Testing tool, blood cell separation method, immunological measurement method, hemolysis inhibitor, and kit

Abstract

This testing tool includes at least a flow channel through which a sample containing red blood cells and a substance to be measured is circulated. The flow channel includes at least a sample supply part to which the sample is supplied, a blood cell separation membrane for separating the red blood cells from the sample, and a detection part for detecting the substance to be measured in the sample. The detection part is located downstream of the blood cell separation membrane in a flow direction of the sample, and at least one polymer of a poly(meth)acrylic acid having a weight average molecular weight of 35,000 to 1,000,000 inclusive and a poly(meth)acrylic acid inorganic salt having a weight average molecular weight of 35,000 to 1,000,000 inclusive is contained in at least a part of the region lying between the sample supply part and the blood cell separation membrane in the flow direction of the sample in the flow channel.

Description

Testing instrument, blood cell separation method, immunological measurement method, hemolysis inhibitor, and kit

[0001] The present invention relates to a testing instrument, a blood cell separation method, an immunological measurement method, a hemolysis inhibitor, and a kit. This application claims priority from Japanese Patent Application No. 2024-189824 filed in Japan on October 29, 2024, the content of which is incorporated herein by reference.

[0002] With the diversification of clinical tests, point-of-care testing (POCT) has attracted attention. POCT is a test performed by medical staff beside the subject using a small analyzer or a rapid diagnostic kit, etc. Compared with tests using a large analyzer, POCT has the advantages of shortening the test time and allowing the subject to confirm the test results. For POCT testing devices, testing instruments, and test reagents, simplicity in operation and system configuration work and short measurement time are required.

[0003] As one of the testing instruments for POCT, testing instruments for biochemical tests of blood have been developed. From the viewpoints of simplicity of operation and shortening of measurement time, testing instruments capable of measuring whole blood as a specimen are required. As a blood component analyzer using whole blood as a specimen, for example, Patent Document 1 discloses a biosensor using chromatography with a single-layer or multiple-layer porous material, and is characterized by supporting a cell shrinkage agent on the porous material or the like.

[0004] Japanese Patent No. 3655283

[0005] When a specimen contains red blood cells like whole blood, the red blood cells may rupture and hemolyze during the chromatography process. When hemolysis occurs, the background sensitivity may increase in the detection part of the detection target substance in the testing instrument, and the detection sensitivity may relatively decrease. In Patent Document 1, although a cell shrinkage agent is used, there are cases where hemolysis cannot be sufficiently suppressed, or sensitivity reduction and poor chromatography development occur.

[0006] An object of the present invention is to provide a testing instrument, a blood cell separation method, an immunological measurement method, a hemolysis inhibitor, and a kit that can suppress hemolysis even when using a specimen containing red blood cells.

[0007] The present invention encompasses the following aspects: [1] A testing device comprising at least a flow path for circulating a sample containing red blood cells and a substance to be measured, wherein the flow path comprises at least a sample supply unit for supplying the sample, a blood cell separation membrane for separating the red blood cells from the sample, and a detection unit for detecting the substance to be measured in the sample, wherein the detection unit is located downstream of the blood cell separation membrane in the flow direction of the sample, and at least a portion of the region from the sample supply unit to the blood cell separation membrane in the flow direction of the sample in the flow path contains at least one polymer of poly(meth)acrylic acid with a weight-average molecular weight of 35,000 to 1,000,000 and an inorganic salt of poly(meth)acrylic acid with a weight-average molecular weight of 35,000 to 1,000,000. [1-1] A testing instrument comprising at least a flow path for circulating a sample containing red blood cells and a substance to be measured, wherein the flow path comprises a sample supply unit to which the sample is supplied, a blood cell separation membrane for separating at least the red blood cells from the sample, and a detection unit for detecting the substance to be measured in the sample, wherein the detection unit is located downstream of the blood cell separation membrane in the direction of sample flow, and at least a portion of the blood cell separation membrane and the region upstream of the blood cell separation membrane in the flow path in the direction of sample flow contains at least one polymer of poly(meth)acrylic acid with a weight-average molecular weight of 35,000 to 1,000,000 and an inorganic salt of poly(meth)acrylic acid with a weight-average molecular weight of 35,000 to 1,000,000. [2] The testing instrument according to [1] and [1-1], wherein the flow path further comprises a labeling substance holding unit between the sample supply unit and the detection unit, which contains a labeling substance that binds to the substance to be measured. [3] The testing device according to [2], wherein the labeling substance comprises at least one selected from metal colloid particles, latex particles, cellulose particles, fluorescent particles, and enzymes. [4] The testing device according to [2] or [3], wherein the labeling substance holding portion comprises the polymer. [5] The testing device according to any one of [1-1], [1] to [4], wherein the sample comprises whole blood.[6] A blood cell separation method comprising: contacting a sample containing red blood cells with at least one polymer of poly(meth)acrylic acid having a weight-average molecular weight of 35,000 to 1,000,000 and an inorganic salt of poly(meth)acrylic acid having a weight-average molecular weight of 35,000 to 1,000,000; and separating the red blood cells from the sample using a blood cell separation membrane after contacting the sample with the polymer. [7] The blood cell separation method according to [6], wherein contacting the sample with the polymer is a mixture of the sample and the polymer. [8] The blood cell separation method according to [6], further comprising flowing the sample to the blood cell separation membrane through a channel for flowing the sample, wherein contacting the sample with the polymer is a contact between the sample and the polymer held in the channel as the sample flows through the channel. [9] The blood cell separation method according to any one of [6] to [8], wherein the sample contains whole blood.

[10] An immunological measurement method comprising: contacting a sample containing red blood cells and a substance to be measured with at least one polymer of poly(meth)acrylic acid having a weight-average molecular weight of 35,000 to 1,000,000 and an inorganic salt of poly(meth)acrylic acid having a weight-average molecular weight of 35,000 to 1,000,000; separating the red blood cells from the sample using a blood cell separation membrane after contacting the sample with the polymer; and detecting the substance to be measured in the sample from which the red blood cells have been separated.

[11] The immunological measurement method according to

[10] , further comprising contacting the sample with a labeling substance that binds to the substance to be measured before detecting the substance to be measured.

[12] The immunological measurement method according to

[11] , wherein the labeling substance comprises at least one selected from metal colloid particles, latex particles, cellulose particles, fluorescent particles, and enzymes.

[13] The immunological measurement method according to any one of

[10] to

[12] , wherein the sample comprises whole blood.

[14] A hemolysis inhibitor comprising at least one polymer of poly(meth)acrylic acid having a weight-average molecular weight of 35,000 or more and 1,000,000 or less, and an inorganic salt of poly(meth)acrylic acid having a weight-average molecular weight of 35,000 or more and 1,000,000 or less, as an active ingredient for preventing hemolysis.

[15] A kit comprising a polymer of at least one of poly(meth)acrylic acid having a weight-average molecular weight of 35,000 to 1,000,000 and an inorganic salt of poly(meth)acrylic acid having a weight-average molecular weight of 35,000 to 1,000,000, for mixing with a sample containing red blood cells and a substance to be measured, and a testing instrument, wherein the testing instrument includes a channel for flowing the sample, and the channel includes, in this order, a sample supply unit to which the sample is supplied, a blood cell separation membrane for separating the red blood cells from the sample, and a detection unit for detecting the substance to be measured in the sample, wherein the detection unit is located downstream of the blood cell separation membrane in the flow direction of the sample.

