Agent for improving agglutination reaction, method for improving target antigen measurement method, and reagent for measuring human brain natriuretic peptide

Abstract

Provided is an agent for improving agglutination reaction comprising a copolymer containing 2-methacryloyloxyethyl phosphorylcholine as a monomer unit, wherein the agglutination reaction is an agglutination reaction of insoluble particles having a granular carrier and an antibody against a target antigen carried on the granular carrier, and the target antigen, and the improvement agent is used for suppressing deviation of reactivity of the agglutination reaction in a system including no blood sample and a system including a blood sample.

Description

Agglutination reaction improving agent, method for improving target antigen measurement method, and reagent for measuring human brain natriuretic peptide

[0001] The present invention relates to an agent for improving an agglutination reaction, an improved method for measuring a target antigen, and a reagent for measuring human brain natriuretic peptide.

[0002] One method for measuring target antigens using immune reactions is to use granular carriers such as latex (see, for example, Patent Document 1). When the target antigen is present, the granular carriers, to which antibodies specific to the target antigen are bound, undergo an antigen-antibody reaction. Due to their specificity and affinity, the antibodies bind to each other, using the target antigen as a bridge, and agglutinate. The presence or absence of the resulting agglutinates and the degree of agglutination determine the presence or absence of the target antigen and its quantity.

[0003] Such measurement methods utilizing immune reactions can have the problem of insufficient detection sensitivity. For example, Patent Document 2 discloses that measurement sensitivity can be improved by performing immune reactions in the presence of polyvinylpyrrolidone.

[0004] JP 2017-083440 A JP 04-020859 A

[0005] The above-described method for measuring a target antigen using an immune reaction is applied to the measurement of a target antigen contained in a specimen containing a blood sample. Since the reactivity of an immune reaction-induced agglutination reaction is significantly affected by the blood sample, in order to accurately measure the target antigen, it is necessary to suppress the discrepancy between the reactivity of the agglutination reaction in a system that does not contain a blood sample and a system that contains a blood sample. However, when the present inventors investigated the reactivity of the above-described agglutination reaction in the presence of polyvinylpyrrolidone as described in Patent Document 2, they found a large discrepancy between the reactivity of the agglutination reaction in a system that does not contain a blood sample and a system that contains a blood sample.

[0006] In view of the above circumstances, an object of the present invention is to provide an agent capable of sufficiently suppressing the discrepancy in reactivity of the agglutination reaction between a system not containing a blood sample and a system containing a blood sample, or a method for sufficiently suppressing the discrepancy.

[0007] As a result of extensive research, the present inventors have found that a copolymer containing 2-methacryloyloxyethyl phosphorylcholine as a monomer unit can sufficiently suppress the discrepancy in reactivity of the agglutination reaction due to the antigen-antibody reaction between insoluble particles having a granular carrier and an antibody against a target antigen supported on the granular carrier, and the target antigen, in a system not containing a blood sample and in a system containing a blood sample, and have completed the present invention.

[0008] The present invention provides, for example, the following inventions. [1] An agent for improving an agglutination reaction, comprising a copolymer containing 2-methacryloyloxyethyl phosphorylcholine as a monomer unit, wherein the agglutination reaction is an agglutination reaction of insoluble particles comprising granular carriers and antibodies against a target antigen supported on the granular carriers, resulting from an antigen-antibody reaction between the insoluble particles and the target antigen, and the agent is used to suppress discrepancy in the reactivity of the agglutination reaction between a system not containing a blood sample and a system containing a blood sample. [2] The agent according to [1], wherein the copolymer contains a monomer unit having an anionic group. [3] The agent according to [1] or [2], wherein the copolymer contains a monomer unit having a nonionic group. [4] A method for measuring a target antigen using insoluble particles having a granular carrier and an antibody against the target antigen supported on the granular carrier, based on an agglutination reaction of the insoluble particles due to an antigen-antibody reaction between the antibody and the target antigen, comprising: a method for suppressing a discrepancy between the reactivity of the agglutination reaction in a system not containing a blood sample and the reactivity of the agglutination reaction in a system containing a blood sample, wherein the agglutination reaction is carried out in the presence of a copolymer containing 2-methacryloyloxyethyl phosphorylcholine as a monomer unit. [5] The method according to claim [4], wherein the copolymer is a copolymer containing a monomer unit having an anionic group. [6] The method according to [4] or [5], wherein the copolymer is a copolymer containing a monomer unit having a nonionic group. [7] A reagent for measuring human brain natriuretic peptide, comprising a copolymer containing 2-methacryloyloxyethyl phosphorylcholine as a monomer unit, wherein the measurement is based on an agglutination reaction of the insoluble particles, which have a granular carrier and an anti-human brain natriuretic peptide antibody supported on the granular carrier, due to an antigen-antibody reaction between the anti-human brain natriuretic peptide antibody and the anti-human brain natriuretic peptide, using the reagent.

