Nonspecific reaction inhibitor, reagent for biochemical measurement, reagent kit for biochemical measurement, and copolymer

Abstract

This nonspecific reaction inhibitor is a copolymer containing a first repeating unit represented by formula (1) and a second repeating unit represented by formula (2). In formula (1), R1, R2, and R3 are each independently a hydrogen atom or a methyl group, X is NH or an oxygen atom, Y is SO3 - or COO-, and Yn and m are each independently an integer from 1 to 5. In formula (2), R4 is a hydrogen atom or a methyl group, Z is NH or an oxygen atom, and A is a linear or branched alkyl group with 12 to 40 carbon atoms.

Description

Non-specific reaction inhibitors, biochemical measurement reagents, biochemical measurement reagent kits, copolymers

[0001] The present invention relates to a nonspecific reaction inhibitor, a biochemical measurement reagent, a biochemical measurement reagent kit, and a copolymer. This application claims priority to Japanese Patent Application No. 2024-052987, filed on March 28, 2024, the contents of which are incorporated herein by reference.

[0002] Biochemical assays have been known as a means of detecting various components contained in human biological samples. Among these biochemical assays, immunoassays are highly specific because they utilize specific binding between proteins, such as antigen-antibody reactions.

[0003] However, because various substances other than the target substance are present in biological samples, binding not based on the original specific reaction may occur or the specific immune reaction may be hindered. These are called non-specific reactions, and the substances that cause them are sometimes called non-specific factors. When non-specific reactions occur in biochemical measurements, measurement errors occur. For this reason, various non-specific factors have been elucidated, and methods to suppress non-specific reactions have been investigated.

[0004] For example, Patent Document 1 discloses a method of adding animal-derived antibodies such as anti-human IgG antibodies, anti-human IgA antibodies, and anti-human IgM antibodies to a reagent as a method of suppressing non-specific reactions.

[0005] Japanese Patent Application Publication No. 07-012818

[0006] The method of suppressing nonspecific reactions using animal-derived antibodies or the like, as described in Patent Document 1, is currently in practical use with a variety of reagents. However, the present inventors' tests have revealed that even these antibodies cannot suppress some nonspecific reactions, and there is room for improvement.

[0007] An object of the present invention is to provide a non-specific reaction inhibitor, a reagent for biochemical measurements, a reagent kit for biochemical measurements, and a copolymer.

[0008] The present invention includes the following aspects: [1] A non-specific reaction inhibitor which is a copolymer comprising a first repeating unit represented by formula (1) and a second repeating unit represented by formula (2).

[0009] (In formula (1), R １ , R ２ and R ３ are each independently a hydrogen atom or a methyl group, X is NH or an oxygen atom, and Y is SO ３ － or COO － wherein n and m are each independently an integer of 1 to 5.

[0010] (In formula (2), R ４ is a hydrogen atom or a methyl group, Z is NH or an oxygen atom, and A is a linear or branched alkyl group having 12 to 40 carbon atoms.) [2] The non-specific reaction inhibitor according to [1], wherein A in formula (2) is a linear or branched alkyl group having 12 to 28 carbon atoms. [3] The non-specific reaction inhibitor according to [1] or [2], further comprising a third repeating unit derived from a polymerizable compound having a molecular weight of 1,000 or less. [4] The non-specific reaction inhibitor according to [3], wherein the third repeating unit is a repeating unit derived from one or more of methyl methacrylate, methyl acrylate, methacrylic acid, 2-methoxyethyl acrylate, and 1-vinyl 2-pyrrolidone. [5] The non-specific reaction inhibitor according to any one of [1] to [4], wherein the weight-average molecular weight of the copolymer is 1,000 to 5,000,000. [6] The non-specific reaction inhibitor according to any one of [1] to [5], wherein the molar fraction of the substance amount of the first repeating unit relative to the substance amount of all repeating units constituting the copolymer is 50 to 99 mol %.

[0011] [7] A non-specific reaction inhibition method, which comprises, when measuring a target substance contained in the biological sample, mixing the non-specific reaction inhibitor according to any one of [1] to [6] with the biological sample to suppress a non-specific reaction of the target substance. [8] The non-specific reaction inhibition method according to [7], wherein the biological sample is a chyle specimen.

[0012] [9] A reagent for use in biochemical measurements, the reagent comprising a copolymer including a first repeating unit represented by formula (1) and a second repeating unit represented by formula (2).

[0013] (In formula (1), R １ , R ２ and R ３ are each independently a hydrogen atom or a methyl group, X is NH or an oxygen atom, and Y is SO ３ － or COO － wherein n and m are each independently an integer of 1 to 5.

[0014] (In formula (2), R ４is a hydrogen atom or a methyl group, Z is NH or an oxygen atom, and A is a linear or branched alkyl group having 12 to 40 carbon atoms.)

[10] The reagent for biochemical measurements according to [9], wherein A in formula (2) is a linear or branched alkyl group having 12 to 28 carbon atoms.

[11] The reagent for biochemical measurements according to [9] or

[10] , further comprising a third repeating unit derived from a polymerizable compound having a molecular weight of 1,000 or less.

[12] The reagent for biochemical measurements according to

[11] , wherein the third repeating unit is a repeating unit derived from one or more of methyl methacrylate, methyl acrylate, methacrylic acid, 2-methoxyethyl acrylate, and 1-vinyl 2-pyrrolidone.

[13] The reagent for biochemical measurements according to any one of [9] to

[12] , wherein the weight-average molecular weight of the copolymer is 1,000 to 5,000,000.

[14] The reagent for biochemical measurements according to any one of [9] to

[13] , wherein the molar fraction of the amount of substance of the first repeating unit relative to the amount of substance of all repeating units constituting the copolymer is 50 to 99 mol %.

[15] The reagent for biochemical measurements according to any one of [9] to

[14] , wherein the biochemical measurement is a homogeneous method.

[16] The reagent for biochemical measurements according to any one of [9] to

[15] , wherein the biochemical measurement is a latex immunoturbidimetric method.

[17] The reagent for biochemical measurements according to any one of [9] to

[16] , wherein the biochemical measurement is a measurement based on an antigen-antibody reaction.

[18] The reagent for biochemical measurements according to any one of [9] to

[17] , wherein the copolymer is included as a non-specific reaction inhibitor.

[0015]

[19] A reagent kit for biochemical measurements, comprising the reagent for biochemical measurements according to any one of [9] to

[18] .

[0016]

[20] A copolymer comprising a first repeating unit represented by formula (1) and a second repeating unit represented by formula (2).

[0017] (In formula (1), R １ , R ２ and R ３ are each independently a hydrogen atom or a methyl group, X is NH or an oxygen atom, and Y is SO ３ －or COO － wherein n and m are each independently an integer of 1 to 5.

[0018] (In formula (2), R ４ is a hydrogen atom or a methyl group, Z is NH or an oxygen atom, and A is a linear or branched alkyl group having 12 to 40 carbon atoms.)

[21] A copolymer comprising a first repeating unit represented by formula (1-1) and a second repeating unit represented by formula (2-1), or a copolymer comprising a first repeating unit represented by formula (1-2) and the second repeating unit represented by formula (2-1).

[0019]

[0020]

[0021] (In formula (2-1), A is a linear or branched alkyl group having 12 to 40 carbon atoms.)

[22] A copolymer comprising a first repeating unit represented by formula (1) and a second repeating unit represented by formula (3).

[0022] (In formula (1), R １ , R ２ and R ３ are each independently a hydrogen atom or a methyl group, X is NH or an oxygen atom, and Y is SO ３ － or COO － wherein n and m are each independently an integer of 1 to 5.

[0023] (In formula (3), R ４ is a hydrogen atom or a methyl group, Z is an NH or an oxygen atom, and R ５ and R ６ are each independently a linear or branched alkyl group having 1 to 30 carbon atoms, and R ５ and R ６ The total number of carbon atoms is 10 to 38.

[0024]

[23] A copolymer comprising a first repeating unit represented by formula (1-3), a first repeating unit represented by formula (1-4), and a second repeating unit represented by formula (2).

[0025] (In formula (1-3), R １ , R ２ and R ３ are each independently a hydrogen atom or a methyl group, X is NH or an oxygen atom, and n and m are each independently an integer of 1 to 5.

[0026] (In formula (1-4), R １ , R ２ and R ３ are each independently a hydrogen atom or a methyl group, X is NH or an oxygen atom, and n and m are each independently an integer of 1 to 5.

[0027] (In formula (2), R ４ is a hydrogen atom or a methyl group, Z is an NH or an oxygen atom, and A is a linear or branched alkyl group having 12 to 40 carbon atoms.

[0028]

[24] The copolymer according to any one of

[20] to

[23] , further comprising a third repeating unit derived from a polymerizable compound having a molecular weight of 1,000 or less.

[25] The copolymer according to

[24] , wherein the third repeating unit is a repeating unit derived from one or more of methyl methacrylate, methyl acrylate, methacrylic acid, 2-methoxyethyl acrylate, and 1-vinyl 2-pyrrolidone.

[26] The copolymer according to any one of

[20] to

[25] , having a weight average molecular weight of 1,000 to 5,000,000.

[27] The copolymer according to any one of

[20] to

[26] , wherein the molar fraction of the amount of substance of the first repeating unit relative to the amount of substance of all repeating units constituting the copolymer is 50 to 99 mol %.

[0029] According to the above aspects, it is possible to provide a non-specific reaction inhibitor, a reagent for biochemical measurements, a reagent kit for biochemical measurements, and a copolymer.

[0030] The phrase "suppressing nonspecific reactions" used herein means reducing nonspecific reactions originating from the sample that occur for some reason, so that the measured value approaches the original measured value (true value). For a target component for which a standard measurement method exists, the true value is the measurement value obtained by the standard measurement method. For a target component for which no standard measurement method exists, measurements using a heterogeneous method (a measurement method that includes a washing step and a B / F (Bound / Free) separation step during measurement) are generally considered to be less prone to nonspecific reactions than homogeneous methods (a measurement method that does not include a washing step and a B / F separation step during measurement). Even heterogeneous methods may be affected by nonspecific reactions and may fail to obtain true values. In such cases, the sample may be diluted with a component that does not contain the target component, such as saline, and then measured again to determine whether the measured value corresponds to the dilution ratio, thereby determining the extent of the influence of nonspecific reactions. To suppress nonspecific reactions, a method of covering all components involved in the reaction with some kind of component may be considered. For example, this applies when a non-specific adsorption inhibitor is used, but in this case, the desired biochemical reaction may not occur easily, resulting in a decrease in measurement sensitivity. In this specification, it is intended to suppress non-specific reactions without causing such a decrease in measurement sensitivity.

[0031] <Non-specific reaction inhibitor> The non-specific reaction inhibitor of this embodiment is a copolymer containing a first repeating unit represented by formula (1) and a second repeating unit represented by formula (2). In this specification, the term "copolymer" may be simply referred to as a polymer.

[0032] (In formula (1), R １ , R ２ and R ３ are each independently a hydrogen atom or a methyl group, X is NH or an oxygen atom, and Y is SO ３ － or COO － wherein n and m are each independently an integer of 1 to 5.

[0033] (In formula (2), R ４is a hydrogen atom or a methyl group, Z is an NH or an oxygen atom, and A is a linear or branched alkyl group having 12 to 40 carbon atoms.

[0034] The first repeating unit is represented by formula (1): １ , R ２ and R ３ are each independently a hydrogen atom or a methyl group. X is NH or an oxygen atom. R ２ or R ３ is preferably a methyl group. n and m each independently represent an integer of 1 to 5, preferably 1 to 4.

