Sensitizer for immunological measurement, kit for immunological measurement, and immunological measurement method

Abstract

The present invention addresses the problem of providing: a sensitizer for immunological measurement with which the problem of salting out in immunological measurement is solved; a kit for immunological measurement, the kit comprising said sensitizer for immunological measurement; and an immunological measurement method using said sensitizer for immunological measurement. The present invention provides a sensitizer for immunological measurement, the sensitizer comprising a copolymer that contains a repeating unit represented by formula [1] and a repeating unit represented by formula [2] or [3]. In the formulae, each group has the definition described in the specification.

Description

Sensitizer for immunoassay, immunoassay kit and immunoassay method

[0001] The present invention relates to a sensitizer for immunoassays, which comprises a copolymer containing a predetermined repeating unit. The present invention also relates to an immunoassay kit and an immunoassay method.

[0002] Known methods for measuring target substances in biological samples such as serum, plasma, and urine include separation analysis using chemical analytical equipment (e.g., HPLC (high-performance liquid chromatography), LC-MS / MS (liquid chromatography tandem mass spectrometry)), and immunoassay using automated analyzers. Immunoassays are widely used in clinical testing because they are capable of highly sensitive measurement of a wide range of substances, from low to high molecular weight. One immunoassay method that can achieve high sensitivity is the two-step sandwich method, in which an antibody is immobilized, reacted with an antigen in the sample, and an antigen-antibody reaction is carried out using an enzyme-labeled antibody. This can be combined with a chemiluminescence reaction to enable highly sensitive measurement. In the two-step sandwich method, a sensitizer is usually used to improve sensitivity. Polyethylene glycol (PEG) is a known sensitizer.

[0003] Patent Document 1 describes a blocking agent for biochemical analysis, which is characterized by containing a water-soluble vinyl polymer having a specific betaine structure. Patent Document 2 describes a cosmetic containing a polymer obtained by copolymerizing a fluorine atom-containing monomer, a betaine monomer, and an alkyl (meth)acrylate.

[0004] JP 2018-72148 A JP 2016-160205 A

[0005] Polyethylene glycol (PEG) is known as a sensitizer for use in immunoassays, but PEG salts out in solutions with high salt concentrations, resulting in poor measurement accuracy. Because of the above-mentioned problems with PEG, the development of an alternative sensitizer has been desired.

[0006] An object of the present invention is to provide a sensitizer for immunoassays that overcomes the problem of salting out in immunoassays.A further object of the present invention is to provide an immunoassay kit containing the above-mentioned sensitizer for immunoassays, and an immunoassay method using the above-mentioned sensitizer for immunoassays.

[0007] As a result of extensive research into solving the above problems, the present inventors have found that the problem of salting out of sensitizers in immunoassays can be solved by using a copolymer obtained by polymerizing a carboxybetaine monomer with a specific monomer, and have thus completed the present invention.

[0008] That is, the present invention provides the following inventions: <1> A sensitizer for immunoassays, comprising a copolymer containing a repeating unit represented by formula [1] and a repeating unit represented by formula [2] or formula [3]. In formula [1], X １ represents an oxygen atom or an —NH— group, and R １ and R ２ each independently represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms; R ３ represents a hydrogen atom or an alkyl group having 1 to 3 carbon atoms; m represents an integer of 1 to 6; and n represents an integer of 1 to 3. In formula [2], X ２ represents an oxygen atom or an —NH— group, and R ４ represents a fluoroalkyl group having 1 to 10 carbon atoms, and R ５ represents a hydrogen atom or an alkyl group having 1 to 3 carbon atoms. In formula [3], R ６ represents an aryl group which may have a carboxyl group or a sulfo group as a substituent, R ７represents a hydrogen atom or an alkyl group having 1 to 3 carbon atoms. <2> The sensitizer for immunoassays according to <1>, wherein the mass ratio of the repeating unit (A) represented by formula [1] to the repeating unit (B) represented by formula [2] or formula [3] is (A) / (B) = 70 / 30 to 90 / 10. <3> The sensitizer for immunoassays according to <1> or <2>, wherein the weight-average molecular weight of the copolymer is 100,000 to 3,000,000. <4> The sensitizer for immunoassays according to any one of <1> to <3>, wherein the weight-average molecular weight of the copolymer is 140,000 to 900,000. <5> A kit for immunoassays comprising the sensitizer for immunoassays according to any one of <1> to <4>, an immobilized first analyte-binding substance, and a labeled second analyte-binding substance. <6> An immunoassay method, comprising a step of carrying out an antigen-antibody reaction in the presence of the sensitizer for immunoassay according to any one of <1> to <4>.

[0009] According to the present invention, the problem of salting out of a sensitizer in immunoassay can be solved.

[0010] The present invention will be described in detail below. In this specification, the word "to" is used to mean that the numerical values ​​before and after it are included as the lower limit and upper limit.