[0009] According to the present invention, it is possible to provide an agent that can sufficiently suppress the discrepancy in reactivity of an agglutination reaction due to an antigen-antibody reaction between insoluble particles having a granular carrier and an antibody against a target antigen supported on the granular carrier, and the target antigen, in a system that does not contain a blood sample and in a system that contains a blood sample, or a method that can sufficiently suppress the discrepancy.

[0010] 1 is a graph showing the ratio of the change in absorbance in plasma to the change in absorbance due to the agglutination reaction of anti-human BNP antibody-sensitized latex in a 2% by mass BSA solution.

[0011] Hereinafter, embodiments of the present disclosure will be described in detail.

[0012] The agglutination reaction improver according to one aspect of this embodiment comprises a copolymer (hereinafter also referred to as "MPC polymer") containing 2-methacryloyloxyethyl phosphorylcholine as a monomer unit, and the agglutination reaction is an agglutination reaction of insoluble particles (hereinafter also simply referred to as "agglutination reaction of insoluble particles") due to an antigen-antibody reaction between the target antigen and insoluble particles having a granular carrier and an antibody against the target antigen supported on the granular carrier, and the target antigen, and is used to suppress the discrepancy in the reactivity of the agglutination reaction between a system that does not contain a blood sample and a system that contains a blood sample.

[0013] The reason why the MPC polymer suppresses the discrepancy in the reactivity of the agglutination reaction of insoluble particles between a system that does not contain a blood sample and a system that contains a blood sample is not entirely clear, but the inventors believe that one reason is that the blood sample inhibits the agglutination reaction of insoluble particles, and the MPC polymer suppresses this inhibition.

[0014] The reactivity of the agglutination reaction of insoluble particles is evaluated by measuring the amount of change (endpoint method) or rate of change (rate method) in absorbance of a system in which the agglutination reaction is occurring. For example, a suspension containing insoluble particles, a test sample containing a target antigen, and an MPC polymer are mixed and placed in a cell, and the cell is irradiated from the outside with visible light to near-infrared light, typically 300 nm to 1000 nm, preferably 500 nm to 900 nm, and the amount or rate of change in absorbance after the mixing is measured. The time for measuring the amount or rate of change in absorbance can be 1 minute to 30 minutes, preferably 1 minute to 10 minutes, but is not limited thereto. The temperature at which the agglutination reaction is performed after the mixing can be 30°C to 40°C, or can also be 33°C to 37°C, but is not limited thereto. The reactivity of the agglutination reaction of insoluble particles can be evaluated, specifically, by the method described in the Examples below.

[0015] The MPC polymer may be a copolymer containing a monomer unit having an anionic group, a monomer unit having a nonionic group, a monomer unit having a cationic group, or the like. From the viewpoint of further suppressing the discrepancy in reactivity of the agglutination reaction of insoluble particles in a system that does not contain a blood sample and a system that contains a blood sample, the MPC polymer is preferably a copolymer containing a monomer unit having an anionic group and / or a monomer unit having a nonionic group, and more preferably a copolymer containing a monomer unit having an anionic group.

[0016] From the viewpoint of further suppressing the discrepancy in the reactivity of the agglutination reaction of insoluble particles between a system containing a blood sample and a system not containing a blood sample, a compound in which the hydrogen atom of the carboxyl group of methacrylic acid is substituted with an anionic group is preferred as the monomer unit having the anionic group. Examples of MPC polymers containing the above compound as a monomer unit include the LIPIDURE (registered trademark)-BL400 series, such as LIPIDURE (registered trademark)-BL405.