[16] The kit according to

[15] , wherein the testing instrument further includes a labeling substance holding unit between the sample supply unit and the detection unit, the labeling substance holding unit containing a labeling substance that binds to the substance to be measured.

[17] The kit according to

[16] , wherein the labeling substance comprises at least one selected from metal colloid particles, latex particles, cellulose particles, fluorescent particles, and enzymes.

[18] The kit according to any one of

[15] to

[17] , wherein the sample comprises whole blood.

[0008] According to the above embodiment, it is possible to provide a testing device, a blood cell separation method, an immunoassay method, a hemolysis inhibitor, and a kit that can suppress hemolysis even when using a sample containing red blood cells.

[0009] Figure 1 is a schematic top view of an inspection device according to one embodiment of the present invention. Figure 2 is a schematic side view of an inspection device according to one embodiment of the present invention. Figure 3 is a schematic top view of a modified example of the inspection device according to one embodiment of the present invention. Figure 4 is a schematic top view of a modified example of the inspection device according to one embodiment of the present invention. Figure 5 is a graph showing the detection sensitivity evaluation results using the polymers of Examples 1 to 5 and Comparative Example 1.

[0010] In this invention, the terms "react" and "bind" are used synonymously to describe the reactivity between an antibody and an antigen.

[0011] When a numerical range is described as, for example, "1 to 10 μm," it means a range from 1 μm to 10 μm, including the lower limit of 1 μm and the upper limit of 10 μm. Furthermore, the upper and lower limits of a numerical range can be combined in any way. In addition, the numerical ranges for each physical property, composition, and measurement process can be combined in any way.

[0012] In this specification, acrylic acid and methacrylic acid are collectively referred to as (meth)acrylic acid.

[0013] <Testing device> In one aspect of this embodiment, the testing device includes at least a flow path for circulating a sample containing red blood cells and a substance to be measured, the flow path including at least a sample supply unit for supplying the sample, a blood cell separation membrane for separating the red blood cells from the sample, and a detection unit for detecting the substance to be measured in the sample, wherein the detection unit is located downstream of the blood cell separation membrane in the flow direction of the sample, and at least a portion of the region from the sample supply unit to the blood cell separation membrane in the flow direction of the sample in the flow path contains at least one polymer of poly(meth)acrylic acid with a weight-average molecular weight of 35,000 to 1,000,000 and an inorganic salt of poly(meth)acrylic acid with a weight-average molecular weight of 35,000 to 1,000,000.

[0014] The embodiments will be described in detail below with reference to the drawings. Below, an example of an immunochromatographic test instrument will be described, but the test instrument is not limited to an immunochromatographic test instrument.

[0015] Figure 1 is a schematic top view of the testing device according to this embodiment. Figure 2 is a schematic side view of the testing device according to this embodiment. The testing device 1 includes a flow path 13 through which the sample 20 moves. The flow path 13 includes a sample supply unit 11, a detection unit 12, and a blood cell separation membrane 14. The testing device 1 may also include a labeling substance holding unit 15. The testing device 1 may also include a control line 18 for confirming whether the test has been performed properly. The testing device 1 may also include an absorbent pad 16 for absorbing excess sample 20.

[0016] The sample supply unit 11 is the part to which the sample 20 containing red blood cells and the substance to be measured is supplied. Examples of materials for the sample supply unit 11 include nonwoven fabric, cellulose filter paper, paper, glass fiber, fiberglass, acrylic fiber, nylon fiber, and various woven fabrics. The sample supply unit 11 may also optionally contain trehalose and polyethylene glycol (PEG) for sample pretreatment.

[0017] Sample 20 may contain blood cells other than red blood cells, or it may contain whole blood. Sample 20 may be whole blood, or it may contain a hypotonic solution, specifically a preservative or surfactant. Sample 20 may be a liquid obtained by diluting whole blood with a buffer solution or the like. Sample 20 may contain blood collected from a living organism. The living organism is preferably a mammal such as a human, mouse, rat, or monkey, with humans being more preferred.

[0018] The substance to be measured contained in sample 20 is the substance detected by the detection unit 12. The detection unit 12 in this embodiment contains a first antibody for detecting the substance to be measured. The first antibody contained in the detection unit 12 may be bound to the substance to be measured, or it may be bound to the substance to be measured in a state where it is bound to a labeled second antibody. Thus, it is preferable that the substance to be measured is a substance detected by an immunological assay method. Details of the immunological assay method will be described later.

[0019] The testing device 1 may optionally include a labeling substance holding section 15. The labeling substance holding section 15 is located between the sample supply section 11 and the detection section 12. The labeling substance holding section 15 may be located upstream of the blood cell separation membrane 14 in the sample flow direction. The labeling substance holding section 15 contains a labeling substance that specifically binds to the substance to be measured. The labeling substance includes a labeling section and a second antibody that binds to the labeling section and specifically binds to the substance to be measured. The labeling section may be particles that are insoluble in water and buffer solutions. The particles are not particularly limited, but examples include metal colloid particles such as gold colloid, platinum colloid, silver colloid, and selenium colloid. The particles may also be colored latex particles obtained by coloring styrene-based latex such as polystyrene latex, colored latex particles obtained by coloring latex particles such as acrylic acid-based latex, colored silica particles obtained by coloring silica consisting of a three-dimensional structure made up of silicon atoms and oxygen atoms, colored cellulose particles obtained by coloring cellulose particles, or carbon black. The labeling substance may contain one or more types of labeling sections.

[0020] The labeling substance may exist in the form of a conjugate pad impregnated with a dedicated pad, or it may exist as a labeling substance holder in a part of the sample supply unit. Furthermore, it may exist as a separate labeling substance reagent, mixed with the sample, separate from the testing instrument.

[0021] The labeling portion may also be fluorescent particles. Examples of fluorescent particles include particles containing europium complexes, rhodamine-based dye molecules, squarylium-based dye molecules, cyanine-based dye molecules, aromatic hydrocarbon-based dye molecules, oxazine-based dye molecules, carbopyronine-based dye molecules, pyromecene-based dye molecules, diketopyrrolopyrrole-based dye molecules, phthalocyanine-based dye molecules, as well as Alexa Fluor® (registered trademark, manufactured by Invitrogen), BODIPY® (registered trademark, manufactured by Invitrogen), Cy® (registered trademark, manufactured by GE Healthcare), DY® (registered trademark, manufactured by DYOMICS), HiLyte® (registered trademark, manufactured by Anaspec), DyLight® (registered trademark, manufactured by ThermoScientific), ATTO® (registered trademark, manufactured by ATTO-TEC), MFP® (registered trademark, manufactured by Mobitec), and fluorescent proteins.

[0022] The labeling portion may be an enzyme. Examples of enzymes include horseradish peroxidase or alkaline phosphatase.

[0023] The method for binding the second antibody to the labeled portion is not particularly limited. Examples include binding by physical adsorption, binding by covalent bonding, and binding by a combination thereof. Furthermore, the second antibody may be directly bound to the labeled portion, or it may be bound indirectly via a linker or the like.