[0011] <Sensitizer for Immunological Assay> The sensitizer for immunological assay of the present invention includes a copolymer containing a repeating unit represented by formula [1] and a repeating unit represented by formula [2] or formula [3]. In formula [1], X １ represents an oxygen atom or an —NH— group, and R １ and R ２ each independently represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms; R ３ represents a hydrogen atom or an alkyl group having 1 to 3 carbon atoms; m represents an integer of 1 to 6; and n represents an integer of 1 to 3. In formula [2], X ２ represents an oxygen atom or an —NH— group, and R ４ represents a fluoroalkyl group having 1 to 10 carbon atoms, and R ５ represents a hydrogen atom or an alkyl group having 1 to 3 carbon atoms. In formula [3], R ６ represents an aryl group which may have a carboxyl group or a sulfo group as a substituent, R ７ represents a hydrogen atom or an alkyl group having 1 to 3 carbon atoms.

[0012] Polyethylene glycol (PEG) has been well known as a sensitizer for immunoassays, but PEG salts out in solutions with high salt concentrations, resulting in high blank values ​​and poor measurement accuracy. In contrast, the sensitizer of the present invention overcomes problems such as salting out, does not affect the immunoassay system, and enables highly sensitive measurements.

[0013] Examples of alkyl groups having 1 to 6 carbon atoms include methyl, ethyl, propyl, butyl, pentyl, and hexyl groups, which may be straight-chain or branched. Examples of alkyl groups having 1 to 3 carbon atoms include methyl, ethyl, and propyl groups. Examples of aryl groups include phenyl and naphthyl groups.

[0014] X １ preferably represents an oxygen atom. １ and R ２ Preferably, each independently represents an alkyl group having 1 to 3 carbon atoms. １ and R ２ More preferably, R represents a methyl group. ３ represents preferably an alkyl group having 1 to 3 carbon atoms, more preferably a methyl group. m represents preferably an integer of 1 to 3, more preferably 2. n represents preferably 1.

[0015] X ２ preferably represents an oxygen atom. ４ represents preferably a fluoroalkyl group having 1 to 5 carbon atoms, more preferably a fluoroalkyl group having 1 to 3 carbon atoms, and particularly preferably —CH ２ -CF ３ Represents R ５ preferably represents an alkyl group having 1 to 3 carbon atoms, and more preferably represents a methyl group.

[0016] R ６R preferably represents an aryl group having a sulfo group as a substituent, and more preferably represents a phenyl group having a sulfo group as a substituent. ７ preferably represents a hydrogen atom.

[0017] Specific examples of the repeating unit represented by formula [1] are shown below.

[0018] Specific examples of the repeating unit represented by formula [2] are shown below.

[0019] Specific examples of the repeating unit represented by formula [3] are shown below.

[0020] The mass ratio of the repeating unit (A) represented by formula [1] to the repeating unit (B) represented by formula [2] or formula [3] is preferably (A) / (B)=70 / 30 to 90 / 10, more preferably (A) / (B)=80 / 20 to 90 / 10.

[0021] The weight average molecular weight of the copolymer is preferably 100,000 to 3,000,000, more preferably 100,000 to 1,000,000, still more preferably 140,000 to 900,000, and particularly preferably 140,000 to 420,000.

[0022] The immunoassay sensitizer of the present invention can be preferably used to improve the sensitivity in immunoassay.

[0023] The sensitizer for immunoassay of the present invention may further contain other components, including, but not limited to, a buffer, an inorganic salt, an inorganic acid or an inorganic base, a stabilizer, a non-specific reaction inhibitor, an adsorption inhibitor, a preservative, a solvent, etc.

[0024] Examples of the buffer include N-(2-acetamido)-2-aminoethanesulfonic acid (ACES), N-(2-acetamido)iminodiacetic acid (ADA), N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES), N,N-bis(2-hydroxyethyl)glycine (Bicine), bis(2-hydroxyethyl)iminotris(hydroxyethyl)methane (Bis-Tris), N-cyclohexyl-3-aminopropanesulfonic acid (CA PS), N-cyclohexyl-2-hydroxy-3-aminopropanesulfonic acid (CAPSO), N-cyclohexyl-2-aminoethanesulfonic acid (CHES), 3-[N,N-bis(2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid (DIPSO), 3-[4-(2-hydroxyethyl)-1-piperazinyl]propanesulfonic acid (EPPS), 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES) , 2-hydroxy-3-[4-(2-hydroxyethyl)-1-piperazinyl]propanesulfonic acid (HEPPSO), 2-(N-morpholino)ethanesulfonic acid (MES), 3-(N-morpholino)propanesulfonic acid (MOPS), 2-hydroxy-3-(N-morpholino)propanesulfonic acid (MOPSO), piperazine-1,4-bis(2-ethanesulfonic acid) (PIPES), piperazine-1,4-bis(2-hydroxy-3-propanesulfonic acid) (P OPSO), N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS), 2-hydroxy-N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPSO), N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES), N-[tris(hydroxymethyl)methyl]glycine (Tricine), phosphate, acetate, citrate, tris(hydroxymethyl)aminomethane, and the like.

[0025] Examples of inorganic salts include those known in the art, such as sodium chloride, potassium chloride, calcium chloride, magnesium sulfate, sodium bicarbonate, and sodium dihydrogen phosphate.

[0026] Examples of inorganic acids or inorganic bases include those known in the art, such as hydrochloric acid, sulfuric acid, boric acid, phosphoric acid, sodium hydroxide, and potassium hydroxide.

[0027] Examples of the stabilizer include known chelating agents and protease inhibitors.

