A glycated protein measurement reagent containing a protease stabilizer that increases the redox potential of ferrocyanide, a method for measuring glycated protein, a method for storing the glycated protein measurement reagent, and a method for stabilizing the glycated protein measurement reagent

Abstract

To provide a glycated protein assay reagent with suppressed reagent blank.SOLUTION: Provided is a glycated protein assay reagent containing at least a Trinder reagent, 4-aminoantipyrine, protease, a stabilizer of the protease, and ferrocyanide, wherein at least the Trinder reagent is contained in a Trinder reagent-containing partial composition, at least the 4-aminoantipyrine is contained in a 4-aminoantipyrine-containing partial composition, the stabilizer of the protease is a stabilizer that increases the oxidation-reduction potential of the ferrocyanide above 0.058 V when the stabilizer of the protease and the ferrocyanide are mixed, and the oxidation-reduction potential is an oxidation-reduction potential in a reaction system containing the stabilizer of the protease and the ferrocyanide and not containing glycated protein.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to clinical test drugs, and relates to a glycated protein measurement reagent, a method for measuring glycated protein, a method for storing a glycated protein measurement reagent, and a method for stabilizing a glycated protein measurement reagent.

Background Art

[0002] In the diagnosis and management of diabetes, the measurement of glycated proteins is important. Glycated hemoglobin (such as HbA1c, etc.) that reflects the average blood glucose level in the past about 1 to 2 months, glycated albumin (GA) that reflects the average blood glucose level in the past about 2 weeks, and fructosamine (FRA), which is a general term for glycated proteins in serum, etc. are routinely measured.

[0003] As a method for measuring glycated proteins with high accuracy, low cost, and simplicity, the enzyme method can be mentioned. For example, Patent Documents 1, 2, and 3 describe that a protease is allowed to act on a glycated protein, hydrogen peroxide is generated using a ketoamine oxidase that acts on the resulting glycated amino acid or glycated peptide, and the glycated protein in serum can be measured by colorimetric quantification using peroxidase, a Trinder reagent, and 4-aminoantipyrine. Patent Document 4 describes that the glycated protein in serum can be measured by measuring the oxygen consumption amount when a ketoamine oxidase that acts on the glycated amino acid or glycated peptide generated by allowing a protease to act on a glycated protein is used, by a color reaction or an oxygen electrode. Patent Documents 5 and 6 describe that glycated hemoglobin and glycated albumin can be measured by allowing a protease to act on glycated hemoglobin or glycated albumin, generating hydrogen peroxide using a ketoamine oxidase that acts on the resulting glycated amino acid or glycated peptide, and performing colorimetric quantification using peroxidase, a Trinder reagent, and 4-aminoantipyrine.

[0004] These glycated protein measurement reagents contain protease stabilizers to improve the stability of the reagents. For example, Patent Document 6 discloses dimethyl sulfoxide, alcohol, water-soluble calcium salts, sodium chloride, quaternary ammonium salts, or quaternary ammonium salt-type cationic surfactants as protease stabilizers. Also, although there is no mention of measurement reagents, Patent Document 7 describes that the storage stability of protease in a liquid detergent can be improved by containing boric acid, boronic acid, or phenylboronic acid as a protease inhibitor. Patent Document 8 describes that polyols are effective for stabilizing protease as an enzyme in an oil-in-water emulsion. Patent Documents 9 and 10 describe that peptide aldehyde, calcium ions, boron compositions, polyols, and benzamidine hydrochloride in a liquid detergent composition can be used as protease stabilizers.

[0005] On the other hand, in reagents for colorimetric quantification of hydrogen peroxide using Trinder reagents and dyes such as 4-aminoantipyrine, non-specific condensation coloring of the Trinder reagent and 4-aminoantipyrine may occur due to components other than the measurement components in the reagent or the sample, which is called a reagent blank. If this reagent blank is large, the ratio of the reagent blank (noise) to the measurement component (signal) in the sample becomes large, causing measurement errors. Therefore, a lower reagent blank is preferable. For example, Patent Document 11 describes a method for suppressing the reagent blank by co-existing catalase in a protease-containing reagent and eliminating peroxides in the reagent in a glycated protein measurement reagent. Patent Document 12 describes that the reagent blank can be suppressed by blending an amphoteric surfactant in a reagent containing a hydrogen donor such as ferrocyanide and a Trinder reagent.

Prior Art Documents

Patent Documents

[0006]

Patent Document 1

Patent Document 2

[0007] As described above, various reagent blank suppression methods have been proposed. However, in a glycated protein measurement reagent containing a Trinder reagent, 4-aminoantipyrine, protease, a stabilizer for the protease, and ferricyanide, a method for sufficiently suppressing the reagent blank has not been disclosed. Therefore, an object of the present invention is to provide a glycated protein measurement reagent with suppressed reagent blank, a method for measuring glycated protein, a method for storing a glycated protein measurement reagent, and a method for stabilizing a glycated protein measurement reagent as part of the problems. [Means for Solving the Problems]

[0008] According to the findings of the present inventors, when ferricyanide is included in a glycated protein measurement reagent for the purpose of preventing measurement errors caused by reducing substances such as bilirubin, the ferricyanide in the reagent may be oxidized to ferricyanide during storage, which may cause a reagent blank. In contrast, as a result of intensive research by the present inventors, in a reagent containing a Trinder reagent, 4-aminoantipyrine, protease, a stabilizer for the protease, and ferricyanide, when the stabilizer for the protease and ferricyanide are mixed, by using a stabilizer for the protease that makes the redox potential of ferricyanide greater than 0.058 V, it has been found that the reagent blank can be significantly suppressed, and the present invention has been completed.

[0009] [1] A glycated protein measurement reagent comprising at least a Trinder reagent, 4-aminoantipyrine, protease, a stabilizer for the protease, and ferricyanide, wherein at least the Trinder reagent is contained in a Trinder reagent-containing partial composition, wherein at least the 4-aminoantipyrine is contained in a 4-aminoantipyrine-containing partial composition, the stabilizer for the protease is a stabilizer that makes the redox potential of the ferricyanide greater than 0.058 V when mixed with the stabilizer for the protease, the redox potential is the redox potential in a reaction system containing the stabilizer for the protease and the ferricyanide and not containing glycated protein, A glycated protein measurement reagent.

[0010] [2] At least one of the Trinder reagent-containing partial composition and the 4-aminoantipyrine-containing partial composition further contains the protease and the stabilizer for the protease, The glycated protein measurement reagent according to [1].

[0011] [3] The Trinder reagent-containing partial composition further contains the protease and the stabilizer for the protease, the glycated protein measurement reagent according to [2].

[0012] [4] The glycated protein measurement reagent according to any one of [1] to [3], wherein the 4-aminoantipyrine-containing partial composition further contains glycated amino acid oxidase.

[0013] [5] The glycated protein measurement reagent according to any one of [1] to [4], wherein at least one of the Trinder reagent-containing partial composition and the 4-aminoantipyrine-containing partial composition contains the ferricyanide.

[0014] [6] The glycated protein measurement reagent according to [2], wherein the 4-aminoantipyrine-containing partial composition further contains the protease and a stabilizer for the protease.

[0015] [7] The glycated protein measurement reagent according to any one of [1] to [3] and [6], wherein the Trinder reagent-containing partial composition further contains glycated amino acid oxidase.

[0016] [8] The glycated protein measurement reagent according to [6] or [7], wherein at least one of the Trinder reagent-containing partial composition and the 4-aminoantipyrine-containing partial composition contains the ferricyanide.

[0017] [9] The glycated protein measurement reagent according to [1], wherein either the Trinder reagent-containing partial composition or the 4-aminoantipyrine-containing partial composition further contains the protease, a stabilizer for the protease, and the ferricyanide.

[0018]

[10] The glycated protein measurement reagent according to [1], wherein the 4-aminoantipyrine-containing partial composition further contains the protease, a stabilizer for the protease, and the ferricyanide.

[0019]

[11] The concentration of the protease stabilizer is a concentration that makes the redox potential of the ferrocyanide greater than 0.058 V when the protease stabilizer and the ferrocyanide are mixed. The glycated protein measurement reagent according to any one of [1] to

[10] .

[0020]

[12] The Trinder reagent-containing partial composition contains the protease stabilizer and the ferrocyanide, and the concentration of the protease stabilizer in the Trinder reagent-containing partial composition is a concentration that makes the redox potential of the ferrocyanide greater than 0.058 V. The glycated protein measurement reagent according to any one of [1] to [5] and [9].

[0021]

[13] The 4-aminoantipyrine-containing partial composition contains the protease stabilizer and the ferrocyanide, and the concentration of the protease stabilizer in the 4-aminoantipyrine-containing partial composition is a concentration that makes the redox potential of the ferrocyanide greater than 0.058 V. The glycated protein measurement reagent according to any one of [1], [2], [6] to

[10] .

[0022]

[14] The 4-aminoantipyrine-containing partial composition further contains a chelating agent. The glycated protein measurement reagent according to any one of [1] to

[13] .

[0023]

[15] The chelating agent is selected from the group consisting of ethylenediaminetetraacetic acid, glycol ether diamine tetraacetic acid, N-(2-hydroxyethyl)iminodiacetic acid, nitrilotris(methylphosphonic acid), nitrilotriacetic acid, trans-1,2-diaminocyclohexane-N,N,N,N-tetraacetic acid, iminodiacetic acid, diethylenetriamine-N,N,N’,N”,N”-pentaacetic acid, 1,3-diamino-2-propanol-N,N,N’,N’-tetraacetic acid, gluconic acid, malic acid, succinic acid, citric acid, salicylic acid, tartaric acid, N-[tris(hydroxymethyl)methyl]glycine, N,N-bis(2-hydroxyethyl)glycine, and salts thereof. The glycated protein measurement reagent according to

[14] .

[0024]

[16] The glycated protein measurement reagent according to

[14] , wherein the chelating agent is citric acid or a salt thereof.

[0025]

[17] The glycated protein measurement reagent according to any one of

[14] to

[16] , wherein the concentration of the chelating agent is 5 μmol / L or more and 1000 mmol / L or less.

[0026]

[18] The glycated protein measurement reagent according to

[17] , wherein the concentration of the chelating agent is 10 μmol / L or more.

[0027]

[19] The glycated protein measurement reagent according to

[17] , wherein the concentration of the chelating agent is 50 μmol / L or more.

[0028]

[20] The glycated protein measurement reagent according to

[17] , wherein the concentration of the chelating agent is 100 mmol / L or less.

[0029]

[21] The glycated protein measurement reagent according to

[17] , wherein the concentration of the chelating agent is 25 mmol / L or less.

[0030]

[22] A glycated protein measurement reagent comprising at least a Trinder reagent, 4-aminoantipyrine, a protease, a stabilizer for the protease, and a ferrocyanide, at least the Trinder reagent is contained in a Trinder reagent-containing partial composition, at least the 4-aminoantipyrine is contained in a 4-aminoantipyrine-containing partial composition, at least the ferrocyanide is contained in a ferrocyanide-containing partial composition, the stabilizer for the protease is a stabilizer for the protease that makes the redox potential of the ferrocyanide greater than 0.058 V when mixed with the stabilizer for the protease, the redox potential is the redox potential in a reaction system containing the stabilizer for the protease and the ferrocyanide and not containing glycated protein. Glycated protein measurement reagent.

[0031]

[23] The glycated protein measurement reagent according to

[22] , wherein any one of the Trinder reagent-containing partial composition, the 4-aminoantipyrine-containing partial composition, and the ferricyanide-containing partial composition further contains the protease and a stabilizer for the protease.

[0032]

[24] The glycated protein measurement reagent according to

[23] , wherein the Trinder reagent-containing partial composition further contains the protease and a stabilizer for the protease.

[0033]

[25] The glycated protein measurement reagent according to any one of

[22] to

[24] , wherein the 4-aminoantipyrine-containing partial composition further contains glycated amino acid oxidase.

[0034]

[26] The glycated protein measurement reagent according to

[23] , wherein the 4-aminoantipyrine-containing partial composition further contains the protease and a stabilizer for the protease.

[0035]

[27] The glycated protein measurement reagent according to any one of

[22] to

[24] and

[26] , wherein the Trinder reagent-containing partial composition further contains glycated amino acid oxidase.

[0036]

[28] The glycated protein measurement reagent according to

[23] , wherein the ferricyanide-containing partial composition further contains the protease and a stabilizer for the protease.

[0037]

[29] The glycated protein measurement reagent according to any one of

[22] to

[28] , wherein the concentration of the stabilizer for the protease is a concentration that makes the redox potential of the ferricyanide greater than 0.058V when the stabilizer for the protease and the ferricyanide are mixed.

[0038]

[30] The 4-aminoantipyrine-containing partial composition contains a stabilizer for the protease, and the concentration of the stabilizer for the protease when the 4-aminoantipyrine-containing partial composition and the cyanide-containing partial composition are mixed is a concentration that makes the redox potential of the cyanide greater than 0.058V. The glycated protein measurement reagent according to any one of

[22] ,

[23] , and

[26] .

[0039]

[31] The Trinder reagent-containing partial composition contains a stabilizer for the protease, and the concentration of the stabilizer for the protease when the Trinder reagent-containing partial composition and the cyanide-containing partial composition are mixed is a concentration that makes the redox potential of the cyanide greater than 0.058V. The glycated protein measurement reagent according to any one of

[22] to

[25] .

[0040]

[32] The cyanide-containing partial composition contains a stabilizer for the protease, and the concentration of the stabilizer for the protease in the cyanide-containing partial composition is a concentration that makes the redox potential of the cyanide greater than 0.058V. The glycated protein measurement reagent according to any one of

[22] ,

[23] , and

[28] .

[0041]

[33] The 4-aminoantipyrine-containing partial composition and / or the cyanide-containing partial composition further contains a chelating agent. The glycated protein measurement reagent according to any one of

[22] to

[32] .