[0035] Specific examples of the first repeating unit include 4-[(3-methacrylamidopropyl)dimethylammonio]butane-1-sulfonic acid, 3-[[2-(methacryloyloxy)ethyl]dimethylammonio]propane-1-sulfonic acid, 2-[[2-(methacryloyloxy)ethyl]dimethylammonio]acetic acid, 3-[[2-(methacryloyloxy)ethyl]dimethylammonio]propionate, 3-[[2-(acryloyloxy)ethyl]dimethylammonio]propionate, and 2-[[2-(acryloyloxy)ethyl]dimethylammonio]propane-1-sulfonic acid. [(2-(methacryloyloxy)ethyl]dimethylammonio]propane-1-sulfonic acid, 4-[[2-(methacryloyloxy)ethyl]dimethylammonio]butane-1-sulfonic acid, 3-[(3-methacrylamidopropyl)dimethylammonio]propane-1-sulfonic acid, 3-[(3-acrylamidopropyl)dimethylammonio]propane-1-sulfonic acid, and 3-[(3-acrylamidopropyl)dimethylammonio]propanoate. Among these, 4-[(3-methacrylamidopropyl)dimethylammonio]butane-1-sulfonic acid, 3-[[2-(methacryloyloxy)ethyl]dimethylammonio]propane-1-sulfonic acid, 3-[[2-(methacryloyloxy)ethyl]dimethylammonio]propionate, and 3-[[2-(acryloyloxy)ethyl]dimethylammonio]propane-1-sulfonic acid are preferred as the first repeating unit.

[0036] The second repeating unit is represented by formula (2): ４is a hydrogen atom or a methyl group. Z is NH or an oxygen atom. A is a linear or branched alkyl group having 12 to 40 carbon atoms, preferably 12 to 28 carbon atoms. When A is a linear alkyl group, A preferably has 12 to 40 carbon atoms, more preferably 12 to 28 carbon atoms, and even more preferably 12 to 22 carbon atoms. When A is a branched alkyl chain, A preferably has 12 to 40 carbon atoms, more preferably 12 to 30 carbon atoms. When A is a linear or branched alkyl group having 12 to 40 carbon atoms, the effect of suppressing non-specific reactions is high.

[0037] Specific examples of the second repeating unit in which A is a linear alkyl group and Z is an oxygen atom include lauryl (meth)acrylate, tetradecyl (meth)acrylate, cetyl (meth)acrylate, stearyl (meth)acrylate, oleyl (meth)acrylate, icosyl (meth)acrylate, and behenyl (meth)acrylate. Furthermore, lauryl (meth)acrylate, stearyl (meth)acrylate, and behenyl (meth)acrylate are preferred as the second repeating unit. Furthermore, when A of the second repeating unit is linear and Z is NH, the second repeating unit is preferably lauryl(meth)acrylamide, tetradecyl(meth)acrylamide, cetyl(meth)acrylamide, stearyl(meth)acrylamide, oleyl(meth)acrylamide, icosyl(meth)acrylamide, or behenyl(meth)acrylamide, and more preferably lauryl(meth)acrylamide, stearyl(meth)acrylamide, or behenyl(meth)acrylamide.

[0038] When A is a branched alkyl chain, the structure shown in formula (3) is preferred. Furthermore, the second repeating unit is preferably decyltetradecyl methacrylate or dodecylhexadecyl methacrylate.

[0039] (In formula (3), R ４ is a hydrogen atom or a methyl group, Z is an NH or an oxygen atom, and R ５ and R ６are each independently a linear or branched alkyl group having 1 to 30 carbon atoms, and R ５ and R ６ The total number of carbon atoms is 10 to 38.

[0040] R ５ and R ６ are each independently preferably a linear or branched alkyl group having 1 to 30 carbon atoms, more preferably 8 to 22 carbon atoms, and even more preferably 10 to 14 carbon atoms. ５ and R ６ The total number of carbon atoms is preferably 10 to 38, more preferably 20 to 30, and even more preferably 22 to 26.

[0041] The copolymer of this embodiment may have a third repeating unit derived from a polymerizable compound having a molecular weight of 1,000 or less. Examples of the third repeating unit include polymerizable compounds having a carbon-carbon double bond, and specific examples thereof include methyl methacrylate, methyl acrylate, methacrylic acid, 2-methoxyethyl acrylate, 1-vinyl 2-pyrrolidone, 3-methoxypropyl acrylate, ethyl acrylate, propyl acrylate, butyl acrylate, isobutyl acrylate, cyclohexyl acrylate, 1-methylcyclopentyl acrylate, phenyl acrylate, benzyl acrylate, 2-(dimethylamino)ethyl acrylate, 2-(diethylamino)ethyl acrylate, potassium acrylate, mono(2-acryloyloxyethyl) succinate, 2-hydroxyethyl acrylate, 4-hydroxybutyl acrylate, 2-(2-oxopyrrolidin-1-yl)ethyl acrylate, glycidyl acrylate, 2-cyanoethyl acrylate, 2-methoxyethyl methacrylate, 2-ethoxyethyl methacrylate, ethyl methacrylate, propyl methacrylate, butyl methacrylate, and methacrylic acid. Isobutyl, cyclohexyl methacrylate, 1-methylcyclopentyl methacrylate, 2-oxotetrahydrofuran-3-yl methacrylate, 5-oxotetrahydrofuran-3-yl methacrylate, phenyl methacrylate, benzyl methacrylate, furfuryl methacrylate, 2-aminoethyl methacrylate hydrochloride, 2-(dimethylamino)ethyl methacrylate, 2-(diethylamino)ethyl methacrylate, 2-(tert-butylamino)ethyl methacrylate, 2-(meth methacryloyloxy)-N,N,N-trimethylethanaminium methyl sulfate, 1-[2-(methacryloyloxy)ethyl] succinate, 2-hydroxyethyl methacrylate, methacryloylcholine chloride, 2-morpholinoethyl methacrylate, 2-(2-oxopyrrolidin-1-yl)ethyl methacrylate, glycidyl methacrylate, potassium 3-sulfopropyl methacrylate, 2-hydroxyethyl methacrylate phosphate, N,N-dimethylacrylamide, N,N-Diethylacrylamide, N-Isopropylacrylamide, N-Propylacrylamide, N-Butylacrylamide, 3-Acryloyl-2-oxazolidinone, N-Phenylacrylamide, N-Benzylacrylamide, N-(Methoxymethyl)acrylamide, N-(Isobutoxymethyl)acrylamide, N-(Butoxymethyl)acrylamide, N-[3-(Dimethylamino)propyl]acrylamide, N-[2-(Dimethylamino)ethyl]acrylamide, N-[2-(Diethylamino)ethyl]acrylamide, 6-Acrylamidehexanoic acid, (3-Acrylamidopropyl)trimethylammonium chloride, N-(Hydroxymethyl)acrylamide, N-(2-Hydroxyethyl)acrylamide, N-(2-Amino-2-oxoethyl)acrylamide, N-Me Examples of the repeating unit include methyl methacrylamide, N,N-dimethyl methacrylamide, N-isopropyl methacrylamide, N-phenyl methacrylamide, N-(methoxymethyl)methacrylamide, N-(3-aminopropyl)methacrylamide hydrochloride, N-(3-dimethylaminopropyl)methacrylamide, N-[2-(dimethylamino)ethyl]methacrylamide, N-(hydroxymethyl)methacrylamide, N-(2-hydroxypropyl)methacrylamide, maleic acid, fumaric acid, itaconic acid, citraconic acid, maleic anhydride, itaconic anhydride, and citraconic anhydride. The repeating unit may be derived from one or more of methyl methacrylate, methyl acrylate, methacrylic acid, 2-methoxyethyl acrylate, and 1-vinyl 2-pyrrolidone, with methyl methacrylate being preferred. When the copolymer contains a third repeating unit, the solubility of the nonspecific reaction inhibitor may be increased.

[0042] The copolymer of this embodiment may contain one, two, or three types of first repeating units represented by formula (1). The copolymer of this embodiment may contain one, two, or three types of second repeating units represented by formula (2). The copolymer of this embodiment preferably contains one type of first repeating unit represented by formula (1) and one type of second repeating unit represented by formula (2). Alternatively, the copolymer of this embodiment may contain two types of first repeating units represented by formula (1) and one type of second repeating unit represented by formula (2).

[0043] The weight-average molecular weight of the copolymer is preferably 1,000 to 5,000,000, more preferably 1,000 to 1,000,000, and most preferably 1,000 to 500,000. A weight-average molecular weight of 1,000 or more increases the affinity with non-specific reaction substances, making it easier for the copolymer to fully exhibit its effect as a non-specific reaction inhibitor. Furthermore, a weight-average molecular weight of 5,000,000 or less prevents the viscosity of the reagent according to the present invention containing the copolymer from becoming too high, making it easier for the intended measurement to be carried out smoothly.

[0044] The weight-average molecular weight of the copolymer is measured by gel filtration chromatography, and reference can be made to WO 2022 / 124288. Specifically, the weight-average molecular weight can be measured by gel filtration chromatography under the following conditions using polyethylene oxide and polyethylene glycol as standard samples. <Measurement device> Device: HLC-8420GPC (manufactured by Tosoh Corporation) Detector: HLC-8420GPC built-in RI detector (manufactured by Tosoh Corporation) Sample column: TSKgel SuperMultiporePW-H (6.0 mm I.D. x 15 cm x 2) (manufactured by Tosoh Corporation) Reference column: TSKgel SuperH-RC (manufactured by Tosoh Corporation) Guard column: TSKgel guardcolumn SuperMP(PW)-H (manufactured by Tosoh Corporation) <Measurement conditions> Eluent: 0.2 M aqueous sodium nitrate solution / methanol = 80 / 20 (volume ratio) Flow rate: 0.4 mL / min Injection volume: 40 μL Column temperature: 40°C Standard samples: polyethylene oxide (Tosoh Corporation), polyethylene glycol (Fujifilm Wako Pure Chemical Industries, Ltd.)

[0045] The molar fraction of the substance amount of the first repeating unit relative to the substance amount of all repeating units constituting the copolymer is preferably 50 to 99 mol%, more preferably 60 to 97 mol%, and even more preferably 60 to 85 mol%, with the total of the substance amount of the first repeating unit and the other repeating units being 100 mol%. When the molar fraction of the substance amount of the first repeating unit relative to the substance amount of all repeating units constituting the copolymer is 50 to 99 mol%, the water solubility of the copolymer is improved, making it easier to handle. Note that the molar fraction of each repeating unit is equivalent to the blending ratio of each monomer, which is a raw material for polymerization of the copolymer.

[0046] The molar fraction of the amount of substance of the second repeating unit relative to the amount of substance of all repeating units constituting the copolymer is preferably 1 to 50 mol %, more preferably 1 to 40 mol %, and most preferably 3 to 40 mol %, with the total amount of substance including the other repeating units being 100 mol %. When the molar fraction of the amount of substance of the second repeating unit relative to the amount of substance of all repeating units constituting the copolymer is 1 to 50 mol %, the effect of favorably suppressing non-specific reactions can be obtained.

[0047] The molar fraction of the amount of substance of the third repeating unit relative to the amount of substance of all repeating units constituting the copolymer is preferably 0 to 49 mol %, and more preferably 1 to 25 mol %, with the total amount of the third repeating unit and the other repeating units being 100 mol %. When the molar fraction of the amount of substance of the third repeating unit relative to the amount of substance of all repeating units constituting the copolymer is 0 to 49 mol %, the effect of suitably suppressing nonspecific reactions can be obtained.

[0048] In another aspect, the copolymer of the present embodiment includes a copolymer including a first repeating unit represented by formula (1-1) and a second repeating unit represented by formula (2-1), or a copolymer including a first repeating unit represented by formula (1-2) and a second repeating unit represented by formula (2-1).

[0049]

[0050]

[0051] (In formula (2-1), R ４ is a hydrogen atom or a methyl group, and A is a linear or branched alkyl group having 12 to 40 carbon atoms.