[0028] As the non-specific reaction inhibitor, any of those known in the art can be used. For example, milk proteins such as skim milk, egg albumin, commercially available blocking agents (e.g., Block Ace (manufactured by Sumitomo Dainippon Pharma Co., Ltd.), Blockmaster TM PA series (manufactured by JSR Corporation), RNase, etc.

[0029] Examples of the adsorption inhibitor include surfactants and inhibitors of peptide adsorption such as albumin (e.g., bovine serum albumin).

[0030] Examples of preservatives include salicylic acid and benzoic acid.

[0031] Examples of the solvent include water and water-soluble organic solvents.

[0032] <Immunological Assay Kit> The immunological assay kit of the present invention comprises the above-mentioned immunological assay sensitizer of the present invention, a solid-phased first analyte-binding substance, and a labeled second analyte-binding substance.

[0033] In the present invention, the substance to be measured is not particularly limited, and examples thereof include all substances that are considered to be measurable by ordinary immunological assays, such as steroids, peptides, hormones, antibodies, proteins, drugs, metabolites, vitamins, etc. contained in biological samples. Specific examples of substances related to endocrine function include thyroid stimulating hormone (TSH), parathyroid hormone (iPTH), growth hormone (GH), somatomedin C (IGF-1), luteinizing hormone (LH), follicle stimulating hormone (FSH), prolactin (PRL), adrenocorticotropic hormone (ACTH), vasopressin, oxytocin, somatostatin, enkephalin, β-endorphin, thyroxine, triiodothyronine, thyroglobulin, antithyroglobulin antibody, anti-T3 antibody, and the like. Body, anti-T4 antibody, anti-TSH antibody, calcitonin, catecholamine, dopamine, serotonin, aldosterone, renin, angiotensin, cortisol, deoxycortisol, cortisone, corticosterone, deoxycorticosterone, androsterone, progesterone, pregnenolone, estrogen, estrone, estriol, estradiol, testosterone, gonadotropins, insulin, anti-insulin antibody, C-peptide, glucagon, gastrin, sex Examples of the anti-inflammatory drugs include retin, cyclic AMP, cyclic GMP, prostaglandins, thromboxane, erythropoietin, and histamine. Examples of tumor-related substances include CEA, ferritin, β2-microglobulin, elastase, α-fetoprotein, nerve-specific enolase, prostate-specific antigen, and CA19-9. Examples of drugs and vitamin-related substances include phenobarbital, phenytoin, carbamazepine, primidone, ethosuximide, valproic acid, and acetazole. Amides, sulthiame, glutethimide, clonazepam, nitrazepam, diazepam, pentobarbital, secobarbital, bupivacaine, mepivacaine, lidocaine, procainamide, quinidine, digoxin, digitoxin, theophylline, amitriptyline, imipramine, amikacin, gentamicin, tobramycin, cephalexin, sulfamethoxazole, methotrexate, cyclosporine, methylprednisolone, salicylic acid, acetaminophen, indomethacin,Examples of the target substance include allopurinol, vitamin A, carotene, vitamin B1, vitamin B2, vitamin B6, vitamin B12, folic acid, vitamin C, vitamin D, and vitamin E. Examples of serum or plasma protein-related substances include albumin, α1-microglobulin, α1-antitrypsin, α2-macroglobulin, haptoglobulin, hemopexin, transferrin, myoglobin, IgG, IgM, IgA, IgD, IgE, fibrinogen, antithrombin, plasminogen, antiplasmin, protein C, rheumatoid factor, anti-DNA antibody, and C-reactive protein. Examples of virus and infectious disease-related substances include HBs antigen, HBs antibody, HBc antibody, HTLV-I antibody, HTLV-III antibody, TPHA, various viral antigens, and various viral antibodies. An example of the target substance to be measured is HBs antigen, as measured in the Examples below, but is not limited to this.

[0034] The analyte-binding substance is a substance that can bind to the analyte. Examples of the analyte-binding substance include antibodies that bind to the analyte, peptides such as cyclic peptides, and aptamers. Antibodies that bind to the analyte include Fab, Fab', F(ab')2, Fv, Fd, single-chain Fv (scFv), disulfide-bonded Fv (sdFv), and V. L , V H , Fv-clasp, diabody ((V L -V H )2 or (V H -V L )2), triabody (trivalent antibody), tetrabody (tetravalent antibody), minibody ((scFV-CH3)2), IgG-delta-CH2, scFv-Fc, (scFv)2-Fc fragment, or a modified form of an antibody.

[0035] Examples of solid-phase carriers for immobilizing the analyte-binding substance include insoluble solid-phase carriers used in conventional immunoassays. Specifically, substrates used in conventional protein immobilization methods, such as slide glasses, ELISA plates, microtiter plates, beads (e.g., magnetic beads), microparticles (e.g., latex particles), filters, tubes, films, and membranes, can be used. Examples of substrate materials include synthetic polymer compounds such as polycarbonate, polystyrene, polyurethane, polypropylene, polyacrylic acid, polymethacrylic acid, polyacrylamide, polyglycidyl methacrylate, polyvinyl chloride, polyethylene, polychlorocarbonate, silicone resin, and silicone rubber, as well as inorganic materials such as glass (e.g., porous glass), ground glass, silicon, ceramics, alumina, silica gel, activated carbon, and metal oxides.