[0042]

[34] The chelating agent is selected from the group consisting of ethylenediaminetetraacetic acid, glycol ether diamine tetraacetic acid, N-(2-hydroxyethyl)iminodiacetic acid, nitrilotris(methylphosphonic acid), nitrilotriacetic acid, trans-1,2-diaminocyclohexane-N,N,N,N-tetraacetic acid, iminodiacetic acid, diethylenetriamine-N,N,N’,N”,N”-pentaacetic acid, 1,3-diamino-2-propanol-N,N,N’,N’-tetraacetic acid, gluconic acid, malic acid, succinic acid, citric acid, salicylic acid, tartaric acid, N-[tris(hydroxymethyl)methyl]glycine, N,N-bis(2-hydroxyethyl)glycine, and salts thereof, and is the glycated protein measurement reagent according to

[33] .

[0043]

[35] The chelating agent is citric acid or a salt thereof, and is the glycated protein measurement reagent according to

[33] .

[0044]

[36] The concentration of the chelating agent is 5 μmol / L or more and 1000 mmol / L or less, and is the glycated protein measurement reagent according to any one of

[33] to

[35] .

[0045]

[37] The concentration of the chelating agent is 10 μmol / L or more, and is the glycated protein measurement reagent according to

[36] .

[0046]

[38] The concentration of the chelating agent is 50 μmol / L or more, and is the glycated protein measurement reagent according to

[36] .

[0047]

[39] The concentration of the chelating agent is 100 mmol / L or less, and is the glycated protein measurement reagent according to

[36] .

[0048]

[40] The concentration of the chelating agent is 25 mmol / L or less, and is the glycated protein measurement reagent according to

[36] .

[0049]

[41] The reaction system containing the protease stabilizer and the ferrocyanide and not containing glycated protein further contains N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid and sodium chloride, and is the glycated protein measurement reagent according to any one of [1] to

[40] .

[0050]

[42] The glycated protein measurement reagent according to any one of [1] to

[41] , wherein the redox potential is the redox potential measured using a silver / silver chloride electrode as a reference electrode.

[0051]

[43] The glycated protein measurement reagent according to any one of [1] to

[42] , wherein the redox potential is greater than 0.058V and not more than 0.400V.

[0052]

[44] The glycated protein measurement reagent according to

[43] , wherein the redox potential is 0.070V or more.

[0053]

[45] The glycated protein measurement reagent according to

[43] , wherein the redox potential is 0.084V or more.

[0054]

[46] The glycated protein measurement reagent according to

[43] , wherein the redox potential is 0.112V or more.

[0055]

[47] The glycated protein measurement reagent according to

[43] , wherein the redox potential is not more than 0.300V.

[0056]

[48] The glycated protein measurement reagent according to

[43] , wherein the redox potential is not more than 0.250V.

[0057]

[49] The glycated protein measurement reagent according to

[43] , wherein the redox potential is not more than 0.235V.

[0058]

[50] The glycated protein measurement reagent according to any one of [1] to

[49] , wherein the stabilizer of the protease is selected from the group consisting of propylene glycol, trimethylene glycol, ethylene glycol, and carboxyphenylboronic acid.

[0059]

[51] The glycated protein measurement reagent according to any one of [1] to

[49] , wherein the stabilizer of the protease is propylene glycol.

[0060]

[52] The glycated protein measurement reagent according to

[50] , wherein the carboxyphenylboronic acid is 2-carboxyphenylboronic acid.

[0061]

[53] The 4-aminoantipyrine-containing partial composition or the Trinder reagent-containing partial composition contains the stabilizer of the protease, the stabilizer of the protease is propylene glycol, and the concentration of propylene glycol in the 4-aminoantipyrine-containing partial composition or the Trinder reagent-containing partial composition is 7.5 wt / vol% or more and 80 wt / vol% or less. The glycated protein measurement reagent according to any one of [1] to

[51] .

[0062]

[54] The glycated protein measurement reagent according to

[53] , wherein the concentration of propylene glycol in the 4-aminoantipyrine-containing partial composition or the Trinder reagent-containing partial composition is 20 wt / vol% or more.

[0063]

[55] The glycated protein measurement reagent according to

[53] , wherein the concentration of propylene glycol in the 4-aminoantipyrine-containing partial composition or the Trinder reagent-containing partial composition is 25 wt / vol% or more.

[0064]

[56] The glycated protein measurement reagent according to

[53] , wherein the concentration of propylene glycol in the 4-aminoantipyrine-containing partial composition or the Trinder reagent-containing partial composition is 62.5 wt / vol% or less.

[0065]

[57] The glycated protein measurement reagent according to

[53] , wherein the concentration of propylene glycol in the 4-aminoantipyrine-containing partial composition or the trinder reagent-containing partial composition is 60 wt / vol% or less.

[0066]

[58] The glycated protein measurement reagent according to any one of

[22] to

[40] , wherein the cyanide-containing partial composition contains a stabilizer for the protease, the stabilizer for the protease is propylene glycol, and the concentration of propylene glycol in the cyanide-containing partial composition is 7.5 wt / vol% or more and 80 wt / vol% or less.

[0067]

[59] The glycated protein measurement reagent according to

[58] , wherein the concentration of propylene glycol in the cyanide-containing partial composition is 20 wt / vol% or more.

[0068]

[60] The glycated protein measurement reagent according to

[58] , wherein the concentration of propylene glycol in the cyanide-containing partial composition is 25 wt / vol% or more.

[0069]

[61] The glycated protein measurement reagent according to

[58] , wherein the concentration of propylene glycol in the cyanide-containing partial composition is 62.5 wt / vol% or less.

[0070]

[62] The glycated protein measurement reagent according to

[58] , wherein the concentration of propylene glycol in the cyanide-containing partial composition is 60 wt / vol% or less.

[0071]

[63] The 4 - aminoantipyrine - containing partial composition or the Trinder reagent - containing partial composition contains a stabilizer for the protease, the stabilizer for the protease is trimethylene glycol, and the concentration of trimethylene glycol in the 4 - aminoantipyrine - containing partial composition or the Trinder reagent - containing partial composition is 40 wt / vol% or more and 80 wt / vol% or less. The glycated protein measurement reagent according to any one of [1] to

[50] .

[0072]

[64] The concentration of trimethylene glycol in the 4 - aminoantipyrine - containing partial composition or the Trinder reagent - containing partial composition is 42.5 wt / vol% or more. The glycated protein measurement reagent according to

[63] .

[0073]

[65] The concentration of trimethylene glycol in the 4 - aminoantipyrine - containing partial composition or the Trinder reagent - containing partial composition is 45 wt / vol% or more. The glycated protein measurement reagent according to

[63] .

[0074]

[66] The concentration of trimethylene glycol in the 4 - aminoantipyrine - containing partial composition or the Trinder reagent - containing partial composition is 75 wt / vol% or less. The glycated protein measurement reagent according to

[63] .

[0075]

[67] The concentration of trimethylene glycol in the 4 - aminoantipyrine - containing partial composition or the Trinder reagent - containing partial composition is 70 wt / vol% or less. The glycated protein measurement reagent according to

[63] .

[0076]

[68] The ferrocyanide - containing partial composition contains a stabilizer for the protease, the stabilizer for the protease is trimethylene glycol, and the concentration of trimethylene glycol in the ferrocyanide - containing partial composition is 40 wt / vol% or more and 80 wt / vol% or less. The glycated protein measurement reagent according to any one of

[22] to

[40] .

[0077]

[69] The glycated protein measurement reagent according to

[68] , wherein the concentration of the trimethylene glycol in the ferrocyanide-containing partial composition is 42.5 wt / vol% or more.

[0078]

[70] The glycated protein measurement reagent according to

[68] , wherein the concentration of the trimethylene glycol in the ferrocyanide-containing partial composition is 45 wt / vol% or more.

[0079]

[71] The glycated protein measurement reagent according to

[68] , wherein the concentration of the trimethylene glycol in the ferrocyanide-containing partial composition is 75 wt / vol% or less.

[0080]

[72] The glycated protein measurement reagent according to

[68] , wherein the concentration of the trimethylene glycol in the ferrocyanide-containing partial composition is 70 wt / vol% or less.

[0081]

[73] The glycated protein measurement reagent according to any one of [1] to

[50] , wherein the 4-aminoantipyrine-containing partial composition or the Trinder reagent-containing partial composition contains a stabilizer for the protease, the stabilizer for the protease is ethylene glycol, and the concentration of the ethylene glycol in the 4-aminoantipyrine-containing partial composition or the Trinder reagent-containing partial composition is 40 wt / vol% or more and 80 wt / vol% or less.

[0082]

[74] The glycated protein measurement reagent according to

[73] , wherein the concentration of the ethylene glycol in the 4-aminoantipyrine-containing partial composition or the Trinder reagent-containing partial composition is 42.5 wt / vol% or more.

[0083]

[75] The glycated protein measurement reagent according to

[73] , wherein the concentration of the ethylene glycol in the 4-aminoantipyrine-containing partial composition or the Trinder reagent-containing partial composition is 45 wt / vol% or more.

[0084]

[76] The glycated protein measurement reagent according to

[73] , wherein the concentration of ethylene glycol in the 4-aminoantipyrine-containing partial composition or the Trinder reagent-containing partial composition is 75 wt / vol% or less.

[0085]

[77] The glycated protein measurement reagent according to

[73] , wherein the concentration of ethylene glycol in the 4-aminoantipyrine-containing partial composition or the Trinder reagent-containing partial composition is 70 wt / vol% or less.

[0086]

[78] The glycated protein measurement reagent according to any one of

[22] to

[40] , wherein the cyanide-containing partial composition contains a stabilizer for the protease, the stabilizer for the protease is ethylene glycol, and the concentration of ethylene glycol in the cyanide-containing partial composition is 40 wt / vol% or more and 80 wt / vol% or less.

[0087]

[79] The glycated protein measurement reagent according to

[78] , wherein the concentration of ethylene glycol in the cyanide-containing partial composition is 42.5 wt / vol% or more.

[0088]

[80] The glycated protein measurement reagent according to

[78] , wherein the concentration of ethylene glycol in the cyanide-containing partial composition is 45 wt / vol% or more.

[0089]

[81] The glycated protein measurement reagent according to

[78] , wherein the concentration of ethylene glycol in the cyanide-containing partial composition is 75 wt / vol% or less.

[0090]

[82] The glycated protein measurement reagent according to

[78] , wherein the concentration of ethylene glycol in the cyanide-containing partial composition is 70 wt / vol% or less.

[0091]

[83] The 4-aminoantipyrine-containing partial composition or the Trinder reagent-containing partial composition contains a stabilizer for the protease, the stabilizer for the protease is 2-carboxyphenylboronic acid, and the concentration of 2-carboxyphenylboronic acid in the 4-aminoantipyrine-containing partial composition or the Trinder reagent-containing partial composition is 0.2 wt / vol% or more and 10 wt / vol% or less. The glycated protein measurement reagent according to any one of [1] to

[50] and

[52] .

[0092]

[84] The concentration of 2-carboxyphenylboronic acid in the 4-aminoantipyrine-containing partial composition or the Trinder reagent-containing partial composition is 0.45 wt / vol% or more. The glycated protein measurement reagent according to

[83] .

[0093]

[85] The concentration of 2-carboxyphenylboronic acid in the 4-aminoantipyrine-containing partial composition or the Trinder reagent-containing partial composition is 0.5 wt / vol% or more. The glycated protein measurement reagent according to

[83] .

[0094]

[86] The concentration of 2-carboxyphenylboronic acid in the 4-aminoantipyrine-containing partial composition or the Trinder reagent-containing partial composition is 7.5 wt / vol% or less. The glycated protein measurement reagent according to

[83] .

[0095]

[87] The concentration of 2-carboxyphenylboronic acid in the 4-aminoantipyrine-containing partial composition or the Trinder reagent-containing partial composition is 5 wt / vol% or less. The glycated protein measurement reagent according to

[83] .

[0096]

[88] The ferrocyanide-containing partial composition contains a stabilizer for the protease, the stabilizer for the protease is 2-carboxyphenylboronic acid, and the concentration of 2-carboxyphenylboronic acid in the ferrocyanide-containing partial composition is 0.2 wt / vol% or more and 10 wt / vol% or less. The glycated protein measurement reagent according to any one of

[22] to

[40] .

[0097]

[89] The glycated protein measurement reagent according to

[88] , wherein the concentration of the 2-carboxyphenylboronic acid in the ferrocyanide-containing partial composition is 0.45 wt / vol% or more.

[0098]

[90] The glycated protein measurement reagent according to

[88] , wherein the concentration of the 2-carboxyphenylboronic acid in the ferrocyanide-containing partial composition is 0.5 wt / vol% or more.

[0099]

[91] The glycated protein measurement reagent according to

[88] , wherein the concentration of the 2-carboxyphenylboronic acid in the ferrocyanide-containing partial composition is 7.5 wt / vol% or less.

[0100]

[92] The glycated protein measurement reagent according to

[88] , wherein the concentration of the 2-carboxyphenylboronic acid in the ferrocyanide-containing partial composition is 5 wt / vol% or less.

[0101]

[93] The glycated protein measurement reagent according to any one of [1] to

[92] , wherein the trinder reagent-containing partial composition and the 4-aminoantipyrine-containing partial composition are in a liquid state.

[0102]

[94] The glycated protein measurement reagent according to any one of

[22] to

[40] , wherein the trinder reagent-containing partial composition, the 4-aminoantipyrine-containing partial composition, and the ferrocyanide-containing partial composition are in a liquid state.

[0103]

[95] The glycated protein measurement reagent according to any one of [1] to

[94] , wherein the ferrocyanide is potassium ferrocyanide.

[0104]

[96] The glycated protein measurement reagent according to any one of [1] to

[95] , wherein the trinder reagent is N,N-bis(4-sulfobutyl)-3-methylaniline.

[0105] The glycated protein measurement reagent according to any one of [1] to

[96] , wherein the glycated protein is glycated albumin.