[0024] The sample supply unit 11 and the detection unit 12 are arranged within the flow path 13. If the testing instrument 1 is an immunochromatographic testing instrument, the detection unit 12 may be formed on a membrane such as a nitrocellulose membrane or a cellulose mixed ester arranged within the flow path 13.

[0025] The blood cell separation membrane 14 separates blood cells, such as red blood cells, contained in the sample 20 from the sample. The blood cell separation membrane may be used alone to separate blood cells, or it may be used in combination with other components that separate blood cells. The blood cell separation membrane 14 is a porous membrane having pores of a diameter sufficient to separate blood cells. As an example, the dimensions of blood cells are as follows: red blood cells have a diameter of 7-8 μm and a thickness of approximately 2 μm, white blood cells have a diameter of 6-30 μm, and platelets have a diameter of 2-3 μm. As an example, the pore diameter of the porous membrane of the blood cell separation membrane 14 is 2-100 μm, preferably 2-30 μm, and more preferably 2-5 μm. The blood cell separation membrane may be an asymmetric porous membrane in which the pore diameter decreases in the thickness direction, in which case the pore diameter of the part with the smallest pore diameter should be within the above range. The material or raw material for the blood cell separation membrane can be any material that satisfies the above-mentioned pore size characteristics, and examples include, but are not limited to, polysulfone, polyethersulfone, and filter paper.

[0026] Of the blood cell separation membrane 14 and the flow channel 13, at least a portion of the region upstream of the blood cell separation membrane 14 in the flow direction of the sample 20 indicated by the arrow in Figure 1 contains at least one polymer (hereinafter sometimes simply referred to as polymer A) of poly(meth)acrylic acid with a weight-average molecular weight of 35,000 to 1,000,000 and an inorganic salt of poly(meth)acrylic acid with a weight-average molecular weight of 35,000 to 1,000,000. At least a portion of the region upstream of the blood cell separation membrane 14 in the flow direction of the sample 20 of the flow channel 13 may be the sample supply unit 11.

[0027] Polymer A is a polymer obtained by polymerizing (meth)acrylic acid or an inorganic salt of (meth)acrylic acid as a monomer. Examples of inorganic salts of (meth)acrylic acid include sodium (meth)acrylate, potassium (meth)acrylate, or lithium (meth)acrylate. Sodium (meth)acrylate is preferred as the inorganic salt of (meth)acrylic acid.

[0028] When polymer A is brought into contact with sample 20, blood cells such as red blood cells contained in the sample agglutinate. When blood cells agglutinate, it becomes more difficult for them to penetrate the inside of the blood cell separation membrane 14, and it is thought that they become less likely to be destroyed inside the porous membrane of the blood cell separation membrane 14. In other words, it is thought that hemolysis is less likely to occur because the blood cells are captured near the surface of the blood cell separation membrane 14. Alternatively, when polymer A is brought into contact with sample 20, the blood cells are coated with polymer A, and it is thought that hemolysis is less likely to occur even if they penetrate the blood cell separation membrane 14.

[0029] The weight-average molecular weight of polymer A is 35,000 to 1,000,000, preferably 35,000 to 900,000, more preferably 35,000 to 800,000, and even more preferably 35,000 to 750,000. When the weight-average molecular weight of polymer A is below the above upper limit, the viscosity of the sample 20 after contact with polymer A does not become too high, and does not hinder the movement of the sample 20 from the sample supply unit 11 to the detection unit 12. Also, when the weight-average molecular weight of polymer A is below the above upper limit, there is sufficient interaction with blood cells such as red blood cells, and the blood cells tend to aggregate. When the weight-average molecular weight of polymer A is above the above lower limit, there is sufficient interaction with blood cells such as red blood cells, and the blood cells tend to aggregate.

[0030] The number-average molecular weight of polymer A is preferably 15,000 to 300,000, and more preferably 18,000 to 250,000. It is more preferable that the number-average molecular weight of polymer A satisfies the above-mentioned range of weight-average molecular weight of polymer A and is 15,000 to 300,000.

[0031] The weight-average molecular weight and number-average molecular weight of polymer A are determined from the results of gel permeation chromatography using polyethylene oxide with a known molecular weight as a standard sample. Specifically, they are determined by the method described in the examples.

[0032] Polymer A is contained in at least a portion of the region from the sample supply unit 11 to the blood cell separation membrane 14 in the flow direction of the sample 20 in the channel 13. The region from the sample supply unit 11 to the blood cell separation membrane 14 includes the sample supply unit 11 and the blood cell separation membrane 14. That is, polymer A may be contained in the sample supply unit 11 or in the blood cell separation membrane 14. Polymer A may be located in either the blood cell separation membrane 14 or the region upstream of the blood cell separation membrane 14 in the flow direction of the sample 20 indicated by the arrow in Figure 1, among the blood cell separation membrane 14 and the channel 13. That is, polymer A may be located in any of the region upstream of the blood cell separation membrane 14 in the flow direction of the sample 20, among the sample supply unit 11, the blood cell separation membrane 14, the labeling substance holding unit 15 and the other channels 13. For example, polymer A may be located on the surface or inside the sample supply unit 11. Also, polymer A may be held in the region of the channel 13 between the sample supply unit 11 and the blood cell separation membrane 14. Furthermore, polymer A may be located on or inside the labeling substance holding portion 15. Polymer A may be located on or inside the blood cell separation membrane 14. It is preferable that polymer A is located on or inside the labeling substance holding portion 15. As a method for holding polymer A, for example, an aqueous solution containing polymer A may be applied to the surface of a part of the flow channel 13, or an aqueous solution containing polymer A may be impregnated into the interior of a part of the flow channel 13 and then dried.

[0033] The mass of polymer A contained in the testing instrument 1 is set taking into account the volume of the sample supplied to the testing instrument. For example, it is preferably 1 to 4.5 mg / mL, and more preferably 2 to 3 mg / mL, relative to the total volume of the supplied sample.

[0034] As an example of the testing apparatus of this embodiment, as shown in Figure 2, the testing apparatus 1 is arranged in the following order from upstream to downstream in the flow direction indicated by the arrow of the sample 20: a sample supply unit 11, a labeled substance holding unit 15, a blood cell separation membrane 14, and a membrane comprising a detection unit 12, with each layer overlapping at least partially with the upper and lower layers. Furthermore, it is preferable to place a substrate 17 to support the membrane comprising the detection unit 12. An absorbent pad 16 is placed on the membrane comprising the detection unit 12 at a position opposite to the sample supply unit 11 in the flow direction of the sample. A first antibody is immobilized on a portion of the membrane between the blood cell separation membrane 14 and the absorbent pad 16, and functions as the detection unit 12.

[0035] The absorption pad 16 absorbs any excess sample that has passed through the detection unit 12. Examples of materials that can be used for the absorption pad 16 include, but are not limited to, cellulose filter paper, paper, glass fiber, fiberglass, acrylic fiber, nylon fiber, and various textiles.