[0036] The method for immobilizing the substance that binds to the analyte on the solid phase carrier is not particularly limited, and known methods such as chemical binding methods (methods for immobilizing by covalent bonding) and physical adsorption methods can be applied.

[0037] As the labeling substance used to label the substance that binds to the measured substance, for example, the following substances can be used, but there is no particular limitation, and any labeling substance that is normally used in this field can be used.

[0038] Enzymes such as peroxidase, horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, microperoxidase, glucose oxidase, glucose-6-phosphate dehydrogenase, acetylcholinesterase, malate dehydrogenase, and luciferase; ９９ｍ Tc, １３１ I, １２５ I, １４ C. ３ H. ３２ P, ３５ Radioactive isotopes such as S;

[0039] Fluorescent substances such as HiLyte 647 (manufactured by AhaSpec), fluorescein, dansyl, fluorescamine, coumarin, naphthylamine, fluorescein isothiocyanate (FITC), rhodamine, rhodamine X isothiocyanate, sulforhodamine 101, Lucifer Yellow, acridine, acridine isothiocyanate, riboflavin, or derivatives thereof; luminescent substances such as luciferin, isoluminol, luminol, 8-amino-5-chloro-7-phenylpyrido[3,4-d]pyridazine-1,4(2H,3H)-dione sodium salt (L-012);

[0040] Substances that absorb in the ultraviolet region, such as phenol, naphthol, anthracene, or derivatives thereof; substances that have properties as spin labeling agents, such as compounds having an oxyl group, such as 4-amino-2,2,6,6-tetramethylpiperidine-1-oxyl, 3-amino-2,2,5,5-tetramethylpyrrolidine-1-oxyl, 2,6-di-t-butyl-α-(3,5-di-t-butyl-4-oxo-2,5-cyclohexadien-1-ylidene)-p-tolyloxyl;

[0041] HiLyte dyes such as HiLyte Fluor 647, HiLyte Fluor 488, HiLyte Fluor 555, HiLyte Fluor 680, and HiLyte Fluor 750 (all trade names of HiLyte Bioscience, Inc.); Alexa Fluor Dye 350, Alexa Fluor Dye 430, Alexa Fluor Dye 488, Alexa Fluor Dye 532, Alexa Fluor Dye 546, Alexa Fluor Dye 555, and Alexa Fluor Dye Alexa dyes such as Alexa Fluor Dye 568, Alexa Fluor Dye 594, Alexa Fluor Dye 633, Alexa Fluor Dye 647, Alexa Fluor Dye 660, Alexa Fluor Dye 680, Alexa Fluor Dye 700, and Alexa Fluor Dye 750 (all trade names of Molecular Probes);

[0042] CyDye dyes such as Cy3, Cy3.5, Cy5, Cy5.5, and Cy7 (all trade names of Amersham Biosciences); dyes such as Coomassie Brilliant Blue R250 and Methyl Orange

[0043] As the labeling substance used to label the substance that binds to the analyte, enzymes such as peroxidase and HPR are preferred.

[0044] The method for binding a labeled substance to an analyte-binding substance can be carried out by appropriately utilizing a labeling method used in conventional immunoassays, etc., and examples thereof include those described in "Medical Chemistry Experiment Lectures," Vol. 8, edited by Yamamura Yuichi, 1st Edition, Nakayama Shoten, 1971; "Illustrated Fluorescent Antibodies," by Kawao Akira, 1st Edition, Soft Science Co., Ltd., 1983; and "Enzyme Immunoassay," edited by Ishikawa Eiji, Kawai Tadashi, and Muroi Kiyoshi, 2nd Edition, Igaku Shoin, 1982. The labeled substance may be directly bound to the analyte-binding substance or the analyte, or may be indirectly bound to the analyte-binding substance via a suitable spacer (e.g., via a combination of one or several amino acids, one or several amino acids and a linker, or avidins (e.g., streptavidin, tamavidin) and a substance having affinity for biotin).

[0045] An example of the kit of the present invention may include the following first to fifth reagents.

[0046] The first reagent is a reagent containing particles to which a substance binding to a substance to be measured is bound. The substance to be measured can be bound to the particles by a known method. It is preferable to use magnetic particles as the particles. The first reagent may contain a dispersion medium such as water, or may be used in the form of particles without containing a dispersion medium.

[0047] The second reagent is an immune reaction buffer solution containing the immunoassay sensitizer of the present invention. The second reagent preferably contains a buffer. Examples of the buffer include those described in <Immunoassay sensitizer>. In the second reagent, the concentration of the copolymer containing the repeating unit represented by formula [1] and the repeating unit represented by formula [2] or [3] is not particularly limited, but is preferably 0.1% by mass or more and 10% by mass or less, more preferably 0.2% by mass or more and 8% by mass or less, even more preferably 0.3% by mass or more and 5% by mass or less, and particularly preferably 0.5% by mass or more and 2% by mass or less.

[0048] The third reagent is a reagent containing a labeled analyte-binding substance. The third reagent preferably contains a labeled analyte-binding substance, a buffer, and the like. Examples of the buffer include those described in <Sensitizers for immunological assays>.

