that specifically binds to a target substance is immobilized are hereinafter also referred to as affinity particles, and particles before a ligand that specifically binds to a target substance is immobilized are also referred to as pre-sensitized particles.

[0011] In response to the above problems, the present inventors have advanced studies to improve the detection sensitivity for low-concentration target substances in the immunoturbidimetry using particles. As a result, it has been found that especially when a protein that forms a complex of hexamer or more is used as the target substance, the higher the particle concentration contained in the reagent, the greater the change in absorbance, and it is excellent in the detection sensitivity for low-concentration target substances. In particular, it has been found that it is even more excellent when the target substance is ferritin that forms a complex of hexamer or more.

[0012] This is thought to be due to the number of ligand recognition sites of the target substance. In the case of proteins that form complexes only as monomers or pentamers or less, the number of ligand recognition sites is also small. Therefore, when the target substance and the reagent are mixed, the ligand recognition sites are easily consumed by reacting with the target substance. As a result, as the reaction progresses, the ligand recognition sites decrease, so even if the particle concentration in the reaction system is high, the amount of particle aggregation hardly changes. On the other hand, in the case of proteins that form complexes of hexamers or more, there are many ligand recognition sites. For example, ferritin is a complex of 24 proteins and can have 24 antibody recognition sites. Even if the reaction progresses, the ligand recognition sites tend to remain, and if the particle concentration in the reaction system is high, multi-particle aggregation in which multiple particles react with one target substance is likely to occur. As a result, it is considered that the higher the particle concentration contained in the reagent, the greater the change in absorbance, and the better the detection sensitivity for low-concentration target substances.

[0013] However, it has been found that when the particle concentration is increased in the measurement of proteins that form complexes of hexamers or more, the measurement reproducibility in the high-concentration region decreases. This is thought to be due to the fact that the measurement error becomes large because the measured absorbance rises too much when measuring high-concentration target substances. In particular, when a protein that forms a complex of hexamers or more is used as the target substance, it is considered that multi-particle aggregation via multiple target substances and multiple particles has occurred. Since the number of particles involved in the aggregation is not stable, and in addition, the measurement error is amplified due to the high measured absorbance, it is considered that this leads to a further decrease in measurement reproducibility.

[0014] Based on the above concept, when the reagent design was carried out to deliberately suppress the change in absorbance in the high-concentration region so as to be within a certain range, it was found that the high-concentration region could also be measured and highly reproducible measurements could be performed. These inventions are issues and design concepts that were first discovered by delving into the study of improving the detection sensitivity for low-concentration target substances using immunoturbidimetry using particles for the measurement of proteins that form complexes of hexamers or more, and can be said to be a unique design different from the conventional concept.

[0015] In immunoturbidimetric methods using particles, when targeting proteins with hexamers or more, by increasing the particle concentration above a certain level to enlarge the change in absorbance in the low-concentration range, and then keeping the change in absorbance at high concentrations within a certain range, it becomes possible to measure even in the high-concentration range, achieving highly reproducible measurements. As a result, it is believed that a wide concentration range from low to high concentrations can be measured with high reproducibility.

[0016] Based on the above considerations, this disclosure is intended to 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. The first mixing step involves mixing the sample with the first reagent to obtain the first mixture, A second mixing step involves mixing the 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 absorbance at a point 35 seconds to 45 seconds after mixing in the second mixing step is 1.40 or higher. The change in absorbance of sample A containing the target substance at Ang / mL is expressed as ΔODA. Let ΔODB(B>A) be the change in absorbance of sample B containing the target substance at a concentration of Bng / mL. When (ΔODB-ΔODA) / (BA)=X, This measurement method is characterized by the existence of an X that is 0.00100 or greater in the range where both A and B are between 0 and 100, and an X that is 0.00003 or greater and 0.00010 or less in the range where both A and B are between 1000 and 1500.

[0017] In this context, the absorbance change refers to the change in absorbance of the second mixed solution from the mixing step to the measurement step.

[0018] 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. Furthermore, "from mixing" in this embodiment 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 absorbance at a time of 35 seconds to 45 seconds after mixing is 1.40 or higher means that there are cases in which the absorbance measured at a time of 35 seconds to 45 seconds after the time when the stirring is completed is 1.40 or higher. The same applies to "what mixing is" and other mixing in this specification.

[0019] Hereafter, X will also be referred to as the magnitude of the change in absorbance with respect to concentration.

[0020] An absorbance of 1.40 or higher in the second mixed solution at a time between 35 and 45 seconds after mixing in the second mixing step indicates a high particle concentration. In this case, excellent detection sensitivity can be obtained when a protein with hexamers or more is used as the target substance. On the other hand, if this measured absorbance is low, it can be said that the particle concentration is low, so the problems described in this disclosure do not occur, and even when the absorbance changes significantly due to sufficient reaction with a high-concentration sample, the measured absorbance does not rise too high.