[0036] The base material 17 may be a plastic base material, or a plastic adhesive sheet having an adhesive layer on a plastic base material. Examples of materials for the plastic base material and the plastic adhesive sheet include, but are not limited to, polyester, polystyrene, polypropylene, and polyvinyl chloride.

[0037] The testing device 1 may further have a housing (not shown) having a sample addition hole and a detection window. The housing houses a sample supply unit 11, a detection unit 12, a blood cell separation membrane 14, and a labeling substance holding unit 15, and is arranged such that the positions of the sample supply unit 11 and the sample addition hole, and the positions of the detection unit 12 and the detection window, coincide.

[0038] In the testing device with the above configuration, hemolysis can be suppressed because the sample containing red blood cells passes through the blood cell separation membrane after coming into contact with polymer A.

[0039] The substances detectable by the testing equipment are not particularly limited, but include, for example, cancer markers, hormones, infectious diseases, autoimmune diseases, plasma proteins, TDM, coagulation / fibrinolysis, amino acids, peptides, proteins, genes, and cells. More specifically, CEA, AFP, ferritillin, β2 micro, PSA, CA19-9, CA125, BFP, elastase 1, pepsinogen 1 and 2, fecal occult blood, urinary β2 micro, PIVKA-2, urinary BTA, insulin, E3, hCG, HPL, LH, HCV antigen, HBs antigen, HBs antibody, HBc antibody, HBe antigen, HBe antibody, HTLV-1 antibody, HIV antibody, toxoplasma antibody, syphilis, A SO, influenza A antigen, influenza A antibody, influenza B antigen, influenza B antibody, coronavirus 229E, coronavirus OC43, and coronavirus NL63, etc., rota antigen, adenovirus antigen, rota-adenovirus antigen, group A streptococcus, group B streptococcus, Candida antigen, C. difficile, Cryptococcus antigen, Vibrio cholerae, Neisseria meningitidis antigen, granulobacter elastase, Helicobacter pylori antibody, O157 antibody, O157 antigen, leptospirosis antibody, Aspergillus antigen, MRSA, RF, total IgE, LE test, CRP, IgG, IgA, IgM, IgD, transferrin, urinary albumin, urinary transferrin, myoglobin, C3 / C4, SAA, LP(a), α1-AC, α1-M, haptoglobin, microtransferrin, APR score, FDP Examples include D-dimer, plasminogen, AT3, α2PI, PIC, PAI-1, protein C, coagulation factor X3, type IV collagen, hyaluronic acid, GHbA1c, NT-proBNP, BNP, PCT, troponin, various other antigens, various antibodies, various viruses, various bacteria, various amino acids, various peptides, various proteins, various DNA, various cells, various allergens, various pesticide residues, and various harmful substances.

[0040] <Modifications of the testing device> The testing device is not limited to the immunochromatographic testing device shown in Figure 1, but may also be, for example, a microfluidic chip equipped with a blood cell separation membrane. Figure 3 is a schematic top view showing an example of a microfluidic chip.

[0041] In the microchannel chip 2 shown in FIG. 3, the flow channel 23 is a path through which a sample formed inside the base material 31 is fed. The sample supply unit 21, the detection unit 22, the blood cell separation unit 24, and the polymer A holding unit 29 are formed inside the base material 31. The sample supply unit 21, the polymer A holding unit 29, the blood cell separation unit 24, and the detection unit 22 are connected in this order. That is, the sample supply unit 21, the polymer A holding unit 29, the blood cell separation unit 24, and the detection unit 22 are arranged in the flow channel 23.

[0042] The sample supply unit 21 is a part to which a sample 20 containing red blood cells and a measurement target substance is supplied. Further, the sample supply unit 21 may optionally contain trehalose or PEG or the like for pre-treatment of the specimen or the like.

[0043] The blood cell separation unit 24 separates blood cells such as red blood cells contained in the sample from the sample. The above-described blood cell separation membrane is arranged in the blood cell separation unit 24.

[0044] Polymer A is held in the polymer A holding unit 29. The position of the polymer A holding unit 29 is not limited as long as it is arranged between the sample supply unit 21 and the blood cell separation unit 24. In addition to the configuration of the microchannel chip 2, for example, polymer A may be arranged in the sample supply unit 21, and the sample supply unit 21 and the polymer A holding unit 29 may be in the same region. Alternatively, in the flow direction of the sample, polymer A may be located in the flow channel on the sample supply unit 21 side of the blood cell separation unit 24 or inside the blood cell separation unit 24.

[0045] Examples of the base material 31 include a glass base material, a plastic base material such as polydimethylsiloxane (PDMS) and polymethyl methacrylate (PMMA).

[0046] The microchannel chip 2 can be manufactured, for example, by injection molding, sheet molding, transfer molding, or the like of a resin base material.

[0047] When a sample is supplied from the sample supply unit 21 to the microchannel chip 2, the sample reaches the polymer A holding unit 29 and comes into contact with the polymer A. As a result, blood cells such as red blood cells contained in the sample aggregate. When the blood cells aggregate, it becomes difficult for the blood cells to enter the inside of the blood cell separation unit 24, and it is considered that the blood cells are hardly destroyed inside the blood cell separation unit 24 where the blood cell separation membrane, which is a porous membrane, is disposed.

[0048] The sample that has passed through the blood cell separation unit 24 reaches the detection unit 22, and when the measurement target substance is contained in the sample, the measurement target substance is detected in the detection unit 22.

[0049] The microchannel chip 2 may include a labeling substance holding unit. The labeling substance holding unit may be disposed at any position as long as it is between the sample supply unit 21 and the detection unit 22. The labeling substance holding unit 15 may be located upstream of the blood cell separation unit 24 in the flow direction of the sample. The polymer A may be disposed in the labeling substance holding unit, and the labeling substance holding unit 15 and the polymer A holding unit 29 may be in the same region.

[0050] The microchannel chip 2 may optionally include an excess sample holding unit 26 downstream of the detection unit 22 in the flow direction of the sample. The excess sample holding unit 26 may include the above-described absorption pad, may be a space capable of holding the excess sample, or may be an extended flow path.

[0051] FIG. 4 is a schematic top view showing another example of the microchannel chip. For the same configuration as that of the microchannel chip 2 in the microchannel chip 2A shown in FIG. 4, the description thereof is omitted. The microchannel chip 2A includes a sample supply unit 21, a detection unit 22, a blood cell separation unit 24, and a polymer A supply unit 29A inside a base material 31. The sample supply unit 21, the blood cell separation unit 24, and the detection unit 22 are disposed in a flow path 23A. The polymer A supply unit 29A is disposed in a branch flow path 30 that is joined to the flow path between the sample supply unit 21 and the blood cell separation unit 24.

[0052] The sample supply unit 21, the blood cell separation unit 24, and the detection unit 22 are connected in this order. The flow path 23A between the sample supply unit 21 and the blood cell separation unit 24, and the branched flow path 30 in which the polymer A supply unit 29A is located, are connected upstream of the blood cell separation unit 24.