[0052] When A is a linear alkyl chain, lauryl (meth)acrylate, tetradecyl (meth)acrylate, cetyl (meth)acrylate, stearyl (meth)acrylate, oleyl (meth)acrylate, icosyl (meth)acrylate, and behenyl (meth)acrylate are preferred. Furthermore, lauryl (meth)acrylate, stearyl (meth)acrylate, and behenyl (meth)acrylate are most preferred. When A is a branched alkyl chain, decyltetradecyl methacrylate and dodecylhexadecyl methacrylate are most preferred.

[0053] In another aspect, the copolymer of this embodiment includes a copolymer including a first repeating unit represented by formula (1) and a second repeating unit represented by formula (3).

[0054] (In formula (1), R １ , R ２ and R ３are each independently a hydrogen atom or a methyl group, X is NH or an oxygen atom, and Y is SO ３ － or COO － wherein n and m are each independently an integer of 1 to 5.

[0055] (In formula (3), R ４ is a hydrogen atom or a methyl group, Z is an NH or an oxygen atom, and R ５ and R ６ are each independently a linear or branched alkyl group having 1 to 30 carbon atoms, and R ５ and R ６ The total number of carbon atoms is 10 to 38.

[0056] In formula (3), R ５ and R ６ are each independently preferably a linear or branched alkyl group having 1 to 30 carbon atoms, more preferably 8 to 22 carbon atoms, and most preferably 10 to 14 carbon atoms. ５ and R ６ The total number of carbon atoms is preferably 10 to 38, more preferably 20 to 30, and most preferably 22 to 26.

[0057] Formula (3) is preferably decyltetradecyl methacrylate or dodecylhexadecyl methacrylate.

[0058] In another aspect, the copolymer may include a first repeating unit represented by formula (1-3), a first repeating unit represented by formula (1-4), and a second repeating unit represented by formula (2).

[0059] (In formula (1-3), R １ , R ２ and R ３ are each independently a hydrogen atom or a methyl group, X is NH or an oxygen atom, and n and m are each independently an integer of 1 to 5.

[0060] (In formula (1-4), R １ , R ２ and R ３are each independently a hydrogen atom or a methyl group, X is NH or an oxygen atom, and n and m are each independently an integer of 1 to 5.

[0061] (In formula (2), R ４ is a hydrogen atom or a methyl group, Z is an NH or an oxygen atom, and A is a linear or branched alkyl group having 12 to 40 carbon atoms.

[0062] The copolymer may contain a third repeating unit, which is as described above and therefore will not be described further.

[0063] The above also applies to the weight average molecular weight of the copolymer, the molar fraction of the substance amount of the first repeating unit relative to the substance amount of all repeating units constituting the copolymer, the molar fraction of the substance amount of the second repeating unit relative to the substance amount of all repeating units constituting the copolymer, and the molar fraction of the substance amount of the third repeating unit relative to the substance amount of all repeating units constituting the copolymer, and therefore explanations of these will be omitted.

[0064] The copolymer of this embodiment can be produced by polymerizing the monomer that will form the first repeating unit, the monomer that will form the second repeating unit, and, if necessary, the monomer that will form the third repeating unit. For example, the monomer that will form the first repeating unit, the monomer that will form the second repeating unit, and, if necessary, the monomer that will form the third repeating unit are dissolved in a solvent, heated under a nitrogen atmosphere, stirred, and then a polymerization initiator is added to obtain the copolymer. The monomer refers to a compound that has a polymerizable functional group and constitutes the minimum repeating unit in the synthesized polymer.

[0065] Examples of the solvent include methanol, ethanol, 2-propanol, TFE (2,2,2-trifluoroethanol), 1,1,1,3,3,3-hexafluoro-2-propanol, THF (tetrahydrofuran), 2-methyltetrahydrofuran, cyclopentyl methyl ether, dimethyl sulfoxide, N,N-dimethylformamide, N,N-dimethylacetamide, toluene, o-xylene, m-xylene, p-xylene, ethyl acetate, propyl acetate, isopropyl acetate, isobutyl acetate, water, and mixed solvents thereof. Examples of polymerization initiators include AIBN (2,2'-azobisisobutyronitrile), V-601 (dimethyl 2,2'-azobis(2-methylpropionate)), VR-110 (2,2'-azobis(2,4,4-trimethylpentane)), VAm-110 (2,2'-azobis(N-butyl-2-methylpropionamide)), V-40 (1,1'-azobis(cyclohexane-1-carbonitrile)), and VE-073 (dimethyl 2,2'-azobis(2-methylpropionate)). Examples of the hydroxybenzoates include 1,1'-azobis(1-cyclohexanecarboxylate)), V-70 (2,2'-azobis(4-methoxy-2,4-dimethylvaleronitrile)), V-65 (2,2'-azobis(2,4-dimethylvaleronitrile)), and V-59 (2,2'-azobis(2-methylbutylonitrile)).

[0066] <Uses of Nonspecific Reaction Inhibitor> The copolymer of this embodiment is mixed with a biological sample (also referred to as a specimen, hereinafter sometimes simply referred to as a sample) to suppress nonspecific reactions that occur when measuring a substance to be measured contained in the sample. By "suppressing nonspecific reactions," measurement errors can be reduced when measuring a substance to be measured contained in the sample, and measurements can be made closer to the true measured value. Examples of the nonspecific reactions include reactions that reduce or inhibit interactions between desired components, reactions that cause interactions between undesired components, and reactions that promote reactions between components that interact so weakly that they are undetectable.

[0067] The biological sample may be derived from a human or an animal. The sample may be blood, culture supernatant, urine, feces, cerebrospinal fluid, saliva, sweat, ascites, or cell or tissue extract. The blood may be any type, such as whole blood, plasma, serum, or blood that has been subjected to any treatment, such as dilution with an appropriate buffer or purification.

[0068] Examples of the substance to be measured include substances to be measured in immunological assays described below.

[0069] The non-specific reaction inhibitor of this embodiment can suppress non-specific reactions.

[0070] <Non-specific reaction inhibition method> In the non-specific reaction inhibition method of this embodiment, a non-specific reaction inhibitor is mixed with a biological sample, and when measuring a target substance contained in the biological sample, a non-specific reaction of the target substance is inhibited. As the non-specific reaction inhibitor in the non-specific reaction inhibition method can be the non-specific reaction inhibitor described above, a redundant description will be omitted.

[0071] By sufficiently dispersing the nonspecific reaction inhibitor in a mixture obtained by mixing the nonspecific reaction inhibitor with a sample, nonspecific reactions can be sufficiently suppressed when measuring the substance to be measured contained in the sample.

[0072] An example of this embodiment is an analytical method in which at least one of a biochemical reaction and a measurement is carried out in the presence of the non-specific reaction inhibitor of the first embodiment.

[0073] In the nonspecific reaction inhibition method of this embodiment, the method for mixing the nonspecific reaction inhibitor with the biological sample is not particularly limited. For example, a method may be used in which the nonspecific reaction inhibitor is added to a buffer solution at a desired concentration to prepare a reagent S, which is then mixed with the sample. The reagent S may be any of a reagent included in a reagent kit for biochemical measurements, a sample diluent, a sample suspension, and a pretreatment solution.

[0074] When Reagent S is a reagent included in a biochemical measurement reagent kit, Reagent S is generally added in an amount 10 to 100 times the volume of the sample. In this case, the concentration of the nonspecific reaction inhibitor in Reagent S is, for example, preferably 0.001 to 5.00% (w / v), more preferably 0.002 to 2.00% (w / v), even more preferably 0.003 to 1.00% (w / v), and most preferably 0.005 to 0.50% (w / v). Within these preferred ranges, the nonspecific reactions can be sufficiently suppressed.

[0075] When reagent S is a sample dilution solution or a pretreatment solution, reagent S is generally added in an amount 1 to 10 times the volume of the sample. In this case, the concentration of the nonspecific reaction inhibitor in reagent S is, for example, preferably 0.001 to 5.00% (w / v), more preferably 0.002 to 4.50% (w / v), even more preferably 0.003 to 3.00% (w / v), and most preferably 0.005 to 2.00% (w / v). Within these preferred ranges, the nonspecific reactions can be sufficiently suppressed.

[0076] In order to ensure that the nonspecific reaction inhibitor contacts, binds to, or reacts with components contained in the sample in a mixed solution obtained by mixing the nonspecific reaction inhibitor with the sample, the mixed solution is preferably left for 5 minutes or more, for example, at room temperature or 37° C. By allowing such a mixing time, nonspecific reactions can be sufficiently suppressed when measuring the analyte contained in the sample.

[0077] The concentration of the non-specific reaction inhibitor in the mixed solution is, for example, preferably 0.0001 to 5.00% (w / v), more preferably 0.0002 to 2.00% (w / v), even more preferably 0.0003 to 1.00% (w / v), particularly preferably 0.0005 to 0.50% (w / v), and most preferably 0.005 to 0.50% (w / v). Within these preferred ranges, the non-specific reaction can be sufficiently suppressed.

[0078] Furthermore, at least one of a biochemical reaction and a measurement may be performed in the presence of a nonspecific reaction inhibitor. As an example, a reaction product between a target substance and a substance that specifically binds to the target substance (specific affinity substance) may be measured in a liquid phase containing a biological sample in the presence of a nonspecific reaction inhibitor. To perform this measurement, a solution containing a specific affinity substance may be further added to a mixture obtained by mixing the nonspecific reaction inhibitor with the sample.

[0079] Here, the concentration of the nonspecific reaction inhibitor in the liquid phase in which the generated reaction product is measured is, for example, preferably 0.0001 to 4.00% (w / v), more preferably 0.0002 to 1.60% (w / v), even more preferably 0.0003 to 0.80% (w / v), particularly preferably 0.0005 to 0.40% (w / v), and most preferably 0.003 to 0.40% (w / v). Within these preferred ranges, the nonspecific reaction can be sufficiently suppressed. These preferred concentration ranges are also suitable in other examples of this embodiment and other embodiments.

[0080] In biochemical measurements, a substance with specific affinity for the substance to be measured is used. If the substance to be measured is an antibody, the specific affinity substance can be the corresponding antigen. If the substance to be measured is an antigen, the specific affinity substance can be the corresponding antibody. Furthermore, if the substance to be measured is labeled with avidin or biotin, the specific affinity substance can be biotin or avidin. Of these, measurements that utilize antigen-antibody reactions are called immunological measurements.

[0081] The type of immunological measurement is not particularly limited, and various known methods can be applied, such as turbidimetric immunoassay (TIA), latex turbidimetric immunoassay (LTIA), immunochromatography (lateral flow type, flow-through type), electrochemiluminescence assay (ECLIA), chemiluminescence assay (CLIA), chemo-enzyme immunoassay (CLEIA), enzyme-linked immunosorbent assay (ELISA), etc. The inhibition of nonspecific reactions exhibited by the nonspecific reaction inhibitor is as significant in the presence of latex particles as in their absence, and therefore LTIA is preferred.

[0082] When the immunological measurement method is LTIA, the non-specific reaction inhibition method may include contacting a biological sample with insoluble carrier particles carrying a substance with specific affinity for the substance to be measured in a liquid phase in the presence of a non-specific reaction inhibitor, and measuring a signal generated by an agglutination reaction between the substance to be measured and the insoluble carrier particles.

[0083] In general, immunoassays are broadly divided into homogeneous and heterogeneous methods. The homogeneous method does not involve a washing process (so-called binding (B) / non-binding (F) separation process) to separate reaction products generated in a mixed solution of a biological sample and a reagent solution involving the substance to be measured from other substances (e.g., contaminants) contained in the biological sample. The reagent of this embodiment can suppress nonspecific reactions in the sample and improve the detection accuracy of antigen-antibody reactions. Therefore, the reagent of this embodiment is preferably used in a homogeneous method. Furthermore, since the suppression of nonspecific reactions exhibited by the reagent of this embodiment is as significant in the presence of latex particles as in their absence, the reagent for biochemical assay of this embodiment is more preferably used in LTIA.