[0021] Furthermore, the existence of an X value greater than or equal to 0.00100 within the range where both A and B are between 0 and 100 indicates that the magnitude of the change in absorbance with respect to concentration is large in the low-concentration region. Therefore, satisfying the above conditions makes it possible to measure the low-concentration region with higher sensitivity.

[0022] Furthermore, the existence of an X of 0.00003 or greater in the range where both A and B are between 1000 and 1500 means that the magnitude of the change in absorbance with respect to concentration is above a certain level in the high-concentration region. Also, the existence of an X of 0.00010 or less in the range where both A and B are between 1000 and 1500 means that the change in absorbance at high concentrations is suppressed, and that even after sufficient reaction during measurement in the high-concentration region, the measured absorbance does not rise too high. As a result, the measurement error is reduced, and results with excellent measurement reproducibility are obtained. In this disclosure, ΔODA < ΔODB is always true in the range where the difference between A and B is 100 or greater in the range where both A and B are between 1000 and 1500.

[0023] <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.

[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 mixed, and then the temperature is adjusted to a predetermined temperature and maintained 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 mixed, 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. Preferably, the measurement wavelength for absorbance is between 500 nm and 750 nm. Alternatively, this measurement wavelength may be combined with another wavelength. Including wavelengths within this range increases the change in absorbance even for low concentrations of the target substance, improving low-concentration sensitivity. Furthermore, the high accuracy of absorbance measurement results in excellent measurement reproducibility. In this embodiment, the change in absorbance ΔOD may be calculated from the difference between the absorbance measured between 15 seconds and 45 seconds after mixing in the second mixing step, and the absorbance measured between 250 seconds and 300 seconds after mixing in the second mixing step.

[0027] The concentration of affinity particles in the second mixture is preferably between 0.02 mass / volume% and 0.10 mass / volume%. A concentration of 0.02 mass / volume% or higher allows for a large change in absorbance at low concentrations for proteins with hexamers or more. Furthermore, a concentration of 0.10 mass / volume% or lower reduces the number of particles that do not participate in aggregation, resulting in lower measured absorbance and less error in absorbance measurement, thus providing excellent measurement reproducibility.

[0028] It is preferable that the concentration of the sample in the second mixture is between 7.0% by volume and 30.0% by volume. If the concentration of the sample in the second mixture is less than 7.0% by volume, sufficient detection sensitivity cannot be obtained. On the other hand, if it is greater than 30.0% by volume, the aggregation of particles will be affected by components other than the target substance contained in the sample, resulting in poor measurement reproducibility.

[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 that forms a complex of 6 or more units. Preferably, it is a protein with a molecular weight of 300 kDa or more and 600 kDa or less. A molecular weight of 300 kDa or more allows for easier reaction with antibodies on the particle surface, resulting in a stable agglutination reaction. A molecular weight of 600 kDa or less increases the number of antigens per unit concentration, leading to an increase in the number of agglutinated particles and a stable change in measured absorbance. Therefore, the effects of this disclosure are easily obtained. Ferritin, exemplified in this disclosure, forms a complex of 6 or more units and has a molecular weight of 445 kDa, making it more preferable. Furthermore, the target substance in this disclosure is preferably a substance that has multiple reaction sites with respect to antibodies (ligands) that specifically bind to the target substance, and from this viewpoint, ferritin is also suitable.

[0031] <First reagent and second reagent> The immunoturbidimetric reagent using particles used in the measurement method of this disclosure comprises a first reagent containing a buffer and a second reagent containing affinity particles on which ligands that specifically bind to the target substance are immobilized. 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] In this embodiment, When the first mixing step involves mixing the sample and the first reagent in a volume ratio of sample:first reagent = 15:60 to obtain the first mixed solution, and the second mixing step involves mixing the first mixed solution and the second reagent in a volume ratio of first mixed solution:second reagent = 75:30 to obtain the second mixed solution, When using a sample containing 0 ng / mL of the target substance The absorbance of the second mixed solution at a point 35 to 45 seconds after mixing in the second mixing step is 1.40 or higher. When using a sample containing 0 ng / mL of the target substance In the second mixing step, the change in absorbance from 40 seconds to 300 seconds after mixing is defined as ΔOD(0). When using a sample containing 10 ng / mL of the target substance The change in absorbance from 40 seconds to 300 seconds after mixing in the second mixing step is defined as ΔOD(10). When using a sample containing 90 ng / mL of the target substance The change in absorbance from 40 seconds to 300 seconds after mixing in the second mixing step is defined as ΔOD(90). When using a sample containing 100 ng / mL of the target substance The change in absorbance from 40 seconds to 300 seconds after mixing in the second mixing step is defined as ΔOD(100). When using a sample containing 1000 ng / mL of the target substance The change in absorbance from 40 seconds to 300 seconds after mixing in the second mixing step is defined as ΔOD(1000). When using a sample containing 1100 ng / mL of the target substance The change in absorbance from 40 seconds to 300 seconds after mixing in the second mixing step is defined as ΔOD(1100). When using a sample containing 1400 ng / mL of the target substance The change in absorbance from 40 seconds to 300 seconds after mixing in the second mixing step is defined as ΔOD(1400). When using a sample containing 1500 ng / mL of the target substance When the change in absorbance from 40 seconds to 300 seconds after mixing in the second mixing step is defined as ΔOD(1500), (ΔOD(10)-ΔOD(0)) / (10-0) or The value of (ΔOD(100)-ΔOD(90)) / (100-90) is 0.00100 or greater. (ΔOD(1100)-ΔOD(1000)) / (1100-1000) or This reagent has a value of (ΔOD(1500)-ΔOD(1400)) / (1500-1400) that is between 0.00003 and 0.00010.