[0106] The glycated protein measurement reagent according to any one of [1] to

[97] , which is stored at 2°C or higher and 10°C or lower.

[0107] The glycated protein measurement reagent according to any one of [1] to

[98] , wherein the stabilizer of the protease excludes dimethyl sulfoxide.

[0108]

[0100] The glycated protein measurement reagent according to any one of [1] to

[99] , wherein either one of the Trinder reagent-containing partial composition and the 4-aminoantipyrine-containing partial composition further contains peroxidase.

[0109]

[0101] A method for measuring glycated protein, comprising: contacting a sample with a Trinder reagent, 4-aminoantipyrine, a protease, a stabilizer for the protease, and a ferrocyanide; detecting a color development depending on the glycated protein present in the sample; and before at least the contacting, the Trinder reagent is contained in a Trinder reagent-containing partial composition; before at least the contacting, the 4-aminoantipyrine is contained in a 4-aminoantipyrine-containing partial composition; either one of the Trinder reagent-containing partial composition and the 4-aminoantipyrine-containing partial composition contains the protease and the stabilizer for the protease; either one of the Trinder reagent-containing partial composition and the 4-aminoantipyrine-containing partial composition contains the ferrocyanide; the stabilizer for the protease is a stabilizer that makes the redox potential of the ferrocyanide greater than 0.058V when mixed with the stabilizer for the protease. The redox potential is the redox potential in a reaction system containing the protease stabilizer and the ferrocyanide and not containing glycated proteins. Method for measuring glycated proteins.

[0110]

[0102] The method for measuring glycated proteins according to

[0101] , wherein the Trinder reagent-containing partial composition contains the protease and the protease stabilizer.

[0111]

[0103] The method for measuring glycated proteins according to

[0101] or

[0102] , wherein the 4-aminoantipyrine-containing partial composition contains glycated amino acid oxidase.

[0112]

[0104] The method for measuring glycated proteins according to

[0101] , wherein the 4-aminoantipyrine-containing partial composition contains the protease and the protease stabilizer.

[0113]

[0105] The method for measuring glycated proteins according to any one of

[0101] ,

[0102] , and

[0104] , wherein the Trinder reagent-containing partial composition contains glycated amino acid oxidase.

[0114]

[0106] The method for measuring a glycated protein measurement reagent according to

[0101] , wherein either the Trinder reagent-containing partial composition or the 4-aminoantipyrine-containing partial composition contains the protease, the protease stabilizer, and the ferrocyanide.

[0115]

[0107] The method for measuring glycated proteins according to

[0101] , wherein the 4-aminoantipyrine-containing partial composition contains the protease, the protease stabilizer, and the ferrocyanide.

[0116] The method for measuring glycated protein according to any one of

[0101] to

[0107] , wherein the concentration of the protease stabilizer is a concentration that makes the redox potential of the ferrocyanide greater than 0.058V when the protease stabilizer and the ferrocyanide are mixed.

[0117] The method for measuring glycated protein according to any one of

[0101] to

[0103] and

[0106] , wherein the Trinder reagent-containing partial composition contains the protease stabilizer and the ferrocyanide, and the concentration of the protease stabilizer in the Trinder reagent-containing partial composition is a concentration that makes the redox potential of the ferrocyanide greater than 0.058V.

[0118] The method for measuring glycated protein according to any one of

[0101] ,

[0104] ,

[0106] and

[0107] , wherein the 4-aminoantipyrine-containing partial composition contains the protease stabilizer and the ferrocyanide, and the concentration of the protease stabilizer in the 4-aminoantipyrine-containing partial composition is a concentration that makes the redox potential of the ferrocyanide greater than 0.058V.

[0119] The method for measuring glycated protein according to any one of

[0101] to

[0110] , wherein the 4-aminoantipyrine-containing partial composition further contains a chelating agent.

[0120]

[0112] The method for measuring glycated protein according to

[0111] , wherein the chelating agent is selected from the group consisting of ethylenediaminetetraacetic acid, glycol ether diamine tetraacetic acid, N-(2-hydroxyethyl)iminodiacetic acid, nitrilotris(methylphosphonic acid), nitrilotriacetic acid, trans-1,2-diaminocyclohexane-N,N,N,N-tetraacetic acid, iminodiacetic acid, diethylenetriamine-N,N,N’,N”,N”-pentaacetic acid, 1,3-diamino-2-propanol-N,N,N’,N’-tetraacetic acid, gluconic acid, malic acid, succinic acid, citric acid, salicylic acid, tartaric acid, N-[tris(hydroxymethyl)methyl]glycine, N,N-bis(2-hydroxyethyl)glycine, and salts thereof.

[0121]

[0113] The method for measuring glycated protein according to

[0111] , wherein the chelating agent is citric acid or a salt thereof.

[0122]

[0114] The method for measuring glycated protein according to any one of

[0111] to

[0113] , wherein the concentration of the chelating agent is 5 μmol / L or more and 1000 mmol / L or less.

[0123]

[0115] The method for measuring glycated protein according to

[0114] , wherein the concentration of the chelating agent is 10 μmol / L or more.

[0124]

[0116] The method for measuring glycated protein according to

[0114] , wherein the concentration of the chelating agent is 50 μmol / L or more.

[0125]

[0117] The method for measuring glycated protein according to

[0114] , wherein the concentration of the chelating agent is 100 mmol / L or less.

[0126]

[0118] The method for measuring glycated protein according to

[0114] , wherein the concentration of the chelating agent is 25 mmol / L or less.

[0127]

[0119] A method for measuring glycated protein, Contacting the sample with a Trinder reagent, 4-aminoantipyrine, a protease, a stabilizer for the protease, and a ferrocyanide; Detecting a color development dependent on glycated proteins present in the sample; comprising; Before at least said contacting, the Trinder reagent is included in a Trinder reagent-containing partial composition; Before at least said contacting, the 4-aminoantipyrine is included in a 4-aminoantipyrine-containing partial composition; Before at least said contacting, the ferrocyanide is included in a ferrocyanide-containing partial composition; Any one of the Trinder reagent-containing partial composition, the 4-aminoantipyrine-containing partial composition, and the ferrocyanide-containing partial composition contains the protease and the stabilizer for the protease; The stabilizer for the protease is a stabilizer that makes the redox potential of the ferrocyanide greater than 0.058 V when mixed with the stabilizer for the protease; The redox potential is the redox potential in a reaction system containing the stabilizer for the protease and the ferrocyanide and not containing glycated proteins; A method for measuring glycated proteins.

[0128]

[0120] The method for measuring glycated proteins according to

[0119] , wherein the Trinder reagent-containing partial composition contains the protease and the stabilizer for the protease.

[0129]

[0121] The method for measuring glycated proteins according to

[0119] or

[0120] , wherein the 4-aminoantipyrine-containing partial composition contains glycated amino acid oxidase.

[0130]

[0122] The method for measuring glycated proteins according to

[0119] , wherein the 4-aminoantipyrine-containing partial composition contains the protease and the stabilizer for the protease.

[0131] The method for measuring glycated protein according to any one of

[0119] ,

[0120] , and

[0122] , wherein the trinder reagent-containing partial composition contains glycated amino acid oxidase.

[0132] The method for measuring glycated protein according to

[0119] , wherein the ferrocyanide-containing partial composition contains the protease and a stabilizer for the protease.

[0133] The method for measuring glycated protein according to any one of

[0119] to

[0124] , wherein the concentration of the stabilizer for the protease is a concentration that makes the redox potential of the ferrocyanide greater than 0.058V when the stabilizer for the protease and the ferrocyanide are mixed.

[0134] The method for measuring glycated protein according to

[0119] or

[0122] , wherein the 4-aminoantipyrine-containing partial composition contains a stabilizer for the protease, and the concentration of the stabilizer for the protease when the 4-aminoantipyrine-containing partial composition and the ferrocyanide-containing partial composition are mixed is a concentration that makes the redox potential of the ferrocyanide greater than 0.058V.

[0135] The method for measuring glycated protein according to any one of

[0119] to

[0121] , wherein the trinder reagent-containing partial composition contains a stabilizer for the protease, and the concentration of the stabilizer for the protease when the trinder reagent-containing partial composition and the ferrocyanide-containing partial composition are mixed is a concentration that makes the redox potential of the ferrocyanide greater than 0.058V.

[0136] The method for measuring glycated protein according to

[0119] or

[0124] , wherein the ferrocyanide-containing partial composition contains a stabilizer for the protease, and the concentration of the stabilizer for the protease in the ferrocyanide-containing partial composition is a concentration that makes the redox potential of the ferrocyanide greater than 0.058V.

[0137]

[0129] The method for measuring glycated protein according to any one of

[0119] to

[0128] , wherein the 4-aminoantipyrine-containing partial composition and / or the ferrocyanide-containing partial composition further contains a chelating agent.

[0138]

[0130] The method for measuring glycated protein according to

[0129] , wherein the chelating agent is selected from the group consisting of ethylenediaminetetraacetic acid, glycol ether diamine tetraacetic acid, N-(2-hydroxyethyl)iminodiacetic acid, nitrilotris(methylphosphonic acid), nitrilotriacetic acid, trans-1,2-diaminocyclohexane-N,N,N,N-tetraacetic acid, iminodiacetic acid, diethylenetriamine-N,N,N’,N”,N”-pentaacetic acid, 1,3-diamino-2-propanol-N,N,N’,N’-tetraacetic acid, gluconic acid, malic acid, succinic acid, citric acid, salicylic acid, tartaric acid, N-[tris(hydroxymethyl)methyl]glycine, N,N-bis(2-hydroxyethyl)glycine, and salts thereof.

[0139]

[0131] The method for measuring glycated protein according to

[0129] , wherein the chelating agent is citric acid or a salt thereof.

[0140]

[0132] The method for measuring glycated protein according to any one of

[0129] to

[0131] , wherein the concentration of the chelating agent is 5 μmol / L or more and 1000 mmol / L or less.

[0141]

[0133] The method for measuring glycated protein according to

[0132] , wherein the concentration of the chelating agent is 10 μmol / L or more.

[0142]

[0134] The method for measuring glycated protein according to

[0132] , wherein the concentration of the chelating agent is 50 μmol / L or more.

[0143]

[0135] The method for measuring glycated protein according to

[0132] , wherein the concentration of the chelating agent is 100 mmol / L or less.

[0144] The method for measuring glycated protein according to

[0132] , wherein the concentration of the chelating agent is 25 mmol / L or less.

[0145] The method for measuring glycated protein according to any one of

[0101] to

[0136] , wherein the reaction system containing the protease stabilizer and the ferrocyanide and not containing glycated protein further contains N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid and sodium chloride.

[0146] The method for measuring glycated protein according to any one of

[0101] to

[0137] , wherein the redox potential is the redox potential measured using a silver / silver chloride electrode as a reference electrode.

[0147] The method for measuring glycated protein according to any one of

[0101] to

[0138] , wherein the redox potential is greater than 0.058 V and 0.400 V or less.

[0148] The method for measuring glycated protein according to

[0139] , wherein the redox potential is 0.070 V or more.

[0149] The method for measuring glycated protein according to

[0139] , wherein the redox potential is 0.084 V or more.

[0150] The method for measuring glycated protein according to

[0139] , wherein the redox potential is 0.112 V or more.

[0151] The method for measuring glycated protein according to

[0139] , wherein the redox potential is 0.300 V or less.

[0152] The method for measuring glycated protein according to

[0139] , wherein the redox potential is 0.250 V or less.

[0153] The method for measuring glycated protein according to

[0139] , wherein the redox potential is 0.235 V or less.

[0154] The method for measuring glycated protein according to any one of

[0101] to

[0145] , wherein the stabilizer for the protease is selected from the group consisting of propylene glycol, trimethylene glycol, ethylene glycol, and carboxyphenylboronic acid.

[0155] The method for measuring glycated protein according to any one of

[0101] to

[0145] , wherein the stabilizer for the protease is propylene glycol.

[0156] The method for measuring glycated protein according to

[0146] , wherein the carboxyphenylboronic acid is 2-carboxyphenylboronic acid.

[0157] The method for measuring glycated protein according to any one of

[0101] to

[0147] , wherein the 4-aminoantipyrine-containing partial composition or the Trinder reagent-containing partial composition contains the stabilizer for the protease, the stabilizer for the protease is propylene glycol, and the concentration of propylene glycol in the 4-aminoantipyrine-containing partial composition or the Trinder reagent-containing partial composition is 7.5 wt / vol% or more and 80 wt / vol% or less.

[0158] The method for measuring glycated protein according to

[0149] , wherein the concentration of propylene glycol in the 4-aminoantipyrine-containing partial composition or the Trinder reagent-containing partial composition is 20 wt / vol% or more.

[0159] The method for measuring glycated protein according to

[0149] , wherein the concentration of propylene glycol in the 4-aminoantipyrine-containing partial composition or the Trinder reagent-containing partial composition is 25 wt / vol% or more.

[0160] The method for measuring glycated protein according to

[0149] , wherein the concentration of propylene glycol in the 4-aminoantipyrine-containing partial composition or the Trinder reagent-containing partial composition is 62.5 wt / vol% or less.

[0161] The method for measuring glycated protein according to

[0149] , wherein the concentration of propylene glycol in the 4-aminoantipyrine-containing partial composition or the Trinder reagent-containing partial composition is 60 wt / vol% or less.

[0162] The method for measuring glycated protein according to any one of

[0119] to

[0136] , wherein the ferrocyanide-containing partial composition contains a stabilizer for the protease, the stabilizer for the protease is propylene glycol, and the concentration of propylene glycol in the ferrocyanide-containing partial composition is 7.5 wt / vol% or more and 80 wt / vol% or less.

[0163] The method for measuring glycated protein according to

[0154] , wherein the concentration of propylene glycol in the ferrocyanide-containing partial composition is 20 wt / vol% or more.

[0164] The method for measuring glycated protein according to

[0154] , wherein the concentration of propylene glycol in the ferrocyanide-containing partial composition is 25 wt / vol% or more.