[0017] As the monomer unit having the nonionic group, a compound in which the hydrogen atom of the carboxyl group of methacrylic acid is substituted with a nonionic group is preferred from the viewpoint of further suppressing the discrepancy in the reactivity of the agglutination reaction of insoluble particles in a system not containing a blood sample and a system containing a blood sample. The nonionic group means a group that does not have either a cationic functional group or an anionic functional group. The nonionic group may be, for example, a group containing a hydrophilic group, a hydrophobic group, a hydrogen bond donor, or the like. Examples of MPC polymers having the above compounds as monomer units include the LIPIDURE (registered trademark)-BL100 series such as LIPIDURE (registered trademark)-BL103, the LIPIDURE (registered trademark)-BL200 series, the LIPIDURE (registered trademark)-BL700 series, the LIPIDURE (registered trademark)-BL800 series, the LIPIDURE (registered trademark)-BL1000 series, the LIPIDURE (registered trademark)-BL1200 series, and the LIPIDURE (registered trademark)-BL1300 series.

[0018] Examples of the monomer unit having a cationic group include compounds in which the hydrogen atom of the carboxyl group of methacrylic acid is substituted with a cationic group. Examples of MPC polymers having the above compound as a monomer unit include the LIPIDURE (registered trademark)-BL500 series, such as LIPIDURE (registered trademark)-BL502.

[0019] The amount of MPC polymer present in the system in which the aggregation reaction is carried out may be 0.005 to 2% by mass, or may be 0.01 to 1% by mass, based on the total amount of the system.

[0020] The MPC polymer is not particularly limited as long as it can sufficiently suppress the discrepancy in reactivity of the agglutination reaction of insoluble particles, and may be added to a system in which the agglutination reaction is carried out in combination with a stabilizer such as bovine serum albumin, an inorganic salt such as calcium chloride, a preservative such as ProClin (registered trademark) 300, a buffer such as Bicine (2-(bis(2-hydroxyethyl))amino)acetic acid) buffer solution, etc., as necessary.

[0021] The MPC polymer is not particularly limited, and may be one produced by a known method or may be a commercially available polymer.

[0022] Examples of blood samples include human-derived body fluids such as plasma samples, whole blood samples, and serum samples, as well as dilutions thereof, with human-derived plasma samples and dilutions thereof being preferred.

[0023] Target antigens include, for example, protein markers such as CRP (C-reactive protein), prostate-specific antigen, ferritin, β-2 microglobulin, myoglobin, hemoglobin, albumin, and creatinine; immunoglobulins such as IgG, IgE, IgA, and IgM; various tumor markers; lipoproteins such as LDL, HDL, and TG; viral antigens such as influenza A virus, influenza B virus, respiratory syncytial virus (RSV), rhinovirus, rotavirus, norovirus, adenovirus, astrovirus, HAV, HBs, HCV, HIV, and EBV; Chlamydia trachomatis; Examples of the antibacterial agent include, but are not limited to, bacterial antigens such as streptococci, Bordetella pertussis, Helicobacter pylori, Leptospira, Treponema pallidum, Toxoplasma gondii, Borrelia, Legionella, Bacillus anthrax, and MRSA, toxins produced by bacteria, and the like, mycoplasmal lipid antigens, peptide hormones such as human brain natriuretic peptide (hBNP) and human chorionic gonadotropin, steroids such as steroid hormones, physiologically active amines such as epinephrine and morphine, vitamins such as B vitamins, prostaglandins, antibiotics such as tetracycline, pesticides, and environmental hormones.

[0024] Examples of granular carriers include latex particles, ceramic particles, alumina particles, silica-alumina particles, and carbon black particles. Among these particles, latex particles are preferred. Examples of latex materials include polystyrene and divinylbenzene, with polystyrene being preferred. The average particle size of the granular carrier can be 0.1 to 5 μm. The particle size of the granular carrier can be measured by dynamic light scattering. In this specification, the term "average particle size" refers to the particle size (median diameter) when the integrated value from the smallest particle size reaches 50% of the total in a volume-based particle size distribution curve obtained by dynamic light scattering.

[0025] The antibody is not particularly limited as long as it can specifically bind to an antigen, and examples include IgG, IgM, IgA, IgD, and IgE, with IgG being preferred. IgG is composed of two heavy chains (H chains) and two light chains (L chains). The heavy chain is composed of, from the N-terminus, a variable region (VH), a first constant region (CH1), a second constant region (CH2), and a third constant region (CH3). The light chain is composed of, from the N-terminus, a variable region (VL) and a constant region (CL). The portion composed of CH2 and CH3 is the Fc region. One heavy chain and one light chain are bonded by a disulfide bond between a cysteine ​​residue present in CH1 and a cysteine ​​residue present in CL. Furthermore, the heavy chains are bonded to each other by a disulfide bond between cysteine ​​residues present in the hinge region located between CH1 and CH2. The antibody may be an IgG fragment containing a heavy chain, or an rIgG (reduced IgG) consisting of one heavy chain and one light chain, or a fragment consisting of two heavy chains, or a fragment consisting of one heavy chain.