[0033] 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, resulting in superior measurement accuracy. The pH values ​​of the first and second reagents may be different.

[0034] 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.

[0035] 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.

[0036] The first and second reagents preferably further contain a surfactant. 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.

[0037] 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.

[0038] 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.

[0039] <Affinity particles and pre-sensitized particles> The volume-average particle size of the affinity particles in this disclosure is preferably 200 nm or more and 500 nm or less. A volume-average particle size of 200 nm or more reduces the number of aggregated particles required per unit change in absorbance, resulting in a more uniform effect of the sample components on the affinity particles. Furthermore, a volume-average particle size of 500 nm or less reduces the amount of absorbance change caused by aggregation per particle, thus reducing fluctuations in absorbance change due to fluctuations in aggregation. Therefore, excellent measurement reproducibility can be obtained.

[0040] Furthermore, the second reagent of this disclosure preferably contains two types of affinity particles with different volume-average particle sizes, and the difference in volume-average particle sizes between the two types of affinity particles is 250 nm or less. By including two types of affinity particles with different volume-average particle sizes, a wider range of target substance concentrations can be measured. In this case, having a particle size difference of 250 nm or less makes it less likely for the reaction between the target substance and the affinity particles to be biased, and even if there is a slight bias, the difference in absorbance change is less likely to occur, thus providing excellent measurement reproducibility across a wide range of target substance concentrations.

[0041] In this disclosure, when two types of affinity particles are used, the larger one is referred to as the large affinity particle and the smaller one as the small affinity particle. It is preferable that the volume-average particle size of the large affinity particle is 350 nm or more and 500 nm or less. When the large affinity particle is 350 nm or larger, the amount of absorbance change due to aggregation of the large affinity particle becomes large when the target substance is at a low concentration, which improves low-concentration sensitivity. When it is 500 nm or less, the amount of absorbance change per particle becomes small, so the fluctuation in the amount of absorbance change that occurs when the degree of aggregation fluctuates becomes small, and excellent measurement reproducibility is obtained.

[0042] Furthermore, it is preferable that the volume-average particle size of the affinity particles is between 200 nm and 300 nm. When the affinity particles are 200 nm or larger, they are of a size that reacts easily with the size of the target substance, resulting in a stable reaction, reduced fluctuation in absorbance change, and excellent measurement reproducibility. When the particle size is 300 nm or smaller, multiple target substances do not react with a single particle when the target substance is at a high concentration, resulting in a stable absorbance change and excellent measurement reproducibility.

[0043] The zeta potential of the affinity small particles in this disclosure is preferably between -40.0 mV and -25.0 mV, and the zeta potential of the affinity large particles is preferably between -35.0 mV and -15.0 mV. Furthermore, it is preferable that the zeta potential of the affinity small particles is smaller than that of the affinity large particles. When the zeta potential is within the above range, the balance between the electrostatic repulsion and electrostatic attraction of the affinity particles becomes appropriate. As a result, the dispersion of the affinity particles is stabilized, and excellent measurement reproducibility can be obtained when the target substance is at a high concentration.

[0044] In the second reagent of this disclosure, the particle concentration ratio of large affinity particles to small affinity particles is preferably 0.5 times or more and 2.0 times or less. When the particle concentration ratio is within the above range, there is no large difference in particle concentration between large affinity particles and small affinity particles, so the possibility of the target substance reacting unevenly is reduced, the reaction is stable and measurement reproducibility is excellent.

[0045] In the mixed system of the two types of affinity particles of this disclosure, it is preferable that the ratio of the refractive index of the small affinity particles to the refractive index of the large affinity particles is 0.87 or more and 0.97 or less. Having a difference in refractive index between the two types of affinity particles within the above range enables highly sensitive measurements from lower to higher concentrations.

[0046] 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.)

[0047] 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 at least one of them contains a hydroxyl group or a carboxyl group. Y1 represents a sulfur atom or an imino group. *1 indicates the bond position with the structural formula shown in formula (1-A).)

[0048] 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]

[0049] <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.

[0050] 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.

[0051] 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.

[0052] 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.