[0084] LTIA is described in detail below as an example of this type of immunoassay. LTIA is a method for measuring a target substance using latex particles to which a substance with specific affinity for the target substance, such as an antigen or antibody, is immobilized, and is widely used in the field of clinical testing.

[0085] Methods for measuring antigens, which are analytes, using LTIA can be broadly divided into two types: (a) a method in which latex particles onto which antibodies against the analyte are immobilized react with the analyte antigen to form a sandwich-type immune complex, and the analyte (antigen) is measured based on the degree of agglutination of the latex particles associated with immune complex formation; and (b) a method in which the antigen-immobilized latex particles and the antigen (analyte) in a sample compete with free antibodies added to a separate reagent to inhibit the formation of an immune complex between the latex particles and the antibody, and the analyte (antigen) is measured based on the degree of inhibition of agglutination of the latex particles associated with the inhibition of immune complex formation. This method can also use a combination of free antibodies added to the reagent and latex particles onto which antibodies against the analyte are immobilized. Both the antibody and the antigen can be immobilized to latex particles.

[0086] In LTIA, some components contained in the sample may cause agglutination that should not occur (positive measurement error) or agglutination that should occur may not occur (negative measurement error) in latex particles that have immobilized a substance with specific affinity for the substance to be measured. These are called non-specific reactions, and they impair the accuracy and reliability of the measurement.

[0087] By suppressing non-specific reactions, the measurement error can be reduced, and the positive or negative measurement error of the above-mentioned measurement value can be brought closer to the original measurement value (true value). Although the cause of non-specific reactions is not necessarily clear, there are cases in which immunoglobulins such as HAMA and rheumatoid factor, chyle, simple lipids, neutral lipids, and the like are thought to be involved.

[0088] The non-specific reaction suppression method of this embodiment is highly effective in suppressing non-specific reactions when the biological sample is a chyle sample. A chyle sample, as used herein, is a sample that contains a high concentration of lipids and exhibits turbidity. In clinical settings, the turbidity of a sample is generally visually observed to determine whether the sample is a chyle sample.

[0089] In LTIA, the test substance can be measured by optically or electrochemically observing the degree of agglutination. Optical observation methods include methods (endpoint method, rate method, etc.) that measure scattered light intensity, absorbance, or transmitted light intensity using optical equipment. The measured values, such as absorbance, obtained by measuring a sample are compared with the measured values, such as absorbance, obtained by measuring a standard substance (a sample with a known concentration of the substance to be measured), to calculate the concentration (quantitative value) of the substance to be measured contained in the sample. Measurement of absorbance, such as transmitted light or scattered light, may be performed using either a single wavelength or a dual wavelength (the difference or ratio between two wavelengths). Measurement wavelengths are generally selected from 500 to 900 nm.

[0090] The LTIA may be performed using a measuring device. The measuring device may be a general-purpose analyzer or a dedicated automatic measuring device. The LTIA is generally performed in multiple steps, such as a two-step method (two-reagent method).

[0091] The substance to be measured in the non-specific reaction inhibition method of this embodiment is not particularly limited as long as it is a substance that can be measured by a known immunological measurement method, and examples thereof include proteins (antigens, haptens, antibodies, etc.), carbohydrates, lipids, glycoproteins, glycolipids, nucleic acids, chemical substances (hormones, drugs), etc. Among these, antigens or antibodies are preferred, and antigens consisting of proteins are more preferred. Specific examples include soluble interleukin-2 receptor (sIL-2R), CRP, fibrin and fibrinogen degradation products, D-dimer, soluble fibrin (SF), lipoprotein(a) (Lp(a)), matrix metalloproteinase-3 (MMP-3), prostate-specific antigen (PSA), IgG, IgA, IgM, IgE, IgD, antistreptolysin O antibody, rheumatoid factor, transferrin, haptoglobin, α1-antitrypsin, α1-acidoglycoprotein, α2-macroglobulin, hemopexin, and a Antithrombin-III, α-fetoprotein, carcinoembryonic antigen (CEA), ferritin, HBs-Ag (hepatitis B envelope antigen), anti-HBs (anti-hepatitis B envelope antibody), HBe-Ag (hepatitis B e antigen), anti-HBe (anti-hepatitis B e antibody), anti-HBc (anti-hepatitis B core antibody), SARS-Cov-2, human brain natriuretic peptide (BNP), pulmonary surfactant protein D (SP-D), thymus and activation-regulated chemokine (TARC, CCL-17), and procalcitonin.

[0092] Substances that have a specific affinity for the substance to be measured and are carried on insoluble carrier particles such as latex include proteins, peptides, amino acids, lipids, carbohydrates, glycoproteins, glycolipids, nucleic acids, and haptens used in known immunological assays. Generally, antibodies or antigens are often used.

[0093] The antibody may be a polyclonal antibody or a monoclonal antibody. The antibody may be a whole antibody molecule or a functional fragment having antigen-antibody activity. The antibody may be obtained by immunizing an animal such as a mouse, or may be synthesized by protein engineering. Examples of such antibodies include antibodies treated with proteases, F(ab')2, Fab', single-chain antibodies (scFv), chimeric antibodies, humanized antibodies, and bispecific antibodies (BsAb). The antibody may be obtained by known methods.

[0094] The latex particles used in LTIA are not particularly limited, and any latex particles commonly used as reagents for biochemical measurements can be used. The latex particles are formed, for example, by polymerizing one or more of the following monomers: Specific examples of the monomer include polymerizable monomers having a phenyl group such as styrene, α-methylstyrene, o-methylstyrene, p-methylstyrene, p-chlorostyrene, 4-vinylbenzoic acid, divinylbenzene, and vinyltoluene; polymerizable monomers having a phenyl group and a sulfonate such as styrene sulfonate, divinylbenzene sulfonate, o-methylstyrene sulfonate, and p-methylstyrene sulfonate; polymerizable unsaturated aromatics such as polymerizable monomers having a naphthyl group such as 1-vinylnaphthalene, 2-vinylnaphthalene, α-naphthyl (meth)acrylate, and β-naphthyl (meth)acrylate; polymerizable unsaturated carboxylic acids such as (meth)acrylic acid, itaconic acid, maleic acid, and fumaric acid; methyl (meth)acrylate, ethyl (meth)acrylate, n-butyl (meth)acrylate, and methyl (meth)acrylate; polymerizable unsaturated carboxylic acid esters such as 2-hydroxyethyl (meth)acrylate, glycidyl (meth)acrylate, ethylene glycol-di-(meth)acrylate, and tribromophenyl (meth)acrylate; polymerizable unsaturated nitriles such as (meth)acrylonitrile; polymerizable unsaturated aldehydes such as (meth)acrolein; polymerizable unsaturated ketones such as 3-methyl-3-buten-2-one; polymerizable unsaturated carboxylic acid amides such as (meth)acrylamide, N-methylol-(meth)acrylamide, and methylenebis(meth)acrylamide; conjugated dienes such as butadiene and isoprene; vinyl esters such as vinyl acetate; polymerizable unsaturated heterocyclic rings such as vinylpyridine; vinyl amides such as N-vinylpyrrolidone; and vinyl halides such as vinyl chloride, vinylidene chloride, and vinyl bromide.

[0095] The average particle size of the latex particles used in LTIA can be selected from, for example, 0.02 to 1.6 μm, preferably 0.1 to 0.4 μm, taking into consideration the concentration of the substance to be measured in the sample or the detection sensitivity of the measuring instrument, etc. Here, the average particle size can be measured using a particle size distribution analyzer.

[0096] The method for loading one or more of the specific affinity substances onto latex particles used in LTIA is not particularly limited, and loading may be performed by known methods such as physical adsorption (hydrophobic bonding), chemical bonding, etc. For the purpose of preventing nonspecific adsorption of contaminant substances to latex particles loaded with a specific affinity substance, the latex particles may be subjected to a known blocking treatment (masking treatment) in which the latex particles are brought into contact with a protein such as bovine serum albumin (BSA), casein, gelatin, egg albumin or a salt thereof, a polymer compound such as a polysaccharide, a surfactant, or skim milk powder.

[0097] <Reagent for Biochemical Measurement> The reagent for biochemical measurement in this embodiment includes a copolymer containing a first repeating unit represented by formula (1) and a second repeating unit represented by formula (2).

[0098] (In formula (1), R １ , R ２ and R ３ are each independently a hydrogen atom or a methyl group, X is NH or an oxygen atom, and Y is SO ３ － or COO － wherein n and m are each independently an integer of 1 to 5.

[0099] (In formula (2), R ４ is a hydrogen atom or a methyl group, Z is an NH or an oxygen atom, and A is a linear or branched alkyl group having 12 to 40 carbon atoms.

[0100] The biochemical measurement reagent is a reagent used to measure a biochemical reaction that occurs in a biological sample and detects the binding between a biochemical substance to be measured and a substance that has specific affinity for the substance to be measured. The biochemical measurement reagent contains the above-mentioned nonspecific reaction inhibitor, which is included as an active ingredient that suppresses nonspecific reactions in the biological sample. As the nonspecific reaction inhibitor described above can be applied, a redundant description will be omitted. Furthermore, as the biochemical reaction is also as described above, a redundant description will be omitted.

[0101] A typical example of the binding occurring in the sample is an antigen-antibody reaction. In the antigen-antibody reaction, the substance to be measured may be an antigen or an antibody. Examples of binding other than the antigen-antibody reaction include binding between avidin and biotin, binding between protein A and the Fc region of immunoglobulin, binding between protein G and the Fc region of immunoglobulin, and binding between protein L and the κ chain of immunoglobulin.

[0102] (Configuration of Biochemical Measurement Reagent) The configuration of the biochemical measurement reagent of this embodiment may be any configuration containing the nonspecific reaction inhibitor of this embodiment, for example, a two-reagent configuration in which the first reagent contains the nonspecific reaction inhibitor and the second reagent contains latex particles. Furthermore, it may be a multi-reagent configuration containing a third reagent or subsequent nth reagent (n represents an integer of 3 or greater) containing other components. The nonspecific reaction inhibitor may be contained together with other components in the second reagent other than the first reagent or in the nth reagent. It is preferable that the nonspecific reaction inhibitor is contained in the first reagent.

[0103] The biochemical assay reagent of this embodiment may contain other components to the extent that the nonspecific reaction inhibitory effect is not impaired. Examples of other components include buffers, proteins, peptides, amino acids, nucleic acids, lipids, phospholipids, sugars, inorganic salts, polymeric compounds, surfactants, other nonspecific reaction inhibitors, and preservatives. Components that buffer or adjust the pH, ionic strength, osmotic pressure, etc. of the sample may include buffers such as acetic acid, citric acid, phosphoric acid, Tris, glycine, boric acid, carbonate, phthalic acid, succinic acid, maleic acid, and imidazole, as well as Good's buffers and their sodium, potassium, and calcium salts. Polymers such as polyvinylpyrrolidone and phospholipid polymers may also be included as components that enhance agglutination formation.

[0104] A specific example of the reagent configuration is a two-reagent type biochemical measurement reagent in which the first reagent is a buffer solution for diluting the sample to which a non-specific reaction inhibitor has been added, and the second reagent is a similar buffer solution in which latex particles carrying a specific affinity substance are dispersed.

[0105] Furthermore, when a diluent, a pretreatment solution, or the like is added to a sample before subjecting it to biochemical measurement, the nonspecific reaction inhibitor of the first form may be contained in the diluent or the pretreatment solution.

[0106] <Concentration of Non-specific Reaction Inhibitor> In the reagent for biochemical measurements of this embodiment, when the first reagent contains a non-specific reaction inhibitor, the concentration of the inhibitor in the first reagent is, for example, preferably 0.001 to 5.00% (w / v), more preferably 0.002 to 2.00% (w / v), even more preferably 0.003 to 1.00% (w / v), and most preferably 0.005 to 0.50% (w / v). Within these preferred ranges, the effect of inhibiting non-specific reactions is high.