[0053] When a sample is supplied to the microfluidic chip 2A from the sample supply unit 21 and polymer A is supplied from the polymer A supply unit 29A, the sample and polymer A come into contact at the confluence of the branched channel 30 and channel 23A. This causes blood cells such as red blood cells contained in the sample to aggregate. When blood cells aggregate, it becomes more difficult for them to penetrate the inside of the blood cell separation unit 24, and it is thought that they become less likely to be destroyed inside the blood cell separation unit 24 where the porous membrane blood cell separation membrane is located. Alternatively, by bringing polymer A into contact with the sample, the blood cells are coated with polymer A, and it is thought that hemolysis is less likely to occur even if they penetrate the blood cell separation unit 24.

[0054] To facilitate the flow of polymer A, a pump or the like may be installed upstream of the polymer A supply unit 29A in the flow direction of polymer A. Alternatively, a suction device or the like may be installed downstream of the detection unit 22 in the flow direction of the sample.

[0055] The sample that has passed through the blood cell separation unit 24 reaches the detection unit 22, and if the sample contains the substance to be measured, the substance to be measured is detected in the detection unit 22.

[0056] The microfluidic chip 2A may optionally include a labeling substance holder. The labeling substance holder may be positioned anywhere within the flow path 23A between the sample supply unit 21 and the detection unit 22, or within the branched flow path 30.

[0057] <Method for separating blood cells> The method for separating blood cells according to this embodiment includes contacting a sample containing red blood cells with at least one polymer (i.e., polymer A) of poly(meth)acrylic acid with a weight-average molecular weight of 35,000 to 1,000,000 and an inorganic salt of poly(meth)acrylic acid with a weight-average molecular weight of 35,000 to 1,000,000, and separating the red blood cells from the sample using a blood cell separation membrane after contacting the sample with the polymer.

[0058] Contacting a sample containing red blood cells with polymer A may also involve mixing the sample with polymer A. More specifically, a liquid containing polymer A may be mixed with the sample. The liquid containing polymer A may be a mixture of water and polymer.

[0059] When mixing the sample with polymer A, the amount of polymer A is adjusted so that the mass ratio of polymer A to the total volume of the sample is preferably 1 to 4.5 mg / mL, more preferably 2 to 3 mg / mL.

[0060] In another embodiment, the blood cell separation method further includes flowing the sample through a channel to the blood cell separation membrane, and contact between the sample and polymer A may be made by bringing the polymer A held on the channel into contact with the sample as the sample flows through the channel. As the sample flows through the channel, polymer A dissolves in the sample, and the sample and polymer A are mixed. In this case as well, it is preferable to prepare the amount of polymer A such that the mass ratio of polymer A to the total volume of the sample is preferably 1 to 4.5 mg / mL, more preferably 2 to 3 mg / mL.

[0061] Polymer A, the blood cell separation membrane, the sample supply unit, the flow path, and the sample are the same as those described in the <Testing Equipment> section, so their explanation is omitted.

[0062] In this embodiment, the blood cell separation method involves separating the red blood cells from the sample using a blood cell separation membrane after the sample containing red blood cells has come into contact with polymer A, thereby suppressing hemolysis.

[0063] <Immunological Measurement Method> The immunological measurement method of this embodiment includes contacting a sample containing red blood cells and a substance to be measured with at least one polymer (i.e., polymer A) of poly(meth)acrylic acid with a weight-average molecular weight of 35,000 to 1,000,000 and an inorganic salt of poly(meth)acrylic acid with a weight-average molecular weight of 35,000 to 1,000,000; separating the red blood cells from the sample using the blood cell separation membrane after contacting the sample with the polymer; and detecting the substance to be measured in the sample from which the red blood cells have been separated.

[0064] In the immunological measurement method of this embodiment, the same steps as those described in the <blood cell separation method> section will be omitted from the explanation.

[0065] The immunological measurement method of this embodiment includes detecting the target substance in a sample from which red blood cells have been separated. The immunological measurement method may further include contacting the sample with a labeling substance that binds to the target substance.

[0066] Any immunological assay method may be used for detection. For example, chemiluminescent enzyme immunoassay (CLEIA), chemiluminescent immunoassay (CLIA), enzyme immunoassay (ELISA), immunochromatography, or fluorescence immunochromatography are preferred.

[0067] The CLEIA method uses magnetic particles conjugated with a first antibody and a second antibody labeled with an enzyme to form a complex of magnetic particles, the substance to be measured, and the enzyme in a sample solution. Bound-free (B / F) separation is then performed using the magnetism of the magnetic particles. Subsequently, a chemiluminescent substrate is added, which is hydrolyzed by the enzyme in the complex, causing it to emit light. The amount of this light emitted is then detected as a chemical signal.

[0068] The CLIA method uses magnetic particles conjugated with a first antibody and a second antibody labeled with a chemiluminescent substance to form a complex of magnetic particles, the substance to be measured, and the chemiluminescent substance in a sample solution. Bound-free (B / F) separation is then performed using the magnetism of the magnetic particles. Subsequently, the amount of chemical luminescence, which increases in proportion to the amount of the complex (amount of chemiluminescent substance), is detected as a chemical signal. Acridinium can be used as the chemiluminescent substance. The method is similar to the CLEIA method, except that it uses a chemiluminescent substance instead of an enzyme for labeling.

[0069] ELISA is a method of detecting antigens or antibodies, which are the target substances contained in a sample, by first capturing them using immobilized antibodies or antigens against the target substance, and then using an enzymatic reaction.

[0070] The first and second antibodies used in the immunoassay method described above can be those described in the section on "Testing Equipment".

[0071] According to the immunological measurement method of this embodiment, hemolysis can be suppressed because the sample containing red blood cells passes through the blood cell separation membrane after coming into contact with polymer A.

[0072] <Hemolysis Inhibitor> The hemolysis inhibitor of this embodiment contains polymer A, which is at least one of poly(meth)acrylic acid having a weight-average molecular weight of 35,000 to 1,000,000 and an inorganic salt of poly(meth)acrylic acid having a weight-average molecular weight of 35,000 to 1,000,000, as an active ingredient for preventing hemolysis.

[0073] The explanation of polymer A is omitted as it is described in the section on "Testing Equipment." As mentioned above, when polymer A comes into contact with a sample containing red blood cells, the red blood cells and other blood cells in the sample agglutinate. When blood cells agglutinate, it becomes more difficult for them to penetrate the inside of the blood cell separation membrane, and it is thought that they become less likely to be destroyed inside the porous membrane of the blood cell separation membrane. In other words, it is thought that hemolysis is less likely to occur because the blood cells are captured near the surface of the blood cell separation membrane. Alternatively, it is thought that when polymer A comes into contact with the sample, the blood cells are coated with polymer A, and even if they penetrate the blood cell separation membrane, hemolysis is less likely to occur.

[0074] The hemolysis inhibitor containing polymer A may be mixed with a sample containing red blood cells as described above. When mixing the sample with polymer A, the amount of polymer A is adjusted so that the mass ratio of polymer A to the total volume of the sample is preferably 1 to 4.5 mg / mL, more preferably 2 to 3 mg / mL.

[0075] Alternatively, polymer A may be held in a part of the testing instrument, and after supplying the sample to the testing instrument, the sample and polymer A may be mixed within the testing instrument. In this case, it is preferable to prepare the amount of polymer A so that the mass ratio of polymer A to the total volume of the sample is preferably 1 to 4.5 mg / mL, more preferably 2 to 3 mg / mL.