[0049] The fourth reagent is a reagent containing a chemiluminescent substance. Examples of the chemiluminescent substance include known chemiluminescent substances such as luciferin and L-012 (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.). The fourth reagent preferably further contains a buffer or the like. Examples of the buffer include those described in <Sensitizers for Immunological Assays>. In the fourth reagent, the concentration of the chemiluminescent substance (concentration as a reagent) is not particularly limited, but is preferably 0.2 mmol / L or more and 20 mmol / L or less, more preferably 0.4 mmol / L or more and 15.0 mmol / L or less, even more preferably 0.5 mmol / L or more and 10.0 mmol / L or less, and particularly preferably 0.5 mmol / L or more and 5.0 mmol / L or less.

[0050] The fifth reagent is a reagent containing an oxidizing agent (e.g., hydrogen peroxide). The fifth reagent preferably further contains a buffer or the like. Examples of the buffer include those described in <Sensitizer for immunological assays>. In the fifth reagent, the concentration of the oxidizing agent (concentration as a reagent) is not particularly limited, but is preferably 1.0 mmol / L or more and 40 mmol / L or less, more preferably 2 mmol / L or more and 20 mmol / L or less, and even more preferably 2.8 mmol / L or more and 7.0 mmol / L or less.

[0051] The immunoassay kit of the present invention may further contain a washing solution. Known washing solutions such as water and buffer solutions can be used as the washing solution. Alternatively, commercially available washing solutions such as Accura Seed B / F Separation Solution (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) may also be used.

[0052] The kit of the present invention may further include a standard sample. The standard sample is preferably a sample containing a known concentration of the substance to be measured. Standard samples are used as calibrators or controls for creating calibration curves showing the correlation between a known amount (concentration) of the substance to be measured contained in the standard sample and measurements obtained by various analytical methods, for confirming the effectiveness of accuracy control and calibration of analytical instruments, and for investigating the stability of quantitative analysis over time. Examples of the measured values ​​include absorbance, change in absorbance, amount of transmitted light, change in transmitted light, luminescence, and change in luminescence. Furthermore, conversion values ​​such as the amount and concentration of the substance to be measured in a sample calculated from the measured value may also be referred to as measured values ​​in this specification.

[0053] The kit of the present invention may further include an instruction manual, etc. The instruction manual may describe the components of the reagent of the present invention, the operating procedure and principles of the method of detecting a substance to be measured of the present invention, etc.

[0054] <Immunoassay Method> The immunoassay method of the present invention comprises the step of carrying out an antigen-antibody reaction in the presence of the above-described sensitizer for immunoassay of the present invention.

[0055] The concentration of the sensitizer for immunological assays in the reaction solution in which an antigen reaction is carried out, i.e., the concentration of the copolymer containing the repeating unit represented by formula [1] and the repeating unit represented by formula [2] or formula [3] in the reaction solution in which an antigen reaction is carried out, is preferably from 0.01% by mass to 5% by mass, more preferably from 0.05% by mass to 3% by mass, and even more preferably from 0.1% by mass to 2% by mass.

[0056] In one example of the present invention, an antigen-antibody reaction can be carried out using a substance to be measured in a sample as an antigen and an antibody as a substance that binds to the substance to be measured.

[0057] The sample is not particularly limited, but animal-derived samples are preferred, and human-derived samples are more preferred. Examples of samples include, but are not limited to, blood-derived samples (serum, plasma, whole blood, etc.), feces, urine, saliva, lymph, cerebrospinal fluid, pleural effusion, ascites, tears, semen, bladder washings, tissue extracts, tissue sections, tissue biopsy samples, and samples prepared therefrom. Blood-derived samples are preferred, with serum, plasma, or whole blood being particularly preferred. The biological sample may be directly collected or may be pretreated, for example, by collection, concentration, purification, isolation, dilution with a buffer solution, or filtration sterilization. These pretreatments may be performed as appropriate according to conventional methods.

[0058] The measurement principle of the immunoassay is not particularly limited, and may be, for example, a sandwich method or a competitive method. The measurement method in the immunoassay is also not particularly limited, and examples thereof include enzyme-linked immunosorbent assay (ELISA) and chemiluminescent enzyme immunoassay (CLEIA).

[0059] In the sandwich method, for example, a first analyte-binding substance is immobilized on the surface of a solid-phase carrier such as magnetic silica particles. A sample containing the analyte is mixed with the solid-phase carrier and a labeled second analyte-binding substance. The immobilized first analyte-binding substance, the analyte in the sample, and the second analyte-binding substance are then contacted. This results in the formation of a labeled complex, which is a complex of the immobilized first analyte-binding substance, the analyte in the sample, and the labeled second analyte-binding substance. The solid-phase carrier carrying the labeled complex is then subjected to B / F separation to measure the amount of label in the labeled complex, and the amount of analyte in the sample can be determined based on the amount of label in the labeled complex.

[0060] In the above method, the analyte in the sample is reacted with the immobilized first analyte-binding substance, and then the labeled second analyte-binding substance is reacted therewith. Alternatively, the labeled second analyte-binding substance may be reacted with the analyte in the sample, and then the immobilized first analyte-binding substance may be reacted therewith, or these three may be reacted simultaneously.