[0165] The method for measuring glycated protein according to

[0154] , wherein the concentration of propylene glycol in the ferrocyanide-containing partial composition is 62.5 wt / vol% or less.

[0166] The method for measuring glycated protein according to

[0154] , wherein the concentration of propylene glycol in the ferrocyanide-containing partial composition is 60 wt / vol% or less.

[0167]

[0159] The 4-aminoantipyrine-containing partial composition or the Trinder reagent-containing partial composition contains a stabilizer for the protease, the stabilizer for the protease is trimethylene glycol, and the concentration of trimethylene glycol in the 4-aminoantipyrine-containing partial composition or the Trinder reagent-containing partial composition is 40 wt / vol% or more and 80 wt / vol% or less. The method for measuring glycated protein according to any one of

[0101] to

[0146] .

[0168]

[0160] The concentration of trimethylene glycol in the 4-aminoantipyrine-containing partial composition or the Trinder reagent-containing partial composition is 42.5 wt / vol% or more. The method for measuring glycated protein according to

[0159] .

[0169]

[0161] The concentration of trimethylene glycol in the 4-aminoantipyrine-containing partial composition or the Trinder reagent-containing partial composition is 45 wt / vol% or more. The method for measuring glycated protein according to

[0159] .

[0170]

[0162] The concentration of trimethylene glycol in the 4-aminoantipyrine-containing partial composition or the Trinder reagent-containing partial composition is 75 wt / vol% or less. The method for measuring glycated protein according to

[0159] .

[0171]

[0163] The concentration of trimethylene glycol in the 4-aminoantipyrine-containing partial composition or the Trinder reagent-containing partial composition is 70 wt / vol% or less. The method for measuring glycated protein according to

[0159] .

[0172]

[0164] The ferrocyanide-containing partial composition contains a stabilizer for the protease, the stabilizer for the protease is trimethylene glycol, and the concentration of trimethylene glycol in the ferrocyanide-containing partial composition is 40 wt / vol% or more and 80 wt / vol% or less. The method for measuring glycated protein according to any one of

[0119] to

[0136] .

[0173]

[0165] The method for measuring glycated protein according to

[0164] , wherein the concentration of the trimethylene glycol in the ferrocyanide-containing partial composition is 42.5 wt / vol% or more.

[0174]

[0166] The method for measuring glycated protein according to

[0164] , wherein the concentration of the trimethylene glycol in the ferrocyanide-containing partial composition is 45 wt / vol% or more.

[0175]

[0167] The method for measuring glycated protein according to

[0164] , wherein the concentration of the trimethylene glycol in the ferrocyanide-containing partial composition is 75 wt / vol% or less.

[0176]

[0168] The method for measuring glycated protein according to

[0164] , wherein the concentration of the trimethylene glycol in the ferrocyanide-containing partial composition is 70 wt / vol% or less.

[0177]

[0169] The method for measuring glycated protein according to any one of

[0101] to

[0146] , wherein the 4-aminoantipyrine-containing partial composition or the Trinder reagent-containing partial composition contains a stabilizer for the protease, the stabilizer for the protease is ethylene glycol, and the concentration of the ethylene glycol in the 4-aminoantipyrine-containing partial composition or the Trinder reagent-containing partial composition is 40 wt / vol% or more and 80 wt / vol% or less.

[0178]

[0170] The method for measuring glycated protein according to

[0169] , wherein the concentration of the ethylene glycol in the 4-aminoantipyrine-containing partial composition or the Trinder reagent-containing partial composition is 42.5 wt / vol% or more.

[0179] The method for measuring glycated protein according to

[0169] , wherein the concentration of ethylene glycol in the 4-aminoantipyrine-containing partial composition or the Trinder reagent-containing partial composition is 45 wt / vol% or more.

[0180] The method for measuring glycated protein according to

[0169] , wherein the concentration of ethylene glycol in the 4-aminoantipyrine-containing partial composition or the Trinder reagent-containing partial composition is 75 wt / vol% or less.

[0181] The method for measuring glycated protein according to

[0169] , wherein the concentration of ethylene glycol in the 4-aminoantipyrine-containing partial composition or the Trinder reagent-containing partial composition is 70 wt / vol% or less.

[0182] The method for measuring glycated protein according to any one of

[0119] to

[0136] , wherein the ferrocyanide-containing partial composition contains a stabilizer for the protease, the stabilizer for the protease is ethylene glycol, and the concentration of ethylene glycol in the ferrocyanide-containing partial composition is 40 wt / vol% or more and 80 wt / vol% or less.

[0183] The method for measuring glycated protein according to

[0174] , wherein the concentration of ethylene glycol in the ferrocyanide-containing partial composition is 42.5 wt / vol% or more.

[0184] The method for measuring glycated protein according to

[0174] , wherein the concentration of ethylene glycol in the ferrocyanide-containing partial composition is 45 wt / vol% or more.

[0185] The method for measuring glycated protein according to

[0174] , wherein the concentration of ethylene glycol in the ferrocyanide-containing partial composition is 75 wt / vol% or less.

[0186] The method for measuring glycated protein according to

[0174] , wherein the concentration of ethylene glycol in the ferrocyanide-containing partial composition is 70 wt / vol% or less.

[0187] The method for measuring glycated protein according to any one of

[0101] to

[0146] and

[0148] , wherein the 4-aminoantipyrine-containing partial composition or the Trinder reagent-containing partial composition contains a stabilizer for the protease, the stabilizer for the protease is 2-carboxyphenylboronic acid, and the concentration of 2-carboxyphenylboronic acid in the 4-aminoantipyrine-containing partial composition or the Trinder reagent-containing partial composition is 0.2 wt / vol% or more and 10 wt / vol% or less.

[0188] The method for measuring glycated protein according to

[0179] , wherein the concentration of 2-carboxyphenylboronic acid in the 4-aminoantipyrine-containing partial composition or the Trinder reagent-containing partial composition is 0.45 wt / vol% or more.

[0189] The method for measuring glycated protein according to

[0179] , wherein the concentration of 2-carboxyphenylboronic acid in the 4-aminoantipyrine-containing partial composition or the Trinder reagent-containing partial composition is 0.5 wt / vol% or more.

[0190] The method for measuring glycated protein according to

[0179] , wherein the concentration of 2-carboxyphenylboronic acid in the 4-aminoantipyrine-containing partial composition or the Trinder reagent-containing partial composition is 7.5 wt / vol% or less.

[0191] The method for measuring glycated protein according to

[0179] , wherein the concentration of 2-carboxyphenylboronic acid in the 4-aminoantipyrine-containing partial composition or the Trinder reagent-containing partial composition is 5 wt / vol% or less.

[0192]

[0184] The ferrocyanide-containing partial composition contains a stabilizer for the protease, the stabilizer for the protease is 2-carboxyphenylboronic acid, and the concentration of 2-carboxyphenylboronic acid in the ferrocyanide-containing partial composition is 0.2 wt / vol% or more and 10 wt / vol% or less. The method for measuring a glycated protein according to any one of

[0119] to

[0136] .

[0193]

[0185] The concentration of 2-carboxyphenylboronic acid in the ferrocyanide-containing partial composition is 0.45 wt / vol% or more. The method for measuring a glycated protein according to

[0184] .

[0194]

[0186] The concentration of 2-carboxyphenylboronic acid in the ferrocyanide-containing partial composition is 0.5 wt / vol% or more. The method for measuring a glycated protein according to

[0184] .

[0195]

[0187] The concentration of 2-carboxyphenylboronic acid in the ferrocyanide-containing partial composition is 7.5 wt / vol% or less. The method for measuring a glycated protein according to

[0184] .

[0196]

[0188] The concentration of 2-carboxyphenylboronic acid in the ferrocyanide-containing partial composition is 5 wt / vol% or less. The method for measuring a glycated protein according to

[0184] .

[0197]

[0189] The Trinder reagent-containing partial composition and the 4-aminoantipyrine-containing partial composition are in a liquid state. The method for measuring a glycated protein according to any one of

[0101] to

[0188] .

[0198]

[0190] The Trinder reagent-containing partial composition, the 4-aminoantipyrine-containing partial composition, and the ferrocyanide-containing partial composition are in a liquid state. The method for measuring a glycated protein according to any one of

[0119] to

[0136] .

[0199] The method for measuring glycated protein according to any one of

[0101] to

[0190] , wherein the ferrocyanide is potassium ferrocyanide.

[0200] The method for measuring glycated protein according to any one of

[0101] to

[0191] , wherein the Trinder reagent is N,N-bis(4-sulfobutyl)-3-methylaniline.

[0201] The method for measuring glycated protein according to any one of

[0101] to

[0192] , wherein the glycated protein is glycated albumin.

[0202] The method for measuring glycated protein according to any one of

[0101] to

[0193] , wherein the Trinder reagent-containing partial composition is stored at 2°C or higher and 10°C or lower.

[0203] The method for measuring glycated protein according to any one of

[0101] to

[0194] , wherein the 4-aminoantipyrine-containing partial composition is stored at 2°C or higher and 10°C or lower.

[0204] The method for measuring glycated protein according to any one of

[0119] to

[0136] , wherein the ferrocyanide-containing partial composition is stored at 2°C or higher and 10°C or lower.

[0205] The method for measuring glycated protein according to any one of

[0101] to

[0196] , wherein the stabilizer for protease excludes dimethyl sulfoxide.

[0206] The method for measuring glycated protein according to any one of

[0101] to

[0197] , wherein either one of the Trinder reagent-containing partial composition and the 4-aminoantipyrine-containing partial composition further contains peroxidase.

[0207]

[0199] A method for storing a glycated protein measurement reagent, Preparing a Trinder reagent-containing partial composition containing at least a Trinder reagent; Storing the Trinder reagent-containing partial composition; Preparing a 4-aminoantipyrine-containing partial composition containing at least 4-aminoantipyrine; Storing the 4-aminoantipyrine-containing partial composition; including; either one of the Trinder reagent-containing partial composition and the 4-aminoantipyrine-containing partial composition further contains a protease and a stabilizer for the protease; either one of the Trinder reagent-containing partial composition and the 4-aminoantipyrine-containing partial composition further contains a ferrocyanide; the stabilizer for the protease is a stabilizer that makes the redox potential of the ferrocyanide when mixed with the stabilizer for the protease greater than 0.058V; the redox potential is the redox potential in a reaction system containing the stabilizer for the protease and the ferrocyanide and not containing glycated proteins; A method for storing a glycated protein measurement reagent.

[0208]

[0200] The method for storing a glycated protein measurement reagent according to

[0199] , wherein the Trinder reagent-containing partial composition contains the protease and the stabilizer for the protease.

[0209]

[0201] The method for storing a glycated protein measurement reagent according to

[0199] or

[0200] , wherein the 4-aminoantipyrine-containing partial composition contains glycated amino acid oxidase.

[0210]

[0202] The method for storing a glycated protein measurement reagent according to

[0199] , wherein the 4-aminoantipyrine-containing partial composition contains the protease and the stabilizer for the protease.

[0211] The method for preserving a glycated protein measurement reagent according to any one of

[0199] ,

[0200] , and

[0202] , wherein the trinder reagent-containing partial composition contains glycated amino acid oxidase.

[0212] The method for preserving a glycated protein measurement reagent according to

[0199] , wherein either one of the trinder reagent-containing partial composition and the 4-aminoantipyrine-containing partial composition contains the protease, the stabilizer of the protease, and the ferrocyanide.

[0213] The method for preserving a glycated protein measurement reagent according to

[0199] , wherein the 4-aminoantipyrine-containing partial composition contains the protease, the stabilizer of the protease, and the ferrocyanide.

[0214] The method for preserving a glycated protein measurement reagent according to any one of

[0199] to

[0205] , wherein the concentration of the stabilizer of the protease is a concentration that makes the redox potential of the ferrocyanide greater than 0.058V when the stabilizer of the protease and the ferrocyanide are mixed.

[0215] The method for preserving a glycated protein measurement reagent according to any one of

[0199] to

[0201] and

[0204] , wherein the trinder reagent-containing partial composition contains the stabilizer of the protease and the ferrocyanide, and the concentration of the stabilizer of the protease in the trinder reagent-containing partial composition is a concentration that makes the redox potential of the ferrocyanide greater than 0.058V.

[0216] The method for preserving a glycated protein measurement reagent according to any one of

[0199] ,

[0202] ,

[0204] , and

[0205] , wherein the 4-aminoantipyrine-containing partial composition contains the stabilizer of the protease and the ferrocyanide, and the concentration of the stabilizer of the protease in the 4-aminoantipyrine-containing partial composition is a concentration that makes the redox potential of the ferrocyanide greater than 0.058V.

[0217]

[0209] The method for preserving the glycated protein measurement reagent according to any one of

[0199] to

[0208] , wherein the 4-aminoantipyrine-containing partial composition further contains a chelating agent.

[0218]

[0210] The method for preserving the glycated protein measurement reagent according to

[0209] , wherein the chelating agent is selected from the group consisting of ethylenediaminetetraacetic acid, glycol ether diamine tetraacetic acid, N-(2-hydroxyethyl)iminodiacetic acid, nitrilotris(methylphosphonic acid), nitrilotriacetic acid, trans-1,2-diaminocyclohexane-N,N,N,N-tetraacetic acid, iminodiacetic acid, diethylenetriamine-N,N,N’,N”,N”-pentaacetic acid, 1,3-diamino-2-propanol-N,N,N’,N’-tetraacetic acid, gluconic acid, malic acid, succinic acid, citric acid, salicylic acid, tartaric acid, N-[tris(hydroxymethyl)methyl]glycine, N,N-bis(2-hydroxyethyl)glycine, and salts thereof.

[0219]

[0211] The method for preserving the glycated protein measurement reagent according to

[0209] , wherein the chelating agent is citric acid or a salt thereof.

[0220]

[0212] The method for preserving the glycated protein measurement reagent according to any one of

[0209] to

[0211] , wherein the concentration of the chelating agent is 5 μmol / L or more and 1000 mmol / L or less.