[0076] <Kit> The kit of this embodiment comprises a polymer of at least one of poly(meth)acrylic acid with a weight-average molecular weight of 35,000 to 1,000,000 and an inorganic salt of poly(meth)acrylic acid with a weight-average molecular weight of 35,000 to 1,000,000, and a hemolysis inhibitor for mixing with a sample containing red blood cells and a substance to be measured, and a testing instrument, wherein the testing instrument includes a flow path for circulating the sample, and the flow path includes at least a sample supply unit to which the sample is supplied, a blood cell separation membrane for separating the red blood cells from the sample, and a detection unit for detecting the substance to be measured in the sample, in this order, with the detection unit located downstream of the blood cell separation membrane in the flow direction of the sample.

[0077] The inspection equipment in this embodiment is as described in the section on "Inspection Equipment," except that it does not contain polymer A, so its description is omitted.

[0078] The hemolysis inhibitor contains polymer A. The hemolysis inhibitor may also contain any other components besides polymer A. The hemolysis inhibitor is mixed with a sample containing red blood cells and the substance to be measured. The sample mixed with the hemolysis inhibitor is supplied from the sample supply unit of the testing instrument, and the substance to be measured is detected.

[0079] The mass ratio of polymer A contained in the hemolysis inhibitor is preferably 1 to 4.5 mg / mL, and more preferably 2 to 3 mg / mL, relative to the total volume of the hemolysis inhibitor.

[0080] According to the kit of this embodiment, when polymer A contained in the hemolysis inhibitor is brought into contact with a sample containing red blood cells, the red blood cells and other blood cells in the sample agglutinate. When the blood cells agglutinate, they penetrate into the blood cell separation membrane and become less susceptible to destruction upon contact with the porous membrane. Alternatively, by bringing polymer A into contact with the sample, the blood cells are coated with polymer A, making it less likely for hemolysis to occur even if they penetrate the blood cell separation membrane. In other words, since the blood cells are captured near the surface of the blood cell separation membrane, hemolysis can be prevented.

[0081] 1. Preparation of Polymer Aqueous Solution 2.00 g (23.2 mmol) of methacrylic acid was dissolved in 16.4 mL of water. After stirring at 60°C for 30 minutes under a nitrogen atmosphere, 2.00 g (4.82 mmol) of 2,2'-azobis[N-(2-carboxyethyl)-2-methylpropionamidine]n hydrate was added as a polymerization initiator. After stirring at 60°C for 18 hours, the mixture was cooled to room temperature, the reaction solution was added to 99.0 mL of acetone, and the solid was filtered off. 10.0 mL of acetone was added to the obtained solid and stirred, and the solid was filtered off again. Washing with acetone and filtering were repeated two more times, and the obtained solid was dried under reduced pressure to obtain 2.55 g of polymethacrylic acid. Water and a 1 mol / L aqueous sodium hydroxide solution were added to this to obtain a polymer solution with a pH of 7.5. The obtained polymer solution was filtered through a membrane filter (material: glass fiber, pore size: 2.0 μm) to obtain an aqueous solution of polysodium methacrylate with a predicted molecular weight (Mw) of 50,000.

[0082] It is known that the weight-average molecular weight (Mw) of a polymer increases as the concentration of the polymerization initiator decreases. For polymethacrylate sodium with predicted molecular weights (Mw) of 100,000, 278,000, 371,000, and 600,000, the initiator amounts were 0.500 g, 0.065 g, 0.036 g, and 0.014 g, respectively, and the synthesis was carried out in the same manner. Poly(methacrylic acid) (molecular weight: <100,000; Fujifilm Wako Pure Chemical Industries) was dissolved in water, and then a 1 mol / l sodium hydroxide aqueous solution was added to adjust the pH to 7.5. Furthermore, a commercially available poly(methacrylic acid, sodium salt) solution (molecular weight: 4,000-6,000; Sigma-Aldrich) was used.

[0083] Sodium polysulfonate was synthesized using sodium 2-acrylamido-2-methylpropane-1-sulfonate (approximately 50% aqueous solution) instead of methacrylic acid, and the resulting solution was adjusted to pH 7.5.

[0084] Sodium polyphosphonate was synthesized using vinylphosphonic acid instead of methacrylic acid and prepared to a pH of 7.5.

[0085] Sodium polyphosphate was synthesized using Phosmer M (methacryloxyethylphosphate) instead of methacrylic acid, but it could not be used for evaluation because it was insoluble in water and 1 mol / L sodium hydroxide aqueous solution.

[0086] Furthermore, the polyacrylic acid used was prepared by dissolving polyacrylic acid 25000 (molecular weight: 25000; Fujifilm Wako Pure Chemical Industries, Ltd.) and polyacrylic acid 250000 (molecular weight: 250000; Fujifilm Wako Pure Chemical Industries, Ltd.) in water, then adding a 1 mol / L sodium hydroxide aqueous solution to adjust the pH to 7.5.

[0087] The weight-average molecular weight of the polymer was measured by the following method. 50 mg of the polymer was dissolved in 50 mL of the eluent described below, and the weight-average volume was measured by gel permeation chromatography under the following conditions. The results of the measured weight-average molecular weight are shown in Table 1 below. • Instrument: HLC-8420GPC (Tosoh Corporation) • Detector: HLC-8420GPC built-in RI detector (Tosoh Corporation) • Sample column: TSKgel SuperMultiporePW-H (6.0 mm I.D. × 15 cm × 2) (Tosoh Corporation) • Reference column: TSKgel SuperH-RC (Tosoh Corporation) • Guard column: TSKgel guard column SuperMP(PW)-H (Tosoh Corporation) • Eluent: 0.2 M sodium nitrate aqueous solution / methanol = 80 / 20 (volume ratio) • Flow rate: 0.4 mL / min • Injection volume: 40 μL • Column temperature: 40°C • Standard sample: Polyethylene oxide (Tosoh Corporation) • Measurement time: 25 minutes • Calculation of number-average molecular weight (Mn) and weight-average molecular weight (Mw): GPC workstation EcoSEC Elite-WS (data analysis program dedicated to HLC-8420GPC)

[0088] Gel filtration chromatography cannot directly determine molecular weight. The molecular weight obtained by gel filtration chromatography is the molecular weight assuming the polymer has the same structure as the standard sample, and is therefore a molecular weight equivalent to the standard sample. Consequently, it is known that if the molecular structure of the polymer being measured differs significantly from that of the standard sample, the discrepancy with the true molecular weight can be substantial.

[0089] 2. Preparation of Test Equipment 1) Preparation of Gold Colloid-Labeled Anti-NT-ProBNP Monoclonal Antibody (Anti-NT-ProBNP Antibody Conjugate) (i) Preparation of Anti-NT-ProBNP Antibody The anti-NT-ProBNP antibody used in the following tests was obtained by immunizing mice with recombinant NT-ProBNP protein as the antigen and using the method commonly used by those skilled in the art to produce monoclonal antibodies. (ii) Preparation of Gold Colloid Solution To 5 L of purified water heated to 80°C, 10 mL of 5% (w / v) triammonium citrate aqueous solution was added and stirred. Subsequently, 10 mL of 5% (w / v) tetrachlorogold(III) aqueous solution was added and reacted for 10 minutes while stirring, and then the reaction solution was brought to a boil. After this, it was cooled in ice water to prepare a gold colloid solution with an average particle size of 50 nm. This gold colloid solution with an average particle size of 50 nm was adjusted with purified water so that the absorbance (unit: OD / mL) at the maximum absorption wavelength of the gold colloid was 1.