[0061] Alternatively, for example, a first analyte-binding substance is immobilized on the surface of a solid-phase carrier such as magnetic silica particles. A sample containing the analyte is mixed with the solid-phase carrier and a labeled "third binding substance that recognizes the complex between the analyte and the first analyte-binding substance." The immobilized first analyte-binding substance is then contacted with the analyte in the sample and the third binding substance. This allows the formation of a labeled complex, which is a complex between the immobilized first analyte-binding substance, the analyte in the sample, and the labeled third binding substance. The solid-phase carrier carrying the labeled complex is then subjected to B / F separation to measure the amount of label in the labeled complex, and the amount of analyte in the sample can be determined based on the amount of label in the labeled complex.

[0062] In the competitive method, a sample containing the substance to be measured, a solid phase carrier (e.g., magnetic particles) to which the substance to be measured is bound, and a labeled substance that binds to the substance to be measured are reacted, and B / F separation (Bound / Free separation) is performed, after which the label bound to the solid phase is detected.

[0063] The solid phase carrier can be any support (particularly an insoluble support) used in conventional immunoassays, and examples thereof include organic substances such as polystyrene, polyacrylic acid, polymethacrylic acid, polymethyl methacrylate, polyacrylamide, polyglycidyl methacrylate, polypropylene, polyolefin, polyimide, polyurethane, polyester, polyvinyl chloride, polyethylene, polychlorocarbonate, silicone resin, silicone rubber, agarose, dextran, and ethylene-maleic anhydride copolymer; inorganic substances such as glass, silicon oxide, diatomaceous earth, porous glass, ground glass, alumina, silica gel, and metal oxides; magnetic substances such as iron, cobalt, nickel, magnetite, and chromite; and substances prepared from alloys of these magnetic substances.

[0064] Immobilization can be performed on a solid phase (for example, any surface such as beads, magnetic beads, membranes, plates, etc.). Various commercially available magnetic beads can be used. For example, the beads disclosed in WO2012 / 173002A may be used as magnetic beads.

[0065] In the sandwich method, the analyte may be brought into contact with the immobilized first analyte-binding substance by a conventional process such as stirring, mixing, etc. The reaction time may be appropriately set depending on the analyte and the first analyte-binding substance, but is usually 1 minute to 24 hours, preferably 1 minute to 1 hour, more preferably 1 to 10 minutes, and particularly preferably 1 to 5 minutes.

[0066] In the sandwich method, the analyte may be contacted with the labeled second analyte-binding substance by a conventional method such as stirring, mixing, etc. The reaction time may be appropriately set depending on the analyte and the second analyte-binding substance, but is usually 1 minute to 24 hours, preferably 1 minute to 1 hour, more preferably 1 to 10 minutes, and particularly preferably 1 to 5 minutes.

[0067] B / F separation (bond / free separation) refers to the separation of a substance supported on a solid-phase carrier from other substances. Specifically, examples of B / F separation in the present invention include: (1) separation of a "complex of the immobilized first analyte-binding substance and the analyte" from the analyte or other substances in the reaction system that were not involved in the formation of the complex; and (2) separation of a complex of the immobilized first analyte-binding substance, the analyte, and a labeled second analyte-binding substance from the labeled second analyte-binding substance or other substances in the reaction system that were not involved in the formation of the complex.

[0068] B / F separation can be performed, for example, by utilizing the magnetic properties of the solid carriers, by collecting the solid carriers with a magnet or the like from the outside of the reaction vessel, discharging the reaction solution, adding a washing solution, removing the magnet, mixing and dispersing the solid carriers, and washing them. The above procedure may be repeated 1 to 3 times. The washing solution is not particularly limited as long as it is one commonly used in this field.

[0069] In one example of the present invention, after an antigen-antibody reaction has occurred, the label can be measured using a chemiluminescent substance and an oxidizing agent.

[0070] The chemiluminescent substance (chemiluminescent substrate) is preferably a nitrogen-containing heterocyclic compound having an amino group or a salt thereof, and examples thereof include a 2,3-dihydro-1,4-phthalazinedione compound having an amino group or a salt thereof, and 8-amino-5-chloro-7-phenylpyrido[3,4-d]pyridazine-1,4(2H,3H)-dione sodium salt (L-012).

[0071] The structure of L-012 is shown below.

[0072] Examples of the 2,3-dihydro-1,4-phthalazinedione compound that can be used include known 2,3-dihydro-1,4-phthalazinedione compounds described in JP-A Nos. 2-291299, 10-319015, and 2000-279196, as well as mixtures thereof. Of these, luminol, luminol derivatives (e.g., isoluminol, N-aminohexyl-N-ethylisoluminol (AHEI), N-aminobutyl-N-ethylisoluminol (ABEI)), and metal salts thereof (e.g., alkali metal salts) are preferred, luminol and its metal salts are more preferred, and luminol sodium salt is particularly preferred.

[0073] The concentration of the chemiluminescent substance is preferably 0.1 mmol / L to 10 mmol / L, more preferably 0.2 mmol / L to 7.5 mmol / L, even more preferably 0.25 mmol / L to 5.0 mmol / L, and particularly preferably 0.25 mmol / L to 2.5 mmol / L. The concentration referred to here is the concentration in the reaction solution when the chemiluminescent substance is reacted.