[0107] <Method of Using the Reagent> Examples of methods of using the reagent for biochemical measurement of this embodiment include the following methods (1) to (4), in which a biological sample containing a substance to be measured is brought into contact with latex particles carrying a substance with specific affinity for the substance to be measured in the presence of a non-specific reaction inhibitor. (1) A method of mixing a biological sample with a first reagent containing a non-specific reaction inhibitor and a buffer solution containing an optional component, and then mixing this mixture with a second reagent containing latex particles carrying a substance with specific affinity for the substance to be measured and a buffer solution containing an optional component. (2) A method of simultaneously mixing a biological sample with a first reagent containing a non-specific reaction inhibitor and a buffer solution containing an optional component, and a second reagent containing latex particles carrying a substance with specific affinity for the substance to be measured and a buffer solution containing an optional component. (3) A method of mixing a biological sample with a second reagent containing latex particles carrying a substance with specific affinity for the substance to be measured and a buffer solution containing an optional component, and then adding the first reagent containing a non-specific reaction inhibitor and a buffer solution containing an optional component to this mixture and mixing them. (4) A method of mixing a first reagent containing a non-specific reaction inhibitor and a buffer solution containing an optional component, and a second reagent containing latex particles carrying a substance with specific affinity for the substance to be measured and a buffer solution containing an optional component, and then adding the biological sample to this mixture and mixing them.

[0108] Among the above methods, from the viewpoint of suppressing non-specific reactions, method (1), (2) or (4) is preferred, method (1) or (4) is more preferred, and method (1) is even more preferred.

[0109] <Biochemical Measurement Reagent Kit> The biochemical measurement reagent kit of this embodiment is a reagent kit including the biochemical measurement reagent described above. The biochemical measurement reagent in the biochemical measurement reagent kit of this embodiment has been described above, so redundant description will be omitted. Components other than the biochemical measurement reagent that may be included in the biochemical measurement reagent kit of this embodiment include, for example, a reagent instruction manual, blood sample collection tools (collection pipette, syringe, cotton swab, filtration filter, etc.), a detection device, an analysis device, etc.

[0110] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to the examples described below.

[0111] The materials used in this example are listed below. Table 1 shows the compounds used as the first repeating unit, the second repeating unit, and the third repeating unit. The first repeating unit may be referred to as Monomer 1, the second repeating unit as Monomer 2, and the third repeating unit as Monomer 3. Monomers 2-10 and 2-11 were synthesized as in Reference Synthesis Examples 1 and 2 described below.

[0112] AIBN (2,2'-azobisisobutyronitrile), manufactured by Fujifilm Wako Pure Chemical Industries, Ltd. Acetone, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd. TFE (2,2,2-trifluoroethanol), manufactured by Tokyo Chemical Industry Co., Ltd. THF (tetrahydrofuran), manufactured by Fujifilm Wako Pure Chemical Industries, Ltd. Otsuka Saline Injection (0.9 w / v% sodium chloride aqueous solution), manufactured by Otsuka Pharmaceutical Factory, Co., Ltd. Lipidure BL-206, Lipidure BL-802, Lipidure BL-1002, manufactured by NOF Corporation HBR-1, manufactured by SCANTIBODIES LABORATORY, INC.

[0113]

[0114] Experimental Example 1 Synthesis of Copolymers Copolymers were synthesized using two or more monomers containing the first repeating unit shown in Table 1.

[0115] Synthesis of Production Example 2: 1.50 g (4.90 mmol) of Monomer 1-1, 0.104 g (0.306 mmol) of Monomer 2-6, and 97.8 μL (0.918 mmol) of Monomer 3-1 were dissolved in a mixture of 6.36 mL of TFE and 1.59 mL of THF. After stirring at 60°C for 30 minutes under a nitrogen atmosphere, 0.119 g (0.726 mmol) of AIBN was added. After further stirring at 60°C for 20 hours, the mixture was cooled to room temperature, and the reaction solution was added to 99.0 mL of acetone, and the solid was collected by filtration. 10.0 mL of acetone was added to the obtained solid, and the mixture was stirred, and the solid was collected by filtration. Washing with acetone and filtration were performed two more times, and the obtained solid was dried under reduced pressure to obtain 1.61 g of copolymer. The copolymer was dissolved in a 0.9 w / v % aqueous sodium chloride solution and then filtered through a membrane filter (material: polyvinylidene fluoride (PVDF), pore size: 0.45 μm) to obtain a copolymer solution.

[0116] Synthesis of Production Examples 1, 3 to 23, and Comparative Production Examples 1 to 7: Copolymer solutions of Production Examples 1, 3 to 23, and Comparative Production Examples 1 to 7 were obtained in the same manner as Production Example 2, except that the compounds shown in Table 2 were used as Monomer 1, Monomer 2, and Monomer 3 in the molar fractions shown in Table 2. In Production Example 22, Monomer 3 was not used. In Comparative Production Examples 2 and 4 to 6, 4.90 mmol of Monomer 1, 0.612 mmol of Monomer 2, and 0.612 mmol of Monomer 3 were used. In Comparative Production Examples 4 and 7 to 9, 4.90 mmol of Monomer 1, 0.918 mmol of Monomer 2, and 0.306 mmol of Monomer 3 were used. In Production Example 22, 4.90 mmol of Monomer 1 and 0.918 mmol of Monomer 2 were used.

[0117]

[0118] Synthesis of Production Examples 24 to 28: 1.50 g (4.90 mmol) of Monomer 1-1, 89.5 μL (0.306 mmol) of Monomer 2-5, and 97.8 μL (0.918 mmol) of Monomer 3-1 were dissolved in a mixed solution of TFE and THF with a volume listed in Table 3. After stirring at 60°C for 30 minutes under a nitrogen atmosphere, AIBN was added in an amount listed in Table 3. After further stirring at 60°C for 20 hours, the mixture was cooled to room temperature, the reaction solution was added to 300 mL of acetone, and the solid was collected by filtration. 10.0 mL of acetone was added to the obtained solid, and the mixture was stirred. The solid was then collected by filtration. Washing with acetone and filtration were repeated two more times, and the obtained solid was dried under reduced pressure to obtain the copolymers of Production Examples 24 to 28. Each copolymer was dissolved in a 0.9 w / v % aqueous sodium chloride solution, and then filtered through a membrane filter (material: PVDF, pore size: 0.45 μm) to obtain each copolymer solution.

[0119]

[0120] Synthesis of Production Examples 29 to 31: Copolymer solutions of Production Examples 29 to 31 were obtained in the same manner as in Production Example 2, except that Monomer 1, Monomer 2, and Monomer 3 were used in the molar fractions shown in Table 4.

[0121]

[0122] [Experimental Example 2] Verification of the non-specific reaction suppression effect of the copolymer - 1 (LTIA method, procalcitonin measurement reagent) A test was conducted to evaluate the degree of aggregation between components in a sample and one type of antibody-sensitized latex using a buffer solution containing the polymer of the present invention.

[0123] 1. Measurement Reagents 1-1. First Reagent A first reagent base solution was prepared with the following composition: 100 mM Bis-Tris-HCl (pH 6.5) 600 mM NaCl 0.2% BSA The additives listed in Table 5 were added to the above first reagent base solution to the final concentrations listed in Table 5 to prepare the first reagent. In Comparative Example 2, "LPD206" represents Lipidure BL-206, and in Comparative Example 3, "LPD802" represents Lipidure BL-802. The antibody used in Comparative Example 12 was an anti-human immunoglobulin M antibody obtained by the method described in Japanese Patent No. 4625879.

[0124]

[0125] 1-2. Second Reagent: 5 mM MOPS-NaOH (pH 7.0) Anti-human procalcitonin monoclonal antibody-sensitized latex (one type) The anti-human procalcitonin monoclonal antibody was obtained by a method known to those skilled in the art using the commercially available procalcitonin antigen described below as an immunogen. The anti-human procalcitonin monoclonal antibody-sensitized latex was prepared with reference to the method described in JP 2017-181377 A. Specifically, an anti-human procalcitonin monoclonal antibody solution diluted to 0.36 mg / mL with an equivalent amount of 5 mM Tris-HCl (pH 8.5) was added to a 1.0% latex solution (5 mM Tris buffer solution (hereinafter referred to as Tris-HCl or simply Tris) (pH 8.5)) with an average particle size of 0.3 μm, and the mixture was stirred at 4° C. for 2 hours. An equal amount of 5 mM Tris-HCl (pH 8.5) containing 0.5% bovine serum albumin (BSA) was then added, and the mixture was stirred at 4° C. for 1 hour to prepare an anti-human procalcitonin monoclonal antibody-sensitized latex particle solution. The latex particle solution was diluted with 5 mM MOPS-NaOH (pH 7.0) to an absorbance of approximately 4.0 OD at 600 nm, which was used as the second reagent. Commercially available procalcitonin antigen; Procalcitonin: Hytest

[0126] Randomly extracted human serum was mixed to obtain a pooled serum. This pooled serum was mixed with chyle from Interference Check A Plus (Sysmex Corporation) according to the instructions to prepare a blank sample, chyle sample 1: 1500 FTU (formazin turbidity), and chyle sample 2: 3000 FTU.

[0127] 2-2. Serum Samples Samples 1 to 6, 8, 9, 21, and 22 listed in Table 18 of Reference Example 4 below, which showed increased absorbance and nonspecific agglutination when human serum was measured using the first and second reagents by the measurement method described below, were used as samples. Note that the donors of the samples listed in Table 18 were all different.

[0128] 3. Measurement Method: The first and second reagents were combined and the sample was measured using a Hitachi 7180 automatic analyzer. Specifically, 120 μL of the first reagent was added to 15.0 μL of sample, and the mixture was incubated at 37°C for 5 minutes. Then, 40 μL of the second reagent was added and stirred. The absorbance was measured over the next 5 minutes at a dominant wavelength of 570 nm and a secondary wavelength of 800 nm. The difference between the absorbance measured 5 minutes after the addition of the second reagent and the absorbance measured immediately after the addition of the second reagent was evaluated as the change in absorbance (mAbs).

[0129] 4. Measurement Results The results were analyzed according to the following methods, and are shown in Tables 6 to 9. Note that when the relative measurement value was less than 0.01, it was expressed as <0.01.

[0130] 4-1. Method for analyzing measurement results of chyle samples For each of chyle samples 1 and 2, the value (mAbS, blank-subtracted absorbance change) was calculated by subtracting the absorbance change of the blank sample from the absorbance change of the chyle sample. Furthermore, the relative values ​​of the absorbance change of Comparative Examples 2 to 7 and Examples 1 to 23 relative to the blank-subtracted absorbance change of Comparative Example 1 were calculated. "Absorbance change" in Tables 6 to 9 indicates the blank-subtracted absorbance change value (mAbS) for Comparative Example 1.

[0131] 4-2. Method for Analyzing Measurement Results of Serum Samples The relative values ​​of the absorbance change (mAbs.) in Comparative Example 1 for Comparative Examples 2 to 7, Examples 1 to 23, and Examples 39 to 41 were calculated.

[0132] 4-3. Data evaluation The relative values ​​were evaluated for the inhibitory effect on non-specific reactions according to the following criteria. A to C were judged to have the inhibitory effect on non-specific reactions, and D or E was judged to have no inhibitory effect on non-specific reactions. 0.3 or less: A, indicating a strong inhibitory effect on non-specific reactions. 0.3 or more and 0.6 or less: B, indicating a moderate inhibitory effect on non-specific reactions. 0.6 or more and 0.8 or less: C, indicating a weak inhibitory effect on non-specific reactions. 0.8 or more and 1.0 or less: D, no effect on the level of non-specific reactions. 1.0 or more: E, exacerbating non-specific reactions. Note that "N.D." in the table indicates that the measurement was not performed.