[0090] (iii) Preparation of anti-NT-proBNP antibody conjugate To the above 1 OD / mL gold colloid solution (pH 7.0), NT-proBNP monoclonal antibody diluted to 46.2 μg / mL with 2 mM Tris-HCl buffer (pH 7.0) was added and stirred at room temperature for 10 minutes. To this mixture of gold colloid and antibody, purified water containing 10% (w / v) BSA was added and stirred at room temperature for 5 minutes. Then, the mixture was centrifuged at 11900 × g for 45 minutes at 10°C. After removing the supernatant, 1 mL of Conjugate Dilution Buffer (SCRIPS Laboratories) was added to the resulting precipitate to suspend the conjugate and obtain the anti-NT-proBNP antibody conjugate.

[0091] (iv) Preparation of gold colloid-labeled KLH (KLH conjugate) for the control line To 20 mL of the gold colloid solution with an absorbance of 1 (unit: OD / mL) described above, 1 mL of KLH (Sigma-America) dissolved in 2 mmol / L phosphate buffer to a concentration of 620 μg / mL was added and the mixture was stirred at room temperature for 10 minutes. To the mixture of gold colloid and KLH, 1 mL of 10% bovine serum albumin (BSA) aqueous solution was added and the mixture was stirred at room temperature for 5 minutes. Then, the mixture was centrifuged at 10°C for 45 minutes, the supernatant was removed, and 1 mL of conjugate diluent was added to the resulting precipitate to suspend the conjugate and obtain the KLH conjugate.

[0092] 2) Preparation of Conjugate Pads: Prepare a detection reagent by mixing 3 OD / mL anti-NT-proBNP antibody conjugate, 0.75 OD / mL KLH conjugate, 2.4% trehalose, 1.5% NPS, and 20 mM MOPS (pH 7.2) in a solution, and adding a polymer aqueous solution under the conditions described in Table 1. Then, apply 0.78 cm of the solution to a fiberglass pad (Merck Millipore) cut to the required size. ２ The material was impregnated at a liquid ratio of 52.2 μL per pad. It was dried by heating in a dry oven to form a conjugate pad.

[0093] 3) Preparation of anti-NT-proBNP monoclonal antibody immobilized membranes (antibody immobilized membranes): For the test line, solutions were prepared by adding sucrose and anti-NT-proBNP monoclonal antibody to 10 mM PBS pH 7.2 to final concentrations of 2.5% and 2 mg / mL. For the control line, rabbit anti-KLH polyclonal antibody (Bethyl) was diluted and prepared in the same manner as described above.

[0094] A test line was formed by applying the above-mentioned anti-NT-proBNP monoclonal antibody to the inside of one end of the short side of a nitrocellulose membrane (UniSart CN150 white backing, Sartorius) using an immunochromatography dispenser "XYZ3050" (BIO DOT) at a rate of 1 μL / cm. A control line was formed by similarly applying anti-KLH polyclonal antibody approximately 4 mm away from the test line. The membranes were dried in a dry oven to obtain antibody-immobilized membranes.

[0095] 4) Preparation of sample pads A 20 mM MOPS (pH 7.2) containing 0.5% trehalose and 0.05% PEG was used as a sample pad. A fiberglass pad (Lydall) cut to the required size was soaked with 1.5 times the volume of the MOPS, and the pad was dried in a dry oven at 70°C for 45 minutes.

[0096] 5) Preparation of Immunochromatographic Test Specimen The structure of the immunochromatographic test specimen in this embodiment will be explained using Figure 2. First, the antibody-immobilized membrane was attached to a plastic adhesive sheet, which is the base material 17, with the anti-NT-proBNP antibody coated area as the detection area 12 on the upstream side of the unfolding, followed by the anti-KLH antibody coated area as the control line. A blood cell separation membrane 14 (model number: T9EXPNP300R022A, manufacturer: Cytiva) was then attached on top of the membrane. Next, the conjugate pad prepared in 2) above was placed and attached as the labeling substance holding area 15, and the sample pad prepared in 4) above was placed and attached as the sample supply area 11 so as to overlap the conjugate pad, and an absorbent pad 16 was placed and attached to the opposite end of the sample supply area 11. The structure made by overlapping each component in this way was cut to prepare the immunochromatographic test specimen.

[0097] 6) The test specimens were placed in a dedicated plastic housing to create the test device.

[0098] 3. Confirmation of Hemolysis 1) Preparation of Polymer-Containing Pads A polymer aqueous solution prepared in "1. Preparation of Polymer Aqueous Solution" was added to a solution of 2.4% trehalose, 1.5% NPS, and 20 mM MOPS (pH 7.2) under the conditions of Examples 1 to 5 and Comparative Examples 1 to 8 listed in Table 1, to prepare a polymer-containing pad treatment solution. A 0.78 cm solution was then applied to a fiberglass pad (Merck Millipore) cut to the required size as needed. ２ A polymer-containing pad was prepared by soaking the pad with a liquid ratio of 52.2 μL per pad and then drying it.

[0099] 2) Preparation of the testing device: Except for using the polymer-containing pad or polymer-free pad instead of the conjugate pad mentioned above to facilitate hemolysis evaluation, the testing device was prepared in the same manner as the testing device described in "2. Preparation of the testing device".

[0100] 3) Hemolysis Evaluation Method: 120 μL of whole blood sample, adjusted to a hematocrit value of 55%, was added to the sample addition well of the immunochromatographic test detection device prepared above. After 10 minutes, the area near the detection part of the membrane was visually inspected to check for hemolysis. Case A was defined as no hemolysis, case B as slight hemolysis that did not interfere with the test, and case C as hemolysis that interfered with the test.

[0101] Table 1 shows the polymers used in Examples 1-5 and Comparative Examples 1-8, the predicted molecular weight (Mw) of the polymers, the measured weight-average molecular weight (Mw), the number-average molecular weight (Mn), Mw / Mn, the polymer content (mass%) in the polymer-containing pad treatment solution, and the results regarding the presence or absence of hemolysis.

[0102]

[0103] As shown in Examples 1 to 5, hemolysis could be prevented when at least one polymer of poly(meth)acrylic acid with a weight-average molecular weight of 35,000 to 1,000,000 and sodium poly(meth)acrylate salt with a weight-average molecular weight of 35,000 to 1,000,000 was brought into contact with the whole blood sample before the sample came into contact with the blood cell separation membrane.

[0104] On the other hand, hemolysis occurred under the conditions of Comparative Example 1 (no polymer added), Comparative Examples 2-4 (polymer), Comparative Example 5 (polymethacrylate sodium salt with a weight-average molecular weight of less than 35,000), and Comparative Examples 6-8 (poly(meth)acrylic acid and poly(meth)acrylate sodium salt with a weight-average molecular weight of more than 100,000) when whole blood samples were in contact.