[0053] Furthermore, the amount of ligand per 1.0 mg of unsensitized particles in the second reagent is preferably between 10.0 μg / mg and 40.0 μg / mg. A ligand concentration of 10.0 μg / mg or higher stabilizes the aggregation reaction between the target substance and the particles, while a ligand concentration of 40.0 μg / mg or lower limits multi-particle aggregation involving multiple target substances and multiple particles in the high-concentration range, resulting in excellent measurement reproducibility.

[0054] Furthermore, it is preferable that the amount of ligand in large affinity particles relative to 1.0 mg of pre-sensitized particles is 4.0 to 20.0 times the amount of ligand in small affinity particles relative to 1.0 mg of pre-sensitized particles. When the ratio of ligand amount to 1.0 mg of pre-sensitized particles is 4.0 times or more, the large affinity particles have higher reactivity with the target substance than the small affinity particles. Therefore, when mixed with low concentrations of the target substance, the large affinity particles react predominantly, resulting in a larger change in absorbance. When the ratio of ligand amount to 1.0 mg of pre-sensitized particles is 20.0 times or less, the reactivity of small affinity particles with the target substance can be maintained. For these reasons, when the ratio of ligand amount to 1.0 mg of pre-sensitized particles is within the above range, excellent detection sensitivity and measurement reproducibility are achieved for both low and high concentrations of the target substance.

[0055] An example of a method for measuring physical properties in this disclosure is described below.

[0056] <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.

[0057] <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.

[0058] 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.

[0059] <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.

[0060] 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 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 of 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. The ligand amount can be determined using a similar procedure.

[0061] <Method for measuring the refractive index of particles> The refractive index of the particles is measured using an Abbemat (Anton Paar). In this disclosure, the refractive index is measured when the particle dispersion is dispersed to a mass / volume of 5%. The measurement conditions are 25°C and a measurement wavelength of 589.3 nm. The particle refractive index is 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. [Examples]

[0062] The present disclosure will be described in detail below with reference to examples, but the present disclosure is not limited to these examples.

[0063] (Synthesis of particle 1) The synthesis of particle 1 involves the following steps 1 to 3.

[0064] (Step 1) 85.89 g of styrene (St: Kishida Chemical Co., Ltd.), 1.56 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 3.72 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.

[0065] (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.

[0066] (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 420 nm.

[0067] (Synthesis of particles 2 to 9 and commercially available particles) Particles 2 through 9 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, and the amount of GMA in step 2 were changed as shown in Table 1. These particles have a structure on their surface derived from GMA, as shown in formula (1). In addition, polystyrene particles (JSR Life Sciences, carboxy-modified type) IMMUTEX P0307 were prepared as particle 10, and polystyrene particles (JSR Life Sciences, carboxy-modified type) IMMUTEX 0113 were prepared as particle 11. The physical properties of the obtained particles 1 through 9, as well as particles 10 and 11, are summarized in Table 1.

[0068] In all of particles 1 to 9, 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).)

[0069] [Table 1]

[0070] (Fabrication of Affinity Large 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).

[0071] 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.

[0072] To this, 30 μL of a 5.0 mg / mL dispersion of mouse monoclonal anti-ferritin antibody (isoelectric point 7.1) (0.15 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 mol / L of a 1 mol / L trishydroxymethylaminomethane (Tris) solution at pH 8.0 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 large particle 1.

[0073] (Production of affinity small particle 15 from affinity large particle 2) Affinity small particles 15 were prepared from affinity large particles 2 using the same experimental procedure as for affinity large particles 1, except that the particle type and the amount of antibody dispersion added were changed as shown in Table 2. The amount of antibody added per 1.0 mg of pre-sensitized particles and the zeta potential of the obtained affinity large particles 1 and affinity small particles 15 are summarized in Table 2.

[0074] [Table 2]

[0075] (Preparation of Reagent 1) Second reagent 1 was prepared using the prepared affinity large particles 1 and affinity small particles 8. Specifically, affinity large particles 1 and affinity small particles 8 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 5.0% by mass sucrose (viscosity modifier) ​​dissolved in deionized water. Subsequently, the particles were mixed and diluted with HEPES buffer so that affinity large particles 1 amounted to 0.075% by mass / volume, affinity small particles 8 amounted to 0.078% by mass / volume, and the total amounted to 0.153% by mass / volume, to obtain second reagent 1.

[0076] (Preparation of Reagent 2 to Reagent 13) Second reagents 2 through 14 were prepared using the same experimental procedure as for second reagent 1, except that the type of affinity particles and the concentration of affinity particles were changed as shown in Table 3. The physical properties of the obtained second reagents 1 through 14 are summarized in Table 3.

[0077] [Table 3]

[0078] (Preparation of the first reagent) The first reagent was prepared by mixing HEPES, TritonX-100, and NaCl using deionized water to achieve concentrations of 50 mM HEPES, 0.10% by mass of TritonX-100, and 0.90% by mass of NaCl, respectively.