[0133]

[0134]

[0135]

[0136]

[0137] 5. Discussion The antigen procalcitonin is not a polyvalent antigen, meaning it has multiple identical epitopes within the molecule. Therefore, in Experimental Example 2, which used only one type of antibody-sensitized latex, no agglutinates were formed even when the procalcitonin in the sample reacted with the antibody-sensitized latex, and the absorbance remained unchanged. In contrast, an increase in absorbance is interpreted as a nonspecific reaction in which some component present in the sample nonspecifically agglutinated the antibody-sensitized latex.

[0138] In chyle samples 1 and 2, the blank subtraction absorbance change when no additives were added (Comparative Example 1) was 38.0 mAb s. and 56.7 mAb s., respectively, indicating that nonspecific reactions occurred due to chyle. Furthermore, in serum samples (specimens 1 to 6, 8, 9, 21, and 22), the absorbance change (mAb s.) when no additives were added was 4.3 mAb s. or more, indicating that nonspecific reactions occurred.

[0139] In contrast, in Example 1, in which the polymer of Production Example 1 of the present invention was added to a reagent for measurement, the relative value for chyle sample 1 was 0.62, indicating that nonspecific reactions were suppressed. Similarly, in Examples 2 to 23 and 39 to 41, in which the polymers of Production Examples 2 to 23 and 29 to 31 were used as additives, nonspecific reactions were suppressed in both chyle samples and serum samples (Evaluations A to C). It was found that the polymer of the present invention has the effect of suppressing nonspecific reactions.

[0140] Comparative Examples 2 and 3 are commercially available as diagnostic reagent additives and are shown to be polymers with blocking effects. However, Comparative Examples 2 and 3, which used this product, were evaluated as D or E, resulting in no nonspecific reaction inhibitory effect or exacerbating the nonspecific reaction. In other words, it was found that even polymers with blocking effects do not necessarily exhibit nonspecific reaction inhibitory effects.

[0141] HBR-1 of Comparative Example 11 is known to specifically bind to human heterophile antibodies and suppress interference with measurements caused by heterophile antibodies. However, the amount of absorbance change increased in chyle samples 1 and 2 (Evaluation E), and the amount of absorbance change increased in serum samples (Evaluation E) or showed no inhibitory effect on nonspecific reactions (Evaluation D).

[0142] The anti-human IgM antibody of Comparative Example 12 is known to have the same purpose as the present application, i.e., as a component that suppresses nonspecific reactions in biochemical measurements. However, the amount of absorbance change increased in chyle samples 1 and 2 (Evaluation E), and the nonspecific reactions could not be suppressed in the serum sample tested this time (Evaluation D).

[0143] The evaluation of non-specific reaction inhibition for Comparative Examples 4 to 10 was D or E, which resulted in either no or an exacerbated effect of inhibiting non-specific reactions. In Comparative Examples 4 to 7, the alkyl side chain of Monomer 2 had 6 carbon atoms. In Comparative Examples 8 to 10, the alkyl side chain of Monomer 2 had a cyclic structure. From the above, it was revealed that Structure A contained in the second repeating unit (Monomer 2) of the present invention does not exhibit a non-specific reaction inhibitory effect when the alkyl chain length is 6 or less or when it has a cyclic structure. It was shown that a linear or branched alkyl group having 12 or more carbon atoms, as typified by Monomers 2-5 to 2-11, is suitable for Structure A of the second repeating unit (Monomer 2) of the present invention.

[0144] The nonspecific reaction inhibitory effects of Example 22, Example 8, Examples 19 to 21, and 23 were comparable in both chyle and serum samples. This demonstrates that the presence of a third repeating unit (monomer 3) in the copolymer is optional for the effect of inhibiting nonspecific reactions. Furthermore, the structure of the third repeating unit may be any as long as it has a molecular weight of 1,000 or less, and this experimental example demonstrates that the structures shown in Monomers 3-1 to 3-5 are suitable.

[0145] The polymers of Production Examples 29 to 31 of the present invention were found to have the effect of suppressing nonspecific reactions in serum samples 21 and 22 (Examples 39 to 41). It was shown that the effect of suppressing nonspecific reactions is achieved when the ratio of the second repeating unit and the third repeating unit is within the range of the present invention.

[0146] According to Sysmex's support information, chyle from Interference Check A Plus (manufactured by Sysmex Corporation) contains lipid components such as triolein, lecithin, and free fatty acids, and chyle samples 1 and 2 also contain these components. The nonspecific reactions observed in chyle samples 1 and 2 were presumed to be caused by lipid components such as triolein, lecithin, and free fatty acids. Among the serum samples, samples 3, 4, and 8 were chyle. The cause of chyle is thought to be the presence of lipid components in serum due to dietary influences, as well as hyperlipidemia. If nonspecific reactions occur in these samples, it is presumed that lipid components are the causative agent. The polymers of Production Examples 1 to 23 of the present invention were found to be effective in suppressing nonspecific reactions in chyle samples 1 and 2 and serum samples 3, 4, and 8, demonstrating their high effectiveness in suppressing nonspecific reactions caused by lipids. Furthermore, the inhibitory effect on nonspecific reactions was also observed in samples 1, 2, 5, 6, and 9, which are not chyle. Although the cause of the non-specific reaction is not clear, it is extremely significant that this compound was shown to have an inhibitory effect on non-specific reactions that could not be suppressed by HRB-1 (Comparative Example 11) or anti-human IgM antibody (Comparative Example 12), both of which are already known to have the effect of suppressing non-specific reactions. This compound is expected to further improve the accuracy of biochemical measurements and contribute to appropriate medical treatment.

[0147] [Experimental Example 3] Verification of the non-specific reaction suppression effect of the copolymer - 2 (CLEIA method or LTIA method, sIL-2R measurement reagent) 1. Measurement by CLEIA method (Reference Example 1) The CLEIA method involves a B / F separation operation and a washing step. Therefore, it is a measurement method that is less susceptible to the influence of non-specific reactions derived from the sample. The measurement value by this method was used as the control value of the sIL-2R concentration in each specimen.

[0148] 1-1. Measurement Reagent Determiner CL (registered trademark) IL-2R NX (Minaris Medical Co., Ltd.) was used.

[0149] 1-2. Samples Samples 13 to 20 listed in Table 18 of Reference Example 4 below were used as human serum samples.

[0150] 1-3. Measurement Method Measurement was performed using CL-JACK NX (registered trademark) (Minaris Medical Co., Ltd.) according to the instructions attached to the measurement reagent.

[0151] 2. Measurement by LTIA Method (Comparative Example 13, Example 24) 2-1. Measurement Reagents A first reagent and a second reagent were prepared according to the methods described in JP 2017-181377 A and JP 2018-173429 A. In Comparative Example 13, the first reagent described in 1-1 of Experimental Example 2 was used as is. In Example 24, a reagent prepared by adding the polymer of Production Example 2 to the first reagent to a final concentration of 0.10% (w / v) was used.

[0152] 2-2. Sample The same sample as described in 1-2 was used.

[0153] 2-3. Measurement Method: The first and second reagents were combined, and the sIL-2R concentration in each sample was measured using a Hitachi 7180-type automated analyzer. Specifically, 120 μL of the first reagent was added to 5.6 μL of sample, and the mixture was incubated at 37°C for 5 minutes. Then, 40 μL of the second reagent was added and stirred. The absorbance change associated with aggregate formation was measured over the next 5 minutes at a dominant wavelength of 570 nm and a sub-wavelength of 800 nm. The absorbance change was applied to a calibration curve obtained by measuring a standard substance of known concentration to calculate the measured value. For each sample, the measured values ​​in Comparative Example 13 and Example 24 were divided by the measured value in Reference Example 1 to calculate a relative value for evaluation. A relative value between 0.85 and 1.15 was determined to be no deviation from the measured value in Reference Example 1.

[0154] 3. Measurement Results The measurement results are shown in Table 10. The column for Reference Example 1 lists the measured values ​​of each specimen in Reference Example 1. The columns for Comparative Example 13 and Example 24 list the relative values ​​of each measured value to Reference Example 1.

[0155]

[0156] 4. Discussion 4-1. Control Samples Samples 17 to 20, in which the relative values ​​of the LTIA measurement value (Comparative Example 13) without any additives were 0.91 to 1.01 relative to the CLEIA measurement value (Reference Example 1), were evaluated as "control samples" in which no nonspecific reactions occurred in the LTIA method. The relative values ​​of the measurement value (Example 24) of the control samples, in which the polymer of Production Example 2, a copolymer of the present invention, was added, were 0.93 to 1.04, which were roughly equivalent to those of Reference Example 1. It was therefore found that the addition of the copolymer of the present invention did not affect the measurement value of samples that did not exhibit nonspecific reactions.

[0157] 4-2. Deviation Samples Samples 13 to 16, in which the relative value of the measured value by the LTIA method without adding any additive (Comparative Example 13) to the measured value by the CLEIA method (Reference Example 1) was greater than 1.15, were evaluated as "deviation samples" in which a nonspecific reaction occurs in the LTIA method. When the copolymer of the present invention was added to all of the samples, the relative value fell within the range of 0.96 to 1.08, which was found to be close to the measured value by the CLEIA method.

[0158] Experimental Examples 2 and 3 demonstrated that in the LTIA method, a homogeneous immunoassay, the presence of the copolymer of the present invention in the reaction system suppressed nonspecific reactions originating from the sample. Anti-human immune immunoglobulin antibodies, which are known to suppress nonspecific reactions, not only failed to exhibit the effect of inhibiting nonspecific reactions, but actually worsened the nonspecific reactions. Furthermore, polymers prepared by polymerizing monomers with short alkyl side chains or cyclic structures as the second repeating unit structure A, which are similar in structure to the present invention, also failed to exhibit the effect of inhibiting nonspecific reactions or even worsened the effect. In both samples, the only polymer that exhibited the effect of inhibiting nonspecific reactions was the polymer of the present invention.

[0159] [Experimental Example 4] Verification of the non-specific reaction suppression effect of the copolymer - 3 (LTIA method, procalcitonin measurement reagent) A test was conducted to evaluate the degree of aggregation between components in a sample and one type of antibody-sensitized latex using buffer solutions containing various concentrations of the polymer of the present invention.

[0160] 1. Measurement Reagents 1-1. First Reagent The polymer of Production Example 2 was added to the first reagent base solution described in Experimental Example 2-1-1 to the final concentrations shown below to prepare the first reagent. Example 25: 0.005% Example 26: 0.05% Example 27: 0.10% Example 28: 0.20% Example 29: 0.50%

[0161] 1-2. Second Reagent The second reagent described in 1-2 of Experimental Example 2 was used.

[0162] 2. Samples 2-1. Chyle sample The chyle reagent described in 2-1 of Experimental Example 2 was used. 2-2. Serum sample Specimens 1 and 10 described in Table 18 of Reference Example 4 below were used as samples.

[0163] 3. Measurement Method Measurement was carried out using the measurement method described in Experimental Example 2, section 3.

[0164] 4. Measurement Results The results were analyzed using the same method as in Test Example 2. The relative values ​​and evaluation results for Examples 25 to 29 are shown in Table 11. In Table 11, "Absorbance change (mAb)" indicates the absorbance change in Comparative Example 1. The "Absorbance change (mAb)" for Chyle Samples 1 and 2 is the absorbance change subtracted from the blank. When the measured value was less than 0.01, it was expressed as a relative value of <0.01.

[0165]

[0166] 5. Discussion The concentration of the polymer of Production Example 2 added to the first reagent was varied between 0.005% and 0.5%. For chyle samples 1 and 2, and specimens 1 and 10, the relative values ​​to Comparative Example 1 were 0.8 or less, demonstrating that nonspecific reactions were suppressed. When the concentration of the polymer of Production Example 2 added to the first reagent was 0.05 to 0.5%, the relative values ​​to Comparative Example 1 were 0.58 or less, demonstrating a particularly high effect.

[0167] [Experimental Example 5] Verification of the non-specific reaction suppression effect of copolymers - 4 (LTIA method, reagent for measuring procalcitonin) This shows a test to evaluate the degree of aggregation between components in a sample and one type of antibody-sensitized latex using buffer solutions to which polymers with different weight-average molecular weights (Production Examples 24 to 27) were added.