[0026] The antibody can be bound to the particulate carrier by a common method, such as physical adsorption or chemical binding.

[0027] Another aspect of this embodiment is a method for suppressing the discrepancy between the reactivity of the agglutination reaction in a system that does not contain a blood sample and the reactivity of the agglutination reaction in a system that contains a blood sample in a method for measuring a target antigen that uses insoluble particles having a granular carrier and an antibody against the target antigen supported on the granular carrier, and that is based on an agglutination reaction of the insoluble particles due to an antigen-antibody reaction between the antibody and the target antigen, and in which the agglutination reaction is carried out in the presence of an MPC polymer.

[0028] Examples of the measurement method include the following. First, multiple standard samples containing the target antigen to be measured at various known concentrations are prepared, and the amount or rate of change in absorbance is measured for each standard sample using the method described above. Examples of standard samples include a 0.1 to 5% by mass bovine serum albumin solution containing the target antigen. A calibration curve is drawn by plotting the concentration of the antigen to be measured in the standard sample on the horizontal axis and the measured amount or rate of change in absorbance on the vertical axis. The amount or rate of change in absorbance for an unknown test sample is also measured using the same method, and the measurement results are applied to the calibration curve, allowing for quantitative evaluation of the target antigen in the test sample. Alternatively, a threshold value for the amount or rate of change in absorbance can be set in advance, and the presence of the target antigen in the test sample can be qualitatively assessed when the threshold value is exceeded.

[0029] A reagent for measuring hBNP according to yet another aspect of the present embodiment comprises a copolymer containing 2-methacryloyloxyethyl phosphorylcholine as a monomer unit, and the measurement is based on an agglutination reaction of the insoluble particles, which have a granular carrier and an anti-hBNP antibody supported on the granular carrier, due to an antigen-antibody reaction between the anti-hBNP antibody and the hBNP.

[0030] In the measurement of hBNP using the above reagent, the discrepancy between the reactivity of the agglutination reaction in a system not containing a blood sample and the reactivity of the agglutination reaction in a system containing a blood sample is suppressed.

[0031] The method for measuring hBNP using the above-mentioned reagent may be the same as the measurement method in the method according to another aspect of the present embodiment described above.

[0032] The reagent may include insoluble particles having a particulate carrier and an anti-hBNP antibody supported on the particulate carrier.

[0033] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0034] <Preparation of stabilizing solution> To a 120 mM bicine (2-(bis(2-hydroxyethyl))amino)acetic acid) buffer solution containing 300 mM potassium chloride, 1% by mass of bovine serum albumin (hereinafter also referred to as "BSA"), and 0.05% by mass of ProClin (registered trademark) 300, polyvinylpyrrolidone (hereinafter also referred to as "PVP"), polyethylene glycol 20000 (hereinafter also referred to as "PEG"), Lipidure (registered trademark)-BL103 (manufactured by NOF Corporation, hereinafter also referred to as "BL103"), or Lipidure (registered trademark)-BL405 (manufactured by NOF Corporation, hereinafter also referred to as "BL405") was added to make 1% by mass, to prepare a stabilizing solution.

[0035] <Preparation of antibody-immobilized latex dispersion> A solution of anti-human brain natriuretic peptide (hereinafter also referred to as "hBNP") antibody, a mouse-derived IgG1 antibody, and polystyrene beads (average particle size: 0.3 μm) were mixed and reacted at 25°C for 1 hour. The mixture was then centrifuged, the supernatant removed, and a 1% by mass BSA solution was added for blocking to obtain antibody-immobilized latex. A dispersion medium was added and adjusted so that the final concentration of the obtained antibody-immobilized latex was 0.125% by mass, thereby obtaining an antibody-immobilized latex dispersion. The dispersion medium used contained 5 mM 3-morpholinopropanesulfonic acid (pH 7.0), 5% by mass sucrose, and 0.4% by mass BSA.