[0079] (Magnitude of change in absorbance with respect to the concentration of low-concentration and high-concentration standard solutions) A BIOSPECTROMETER spectrophotometer (Eppendorf Co., Ltd.) was used for measurement, with a wavelength of 572 nm. A mixture was prepared by mixing 15 μL of the sample with 60 μL of the first reagent 1, and the mixture was incubated at 37°C for 290 seconds. Next, 30 μL of the second reagent 1 was mixed into the mixture, stirred, and the absorbance was measured after 42 seconds. Furthermore, this mixture was allowed to stand at 37°C for 253 seconds, and the absorbance was measured again. The difference from the absorbance measured after 42 seconds was defined as the change in absorbance.

[0080] Standard solutions with ferritin concentrations of 0, 10, 90, 100, 1000, 1100, 1400, and 1500 ng / mL were prepared. The change in absorbance was measured for each of these standard solutions, and the absorbance change ΔOD was calculated.

[0081] Here, when the change in absorbance of standard solution A containing ferritin Ang / mL is denoted as ΔODA, and the change in absorbance of standard solution B containing ferritin B ng / mL is denoted as ΔODB(B>A) and (ΔODB-ΔODA) / (BA)=X[AB], the magnitude of the change in absorbance with respect to the concentration of the low-concentration standard solution was calculated as X[0-10] and X[90-100], and the magnitude of the change in absorbance with respect to the concentration of the high-concentration standard solution was calculated as X[1000-1100] and X[1400-1500]. These experiments were performed for each second reagent used in each example and comparative example. Furthermore, in the measurement of the 0 ng / mL ferritin standard solution, the absorbance measured 42 seconds after mixing with the second reagent and stirring was taken as the initial absorbance of 0 ng / mL. These results are summarized in Table 4.

[0082] [Table 4]

[0083] (Method for evaluating the average value of absorbance change ΔOD in low-ferritin concentration samples) A discrete clinical chemistry automated analyzer TBA-120FR (Canon Medical Systems Corporation) was used as the measuring instrument. Although the optical path length of this instrument is 5 mm, the absorbance obtained as the measurement value is converted to the value for an optical path length of 1 cm. PBS (without ferritin) and sample C containing 9.8 ng / mL of ferritin were used as the samples. The assay parameters were 15 μL of sample, 60 μL of the first reagent 1, 30 μL of the second reagent, and a measurement wavelength of 572 nm. The absorbance change ΔOD was calculated by subtracting the absorbance OD(33) from the absorbance OD(33) at photometric point 19 (approximately 40 seconds after mixing the second reagent) and the absorbance OD(33) at photometric point 33 (approximately 300 seconds after mixing the second reagent). Each measurement was performed 20 times, and the average value of the absorbance change ΔOD for PBS and the average value of the absorbance change ΔOD for sample C were calculated. The value obtained by subtracting the average value of the absorbance change ΔOD when using physiological saline from the average value of the absorbance change ΔOD when using sample C was taken as the average value of the absorbance change ΔOD for 9.8 ng / mL.

[0084] The average value of the absorbance change ΔOD for 9.8 ng / mL obtained in this way was evaluated according to the following criteria, and the results are summarized in Table 5 below. A: ΔOD average is 0.00160 or higher B: ΔOD mean is between 0.00130 and 0.00160 C: ΔOD mean is between 0.00100 and less than 0.00130 D: ΔOD mean is between 0.00080 and 0.00100 E: ΔOD mean is less than 0.00080

[0085] (Method for evaluating the reproducibility of measurements in ferritin-low concentration samples) A discrete clinical chemistry automated analyzer TBA-120FR (Canon Medical Systems Corporation) was used as the measuring instrument. For sample C containing 9.8 ng / mL of ferritin, the assay parameters were set to sample: 15 μL, first reagent 1: 60 μL, second reagent: 30 μL, and measurement wavelength: 572 nm. 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. This measurement was performed 20 times, and the standard deviation (hereinafter SD) of the absorbance change ΔOD was calculated.

[0086] The standard deviation (SD) of the absorbance change ΔOD for sample C, which thus contained 9.8 ng / mL, was evaluated according to the following criteria, and the results are summarized in Table 5 below. A: SD is less than 3.0 B: SD is 3.0 or higher but less than 4.0 C:SD is between 4.0 and 5.0 D:SD is 5.0 or higher but less than 7.0 E:SD is between 7.0 and 10.0 F:SD is 10.0 or higher

[0087] (Creating a calibration curve) A discrete clinical chemistry automated analyzer TBA-120FR (Canon Medical Systems Corporation) was used as the measuring instrument. First, standard solutions with ferritin concentrations of 0, 100, 250, 500, 1000, 1500, and 2000 ng / mL were prepared. These standard solutions were used as samples, and measurements were performed under the following assay parameters: sample: 15 μL, first reagent 1: 60 μL, second reagent: 30 μL, measurement wavelength: 572 nm. 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. 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.