[0168] 1. Measurement Reagents 1-1. First Reagent The polymers described below were added to the first reagent base solution described in 1-1 of Experimental Example 2 to a final concentration of 0.10% to prepare first reagents. Example 30: Production Example 24 Example 31: Production Example 25 Example 32: Production Example 26 Example 33: Production Example 27 Example 34: Production Example 28

[0169] 1-2. Second Reagent The second reagent described in 1-2 of Experimental Example 2 was used.

[0170] 2. Samples 2-1. Chyle Samples Blank sample, chyle sample 1 and chyle sample 2 in 2-1 of Experimental Example 2 were used.

[0171] 2-2. Serum Samples Samples 2, 7, and 9 shown in Table 18 of Reference Example 4 below were used as samples.

[0172] 3. Measurement Method Measurement was carried out by the method described in Experimental Example 2, Section 3.

[0173] 4. Measurement Results The results were analyzed using the same method as in Test Example 2, and are shown in Table 12. Measured values ​​less than 0.01 were expressed as <0.01. The "Absorbance Change (mAbs.)" in Table 12 indicates the absorbance change in Comparative Example 1. The absorbance changes for chyle samples 1 and 2 are the absorbance changes subtracted from the blank.

[0174]

[0175] 5. Discussion It is known that the weight-average molecular weight of a polymer increases as the concentration of the polymerization initiator decreases. The weight-average molecular weights of Production Examples 24, 25, and 26 were 27,700, 86,300, and 194,000, respectively. The weight-average molecular weights of Production Examples 27 and 28 are estimated to be 200,000 or more. The change in absorbance when the polymers of Production Examples 24 to 28 were added to the reagent was 0.27 to 0.80 (evaluations A to C) compared to the change in absorbance when no additive was added (Comparative Example 1), demonstrating that all of the polymers suppress nonspecific reactions.

[0176] [Experimental Example 6] Verification of the effect of copolymer on specific reaction of LTIA reagent - 1 (LTIA method, TARC reagent) Using a buffer solution containing the polymer of the present invention, TARC antigen of known concentrations was measured, and the effect of the polymer of the present invention on the specific reaction was evaluated. 1. Measurement Reagents 1-1. First Reagent A first reagent base solution was prepared with the following composition: 100 mM MOPS-NaOH (pH 7.5) 500 mM NaCl 0.5% BSA The polymer described below was added to the first reagent base solution to a final concentration of 0.1%, to prepare the first reagent. Comparative Example 14: Not added Example 35: Production Example 2

[0177] 1-2. Second Reagent: 5 mM MOPS-NaOH (pH 7.0) Anti-human TARC monoclonal antibody-sensitized latex (2 types) The anti-human TARC monoclonal antibody was obtained using the commercially available TARC antigen described below by a method known to those skilled in the art. Furthermore, a combination of monoclonal antibodies that allows sandwich assay of the TARC antigen was selected by a method known to those skilled in the art. The anti-human TARC monoclonal antibody-sensitized latex was prepared with reference to the method described in JP 2017-181377 A. Commercially available TARC antigen: CCL17, thymus, and activation regulated chemokine: Shenandoah Biotechnology, Inc. Company CCL17 / TARC, Human: LifeSpan Biosscience, Inc. Human TARC (CCL17): Abeomics, Inc. company

[0178] 2. Samples The commercially available TARC antigen described above was added to a diluent (PBS (pH 7.2)) at the following concentrations to prepare samples: TARC sample 1: physiological saline; TARC sample 2: TARC antigen equivalent to 650 pg / mL; TARC sample 3: TARC antigen equivalent to 1800 pg / mL; TARC sample 4: TARC antigen equivalent to 4750 pg / mL; TARC sample 5: TARC antigen equivalent to 10000 pg / mL; TARC sample 6: TARC antigen equivalent to 20700 pg / mL.

[0179] 3. Measurement Method The first and second reagents were combined, and the sample described in 2. was measured using a Hitachi automated analyzer 3500. Specifically, 120 μL of the first reagent was added to 2.4 μL of sample, and the mixture was incubated at 37°C for 5 minutes, after which 40 μL of the second reagent was added and stirred. The change in absorbance associated with aggregation formation was measured over the next 5 minutes at a dominant wavelength of 570 nm and a sub-wavelength of 800 nm, and the amount of absorbance change was calculated.

[0180] 4. Measurement Results The absorbance change of TARC sample 1 was used as a blank, and the value (mAbS, blank-subtracted absorbance change) was calculated by subtracting the absorbance change of the blank sample from the absorbance change of each sample. Furthermore, the relative value of the absorbance change of Example 35 to the blank-subtracted absorbance change of Comparative Example 14 was calculated. The results are shown in Table 13. When the relative value was 0.85 or more and 1.15 or less, it was determined that there was no effect on the specific reaction. In Table 13, "Absorbance change (mAbS)" indicates the absorbance change in Comparative Example 14. The numerical values ​​shown are the values ​​of the blank-subtracted absorbance change.

[0181]

[0182] 5. Discussion The relative change in absorbance in Example 35, in which the polymer of the present invention was added, relative to that in Comparative Example 14, in which no additive was added, was 0.99 to 1.11, regardless of the concentration of TARC antigen. Therefore, this experimental example demonstrated that the polymer of the present invention can be used without affecting the specific reaction.

[0183] Experimental Example 7 Verification of the influence of the copolymer on the specific reaction of the LTIA reagent - 2 (LTIA method, reagent for measuring sIL-2R) Using a buffer solution containing the polymer of the present invention, sIL-2R of known concentrations was measured to evaluate the influence of the polymer of the present invention on the specific reaction.

[0184] 1. Measurement Reagents The first and second reagents were prepared in the same manner as in Experimental Examples 3 and 2-1. In Comparative Example 15, the first reagent described above was used as is. In Example 36, a reagent prepared by adding the polymer of Production Example 2 to the first reagent described above to a final concentration of 0.10% (w / v) was used.

[0185] 2. Samples sIL-2R was added to the buffer solution at the following concentrations to prepare samples: IL-2R Sample 1: Physiological saline solution IL-2R Sample 2: Equivalent to 480 U / mL of sIL-2R IL-2R Sample 3: Equivalent to 2000 U / mL of sIL-2R IL-2R Sample 4: Equivalent to 5060 U / mL of sIL-2R IL-2R Sample 5: Equivalent to 10720 U / mL of sIL-2R

[0186] 3. Measurement Method Measurement was carried out in the same manner as in Test Examples 3 and 2-3, and the amount of change in absorbance of each sample was measured.

[0187] 4. Measurement Results The absorbance change of IL-2R sample 1 was used as a blank, and the value (mAbS, blank-subtracted absorbance change) was calculated by subtracting the absorbance change of the blank sample from the absorbance change of each sample. Furthermore, the relative value of the absorbance change of Example 38 relative to the blank-subtracted absorbance change of Comparative Example 15 was calculated. The results are shown in Table 14. In Table 14, "Absorbance change (mAbS)" indicates the absorbance change in Comparative Example 15. The values ​​shown are the blank-subtracted absorbance change values. When the relative value was 0.85 or more and 1.15 or less, it was determined that there was no effect on the measurement sensitivity.

[0188]

[0189] 5. Discussion The relative change in absorbance in Example 36, in which the polymer of the present invention was added, compared to the case in which no additive was added (Comparative Example 15) was 0.98 to 1.00, regardless of the concentration of sIL-2R. Experimental Examples 6 and 7 demonstrated that the polymer of the present invention does not affect specific reactions, regardless of the object being measured. Clinical testing requires the detection of extremely small amounts of target substances contained in serum or other samples being analyzed. The polymer of the present invention was found to be an extremely practical component that suppresses non-specific reactions without affecting specific reactions for a variety of objects being measured.

[0190] [Experimental Example 8] Verification of the effect of adding a copolymer on suppressing non-specific reactions - 5 (LTIA method, reagent for measuring procalcitonin) This shows a test in which the degree of aggregation between components in a sample and one type of antibody-sensitized latex was evaluated using a buffer solution to which a polymer having a structural unit derived from 2-methacryloyloxyethyl phosphorylcholine or a polymer of the present invention was added.

[0191] 1. Measurement Reagents 1-1. First Reagent The polymers described below were added to the first reagent base solution described in 1-1 of Experimental Example 2 to a final concentration of 0.10% to prepare the first reagent. Comparative Example 16: No addition Comparative Example 17: Lipidure-BL1002 Example 37: Production Example 2

[0192] 1-2. Second Reagent The same second reagent as described in 1-2 of Experimental Example 2 was used.

[0193] 2. Samples Specimens 3, 8, 11 and 12 shown in Table 18 of Reference Example 4 below were used as samples.

[0194] 3. Measurement Method Measurement was carried out by the method described in Experimental Example 2, section 3.

[0195] 4. Measurement Results The results were analyzed in the same manner as in Experimental Example 2, and the results are shown in Table 15. That is, the relative values ​​of Comparative Example 17 and Example 37 to the absorbance change (mAb) of Comparative Example 16 were calculated. In Table 15, "Absorbance change (mAb)" indicates the absorbance change in Comparative Example 16. Note that when the relative value was less than 0.01, it was expressed as <0.01.

[0196]

[0197] 5. Discussion The inhibitory effect of Lipidure-BL1002 or the polymer of the present invention on nonspecific reactions was examined using serum samples in which nonspecific reactions were occurring. Lipidure is a polymer having structural units derived from 2-methacryloyloxyethyl phosphorylcholine. Lipidure-BL1002 is a polymer with hydrophobic side chains and is a commercially available diagnostic reagent additive known to have blocking, reagent stabilization, sensitization, improved reproducibility and accuracy, and solubilization effects. In a sample exhibiting a nonspecific reaction, the absorbance change in Comparative Example 17, in which Lipidure-BL1002 was added, was 0.82 to 1.90 (evaluation D or E) compared to the absorbance change in Comparative Example 16, in which Lipidure-BL1002 was not added, resulting in either no inhibition of the nonspecific reaction or an exacerbation of the reaction. In particular, the absorbance change in sample 11 was 1.90 compared to the amount of absorbance change in Comparative Example 16, and the addition of Lipidure-BL1002 resulted in a significant deviation in the measured values. On the other hand, in Example 37, in which the polymer of the present invention was added, the absorbance change was <0.01 to 0.70 (evaluations A to C) compared to the amount of absorbance change in Comparative Example 16, and nonspecific reactions were suppressed in all samples.

[0198] [Experimental Example 9] Verification of the effect of adding a copolymer on inhibiting non-specific reactions - 6 (LTIA method, reagent for measuring procalcitonin) A test was conducted to verify the principle that the polymer of the present invention exhibits an effect of inhibiting non-specific reactions.

[0199] 1. Measurement Reagents 1-1. First Reagent The first reagent base solution described in 1-1 of Experimental Example 2 was used as the first reagent.

[0200] 1-2. Second Reagent Solutions were prepared by adding the additives shown in Table 16 to the second reagent described in 1-2 of Experimental Example 2 to a final concentration of 0.3%. Each prepared solution was incubated at 37°C for 10 minutes, thereby subjecting the antibody-sensitized latex to a blocking treatment. For Comparative Examples 18 and 19, which are labeled "with centrifugation" in Table 16, each solution was centrifuged (15,000 rpm, 20 minutes) after the blocking treatment. The supernatant was removed, and the resulting precipitate was resuspended in 5 mM MOPS-NaOH (pH 7.0) to prepare the second reagent. For Comparative Example 20 and Example 38, which are labeled "without centrifugation" in Table 16, the solution after incubation was used as is as the second reagent.

[0201]

[0202] 2. Samples 2-1. Chyle Sample The sample described in 2-1 of Experimental Example 2 was used.