[0221]

[0213] The method for preserving the glycated protein measurement reagent according to

[0212] , wherein the concentration of the chelating agent is 10 μmol / L or more.

[0222]

[0214] The method for preserving the glycated protein measurement reagent according to

[0212] , wherein the concentration of the chelating agent is 50 μmol / L or more.

[0223]

[0215] The method for preserving the glycated protein measurement reagent according to

[0212] , wherein the concentration of the chelating agent is 100 mmol / L or less.

[0224] The method for preserving a glycated protein measurement reagent according to

[0212] , wherein the concentration of the chelating agent is 25 mmol / L or less.

[0225]

[0217] A method for preserving a glycated protein measurement reagent, comprising: preparing a Trinder reagent-containing partial composition containing at least a Trinder reagent; preserving the Trinder reagent-containing partial composition; preparing a 4-aminoantipyrine-containing partial composition containing at least 4-aminoantipyrine; preserving the 4-aminoantipyrine-containing partial composition; preparing a ferricyanide-containing partial composition containing at least ferricyanide; preserving the ferricyanide-containing partial composition; including wherein any one of the Trinder reagent-containing partial composition, the 4-aminoantipyrine-containing partial composition, and the ferricyanide-containing partial composition contains a protease and a stabilizer for the protease; the stabilizer for the protease is a stabilizer that makes the redox potential of the ferricyanide greater than 0.058V when mixed with the stabilizer for the protease; the redox potential is the redox potential in a reaction system containing the stabilizer for the protease and the ferricyanide and not containing glycated protein; A method for preserving a glycated protein measurement reagent.

[0226] The method for preserving a glycated protein measurement reagent according to

[0217] , wherein the Trinder reagent-containing partial composition contains the protease and the stabilizer for the protease.

[0227] The method for preserving a glycated protein measurement reagent according to

[0217] or

[0218] , wherein the 4-aminoantipyrine-containing partial composition contains glycated amino acid oxidase.

[0228]

[0220] The method for preserving a glycated protein measurement reagent according to

[0217] , wherein the 4-aminoantipyrine-containing partial composition contains the protease and a stabilizer for the protease.

[0229]

[0221] The method for preserving a glycated protein measurement reagent according to any one of

[0217] ,

[0218] , and

[0220] , wherein the Trinder reagent-containing partial composition contains glycated amino acid oxidase.

[0230]

[0222] The method for preserving a glycated protein measurement reagent according to

[0217] , wherein the ferrocyanide-containing partial composition contains the protease and a stabilizer for the protease.

[0231]

[0223] The method for preserving a glycated protein measurement reagent according to any one of

[0217] to

[0222] , wherein the concentration of the stabilizer for the protease is a concentration that makes the redox potential of the ferrocyanide greater than 0.058V when the stabilizer for the protease and the ferrocyanide are mixed.

[0232]

[0224] The method for preserving a glycated protein measurement reagent according to any one of

[0217] to

[0219] , wherein the Trinder reagent-containing partial composition contains a stabilizer for the protease, and the concentration of the stabilizer for the protease when the Trinder reagent-containing partial composition and the ferrocyanide-containing partial composition are mixed is a concentration that makes the redox potential of the ferrocyanide greater than 0.058V.

[0233]

[0225] The method for preserving a glycated protein measurement reagent according to

[0217] or

[0220] , wherein the 4-aminoantipyrine-containing partial composition contains a stabilizer for the protease, and the concentration of the stabilizer for the protease when the 4-aminoantipyrine-containing partial composition and the ferrocyanide-containing partial composition are mixed is a concentration that makes the redox potential of the ferrocyanide greater than 0.058V.

[0234]

[0226] The ferrocyanide-containing partial composition contains a stabilizer for the protease, and the concentration of the stabilizer for the protease in the ferrocyanide-containing partial composition is a concentration that makes the redox potential of the ferrocyanide greater than 0.058 V. The method for storing the glycated protein measurement reagent according to

[0217] or

[0222] .

[0235]

[0227] The 4-aminoantipyrine-containing partial composition and / or the ferrocyanide-containing partial composition further contains a chelating agent. The method for storing the glycated protein measurement reagent according to any one of

[0217] to

[0226] .

[0236]

[0228] The chelating agent is selected from the group consisting of ethylenediaminetetraacetic acid, glycol ether diamine tetraacetic acid, N-(2-hydroxyethyl)iminodiacetic acid, nitrilotris(methylphosphonic acid), nitrilotriacetic acid, trans-1,2-diaminocyclohexane-N,N,N,N-tetraacetic acid, iminodiacetic acid, diethylenetriamine-N,N,N’,N”,N”-pentaacetic acid, 1,3-diamino-2-propanol-N,N,N’,N’-tetraacetic acid, gluconic acid, malic acid, succinic acid, citric acid, salicylic acid, tartaric acid, N-[tris(hydroxymethyl)methyl]glycine, N,N-bis(2-hydroxyethyl)glycine, and salts thereof. The method for storing the glycated protein measurement reagent according to

[0227] .

[0237]

[0229] The chelating agent is citric acid or a salt thereof. The method for storing the glycated protein measurement reagent according to

[0227] .

[0238]

[0230] The concentration of the chelating agent is 5 μmol / L or more and 1000 mmol / L or less. The method for storing the glycated protein measurement reagent according to any one of

[0227] to

[0229] .

[0239]

[0231] The concentration of the chelating agent is 10 μmol / L or more. The method for storing the glycated protein measurement reagent according to

[0230] .

[0240] The method for storing the glycated protein measurement reagent according to

[0230] , wherein the concentration of the chelating agent is 50 μmol / L or more.

[0241] The method for storing the glycated protein measurement reagent according to

[0230] , wherein the concentration of the chelating agent is 100 mmol / L or less.

[0242] The method for storing the glycated protein measurement reagent according to

[0230] , wherein the concentration of the chelating agent is 25 mmol / L or less.

[0243] The method for storing the glycated protein measurement reagent according to any one of

[0199] to

[0234] , wherein the reaction system containing the protease stabilizer and the ferrocyanide and not containing glycated protein further contains N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid and sodium chloride.

[0244] The method for storing the glycated protein measurement reagent according to any one of

[0199] to

[0235] , wherein the redox potential is the redox potential measured using a silver / silver chloride electrode as a reference electrode.

[0245] The method for storing the glycated protein measurement reagent according to any one of

[0199] to

[0236] , wherein the redox potential is greater than 0.058 V and 0.400 V or less.

[0246] The method for storing the glycated protein measurement reagent according to

[0237] , wherein the redox potential is 0.070 V or more.

[0247] The method for storing the glycated protein measurement reagent according to

[0237] , wherein the redox potential is 0.084 V or more.

[0248] The method for storing the glycated protein measurement reagent according to

[0237] , wherein the redox potential is 0.112 V or more.

[0249]

[0241] The method for storing the glycated protein measurement reagent according to

[0237] , wherein the redox potential is 0.300 V or less.

[0250]

[0242] The method for storing the glycated protein measurement reagent according to

[0237] , wherein the redox potential is 0.250 V or less.

[0251]

[0243] The method for storing the glycated protein measurement reagent according to

[0237] , wherein the redox potential is 0.235 V or less.

[0252]

[0244] The method for storing the glycated protein measurement reagent according to any one of

[0199] to

[0243] , wherein the stabilizer for the protease is selected from the group consisting of propylene glycol, trimethylene glycol, ethylene glycol, and carboxyphenylboronic acid.

[0253]

[0245] The method for storing the glycated protein measurement reagent according to any one of

[0199] to

[0243] , wherein the stabilizer for the protease is propylene glycol.

[0254]

[0246] The method for storing the glycated protein measurement reagent according to

[0244] , wherein the carboxyphenylboronic acid is 2-carboxyphenylboronic acid.

[0255]

[0247] The 4-aminoantipyrine-containing partial composition or the Trinder reagent-containing partial composition contains the stabilizer for the protease, the stabilizer for the protease is propylene glycol, and the concentration of propylene glycol in the 4-aminoantipyrine-containing partial composition or the Trinder reagent-containing partial composition is 7.5 wt / vol% or more and 80 wt / vol% or less. The method for storing the glycated protein measurement reagent according to any one of

[0199] to

[0245] .

[0256] The method for storing a glycated protein measurement reagent according to

[0247] , wherein the concentration of propylene glycol in the 4-aminoantipyrine-containing partial composition or the Trinder reagent-containing partial composition is 20 wt / vol% or more.

[0257] The method for storing a glycated protein measurement reagent according to

[0247] , wherein the concentration of propylene glycol in the 4-aminoantipyrine-containing partial composition or the Trinder reagent-containing partial composition is 25 wt / vol% or more.

[0258] The method for storing a glycated protein measurement reagent according to

[0247] , wherein the concentration of propylene glycol in the 4-aminoantipyrine-containing partial composition or the Trinder reagent-containing partial composition is 62.5 wt / vol% or less.

[0259] The method for storing a glycated protein measurement reagent according to

[0247] , wherein the concentration of propylene glycol in the 4-aminoantipyrine-containing partial composition or the Trinder reagent-containing partial composition is 60 wt / vol% or less.

[0260] The method for storing a glycated protein measurement reagent according to any one of

[0217] to

[0234] , wherein the ferrocyanide-containing partial composition contains a stabilizer for the protease, the stabilizer for the protease is propylene glycol, and the concentration of propylene glycol in the ferrocyanide-containing partial composition is 7.5 wt / vol% or more and 80 wt / vol% or less.

[0261] The method for storing a glycated protein measurement reagent according to

[0252] , wherein the concentration of propylene glycol in the ferrocyanide-containing partial composition is 20 wt / vol% or more.

[0262] The preservation method of the glycated protein measurement reagent according to

[0252] , wherein the concentration of the propylene glycol in the ferrocyanide-containing partial composition is 25 wt / vol% or more.

[0263] The preservation method of the glycated protein measurement reagent according to

[0252] , wherein the concentration of the propylene glycol in the ferrocyanide-containing partial composition is 62.5 wt / vol% or less.

[0264] The preservation method of the glycated protein measurement reagent according to

[0252] , wherein the concentration of the propylene glycol in the ferrocyanide-containing partial composition is 60 wt / vol% or less.

[0265] The preservation method of the glycated protein measurement reagent according to any one of

[0199] to

[0244] , wherein the 4-aminoantipyrine-containing partial composition or the Trinder reagent-containing partial composition contains a stabilizer for the protease, the stabilizer for the protease is trimethylene glycol, and the concentration of the trimethylene glycol in the 4-aminoantipyrine-containing partial composition or the Trinder reagent-containing partial composition is 40 wt / vol% or more and 80 wt / vol% or less.

[0266] The preservation method of the glycated protein measurement reagent according to

[0257] , wherein the concentration of the trimethylene glycol in the 4-aminoantipyrine-containing partial composition or the Trinder reagent-containing partial composition is 42.5 wt / vol% or more.

[0267] The preservation method of the glycated protein measurement reagent according to

[0257] , wherein the concentration of the trimethylene glycol in the 4-aminoantipyrine-containing partial composition or the Trinder reagent-containing partial composition is 45 wt / vol% or more.

[0268] The method for storing a glycated protein measurement reagent according to

[0257] , wherein the concentration of the trimethylene glycol in the 4-aminoantipyrine-containing partial composition or the Trinder reagent-containing partial composition is 75 wt / vol% or less.

[0269] The method for storing a glycated protein measurement reagent according to

[0257] , wherein the concentration of the trimethylene glycol in the 4-aminoantipyrine-containing partial composition or the Trinder reagent-containing partial composition is 70 wt / vol% or less.

[0270] The method for storing a glycated protein measurement reagent according to any one of

[0217] to

[0234] , wherein the cyanide-containing partial composition contains a stabilizer for the protease, the stabilizer for the protease is trimethylene glycol, and the concentration of the trimethylene glycol in the cyanide-containing partial composition is 40 wt / vol% or more and 80 wt / vol% or less.

[0271] The method for storing a glycated protein measurement reagent according to

[0262] , wherein the concentration of the trimethylene glycol in the cyanide-containing partial composition is 42.5 wt / vol% or more.

[0272] The method for storing a glycated protein measurement reagent according to

[0262] , wherein the concentration of the trimethylene glycol in the cyanide-containing partial composition is 45 wt / vol% or more.

[0273] The method for storing a glycated protein measurement reagent according to

[0262] , wherein the concentration of the trimethylene glycol in the cyanide-containing partial composition is 75 wt / vol% or less.

[0274] The method for storing a glycated protein measurement reagent according to

[0262] , wherein the concentration of the trimethylene glycol in the cyanide-containing partial composition is 70 wt / vol% or less.

[0275]

[0267] The 4 - aminoantipyrine - containing partial composition or the Trinder reagent - containing partial composition contains a stabilizer for the protease, the stabilizer for the protease is ethylene glycol, and the concentration of ethylene glycol in the 4 - aminoantipyrine - containing partial composition or the Trinder reagent - containing partial composition is 40 wt / vol% or more and 80 wt / vol% or less. The method for storing a glycated protein measurement reagent according to any one of

[0199] to

[0244] .

[0276]

[0268] The method for storing a glycated protein measurement reagent according to

[0267] , wherein the concentration of ethylene glycol in the 4 - aminoantipyrine - containing partial composition or the Trinder reagent - containing partial composition is 42.5 wt / vol% or more.

[0277]

[0269] The method for storing a glycated protein measurement reagent according to

[0267] , wherein the concentration of ethylene glycol in the 4 - aminoantipyrine - containing partial composition or the Trinder reagent - containing partial composition is 45 wt / vol% or more.

[0278]

[0270] The method for storing a glycated protein measurement reagent according to

[0267] , wherein the concentration of ethylene glycol in the 4 - aminoantipyrine - containing partial composition or the Trinder reagent - containing partial composition is 75 wt / vol% or less.