[0088] (Method for evaluating the reproducibility of measurements in ferritin-high concentration samples) A discrete clinical chemistry automated analyzer TBA-120FR (Canon Medical Systems Corporation) was used as the measuring instrument. For sample D containing 1515.0 ng / mL of ferritin, the assay parameters were set to: sample: 15 μL, first reagent 1: 60 μL, second reagent: 30 μL, and measurement wavelength: 572 nm. The absorbance change ΔOD was calculated as the difference between the absorbance OD(19) at photometric point 19 and the absorbance OD(33) at photometric point 33, OD(33)-OD(19). This measurement was performed 20 times, and the ferritin concentration was quantified using a pre-prepared calibration curve, and the standard deviation (SD) of the obtained ferritin-equivalent concentration was calculated.

[0089] The standard deviation (SD) of the ferritin-equivalent concentration for sample D, which thus had a concentration of 1515.0 ng / mL, was evaluated according to the following criteria, and the results are summarized in Table 5 below. A: SD is less than 15.0 B: SD is between 15.0 and 18.0 C:SD is between 18.0 and 21.0 D:SD is between 21.0 and 24.0 E:SD is between 24.0 and 27.0 F:SD is 27.0 or higher

[0090] [Table 5]

[0091] These results indicate that reagents meeting the requirements of this disclosure provide excellent detection limits and highly reproducible measurements even in high-concentration ranges.

[0092] 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, have multiple ligand recognition sites. Therefore, even if the particle aggregation reaction progresses, the ligand recognition sites tend to remain, and if the particle concentration in the reaction system is high, multi-particle aggregation, in which multiple particles react with a single target substance, is likely to occur. From this perspective, the measurement method of this disclosure can be said to target substances that have multiple reaction sites with respect to ligands that specifically bind to the target substance.