[0036] <Evaluation of Agglutination Reactivity> The effects of Lipidure®-BL103 and Lipidure®-BL405 on the discrepancy between the agglutination reactivity in a 2% by weight BSA solution and that in plasma were evaluated using a two-reagent latex agglutination immunoassay using a stabilizing solution and an antibody-immobilized latex dispersion. Specifically, hBNP solution was added to a 2% by weight BSA solution and human plasma so that the hBNP content in each solution was 0 pg / ml, 100 pg / ml, or 1000 pg / ml, to prepare hBNP-containing samples. The amount of the hBNP solution carried over into the hBNP-containing samples was 10% by mass based on the total mass of the hBNP-containing sample. 33 μL of the stabilizing solution was added to 10 μL of the obtained hBNP-containing sample, mixed, and then reacted at 37°C for 5 minutes. Thereafter, 100 μL of the antibody-immobilized latex dispersion was added, stirred, and then reacted at 37°C for 5 minutes. The absorbance was continuously measured for 5 minutes after the addition of the antibody-immobilized latex dispersion using a clinical chemistry analyzer TBA-120FR at photometric points 21-23 and 31-33 and a dominant measurement wavelength of 628 nm, and the change in absorbance over the 5 minutes was calculated. Next, for each stabilizing solution, the percentage of the change in absorbance in human plasma containing 100 pg / ml or 1000 pg / ml of hBNP relative to the change in absorbance in a 2% by mass BSA solution containing the same concentration of hBNP was calculated. The results are shown in Figure 1. In Figure 1, "Signal in plasma / Signal of reference measurement value [%]" indicates the percentage.

[0037] As shown in Figure 1, when a stabilizing solution containing Lipidure (registered trademark)-BL103 or Lipidure (registered trademark)-BL405 was used, the discrepancy between the agglutination reactivity in a 2% by mass BSA solution and the agglutination reactivity in human plasma was improved compared to when a stabilizing solution containing polyvinylpyrrolidone or polyethylene glycol 20000 was used. Furthermore, this improvement effect was more pronounced with a stabilizing solution containing Lipidure (registered trademark)-BL405. From the above, it was demonstrated that the use of a copolymer containing 2-methacryloyloxyethyl phosphorylcholine as a monomer unit can sufficiently suppress the discrepancy between the agglutination reactivity in a system not containing a blood sample and the agglutination reactivity in a system containing a blood sample.

Claims

1. An agent for improving an agglutination reaction, comprising a copolymer containing 2-methacryloyloxyethyl phosphorylcholine as a monomer unit, wherein the agglutination reaction is an agglutination reaction of insoluble particles having a granular carrier and an antibody against a target antigen supported on the granular carrier, by an antigen-antibody reaction between the target antigen and the insoluble particles, and the agent is used to suppress the discrepancy in the reactivity of the agglutination reaction between a system not containing a blood sample and a system containing a blood sample.

2. The improving agent according to claim 1, wherein the copolymer is a copolymer containing a monomer unit having an anionic group.

3. The improving agent according to claim 1 or 2, wherein the copolymer is a copolymer containing a monomer unit having a nonionic group.

4. A method for suppressing the discrepancy between the reactivity of an agglutination reaction in a system not containing a blood sample and the reactivity of an agglutination reaction in a system containing a blood sample in a method for measuring a target antigen using insoluble particles having a granular carrier and an antibody against the target antigen supported on the granular carrier, the method being based on an agglutination reaction of the insoluble particles due to an antigen-antibody reaction between the antibody and the target antigen, the method comprising: carrying out the agglutination reaction in the presence of a copolymer containing 2-methacryloyloxyethyl phosphorylcholine as a monomer unit.

5. The method according to claim 4, wherein the copolymer is a copolymer containing monomer units having an anionic group.

6. The method according to claim 4 or 5, wherein the copolymer is a copolymer containing a monomer unit having a nonionic group.

7. A reagent for measuring human brain natriuretic peptide, comprising a copolymer containing 2-methacryloyloxyethyl phosphorylcholine as a monomer unit, wherein the measurement uses insoluble particles having a granular carrier and an anti-human brain natriuretic peptide antibody supported on the granular carrier, and is based on an agglutination reaction of the insoluble particles due to an antigen-antibody reaction between the anti-human brain natriuretic peptide antibody and the anti-human brain natriuretic peptide.