[0279]

[0271] The method for storing a glycated protein measurement reagent according to

[0267] , wherein the concentration of ethylene glycol in the 4 - aminoantipyrine - containing partial composition or the Trinder reagent - containing partial composition is 70 wt / vol% or less.

[0280]

[0272] The ferrocyanide-containing partial composition contains a stabilizer for the protease, the stabilizer for the protease is ethylene glycol, and the concentration of ethylene glycol in the ferrocyanide-containing partial composition is 40 wt / vol% or more and 80 wt / vol% or less. The method for storing a glycated protein measurement reagent according to any one of

[0217] to

[0234] .

[0281]

[0273] The concentration of ethylene glycol in the ferrocyanide-containing partial composition is 42.5 wt / vol% or more. The method for storing a glycated protein measurement reagent according to

[0272] .

[0282]

[0274] The concentration of ethylene glycol in the ferrocyanide-containing partial composition is 45 wt / vol% or more. The method for storing a glycated protein measurement reagent according to

[0272] .

[0283]

[0275] The concentration of ethylene glycol in the ferrocyanide-containing partial composition is 75 wt / vol% or less. The method for storing a glycated protein measurement reagent according to

[0272] .

[0284]

[0276] The concentration of ethylene glycol in the ferrocyanide-containing partial composition is 70 wt / vol% or less. The method for storing a glycated protein measurement reagent according to

[0272] .

[0285]

[0277] The 4-aminoantipyrine-containing partial composition or the Trinder reagent-containing partial composition contains a stabilizer for the protease, the stabilizer for the protease is 2-carboxyphenylboronic acid, and the concentration of 2-carboxyphenylboronic acid in the 4-aminoantipyrine-containing partial composition or the Trinder reagent-containing partial composition is 0.2 wt / vol% or more and 10 wt / vol% or less. The method for storing a glycated protein measurement reagent according to any one of

[0199] to

[0244] and

[0246] .

[0286] The preservation method of the glycated protein measurement reagent according to

[0277] , wherein the concentration of the 2-carboxyphenylboronic acid in the 4-aminoantipyrine-containing partial composition or the Trinder reagent-containing partial composition is 0.45 wt / vol% or more.

[0287] The preservation method of the glycated protein measurement reagent according to

[0277] , wherein the concentration of the 2-carboxyphenylboronic acid in the 4-aminoantipyrine-containing partial composition or the Trinder reagent-containing partial composition is 0.5 wt / vol% or more.

[0288] The preservation method of the glycated protein measurement reagent according to

[0277] , wherein the concentration of the 2-carboxyphenylboronic acid in the 4-aminoantipyrine-containing partial composition or the Trinder reagent-containing partial composition is 7.5 wt / vol% or less.

[0289] The preservation method of the glycated protein measurement reagent according to

[0277] , wherein the concentration of the 2-carboxyphenylboronic acid in the 4-aminoantipyrine-containing partial composition or the Trinder reagent-containing partial composition is 5 wt / vol% or less.

[0290] The preservation method of the glycated protein measurement reagent according to any one of

[0217] to

[0234] , wherein the cyanide-containing partial composition contains a stabilizer for the protease, the stabilizer for the protease is 2-carboxyphenylboronic acid, and the concentration of the 2-carboxyphenylboronic acid in the cyanide-containing partial composition is 0.2 wt / vol% or more and 10 wt / vol% or less.

[0291] The preservation method of the glycated protein measurement reagent according to

[0282] , wherein the concentration of the 2-carboxyphenylboronic acid in the cyanide-containing partial composition is 0.45 wt / vol% or more.

[0292] The method for storing the glycated protein measurement reagent according to

[0282] , wherein the concentration of the 2-carboxyphenylboronic acid in the ferrocyanide-containing partial composition is 0.5 wt / vol% or more.

[0293] The method for storing the glycated protein measurement reagent according to

[0282] , wherein the concentration of the 2-carboxyphenylboronic acid in the ferrocyanide-containing partial composition is 7.5 wt / vol% or less.

[0294] The method for storing the glycated protein measurement reagent according to

[0282] , wherein the concentration of the 2-carboxyphenylboronic acid in the ferrocyanide-containing partial composition is 5 wt / vol% or less.

[0295] The method for storing the glycated protein measurement reagent according to any one of

[0199] to

[0286] , wherein the trinder reagent-containing partial composition and the 4-aminoantipyrine-containing partial composition are in a liquid state.

[0296] The method for storing the glycated protein measurement reagent according to any one of

[0217] to

[0234] , wherein the trinder reagent-containing partial composition, the 4-aminoantipyrine-containing partial composition, and the ferrocyanide-containing partial composition are in a liquid state.

[0297] The method for storing the glycated protein measurement reagent according to any one of

[0199] to

[0288] , wherein the ferrocyanide is potassium ferrocyanide.

[0298] The method for storing the glycated protein measurement reagent according to any one of

[0199] to

[0289] , wherein the trinder reagent is N,N-bis(4-sulfobutyl)-3-methylaniline.

[0299] The method for storing the glycated protein measurement reagent according to any one of

[0199] to

[0290] , wherein the glycated protein is glycated albumin.

[0300]

[0292] The storage method of the glycated protein measurement reagent according to any one of

[0199] to

[0291] , wherein the Trinder reagent-containing partial composition is stored at 2°C or higher and 10°C or lower.

[0301]

[0293] The storage method of the glycated protein measurement reagent according to any one of

[0199] to

[0292] , wherein the 4-aminoantipyrine-containing partial composition is stored at 2°C or higher and 10°C or lower.

[0302]

[0294] The storage method of the glycated protein measurement reagent according to any one of

[0217] to

[0234] , wherein the ferrocyanide-containing partial composition is stored at 2°C or higher and 10°C or lower.

[0303]

[0295] The storage method of the glycated protein measurement reagent according to any one of

[0199] to

[0294] , wherein the stabilizer of the protease excludes dimethyl sulfoxide.

[0304]

[0296] The storage method of the glycated protein measurement reagent according to any one of

[0199] to

[0295] , wherein either the Trinder reagent-containing partial composition or the 4-aminoantipyrine-containing partial composition further contains peroxidase.

[0305]

[0297] A method for stabilizing a glycated protein measurement reagent, comprising: preparing a Trinder reagent-containing partial composition containing at least a Trinder reagent; preparing a 4-aminoantipyrine-containing partial composition containing at least 4-aminoantipyrine; including: including a protease and a stabilizer of the protease in either the Trinder reagent-containing partial composition or the 4-aminoantipyrine-containing partial composition; including a ferrocyanide in either the Trinder reagent-containing partial composition or the 4-aminoantipyrine-containing partial composition; The stabilizer for the protease is a stabilizer that increases the redox potential of the ferrocyanide to greater than 0.058 V when mixed with the stabilizer for the protease, The redox potential is the redox potential in a reaction system containing the stabilizer for the protease and the ferrocyanide and not containing glycated protein, A method for stabilizing a glycated protein measurement reagent.

[0306]

[0298] The method for stabilizing a glycated protein measurement reagent according to

[0297] , wherein the trinder reagent-containing partial composition contains the protease and the stabilizer for the protease.

[0307]

[0299] The method for stabilizing a glycated protein measurement reagent according to

[0297] or

[0298] , wherein the 4-aminoantipyrine-containing partial composition contains glycated amino acid oxidase.

[0308]

[0300] The method for stabilizing a glycated protein measurement reagent according to

[0297] , wherein the 4-aminoantipyrine-containing partial composition contains the protease and the stabilizer for the protease.

[0309]

[0301] The method for stabilizing a glycated protein measurement reagent according to any one of

[0297] ,

[0298] , and

[0300] , wherein the trinder reagent-containing partial composition contains glycated amino acid oxidase.

[0310]

[0302] The method for stabilizing a glycated protein measurement reagent according to

[0297] , wherein either the trinder reagent-containing partial composition or the 4-aminoantipyrine-containing partial composition contains the protease, the stabilizer for the protease, and the ferrocyanide.

[0311]

[0303] The method for stabilizing a glycated protein measurement reagent according to

[0297] , wherein the 4-aminoantipyrine-containing partial composition contains the protease, the stabilizer for the protease, and the ferrocyanide.

[0312]

[0304] The concentration of the stabilizer for the protease is a concentration that makes the redox potential of the ferrocyanide greater than 0.058 V when the stabilizer for the protease and the ferrocyanide are mixed. The method for stabilizing a glycated protein measurement reagent according to any one of

[0297] to

[0303] .

[0313]

[0305] The stabilizer for the protease and the ferrocyanide are included in the Trinder reagent-containing partial composition, and the concentration of the stabilizer for the protease in the Trinder reagent-containing partial composition is a concentration that makes the redox potential of the ferrocyanide greater than 0.058 V. The method for stabilizing a glycated protein measurement reagent according to any one of

[0297] to

[0299] and

[0302] .

[0314]

[0306] The stabilizer for the protease and the ferrocyanide are included in the 4-aminoantipyrine-containing partial composition, and the concentration of the stabilizer for the protease in the 4-aminoantipyrine-containing partial composition is a concentration that makes the redox potential of the ferrocyanide greater than 0.058 V. The method for stabilizing a glycated protein measurement reagent according to any one of

[0297] ,

[0300] ,

[0302] , and

[0303] .

[0315]

[0307] The 4-aminoantipyrine-containing partial composition further includes a chelating agent. The method for stabilizing a glycated protein measurement reagent according to any one of

[0297] to

[0306] .

[0316]

[0308] The chelating agent is selected from the group consisting of ethylenediaminetetraacetic acid, glycol ether diamine tetraacetic acid, N-(2-hydroxyethyl)iminodiacetic acid, nitrilotris(methylphosphonic acid), nitrilotriacetic acid, trans-1,2-diaminocyclohexane-N,N,N,N-tetraacetic acid, iminodiacetic acid, diethylenetriamine-N,N,N’,N”,N”-pentaacetic acid, 1,3-diamino-2-propanol-N,N,N’,N’-tetraacetic acid, gluconic acid, malic acid, succinic acid, citric acid, salicylic acid, tartaric acid, N-[tris(hydroxymethyl)methyl]glycine, N,N-bis(2-hydroxyethyl)glycine, and salts thereof, and is the method for stabilizing a glycated protein measurement reagent according to

[0307] .

[0317]

[0309] The chelating agent is citric acid or a salt thereof, and is the method for stabilizing a glycated protein measurement reagent according to

[0307] .

[0318]

[0310] The concentration of the chelating agent is 5 μmol / L or more and 1000 mmol / L or less, and is the method for stabilizing a glycated protein measurement reagent according to any one of

[0307] to

[0309] .

[0319]

[0311] The concentration of the chelating agent is 10 μmol / L or more, and is the method for stabilizing a glycated protein measurement reagent according to

[0310] .

[0320]

[0312] The concentration of the chelating agent is 50 μmol / L or more, and is the method for stabilizing a glycated protein measurement reagent according to

[0310] .

[0321]

[0313] The concentration of the chelating agent is 100 mmol / L or less, and is the method for stabilizing a glycated protein measurement reagent according to

[0310] .

[0322]

[0314] The concentration of the chelating agent is 25 mmol / L or less, and is the method for stabilizing a glycated protein measurement reagent according to

[0310] .

[0323]

[0315] A method for stabilizing a glycated protein measurement reagent, comprising: preparing a Trinder reagent-containing partial composition containing at least a Trinder reagent; preparing a 4-aminoantipyrine-containing partial composition containing at least 4-aminoantipyrine; preparing a ferrocyanide-containing partial composition containing at least a ferrocyanide; including: adding a protease and a stabilizer for the protease to any one of the Trinder reagent-containing partial composition, the 4-aminoantipyrine-containing partial composition, and the ferrocyanide-containing partial composition; the stabilizer for the protease being a stabilizer that makes the redox potential of the ferrocyanide greater than 0.058V when mixed with the stabilizer for the protease; the redox potential being the redox potential in a reaction system containing the stabilizer for the protease and the ferrocyanide and not containing glycated protein; A method for stabilizing a glycated protein measurement reagent.

[0324]

[0316] The method for stabilizing a glycated protein measurement reagent according to

[0315] , wherein the protease and the stabilizer for the protease are added to the Trinder reagent-containing partial composition.

[0325]

[0317] The method for stabilizing a glycated protein measurement reagent according to

[0315] or

[0316] , wherein glycated amino acid oxidase is added to the 4-aminoantipyrine-containing partial composition.

[0326]

[0318] The method for stabilizing a glycated protein measurement reagent according to

[0315] , wherein the protease and the stabilizer for the protease are added to the 4-aminoantipyrine-containing partial composition.

[0327] The method for stabilizing a glycated protein measurement reagent according to any one of

[0315] ,

[0316] , and

[0318] , wherein the trinder reagent-containing partial composition contains glycated amino acid oxidase.

[0328] The method for stabilizing a glycated protein measurement reagent according to

[0315] , wherein the ferrocyanide-containing partial composition contains the protease and a stabilizer for the protease.

[0329] The method for stabilizing a glycated protein measurement reagent according to any one of

[0315] to

[0320] , wherein the concentration of the stabilizer for the protease is a concentration that makes the redox potential of the ferrocyanide greater than 0.058V when the stabilizer for the protease and the ferrocyanide are mixed.

[0330] The method for stabilizing a glycated protein measurement reagent according to any one of

[0315] to

[0317] , wherein the trinder reagent-containing partial composition contains the stabilizer for the protease, and the concentration of the stabilizer for the protease when the trinder reagent-containing partial composition and the ferrocyanide-containing partial composition are mixed is a concentration that makes the redox potential of the ferrocyanide greater than 0.058V.

[0331] The method for stabilizing a glycated protein measurement reagent according to any one of

[0315] and

[0318] , wherein the 4-aminoantipyrine-containing partial composition contains the stabilizer for the protease, and the concentration of the stabilizer for the protease when the 4-aminoantipyrine-containing partial composition and the ferrocyanide-containing partial composition are mixed is a concentration that makes the redox potential of the ferrocyanide greater than 0.058V.