[0093] The disclosure of this embodiment includes the following configurations or methods. (Method 1) 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 involves mixing the sample with the first reagent to obtain a first mixed solution, 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 absorbance at a time of 35 seconds to 45 seconds after the mixing in the second mixing step is 1.40 or higher. The change in absorbance of sample A containing the target substance at Ang / mL is expressed as ΔODA. Let ΔODB(B>A) be the change in absorbance of sample B containing the target substance at Bng / mL. When (ΔODB-ΔODA) / (BA)=X, A measurement method characterized in that, in the range where both A and B are between 0 and 100, there exists an X that is 0.00100 or greater, and in the range where both A and B are between 1000 and 1500, there exists an X that is 0.00003 or greater and 0.00010 or less. (Method 2) The measurement method according to Method 1, characterized in that the sample is selected from the group consisting of blood, serum, and plasma containing the target substance. (Method 3) The measurement method according to method 1 or 2, characterized in that the molecular weight of the target substance is 300 kDa or more and 600 kDa or less. (Method 4) A measurement method according to any one of methods 1 to 3, characterized in that the target substance is ferritin. (Method 5) A measurement method according to any one of methods 1 to 4, characterized in that the volume-average particle size of the particles is 200 nm or more and 500 nm or less. (Method 6) The measurement method according to any one of methods 1 to 5, 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. (Method 7) The measurement method according to any one of methods 1 to 6, wherein the particle is a mixture of two types of particles with different volume-average particle sizes, and the volume-average particle size of the particle with the larger volume-average particle size is 350 nm or more and 500 nm or less. (Method 8) The measurement method according to any one of methods 1 to 7, wherein the particle is a mixture of two types of particles with different volume-average particle sizes, and the volume-average particle size of the particle with the smaller volume-average particle size is 200 nm or more and 300 nm or less. (Method 9) The measurement method according to any one of methods 1 to 8, characterized in that the concentration of the particles in the second mixed solution is 0.02 mass / volume% or more and 0.10 mass / volume% or less. (Method 10) The measurement method according to any one of methods 1 to 9, characterized in that the particles are a mixture of two types of particles with different volume-average particle sizes, and the concentration of the particles with larger volume-average particle size in the second reagent is 0.5 times or more and 2.0 times or less than the concentration of the particles with smaller volume-average particle size. (Method 11) The measurement method according to any one of methods 1 to 10, characterized in that the amount of ligand in the second reagent per 1.0 mg of pre-sensitized particles from which the ligand has been removed is 10.0 μg / mg or more and 40.0 μg / mg or less. (Method 12) The measurement method according to Method 11, characterized in that the particles are a mixture of two types of particles with different volume-average particle sizes, and the amount of ligand per 1.0 mg of the pre-sensitization particles with larger volume-average particle size is 4.0 times or more and 20.0 times or less the amount of ligand per 1.0 mg of the pre-sensitization particles with smaller volume-average particle size. (Method 13) The measurement method according to any one of methods 1 to 12, characterized in that the ligand is an antibody having an isoelectric point of 5.0 or higher and 8.0 or lower. (Method 14) The measurement method according to any one of methods 1 to 13, characterized in that the particles are a mixture of two types of particles with different volume-average particle sizes, and the ratio of the refractive index of the larger volume-average particle size to the smaller volume-average particle size is 0.87 or more and 0.97 or less. (Method 15) The measurement method according to any one of methods 1 to 14, characterized in that the particles are a mixture of two types of particles with different volume-average particle sizes, and the zeta potential of the particle with the larger volume-average particle size is -35.0 mV or more and -15.0 mV or less. (Method 16) The measurement method according to methods 1 to 15, characterized in that the particles are a mixture of two types of particles with different volume-average particle sizes, and the zeta potential of the particle with the smaller volume-average particle size is -40.0 mV or more and -25.0 mV or less. (Method 17) A measurement method according to any one of methods 1 to 16, characterized in that the particles contain a polymer having a structure 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.) (Method 18) The measurement method according to method 17, 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).) (Method 19) A measurement method according to any one of methods 1 to 18, characterized in that the measurement wavelength of the absorbance includes 500 nm or more and 750 nm or less. (Method 20) The measurement method according to any one of methods 1 to 19, characterized in that, in the measurement step, the absorbance change amount ΔOD is calculated from the difference between the absorbance in the second mixing step within a range of 15 seconds to 45 seconds after the mixing and the absorbance in the second mixing step within a range of 250 seconds to 300 seconds after the mixing. (Method 21) The measurement method according to any one of methods 1 to 20, 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 22) The measurement method according to any one of methods 1 to 21, characterized in that the first reagent and the second reagent contain at least one of sugars and sugar alcohols. (Method 23) The measurement method according to any one of methods 1 to 22, 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. (Method 24) The measurement method according to any one of methods 1 to 23, characterized in that the target substance is a substance having multiple reaction sites with respect to a ligand that specifically binds to the target substance. (Composition 25) 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. The reagent is obtained in a first mixing step, which involves mixing the sample and the first reagent in a volume ratio of sample:first reagent = 15:60 to obtain a first mixed solution. When the second mixing step involves mixing the first mixture and the second reagent in a volume ratio of first mixture:second reagent = 75:30 to obtain a second mixture, When using the sample containing 0 ng / mL of the target substance The absorbance of the second mixed liquid at a time of 35 seconds to 45 seconds after the mixing in the second mixing step is 1.40 or higher. When using the sample containing 0 ng / mL of the target substance In the second mixing step, the change in absorbance from 40 seconds to 300 seconds after mixing is defined as ΔOD(0). When using the sample containing 10 ng / mL of the target substance The change in absorbance from 40 seconds to 300 seconds after mixing in the second mixing step is defined as ΔOD(10). When using the sample containing 90 ng / mL of the target substance The change in absorbance between 40 seconds and 300 seconds after mixing in the second mixing step is defined as ΔOD(90). When using the sample containing 100 ng / mL of the target substance The change in absorbance from 40 seconds to 300 seconds after mixing in the second mixing step is defined as ΔOD(100). When using the sample containing 1000 ng / mL of the target substance The change in absorbance from 40 seconds to 300 seconds after mixing in the second mixing step is defined as ΔOD(1000). When using the sample containing 1100 ng / mL of the target substance The change in absorbance between 40 seconds and 300 seconds after mixing in the second mixing step is defined as ΔOD(1100). When using the sample containing 1400 ng / mL of the target substance The change in absorbance from 40 seconds to 300 seconds after mixing in the second mixing step is defined as ΔOD(1400). When using the sample containing 1500 ng / mL of the target substance When the change in absorbance from 40 seconds to 300 seconds after mixing in the second mixing step is defined as ΔOD(1500), (ΔOD(10)-ΔOD(0)) / (10-0) or The value of (ΔOD(100)-ΔOD(90)) / (100-90) is 0.00100 or greater. (ΔOD(1100)-ΔOD(1000)) / (1100-1000) or A reagent used in the measurement method described in any one of methods 1 to 24, characterized in that the value of (ΔOD(1500)-ΔOD(1400)) / (1500-1400) is 0.00003 or more and 0.00010 or less.

Claims

1. 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 involves mixing the sample with the first reagent to obtain a first mixed solution, 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 absorbance at a time of 35 seconds to 45 seconds after the mixing in the second mixing step is 1.40 or higher. The change in absorbance of sample A containing the target substance at Ang / mL is expressed as ΔODA. Let ΔODB (B > A) be the change in absorbance of sample B containing the target substance at Bng / mL. When (ΔODB - ΔODA) / (B - A) = X, A measurement method characterized in that, in the range where both A and B are between 0 and 100, there exists an X that is 0.00100 or greater, and in the range where both A and B are between 1000 and 1500, there exists an X that is 0.00003 or greater and 0.00010 or less.

2. The measurement method 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 measurement method according to claim 1, characterized in that the molecular weight of the target substance is 300 kDa or more and 600 kDa or less.