[0074] Examples of the oxidizing agent include aqueous solutions of known oxidizing agents such as those described in JP-A-8-261943 and JP-A-2000-279196 (inorganic peroxides (such as hydrogen peroxide, sodium perborate, and potassium perborate), organic peroxides (such as dialkyl peroxides and acyl peroxides), and peroxyacid compounds (such as peroxosulfuric acid and peroxophosphoric acid). Among these, from the viewpoint of storage stability, hydrogen peroxide, sodium perborate, and potassium perborate are preferred, and hydrogen peroxide is more preferred.

[0075] The concentration of the oxidizing agent is set appropriately depending on the type thereof, the measurement method and conditions to be applied, etc., but is preferably 0.5 mmol / L to 20 mmol / L, more preferably 1 mmol / L to 10 mmol / L, and even more preferably 1.4 mmol / L to 3.5 mmol / L. The concentration referred to here is the concentration in the reaction solution when the oxidizing agent is reacted.

[0076] The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples. In this specification, mM means mmol / L.

[0077] Example 1 Synthesis of Polymer (P1-1) Synthesis of N-[2-(carboxymethyldimethylamino)ethyl]methacrylic acid 350 g (6.2 mol) of potassium hydroxide was dissolved in 2.3 L of ethanol. 500 g (5.3 mol) of chloroacetic acid (manufactured by FUJIFILM Wako Pure Chemical Industries, Ltd.) was added to the resulting solution, and the mixture was stirred at room temperature for 1 hour. Crystals precipitated by the reaction were then collected by filtration and washed with isopropanol. The washed crystals were then mixed with 1.7 L of isopropanol to obtain a suspension. 557 g (3.5 mol) of N-[2-(dimethylamino)ethyl]methacrylic acid (manufactured by FUJIFILM Wako Pure Chemical Industries, Ltd.) was added to the resulting suspension, and the mixture was stirred under reflux for 12 hours. After the reaction was completed, insoluble matter was filtered off, washed with isopropanol, and the washings were recovered. The filtrate and washings were mixed and dried under reduced pressure, and then acetone was added to the resulting residue to precipitate crystals, which were then filtered and dried under reduced pressure to obtain 350 g of N-[2-(carboxymethyldimethylamino)ethyl]methacrylic acid (hereinafter also referred to as "Monomer A").

[0078]

[0079] <Synthesis of Polymer (P1-1)> 11 g of Monomer A obtained in the above <Synthesis of N-[2-(carboxymethyldimethylamino)ethyl]methacrylic acid> and 1.0 g of sodium p-styrenesulfonate (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) were dissolved in a mixed solvent of 50 mL of ion-exchanged water and 40 mL of ethanol, and the atmosphere was purged with argon gas for 90 minutes. Next, 2 mL of 10% ammonium peroxodisulfate solution was added to this solution, and the mixture was stirred at 60°C for 1 hour. After completion of the reaction, the reaction solution was purified using a dialysis tube [Spectrapore 2 (molecular weight cutoff 12K-14K, manufactured by Spectrum), ion-exchanged water; 5 L x 6 times]. After purification, the resulting polymer solution was freeze-dried to obtain 9.6 g of Polymer (P1-1).

[0080]

[0081] Example 2 Synthesis of Polymer (P1-2) 11 g of Monomer A obtained in Example 1 (Synthesis of N-[2-(carboxymethyldimethylamino)ethyl]methacrylic acid) and 0.4 g of 2,2,2-trifluoroethyl methacrylic acid (Tokyo Chemical Industry Co., Ltd.) were dissolved in a mixed solvent of 60 mL of ion-exchanged water and 30 mL of ethanol, and the atmosphere was purged with argon gas for 90 minutes. Next, 2 mL of 10% ammonium peroxodisulfate solution was added to this solution, and the mixture was stirred at 50°C for 1 hour. After completion of the reaction, the reaction solution was purified using a dialysis tube [Spectrapore 2 (molecular weight cutoff 12K-14K, Spectrum), ion-exchanged water; 5 L x 6 times]. After purification, the resulting polymer solution was freeze-dried to obtain 8.2 g of Polymer (P1-2).

[0082]

[0083] The weight-average molecular weight of the polymer was calculated as a polyethylene oxide equivalent value by gel permeation chromatography (GPC). Approximately 10 mg of polyethylene glycol calibration standards (listed molecular weights: 2,000 (Fujifilm Wako Pure Chemical Industries, Ltd.), 24,000, 50,000, 107,000, 140,000, 250,000, 540,000, and 879,000 (Tosoh Corporation)) were weighed into 5 mL disposable PS test tubes, and mobile phase was added at a ratio of 10 mg / 1 mL to the weighed value. The mixture was vigorously stirred in a vortex mixer to dissolve (sample concentration: 1%).

[0084] Similarly, approximately 10 mg of each of polymer (P1-1) and polymer (P1-2) was weighed into a 5 mL disposable PS test tube, and mobile phase was added at a ratio of 10 mg / 1 mL relative to the weighed value, followed by vigorously stirring with a vortex mixer to dissolve (sample concentration: 1%). 10 μL of each of the above solutions was analyzed using the apparatus and conditions described below, and the retention times were determined. A molecular weight calibration curve was created from the molecular weights and retention times of the polyethylene glycol calibration standards, and the molecular weights of polymer (P1-1) and polymer (P1-2) were calculated from the retention times of polymer (P1-1) and polymer (P1-2).