[0332]

[0324] The ferrocyanide-containing partial composition contains a stabilizer for the protease, and the concentration of the stabilizer for the protease in the ferrocyanide-containing partial composition is a concentration that makes the redox potential of the ferrocyanide greater than 0.058 V. The method for stabilizing a glycated protein measurement reagent according to any one of

[0315] and

[0320] .

[0333]

[0325] The 4-aminoantipyrine-containing partial composition and / or the ferrocyanide-containing partial composition further contains a chelating agent. The method for stabilizing a glycated protein measurement reagent according to any one of

[0315] to

[0324] .

[0334]

[0326] The chelating agent is selected from the group consisting of ethylenediaminetetraacetic acid, glycol ether diamine tetraacetic acid, N-(2-hydroxyethyl)iminodiacetic acid, nitrilotris(methylphosphonic acid), nitrilotriacetic acid, trans-1,2-diaminocyclohexane-N,N,N,N-tetraacetic acid, iminodiacetic acid, diethylenetriamine-N,N,N’,N”,N”-pentaacetic acid, 1,3-diamino-2-propanol-N,N,N’,N’-tetraacetic acid, gluconic acid, malic acid, succinic acid, citric acid, salicylic acid, tartaric acid, N-[tris(hydroxymethyl)methyl]glycine, N,N-bis(2-hydroxyethyl)glycine, and salts thereof. The method for stabilizing a glycated protein measurement reagent according to

[0325] .

[0335]

[0327] The chelating agent is citric acid or a salt thereof. The method for stabilizing a glycated protein measurement reagent according to

[0325] .

[0336]

[0328] The concentration of the chelating agent is 5 μmol / L or more and 1000 mmol / L or less. The method for stabilizing a glycated protein measurement reagent according to any one of

[0325] to

[0327] .

[0337]

[0329] The concentration of the chelating agent is 10 μmol / L or more. The method for stabilizing a glycated protein measurement reagent according to

[0328] .

[0338] The method for stabilizing a glycated protein measurement reagent according to

[0328] , wherein the concentration of the chelating agent is 50 μmol / L or more.

[0339] The method for stabilizing a glycated protein measurement reagent according to

[0328] , wherein the concentration of the chelating agent is 100 mmol / L or less.

[0340] The method for stabilizing a glycated protein measurement reagent according to

[0328] , wherein the concentration of the chelating agent is 25 mmol / L or less.

[0341] The method for stabilizing a glycated protein measurement reagent according to any one of

[0297] to

[0332] , wherein the reaction system containing the protease stabilizer and the ferrocyanide and not containing glycated protein further contains N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid and sodium chloride.

[0342] The method for stabilizing a glycated protein measurement reagent according to any one of

[0297] to

[0333] , wherein the redox potential is the redox potential measured using a silver / silver chloride electrode as a reference electrode.

[0343] The method for stabilizing a glycated protein measurement reagent according to any one of

[0297] to

[0334] , wherein the redox potential is greater than 0.058 V and 0.400 V or less.

[0344] The method for stabilizing a glycated protein measurement reagent according to

[0335] , wherein the redox potential is 0.070 V or more.

[0345] The method for stabilizing a glycated protein measurement reagent according to

[0335] , wherein the redox potential is 0.084 V or more.

[0346] The method for stabilizing a glycated protein measurement reagent according to

[0335] , wherein the redox potential is 0.112 V or more.

[0347]

[0339] The method for stabilizing a glycated protein measurement reagent according to

[0335] , wherein the redox potential is 0.300 V or less.

[0348]

[0340] The method for stabilizing a glycated protein measurement reagent according to

[0335] , wherein the redox potential is 0.250 V or less.

[0349]

[0341] The method for stabilizing a glycated protein measurement reagent according to

[0335] , wherein the redox potential is 0.235 V or less.

[0350]

[0342] The method for stabilizing a glycated protein measurement reagent according to any one of

[0297] to

[0341] , wherein the stabilizer for the protease is selected from the group consisting of propylene glycol, trimethylene glycol, ethylene glycol, and carboxyphenylboronic acid.

[0351]

[0343] The method for stabilizing a glycated protein measurement reagent according to any one of

[0297] to

[0341] , wherein the stabilizer for the protease is propylene glycol.

[0352]

[0344] The method for stabilizing a glycated protein measurement reagent according to

[0342] , wherein the carboxyphenylboronic acid is 2-carboxyphenylboronic acid.

[0353]

[0345] The 4-aminoantipyrine-containing partial composition or the Trinder reagent-containing partial composition contains the stabilizer for the protease, the stabilizer for the protease is propylene glycol, and the concentration of propylene glycol in the 4-aminoantipyrine-containing partial composition or the Trinder reagent-containing partial composition is 7.5 wt / vol% or more and 80 wt / vol% or less. The method for stabilizing a glycated protein measurement reagent according to any one of

[0297] to

[0343] .

[0354] The method for stabilizing a glycated protein measurement reagent according to

[0345] , wherein the concentration of propylene glycol in the 4-aminoantipyrine-containing partial composition or the Trinder reagent-containing partial composition is 20 wt / vol% or more.

[0355] The method for stabilizing a glycated protein measurement reagent according to

[0345] , wherein the concentration of propylene glycol in the 4-aminoantipyrine-containing partial composition or the Trinder reagent-containing partial composition is 25 wt / vol% or more.

[0356] The method for stabilizing a glycated protein measurement reagent according to

[0345] , wherein the concentration of propylene glycol in the 4-aminoantipyrine-containing partial composition or the Trinder reagent-containing partial composition is 62.5 wt / vol% or less.

[0357] The method for stabilizing a glycated protein measurement reagent according to

[0345] , wherein the concentration of propylene glycol in the 4-aminoantipyrine-containing partial composition or the Trinder reagent-containing partial composition is 60 wt / vol% or less.

[0358] The method for stabilizing a glycated protein measurement reagent according to any one of

[0315] to

[0332] , wherein the ferrocyanide-containing partial composition contains a stabilizer for the protease, the stabilizer for the protease is propylene glycol, and the concentration of propylene glycol in the ferrocyanide-containing partial composition is 7.5 wt / vol% or more and 80 wt / vol% or less.

[0359] The method for stabilizing a glycated protein measurement reagent according to

[0350] , wherein the concentration of propylene glycol in the ferrocyanide-containing partial composition is 20 wt / vol% or more.

[0360] The method for stabilizing a glycated protein measurement reagent according to

[0350] , wherein the concentration of propylene glycol in the ferrocyanide-containing partial composition is 25 wt / vol% or more.

[0361] The method for stabilizing a glycated protein measurement reagent according to

[0350] , wherein the concentration of propylene glycol in the ferrocyanide-containing partial composition is 62.5 wt / vol% or less.

[0362] The method for stabilizing a glycated protein measurement reagent according to

[0350] , wherein the concentration of propylene glycol in the ferrocyanide-containing partial composition is 60 wt / vol% or less.

[0363] The method for stabilizing a glycated protein measurement reagent according to any one of

[0297] to

[0342] , wherein the 4-aminoantipyrine-containing partial composition or the Trinder reagent-containing partial composition contains a stabilizer for the protease, the stabilizer for the protease is trimethylene glycol, and the concentration of trimethylene glycol in the 4-aminoantipyrine-containing partial composition or the Trinder reagent-containing partial composition is 40 wt / vol% or more and 80 wt / vol% or less.

[0364] The method for stabilizing a glycated protein measurement reagent according to

[0355] , wherein the concentration of trimethylene glycol in the 4-aminoantipyrine-containing partial composition or the Trinder reagent-containing partial composition is 42.5 wt / vol% or more.

[0365] The method for stabilizing a glycated protein measurement reagent according to

[0355] , wherein the concentration of trimethylene glycol in the 4-aminoantipyrine-containing partial composition or the Trinder reagent-containing partial composition is 45 wt / vol% or more.

[0366] The method for stabilizing a glycated protein measurement reagent according to

[0355] , wherein the concentration of the trimethylene glycol in the 4-aminoantipyrine-containing partial composition or the Trinder reagent-containing partial composition is 75 wt / vol% or less.

[0367] The method for stabilizing a glycated protein measurement reagent according to

[0355] , wherein the concentration of the trimethylene glycol in the 4-aminoantipyrine-containing partial composition or the Trinder reagent-containing partial composition is 70 wt / vol% or less.

[0368] The method for stabilizing a glycated protein measurement reagent according to any one of

[0315] to

[0332] , wherein the ferrocyanide-containing partial composition contains a stabilizer for the protease, the stabilizer for the protease is trimethylene glycol, and the concentration of the trimethylene glycol in the ferrocyanide-containing partial composition is 40 wt / vol% or more and 80 wt / vol% or less.

[0369] The method for stabilizing a glycated protein measurement reagent according to

[0360] , wherein the concentration of the trimethylene glycol in the ferrocyanide-containing partial composition is 42.5 wt / vol% or more.

[0370] The method for stabilizing a glycated protein measurement reagent according to

[0360] , wherein the concentration of the trimethylene glycol in the ferrocyanide-containing partial composition is 45 wt / vol% or more.

[0371] The method for stabilizing a glycated protein measurement reagent according to

[0360] , wherein the concentration of the trimethylene glycol in the ferrocyanide-containing partial composition is 75 wt / vol% or less.

[0372] The method for stabilizing a glycated protein measurement reagent according to

[0360] , wherein the concentration of the trimethylene glycol in the ferrocyanide-containing partial composition is 70 wt / vol% or less.

[0373]

[0365] The 4 - aminoantipyrine - containing partial composition or the Trinder reagent - containing partial composition contains a stabilizer for the protease, the stabilizer for the protease is ethylene glycol, and the concentration of ethylene glycol in the 4 - aminoantipyrine - containing partial composition or the Trinder reagent - containing partial composition is 40 wt / vol% or more and 80 wt / vol% or less. The method for stabilizing a glycated protein measurement reagent according to any one of

[0297] to

[0342] .

[0374]

[0366] The concentration of ethylene glycol in the 4 - aminoantipyrine - containing partial composition or the Trinder reagent - containing partial composition is 42.5 wt / vol% or more. The method for stabilizing a glycated protein measurement reagent according to

[0365] .

[0375]

[0367] The concentration of ethylene glycol in the 4 - aminoantipyrine - containing partial composition or the Trinder reagent - containing partial composition is 45 wt / vol% or more. The method for stabilizing a glycated protein measurement reagent according to

[0365] .

[0376]

[0368] The concentration of ethylene glycol in the 4 - aminoantipyrine - containing partial composition or the Trinder reagent - containing partial composition is 75 wt / vol% or less. The method for stabilizing a glycated protein measurement reagent according to

[0365] .

[0377]

[0369] The concentration of ethylene glycol in the 4 - aminoantipyrine - containing partial composition or the Trinder reagent - containing partial composition is 70 wt / vol% or less. The method for stabilizing a glycated protein measurement reagent according to

[0365] .

[0378]

[0370] The ferrocyanide-containing partial composition contains a stabilizer for the protease, the stabilizer for the protease is ethylene glycol, and the concentration of ethylene glycol in the ferrocyanide-containing partial composition is 40 wt / vol% or more and 80 wt / vol% or less. The method for stabilizing a glycated protein measurement reagent according to any one of

[0315] to

[0332] .

[0379]

[0371] The concentration of ethylene glycol in the ferrocyanide-containing partial composition is 42.5 wt / vol% or more. The method for stabilizing a glycated protein measurement reagent according to

[0370] .

[0380]

[0372] The concentration of ethylene glycol in the ferrocyanide-containing partial composition is 45 wt / vol% or more. The method for stabilizing a glycated protein measurement reagent according to

[0370] .

[0381]

[0373] The concentration of ethylene glycol in the ferrocyanide-containing partial composition is 75 wt / vol% or less. The method for stabilizing a glycated protein measurement reagent according to

[0370] .

[0382]

[0374] The concentration of ethylene glycol in the ferrocyanide-containing partial composition is 70 wt / vol% or less. The method for stabilizing a glycated protein measurement reagent according to

[0370] .

[0383]

[0375] The 4-aminoantipyrine-containing partial composition or the Trinder reagent-containing partial composition contains a stabilizer for the protease, the stabilizer for the protease is 2-carboxyphenylboronic acid, and the concentration of 2-carboxyphenylboronic acid in the 4-aminoantipyrine-containing partial composition or the Trinder reagent-containing partial composition is 0.2 wt / vol% or more and 10 wt / vol% or less. The method for stabilizing a glycated protein measurement reagent according to any one of

[0297] to

[0342] and

[0344] .

[0384] The method for stabilizing a glycated protein measurement reagent according to

[0375] , wherein the concentration of the 2-carboxyphenylboronic acid in the 4-aminoantipyrine-containing partial composition or the Trinder reagent-containing partial composition is 0.45 wt / vol% or more.

[0385] The method for stabilizing a glycated protein measurement reagent according to

[0375] , wherein the concentration of the 2-carboxyphenylboronic acid in the 4-aminoantipyrine-containing partial composition or the Trinder reagent-containing partial composition is 0.5 wt / vol% or more.

[0386] The method for stabilizing a glycated protein measurement reagent according to

[0375] , wherein the concentration of the 2-carboxyphenylboronic acid in the 4-aminoantipyrine-containing partial composition or the Trinder reagent-containing partial composition is 7.5 wt / vol% or less.

[0387] The method for stabilizing a glycated protein measurement reagent according to

[0375] , wherein the concentration of the 2-carboxyphenylboronic acid in the 4-aminoantipyrine-containing partial composition or the Trinder reagent-containing partial composition is 5 wt / vol% or less.

[0388] The method for stabilizing a glycated protein measurement reagent according to any one of

[0315] to

[0332] , wherein the ferrocyanide-containing partial composition contains a stabilizer for the protease, the stabilizer for the protease is 2-carboxyphenylboronic acid, and the concentration of the 2-carboxyphenylboronic acid in the ferrocyanide-containing partial composition is 0.2 wt / vol% or more and 10 wt / vol% or less.