4. The measurement method according to claim 1, characterized in that the target substance is ferritin.

5. The measurement method 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.

6. The measurement method 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.

7. The measurement method according to claim 1, wherein the particles are a mixture of two types of particles with different volume-average particle sizes, and the volume-average particle size of the larger particle is 350 nm or more and 500 nm or less.

8. The measurement method according to claim 1, wherein the particles are a mixture of two types of particles with different volume-average particle sizes, and the volume-average particle size of the smaller particle is 200 nm or more and 300 nm or less.

9. The measurement method according to claim 1, characterized in that the concentration of the particles in the second mixed solution is 0.02 mass / volume% or more and 0.10 mass / volume% or less.

10. The measurement method 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 concentration of the particles with larger volume-average particle size in the second reagent is 0.5 times or more and 2.0 times or less than the concentration of the particles with smaller volume-average particle size.

11. The measurement method according to claim 1, characterized in that the amount of ligand in the second reagent per 1.0 mg of pre-sensitized particles from which the ligand has been removed is 10.0 μg / mg or more and 40.0 μg / mg or less.

12. The measurement method according to claim 11, characterized in that the particles are a mixture of two types of particles with different volume-average particle sizes, and the amount of ligand per 1.0 mg of the pre-sensitized particles with a larger volume-average particle size is 4.0 times or more and 20.0 times or less the amount of ligand per 1.0 mg of the pre-sensitized particles with a smaller volume-average particle size.

13. The measurement method according to claim 1, characterized in that the ligand is an antibody having an isoelectric point of 5.0 or higher and 8.0 or lower.

14. The measurement method 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 ratio of the refractive index of the particle with the larger volume-average particle size to the particle with the smaller volume-average particle size is 0.87 or more and 0.97 or less.

15. The measurement method 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 zeta potential of the particle with the larger volume-average particle size is -35.0 mV or more and -15.0 mV or less.

16. The measurement method 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 zeta potential of the particle with the smaller volume-average particle size is -40.0 mV or more and -25.0 mV or less.

17. The measurement method 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.)

18. The measurement method according to claim 17, characterized in that 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). )

19. The measurement method according to claim 1, characterized in that the measurement wavelength of the absorbance includes 500 nm or more and 750 nm or less.

20. The measurement method according to claim 1, characterized in that, in the measurement step, the absorbance change amount ΔOD is calculated from the difference between the absorbance in the second mixing step within a range of 15 seconds to 45 seconds after the mixing and the absorbance in the second mixing step within a range of 250 seconds to 300 seconds after the mixing.

21. The measurement method according to claim 1, 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.

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

23. The measurement method according to claim 1, 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.

24. The measurement method according to claim 1, characterized in that the target substance is a substance having multiple reaction sites with respect to a ligand that specifically binds to the target substance.

25. 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. The reagent is obtained in a first mixing step, which involves mixing the sample and the first reagent in a volume ratio of sample:first reagent = 15:60 to obtain a first mixed solution. When the second mixing step involves mixing the first mixture and the second reagent in a volume ratio of first mixture:second reagent = 75:30 to obtain a second mixture, When using the sample containing 0 ng / mL of the target substance The absorbance of the second mixed solution at a time of 35 seconds to 45 seconds after mixing in the second mixing step is 1.40 or higher. When using the sample containing 0 ng / mL of the target substance In the second mixing step, the change in absorbance from 40 seconds to 300 seconds after mixing is ΔOD(0). When using the sample containing 10 ng / mL of the target substance In the second mixing step, the change in absorbance from 40 seconds to 300 seconds after mixing is ΔOD(10). When using the sample containing 90 ng / mL of the target substance In the second mixing step, the change in absorbance from 40 seconds to 300 seconds after mixing is ΔOD(90). When using the sample containing 100 ng / mL of the target substance The change in absorbance between 40 seconds and 300 seconds after the mixing in the second mixing step is defined as ΔOD(100). When using the sample containing 1000 ng / mL of the target substance The change in absorbance between 40 seconds and 300 seconds after mixing in the second mixing step is defined as ΔOD(1000). When using the sample containing the target substance at a concentration of 1100 ng / mL The change in absorbance between 40 seconds and 300 seconds after the mixing in the second mixing step is defined as ΔOD(1100). When using the sample containing the target substance at a concentration of 1400 ng / mL The change in absorbance between 40 seconds and 300 seconds after the mixing in the second mixing step is defined as ΔOD(1400). When using the sample containing the target substance at a concentration of 1500 ng / mL When the change in absorbance from 40 seconds to 300 seconds after the mixing in the second mixing step is defined as ΔOD(1500), (ΔOD(10) - ΔOD(0)) / (10 - 0) or The value of (ΔOD(100) - ΔOD(90)) / (100 - 90) is 0.00100 or greater. (ΔOD(1100) - ΔOD(1000)) / (1100 - 1000) or A reagent used in the measurement method according to any one of claims 1 to 24, characterized in that the value of (ΔOD(1500) - ΔOD(1400)) / (1500 - 1400) is 0.00003 or more and 0.00010 or less.

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