[0085] Apparatus: HLC-8320GPC Column: Shodex OHpak SB-806M HQ (8.0 mm i.d. x 300 mm x 3 columns) Column temperature: 35°C Eluent: 100 mmol / L sodium chloride Flow rate: 1.0 ml / min Detector: differential refractometer (cell temperature: 35°C) Content calculation method: area percentage method

[0086] Example 3: Immunochemiluminescence measurement of hepatitis B virus surface antigen (HBs antigen) Immunochemiluminescence measurement of hepatitis B virus surface antigen (HBs antigen) was carried out using a hepatitis B virus surface antigen kit (Accuraseed HBs antigen) (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) The first, third, fourth, and fifth reagents used were those included in the hepatitis B virus surface antigen kit.

[0087] First reagent: anti-HBs mouse monoclonal antibody-bound particles; Third reagent: peroxidase-labeled anti-HBs mouse monoclonal antibody; Fourth reagent: L-012; Fifth reagent: hydrogen peroxide (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.)

[0088] The second reagent (immune reaction buffer) was prepared as follows. Bovine serum albumin (BSA) protein and sodium chloride were added to a pH-adjusting buffer solution and dissolved, creating an immunoreaction buffer solution without a sensitizer (No. 0). Polymer (P1-1) or (P1-2) was added to this immunoreaction buffer solution to a final concentration of 1%, creating immunoreaction buffer solutions containing the sensitizer of the present invention (Nos. 1 to 4). The addition of this polymer did not cause salting out. For comparison, polyethylene glycol (PEG), a known immunoreaction sensitizer, was added at the same concentration to prepare another solution (No. 5).

[0089] To examine the sensitizing effect of the immune reaction buffer prepared above, the first to fifth reagents and Accura Seed B / F separation solution (Fujifilm Wako Pure Chemical Industries, Ltd.) were used to measure the luminescence intensity of each solution using an automated chemiluminescent enzyme immunoassay analyzer, Accura Seed (Fujifilm Wako Pure Chemical Industries, Ltd.), according to the following procedure. An HBs antigen-negative blank sample (0 IU / mL) and a cutoff value sample (0.05 IU / mL) were used as samples.

[0090] 50 μL of the second reagent (immunoreaction buffers No. 0 to 5) was added to the first reagent (antibody-bound particles), followed by 25 μL of the sample, and the reaction was allowed to proceed at 37°C for approximately 3 minutes (first reaction). B / F separation and washing were performed. 50 μL of the third reagent (enzyme-labeled antibody) was added, and the reaction was allowed to proceed at 37°C for approximately 3 minutes (second reaction). B / F separation and washing were performed. 100 μL of the fourth reagent (substrate solution) and 100 μL of the fifth reagent (hydrogen peroxide solution) were added (enzyme reaction), and the luminescence intensity was measured. The difference in luminescence intensity between the blank sample and the cutoff sample when each immune reaction buffer was used was calculated. The ratio of the luminescence intensity difference for No. 1 to No. 5, assuming that the luminescence intensity difference for No. 0 is 1.00, is shown in Table 1.

[0091]

[0092] The addition of polymers (P1-1) and (P1-2) resulted in a sensitization effect of 1.47 to 2.19 times that observed when no sensitizer was added. The sensitization effect tended to increase as the weight-average molecular weight (Mw) of the polymer decreased, but the sensitization effect was consistently observed within the Mw range of 141,137 to 809,817, and was higher than that of polyethylene glycol (Mw: 20,000) used for comparison.

Claims

1. A sensitizer for immunoassays, comprising a copolymer containing a repeating unit represented by formula [1] and a repeating unit represented by formula [2] or formula [3]. In formula [1], X １ represents an oxygen atom or an —NH— group, and R １ and R ２ each independently represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms; R ３ represents a hydrogen atom or an alkyl group having 1 to 3 carbon atoms; m represents an integer of 1 to 6; and n represents an integer of 1 to 3. In formula [2], X ２ represents an oxygen atom or an —NH— group, and R ４ represents a fluoroalkyl group having 1 to 10 carbon atoms, and R ５ represents a hydrogen atom or an alkyl group having 1 to 3 carbon atoms. In formula [3], R ６ represents an aryl group which may have a carboxyl group or a sulfo group as a substituent, R ７ represents a hydrogen atom or an alkyl group having 1 to 3 carbon atoms.

2. The sensitizer for immunoassays according to claim 1, wherein the mass ratio of the repeating unit (A) represented by formula [1] to the repeating unit (B) represented by formula [2] or formula [3] is (A) / (B) = 70 / 30 to 90 / 10.

3. The sensitizer for immunoassays according to claim 1, wherein the weight-average molecular weight of the copolymer is 100,000 to 3,000,000.

4. The sensitizer for immunoassays according to claim 1, wherein the weight-average molecular weight of the copolymer is 140,000 to 900,000.

5. A kit for immunoassay comprising the sensitizer for immunoassay according to any one of claims 1 to 4, a solid-phased first analyte-binding substance, and a labeled second analyte-binding substance.

6. An immunoassay method comprising the step of carrying out an antigen-antibody reaction in the presence of the sensitizer for immunoassays according to any one of claims 1 to 4.

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