[0105] 4. Confirmation of hemolysis 1) Preparation of polymer-containing pads A polymer aqueous solution prepared in "1. Preparation of polymer aqueous solution" was added to a solution of 0.5% trehalose and 20 mM MOPS (pH 7.2) under conditions that resulted in the polymer content of Examples 6 to 8 and Comparative Example 9 as described in Table 2, to prepare a polymer-containing pad treatment solution. A volume 1.5 times the volume of the solution was then impregnated into a fiberglass pad (Lydall) cut to the required size, and dried to prepare a polymer-containing pad.

[0106] 2) Preparation of testing equipment: Except for using the polymer-containing pad or polymer-free pad instead of the sample pad mentioned above to facilitate hemolysis evaluation, the testing equipment was prepared in the same manner as described in "2. Preparation of testing equipment".

[0107] 3) Hemolysis Evaluation Method The presence or absence of hemolysis was confirmed using the same method and criteria as described in "3) Hemolysis Evaluation Method" of "3. Confirmation of Hemolysis".

[0108] Table 2 shows the polymers used in Examples 6-8 and Comparative Example 9, the predicted molecular weight (Mw) of the polymers, the measured weight-average molecular weight (Mw), the number-average molecular weight (Mn), Mw / Mn, the polymer content (mass%) in the polymer-containing pad treatment solution, and the results regarding the presence or absence of hemolysis.

[0109]

[0110] In Comparative Example 9, under conditions without polymer addition, hemolysis occurred. As shown in Examples 6 to 8, hemolysis was prevented when a sodium poly(meth)acrylate polymer with a weight-average molecular weight of 35,000 to 1,000,000 was brought into contact with the whole blood sample before the sample came into contact with the blood cell separation membrane.

[0111] 3. Sensitivity Evaluation Method Using the testing device prepared in "1. Preparation of Testing Device," 120 μL of plasma sample containing NT-proBNP antigen (Hytest Co., Ltd.) was added to the sample addition well, and after 10 minutes, the absorbance of the test line was calculated using RapidPeer (Sekisui Medical). The results of the sensitivity evaluation are shown in Figure 5.

[0112] As shown in Figure 5, the testing equipment using the polymers of Examples 1, 2, 4, and 5 showed higher detection sensitivity than Comparative Example 1 without added polymer. The testing equipment using the polymer of Example 3 showed lower detection sensitivity than Comparative Example 1 without added polymer, but it was shown to be almost equivalent.

[0113] According to the above embodiment, it is possible to provide a testing device, a blood cell separation method, an immunoassay method, a hemolysis inhibitor, and a kit that can suppress hemolysis even when using a sample containing red blood cells.

[0114] 1... Testing instrument, 2... Microfluidic chip, 11... Sample supply unit, 12... Detection unit, 13... Channel, 14... Blood cell separation membrane, 15... Labeling substance holding unit, 16... Absorption pad, 17... Substrate, 18... Control line, 20... Sample, 21... Sample supply unit, 22... Detection unit, 23, 23A... Channel, 24... Blood cell separation unit, 26... Excess sample holding unit, 29, 29A... Polymer A holding unit, 30... Branching channel, 31... Substrate

Claims

1. A testing device comprising at least a flow path for circulating a sample containing red blood cells and a substance to be measured, wherein the flow path comprises at least: a sample supply unit for which the sample is supplied; a blood cell separation membrane for separating the red blood cells from the sample; and a detection unit for detecting the substance to be measured in the sample, wherein the detection unit is located downstream of the blood cell separation membrane in the direction of sample flow, and at least a portion of the region from the sample supply unit to the blood cell separation membrane in the direction of sample flow in the flow path contains at least one polymer of poly(meth)acrylic acid with a weight-average molecular weight of 35,000 to 1,000,000 and an inorganic salt of poly(meth)acrylic acid with a weight-average molecular weight of 35,000 to 1,000,000.

2. The inspection apparatus according to claim 1, wherein the flow path further includes a labeling substance holding section between the sample supply section and the detection section, the labeling substance holding section containing a labeling substance that binds to the substance to be measured.

3. The inspection device according to claim 2, wherein the labeling substance comprises at least one selected from metal colloid particles, latex particles, cellulose particles, fluorescent particles, and enzymes.

4. The inspection device according to claim 2 or 3, wherein the polymer is included in the labeling substance holding portion.

5. A blood cell separation method comprising: contacting a sample containing red blood cells with at least one polymer of poly(meth)acrylic acid having a weight-average molecular weight of 35,000 to 1,000,000 and an inorganic salt of poly(meth)acrylic acid having a weight-average molecular weight of 35,000 to 1,000,000; and separating the red blood cells from the sample using a blood cell separation membrane after contacting the sample with the polymer.

6. The blood cell separation method according to claim 5, wherein contacting the sample with the polymer constitutes mixing the sample with the polymer.

7. The blood cell separation method according to claim 5, comprising flowing the sample to the blood cell separation membrane through a channel for flowing the sample, wherein contacting the sample with the polymer is achieved by the sample flowing through the channel, thereby bringing the sample into contact with the polymer held within the channel.

8. An immunological measurement method comprising: contacting a sample containing red blood cells and a substance to be measured with at least one polymer of poly(meth)acrylic acid having a weight-average molecular weight of 35,000 to 1,000,000 and an inorganic salt of poly(meth)acrylic acid having a weight-average molecular weight of 35,000 to 1,000,000; separating the red blood cells from the sample using a blood cell separation membrane after contacting the sample with the polymer; and detecting the substance to be measured in the sample from which the red blood cells have been separated.

9. The immunological measurement method according to claim 8, further comprising contacting the sample with a labeling substance that binds to the substance to be measured before detecting the substance to be measured.

10. The immunoassay method according to claim 9, wherein the labeling substance comprises at least one selected from metal colloid particles, latex particles, cellulose particles, fluorescent particles, and enzymes.

11. A hemolysis inhibitor comprising at least one polymer of poly(meth)acrylic acid having a weight-average molecular weight of 35,000 to 1,000,000 and an inorganic salt of poly(meth)acrylic acid having a weight-average molecular weight of 35,000 to 1,000,000 as an active ingredient for preventing hemolysis.

12. A kit comprising: a polymer of at least one of poly(meth)acrylic acid having a weight-average molecular weight of 35,000 to 1,000,000 and an inorganic salt of poly(meth)acrylic acid having a weight-average molecular weight of 35,000 to 1,000,000, for mixing with a sample containing red blood cells and a substance to be measured; and a testing instrument, wherein the testing instrument includes a channel for circulating the sample, and the channel includes, in this order, a sample supply unit for supplying the sample; a blood cell separation membrane for separating the red blood cells from the sample; and a detection unit for detecting the substance to be measured in the sample, wherein the detection unit is located downstream of the blood cell separation membrane in the direction of sample flow.

13. The kit according to claim 12, wherein the testing instrument further includes a labeling substance holding unit between the sample supply unit and the detection unit, the labeling substance holding unit containing a labeling substance that binds to the substance to be measured.

14. The kit according to claim 13, wherein the labeling substance comprises at least one selected from metal colloid particles, latex particles, cellulose particles, fluorescent particles, and enzymes.

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