[0203] 2-2. Serum Samples Samples 1 and 2 shown in Table 18 of Reference Example 4 below were used as samples.

[0204] 3. Measurement Method Measurement was carried out by the method described in Experimental Example 2, Section 3.

[0205] 4. Measurement Results The results were analyzed according to the following method, and the results are shown in Table 17.

[0206] 4-1. Method for analyzing measurement results of chyle samples For chyle sample 1, the value (mAbs., blank-subtracted absorbance change) was calculated by subtracting the absorbance change of the blank sample from the absorbance change of the chyle sample. Furthermore, the relative value of the blank-subtracted absorbance change of Comparative Example 19 to the blank-subtracted absorbance change of Comparative Example 18 was calculated. Furthermore, the relative value of the blank-subtracted absorbance change of Example 38 to the blank-subtracted absorbance change of Comparative Example 20 was calculated.

[0207] 4-2. Method for analyzing measurement results of serum samples The relative value of the amount of absorbance change (mAb) in Comparative Example 19 to the amount of absorbance change (mAb) in Comparative Example 18 was calculated. In addition, the relative value of the amount of absorbance change (mAb) in Example 38 to the amount of absorbance change (mAb) in Comparative Example 20 was calculated.

[0208] 4-3. Data Evaluation The non-specific reaction inhibitory effect was evaluated using relative values ​​according to the same criteria as in Experimental Example 2. Among the "change in absorbance (mAbs.)" in Table 17, the values ​​for chyle samples 1 and 2 are the change in absorbance subtracted from the blank.

[0209]

[0210] 5. Discussion It is presumed that by blocking antibody-sensitized latex with the polymer of the present invention and then removing the supernatant by centrifugation, the free polymer during the reaction is removed, and the only polymer that comes into contact with the sample is that bound to the antibody-sensitized latex particles. On the other hand, if centrifugation is not performed, free polymer is present in the reaction solution, and it is presumed that it can come into contact with the sample in a free state.

[0211] In Comparative Example 19, the nonspecific reaction was not suppressed or was exacerbated in both the chyle sample and the serum sample (evaluation D or E). In contrast, in Example 38, the nonspecific reaction was suppressed in both the chyle sample and the serum sample (evaluation B). Therefore, this experimental example demonstrated that in order for the polymer of the present invention to exhibit the nonspecific reaction suppression effect, it is necessary for it to come into contact with the sample in a free state.

[0212] Lipidure BL-206, Lipidure BL-802, and the like are commercially available polymers used as additives for diagnostic reagents and have been shown to have a blocking effect. One example of how these additives are used is as a blocking agent in Western blotting. In Western blotting, adhesion of components other than the target of measurement to the detection membrane can lead to errors in measurement values. Blocking agents are used to prevent this. A common method involves contacting the membrane with a blocking agent, then washing the membrane to remove excess free blocking agent. Therefore, in Western blotting, the target of measurement does not come into contact with the free blocking agent. Therefore, the polymer of the present invention can be said to have a new effect that is different from that of blocking agents.

[0213] Reference Example 2: Synthesis of Monomer 2-10 3.50 g (9.78 mmol) of 2-Decyl-1-tetradecanol (Tokyo Chemical Industry Co., Ltd.) and 2.50 mL (29.6 mmol) of methacrylic acid were dissolved in a mixed solution of 34.5 mL of THF and 14.8 mL of N,N-dimethylformamide (Fujifilm Wako Pure Chemical Industries, Ltd.) To this solution, 5.68 g (29.6 mmol) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimid ehydrochloride (EDCI.HCl) and 1.21 g (9.87 mmol) of 4-dimethylaminopyridine (DMAP) were added, and the mixture was stirred at room temperature for 1 hour and 50 minutes. The reaction solution was concentrated under reduced pressure, and 123 mL of heptane (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), 61.7 mL of acetonitrile, and 6.9 mL of water were added to the resulting residue for separation and washing. 12.3 mL of heptane and 61.7 mL of acetonitrile were added to the resulting heptane layer for separation and washing. This separation and washing with heptane and acetonitrile was performed once more, and the heptane layer was concentrated and dried under reduced pressure to obtain 1.85 g of Monomer 2-10. Mass spectrometry confirmed that the target product had been produced. Electrospray ionization mass spectrometry (ESIMS) (m / z) 445.4 (M+Na)+

[0214] Reference Example 3: Synthesis of Monomer 2-11 Using 3.00 g (7.30 mmol) of 2-dodecylhexadecan-1-ol (Tokyo Chemical Industry Co., Ltd.), 1.85 g of Monomer 2-11 was obtained in the same manner as in Reference Synthesis Example 2. Mass spectrometry confirmed that the target product had been produced. ESIMS (m / z) 501.5 (M+Na)+

[0215] Reference Example 4: Confirmation of chyle in serum samples The presence or absence of chyle in the human serum samples used in this example was determined by the following method.

[0216] 1. Evaluation method The presence or absence of turbidity in each serum sample was visually evaluated.

[0217] 2. Results The presence or absence of chyle in each serum was determined by visual inspection and the results are shown in Table 18. If turbidity was present, the sample was determined to have chyle, and if no turbidity was present, the sample was determined to have no chyle.

[0218]

[0219] According to the present invention, it is possible to provide a non-specific reaction inhibitor, a reagent for biochemical measurements, a reagent kit for biochemical measurements, and a copolymer.

Claims

1. A non-specific reaction inhibitor which is a copolymer comprising a first repeating unit represented by formula (1) and a second repeating unit represented by formula (2). (In formula (1), R1, R2, and R3 each independently represent a hydrogen atom or a methyl group, X represents NH or an oxygen atom, Y represents SO3- or COO-, and n and m each independently represent an integer of 1 to 5.) (In formula (2), R4 is a hydrogen atom or a methyl group, Z is NH or an oxygen atom, and A is a linear or branched alkyl group having 12 to 40 carbon atoms.) 2. The non-specific reaction inhibitor according to claim 1, wherein A in the formula (2) is a linear or branched alkyl group having 12 to 28 carbon atoms.

3. The non-specific reaction inhibitor according to claim 1 or 2, further comprising a third repeating unit derived from a polymerizable compound having a molecular weight of 1,000 or less.

4. The non-specific reaction inhibitor according to claim 3, wherein the third repeating unit is a repeating unit derived from one or more of methyl methacrylate, methyl acrylate, methacrylic acid, 2-methoxyethyl acrylate, and 1-vinyl 2-pyrrolidone.

5. The non-specific reaction inhibitor according to claim 1 or 2, wherein the weight-average molecular weight of the copolymer is 1,000 to 5,000,000.

6. The non-specific reaction inhibitor according to claim 1 or 2, wherein the molar fraction of the substance amount of the first repeating unit relative to the substance amount of all repeating units constituting the copolymer is 50 to 99 mol %.

7. A method for inhibiting non-specific reactions, comprising mixing the non-specific reaction inhibitor according to claim 1 or 2 with a biological sample, and inhibiting non-specific reactions of the object to be measured when measuring the object to be measured contained in the biological sample.

8. The method for inhibiting a non-specific reaction according to claim 7, wherein the biological sample is a chyle specimen.

9. A reagent for biochemical measurement, the reagent comprising a copolymer containing a first repeating unit represented by formula (1) and a second repeating unit represented by formula (2). (In formula (1), R １ , R ２ and R ３ are each independently a hydrogen atom or a methyl group, X is NH or an oxygen atom, and Y is SO ３ － or COO － wherein n and m are each independently an integer of 1 to 5. (In formula (2), R ４ is a hydrogen atom or a methyl group, Z is an NH or an oxygen atom, and A is a linear or branched alkyl group having 12 to 40 carbon atoms.

10. The reagent for biochemical measurements according to claim 9, wherein A in formula (2) is a linear or branched alkyl group having 12 to 28 carbon atoms.

11. The reagent for biochemical measurements according to claim 9 or 10, further comprising a third repeating unit derived from a polymerizable compound having a molecular weight of 1,000 or less.

12. The reagent for biochemical measurements according to claim 11, wherein the third repeating unit is a repeating unit derived from one or more of methyl methacrylate, methyl acrylate, methacrylic acid, 2-methoxyethyl acrylate, and 1-vinyl 2-pyrrolidone.

13. The reagent for biochemical measurements according to claim 9 or 10, wherein the weight-average molecular weight of the copolymer is 1,000 to 5,000,000.

14. The reagent for biochemical measurements according to claim 9 or 10, wherein the molar fraction of the amount of substance of the first repeating unit relative to the amount of substance of all repeating units constituting the copolymer is 50 to 99 mol %.

15. The reagent for biochemical measurement according to claim 9 or 10, wherein the biochemical measurement is a homogeneous method.

16. The reagent for biochemical measurement according to claim 9 or 10, wherein the biochemical measurement is a latex immunoturbidimetric method.

17. The reagent for biochemical measurement according to claim 9 or 10, wherein the biochemical measurement is based on an antigen-antibody reaction.

18. The reagent for biochemical measurements according to claim 9 or 10, wherein the copolymer is contained as a non-specific reaction inhibitor.

19. A reagent kit for biochemical measurements, comprising the reagent for biochemical measurements according to claim 9 or 10.

20. A copolymer comprising a first repeating unit represented by formula (1) and a second repeating unit represented by formula (2). (In formula (1), R １ , R ２ and R ３ are each independently a hydrogen atom or a methyl group, X is NH or an oxygen atom, and Y is SO ３ － or COO － wherein n and m are each independently an integer of 1 to 5. (In formula (2), R ４ is a hydrogen atom or a methyl group, Z is an NH or an oxygen atom, and A is a linear or branched alkyl group having 12 to 40 carbon atoms.

21. A copolymer comprising a first repeating unit represented by formula (1-1) and a second repeating unit represented by formula (2-1), or a copolymer comprising a first repeating unit represented by formula (1-2) and the second repeating unit represented by formula (2-1). (In formula (2-1), A is a linear or branched alkyl group having 12 to 40 carbon atoms.) 22. A copolymer comprising a first repeating unit represented by formula (1) and a second repeating unit represented by formula (3). (In formula (1), R １ , R ２ and R ３ are each independently a hydrogen atom or a methyl group, X is NH or an oxygen atom, and Y is SO ３ － or COO － wherein n and m are each independently an integer of 1 to 5. (In formula (3), R ４ is a hydrogen atom or a methyl group, Z is an NH or an oxygen atom, and R ５ and R ６ are each independently a linear or branched alkyl group having 1 to 30 carbon atoms, and R ５ and R ６ The total number of carbon atoms is 10 to 38.

23. A copolymer comprising a first repeating unit represented by formula (1-3), a first repeating unit represented by formula (1-4), and a second repeating unit represented by formula (2). (In formula (1-3), R １ , R ２ and R ３ are each independently a hydrogen atom or a methyl group, X is NH or an oxygen atom, and n and m are each independently an integer of 1 to 5. (In formula (1-4), R １ , R ２ and R ３ are each independently a hydrogen atom or a methyl group, X is NH or an oxygen atom, and n and m are each independently an integer of 1 to 5. (In formula (2), R ４ is a hydrogen atom or a methyl group, Z is an NH or an oxygen atom, and A is a linear or branched alkyl group having 12 to 40 carbon atoms.

24. The copolymer according to any one of claims 20 to 23, further comprising a third repeating unit derived from a polymerizable compound having a molecular weight of 1,000 or less.

25. The copolymer according to claim 24, wherein the third repeating unit is a repeating unit derived from one or more of methyl methacrylate, methyl acrylate, methacrylic acid, 2-methoxyethyl acrylate, and 1-vinyl 2-pyrrolidone.

26. The copolymer according to any one of claims 20 to 23, having a weight average molecular weight of 1,000 to 5,000,000.

27. The copolymer according to any one of claims 20 to 23, wherein the molar fraction of the amount of substance of the first repeating unit relative to the amount of substance of all repeating units constituting the copolymer is 50 to 99 mol %.

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