[0389] The method for stabilizing a glycated protein measurement reagent according to

[0380] , wherein the concentration of the 2-carboxyphenylboronic acid in the ferrocyanide-containing partial composition is 0.45 wt / vol% or more.

[0390] The method for stabilizing a glycated protein measurement reagent according to

[0380] , wherein the concentration of the 2-carboxyphenylboronic acid in the ferrocyanide-containing partial composition is 0.5 wt / vol% or more.

[0391] The method for stabilizing a glycated protein measurement reagent according to

[0380] , wherein the concentration of the 2-carboxyphenylboronic acid in the ferrocyanide-containing partial composition is 7.5 wt / vol% or less.

[0392] The method for stabilizing a glycated protein measurement reagent according to

[0380] , wherein the concentration of the 2-carboxyphenylboronic acid in the ferrocyanide-containing partial composition is 5 wt / vol% or less.

[0393] The method for stabilizing a glycated protein measurement reagent according to any one of

[0297] to

[0384] , wherein the trinder reagent-containing partial composition and the 4-aminoantipyrine-containing partial composition are in a liquid state.

[0394] The method for stabilizing a glycated protein measurement reagent according to any one of

[0315] to

[0332] , wherein the trinder reagent-containing partial composition, the 4-aminoantipyrine-containing partial composition, and the ferrocyanide-containing partial composition are in a liquid state.

[0395] The method for stabilizing a glycated protein measurement reagent according to any one of

[0297] to

[0386] , wherein the ferrocyanide is potassium ferrocyanide.

[0396] The method for stabilizing a glycated protein measurement reagent according to any one of

[0297] to

[0387] , wherein the trinder reagent is N,N-bis(4-sulfobutyl)-3-methylaniline.

[0397] The method for stabilizing a glycated protein measurement reagent according to any one of

[0297] to

[0388] , wherein the glycated protein is glycated albumin.

[0398]

[0390] The method for stabilizing a glycated protein measurement reagent according to any one of

[0297] to

[0389] , wherein the Trinder reagent-containing partial composition is stored at 2°C or higher and 10°C or lower.

[0399]

[0391] The method for stabilizing a glycated protein measurement reagent according to any one of

[0297] to

[0390] , wherein the 4-aminoantipyrine-containing partial composition is stored at 2°C or higher and 10°C or lower.

[0400]

[0392] The method for stabilizing a glycated protein measurement reagent according to any one of

[0315] to

[0332] , wherein the ferrocyanide-containing partial composition is stored at 2°C or higher and 10°C or lower.

[0401]

[0393] The method for stabilizing a glycated protein measurement reagent according to any one of

[0297] to

[0392] , wherein the stabilizer for the protease excludes dimethyl sulfoxide.

[0402]

[0394] The method for stabilizing a glycated protein measurement reagent according to any one of

[0297] to

[0393] , wherein either the Trinder reagent-containing partial composition or the 4-aminoantipyrine-containing partial composition further contains peroxidase.

[0403]

[0395] The ratio of the measurement sensitivity after storing the Trinder reagent-containing partial composition and the 4-aminoantipyrine-containing partial composition at 5°C for 1 year to the measurement sensitivity after storing the Trinder reagent-containing partial composition and the 4-aminoantipyrine-containing partial composition at 5°C for 28 days is 80% or more and 110% or less. The method for stabilizing a glycated protein measurement reagent according to any one of

[0297] to

[0394] .

[0404] The ratio of the measurement sensitivity of the Trinder reagent-containing partial composition, the 4-aminoantipyrine-containing partial composition, and the ferrocyanide-containing partial composition after storage at 5°C for 28 days to the measurement sensitivity of the Trinder reagent-containing partial composition, the 4-aminoantipyrine-containing partial composition, and the ferrocyanide-containing partial composition after storage at 5°C for 1 year is 80% or more and 110% or less. A method for stabilizing a glycated protein measurement reagent according to any one of

[0315] to

[0332] .

Advantages of the Invention

[0405] According to the present invention, it is possible to provide a glycated protein measurement reagent, a method for measuring glycated protein, a method for storing a glycated protein measurement reagent, and a method for stabilizing a glycated protein measurement reagent that suppress an increase in reagent blank.

Brief Description of the Drawings

[0406]

Figure 1

Figure 2

Figure 3

Figure 4

Modes for Carrying Out the Invention

[0407] Hereinafter, preferred embodiments of the present invention (hereinafter referred to as "embodiments") will be described in detail. Note that the embodiments shown below exemplify devices and methods for embodying the technical idea of this invention, and the technical idea of this invention does not specify combinations of constituent members, etc. to those described below. Various changes can be made to the technical idea of this invention within the scope of the claims.

[0408] The glycated protein measurement reagent according to the embodiment contains at least a Trinder reagent, 4-aminoantipyrine, a protease, a stabilizer for the protease, and a ferrocyanide. At least the Trinder reagent is contained in a Trinder reagent-containing partial composition, at least 4-aminoantipyrine is contained in a 4-aminoantipyrine-containing partial composition, and the stabilizer is a stabilizer that makes the redox potential of the ferrocyanide greater than 0.058 V when mixed with the stabilizer, and the redox potential is the redox potential in a reaction system containing the stabilizer and the ferrocyanide and not containing glycated protein.

[0409] In the present disclosure, a reagent is a reagent for measuring a measurement target, and is, for example, an aggregate of partial compositions. A reagent can detect a measurement target by itself. A partial composition is a composition that constitutes a part of a reagent. A partial composition alone cannot measure a measurement target.

[0410] The ferrocyanide may be contained in at least one of the Trinder reagent-containing partial composition and the 4-aminoantipyrine-containing partial composition. Alternatively, the ferrocyanide may be contained in a ferrocyanide-containing partial composition different from the Trinder reagent-containing partial composition and the 4-aminoantipyrine-containing partial composition.

[0411] The glycated protein measurement reagent according to the embodiment may be stored, distributed, and used in the form of a kit. Examples of the form of the kit include a two-reagent kit, a three-reagent kit, and the like. For example, in the case of a two-reagent kit, ferricyanide is included in at least one of the partial composition containing the Trinder reagent and the partial composition containing 4-aminoantipyrine. For example, in the case of a three-reagent kit, ferricyanide is included in a partial composition containing ferricyanide different from the partial composition containing the Trinder reagent and the partial composition containing 4-aminoantipyrine. The three-reagent kit in which the Trinder reagent, 4-aminoantipyrine, and ferricyanide are stored separately has a higher residual detection sensitivity rate because the decrease in sensitivity is suppressed compared to the two-reagent kit. However, it does not prevent the glycated protein measurement reagent according to the embodiment from being a two-reagent type.

[0412] Glycated proteins are, for example, glycated albumin (GA), glycated hemoglobin (such as HbA1c), etc.

[0413] The partial composition containing the Trinder reagent may further contain at least one of glycated amino acid oxidase, ferricyanide, protease, a stabilizer for protease, and peroxidase. Or the partial composition containing the Trinder reagent may further contain at least one of glycated amino acid oxidase, ferricyanide, and peroxidase. In this case, at least one of the partial composition containing 4-aminoantipyrine and the partial composition containing ferricyanide may further contain protease and a stabilizer for protease. The partial composition containing 4-aminoantipyrine may further contain ferricyanide. Also, at least one of the partial composition containing 4-aminoantipyrine and the partial composition containing ferricyanide may further contain glycated amino acid oxidase or peroxidase.

[0414] The 4-aminoantipyrine-containing partial composition may further contain at least one of glycated amino acid oxidase, ferricyanide, protease, a stabilizer for protease, and peroxidase. Alternatively, the 4-aminoantipyrine-containing partial composition may further contain at least one of glycated amino acid oxidase, ferricyanide, and peroxidase. In this case, at least one of the Trinder reagent-containing partial composition and the ferricyanide-containing partial composition may further contain protease and a stabilizer for protease. The Trinder reagent-containing partial composition may further contain ferricyanide. Also, at least one of the Trinder reagent-containing partial composition and the ferricyanide-containing partial composition may further contain glycated amino acid oxidase or peroxidase.

[0415] When the glycated protein measurement reagent according to the embodiment is brought into contact with a protein (glycated protein) to which glucose contained in a specimen (sample specimen) to be measured is bound, the glycated protein is decomposed into glycated amino acids or glycated peptides by protease. The glycated amino acids or glycated peptides are decomposed into amino acids or peptides and glucosone by glycated amino acid oxidase. At this time, water and oxygen react with each other by glycated amino acid oxidase to generate hydrogen peroxide. The Trinder reagent and 4-aminoantipyrine undergo an oxidative condensation reaction and develop color in the presence of hydrogen peroxide and peroxidase. For example, when N,N-bis(4-sulfobutyl)-3-methylaniline (TODB) is used as the Trinder reagent, a color development reaction that develops a blue-violet color occurs. The blue-violet color development is detected by measuring the absorbance at a wavelength of 546 nm. Based on the absorbance at a wavelength of 546 nm, the concentration of the glycated protein contained in the specimen is quantified.

[0416] In the glycated protein measurement reagent according to the embodiment, when ferrocyanide is contained in at least one of the Trinder reagent-containing partial composition and the 4-aminoantipyrine-containing partial composition, in the normal usage method of the glycated protein measurement reagent according to the embodiment, first, the Trinder reagent-containing partial composition is added to the sample specimen, and the mixture of the sample specimen and the Trinder reagent-containing partial composition is left to stand for several minutes, for example, about 5 to 10 minutes, and then the 4-aminoantipyrine-containing partial composition is added to the mixture, and the glycated protein can be measured by measuring the degree of color development. However, in some cases, when adding to the sample specimen, the Trinder reagent-containing partial composition and the 4-aminoantipyrine-containing partial composition may be added and mixed simultaneously, or the Trinder reagent-containing partial composition and the 4-aminoantipyrine-containing partial composition may be premixed and used immediately, or first, the 4-aminoantipyrine-containing partial composition may be added to the sample specimen, and then the Trinder reagent-containing partial composition may be added.

[0417] In the glycated protein measurement reagent according to the embodiment, when ferrocyanide is included in the ferrocyanide-containing partial composition, for example, the 4-aminoantipyrine-containing partial composition and the ferrocyanide-containing partial composition are mixed in advance, and the 4-aminoantipyrine and ferrocyanide-containing partial composition is prepared. In that case, the Trinder reagent-containing partial composition is first added to the sample specimen, and the mixture of the sample specimen and the Trinder reagent-containing partial composition is left for several minutes, for example, about 5 to 10 minutes, and then the 4-aminoantipyrine and ferrocyanide-containing partial composition is added to the mixture, and the degree of color development is measured, whereby the glycated protein can be measured. However, in some cases, when adding to the sample specimen, the Trinder reagent-containing partial composition and the 4-aminoantipyrine and ferrocyanide-containing partial composition may be added and mixed simultaneously, or the Trinder reagent-containing partial composition and the 4-aminoantipyrine and ferrocyanide-containing partial composition may be mixed in advance and used immediately, or first the 4-aminoantipyrine and ferrocyanide-containing partial composition may be added to the sample specimen, and then the Trinder reagent-containing partial composition may be added. Or, in advance, the Trinder reagent-containing partial composition and the ferrocyanide-containing partial composition may be mixed, and the Trinder reagent and ferrocyanide-containing partial composition may be prepared. In that case, the 4-aminoantipyrine-containing partial composition is first added to the sample specimen, and the mixture of the sample specimen and the 4-aminoantipyrine-containing partial composition is left for several minutes, for example, about 5 to 10 minutes, and then the Trinder reagent and ferrocyanide-containing partial composition is added to the mixture, and the degree of color development is measured, whereby the glycated protein can be measured. However, in some cases, when adding to the sample specimen, the 4-aminoantipyrine-containing partial composition and the Trinder reagent and ferrocyanide-containing partial composition may be added and mixed simultaneously, or the 4-aminoantipyrine-containing partial composition and the Trinder reagent and ferrocyanide-containing partial composition may be mixed in advance and used immediately, or first the Trinder reagent and ferrocyanide-containing partial composition may be added to the sample specimen, and then the 4-aminoantipyrine-containing partial composition may be added.Alternatively, the Trinder reagent-containing partial composition and the 4-aminoantipyrine-containing partial composition may be premixed and used as the Trinder reagent and 4-aminoantipyrine-containing partial composition. In that case, first, the cyanide-containing partial composition and the sample specimen are mixed, and the mixture of the sample specimen and the cyanide-containing partial composition is left standing for several minutes, for example, about 5 to 10 minutes, and then the Trinder reagent and 4-aminoantipyrine-containing partial composition may be mixed. Also, in some cases, when adding to the sample specimen, the cyanide-containing partial composition, the Trinder reagent, and the 4-aminoantipyrine-containing partial composition may be added and mixed simultaneously, or the cyanide-containing partial composition, the Trinder reagent, and the 4-aminoantipyrine-containing partial composition may be premixed and added, or first, the Trinder reagent and 4-aminoantipyrine-containing partial composition may be mixed with the sample specimen, and then the cyanide-containing partial composition may be added. Alternatively, the sample specimen, the Trinder reagent-containing partial composition, the 4-aminoantipyrine-containing partial composition, and the cyanide-containing partial composition may be added and mixed simultaneously, or the Trinder reagent-containing partial composition, the 4-aminoantipyrine-containing partial composition, and the cyanide-containing partial composition may be premixed and added to the sample specimen.

[0418] In order to quantify glycated proteins, usually, one or more calibration substances (calibrators) with known glycated protein concentrations are measured and calibration is often performed. As a method for preparing the calibrator and a calibration method, those skilled in the art can implement based on publicly known information. For example, it can be appropriately implemented with reference to the descriptions in International Publication No. 2001 / 094618, Japanese Patent Laid-Open No. 2005-261383, etc. Also, in order to correct fluctuations in the measured values due to a decrease in sensitivity during storage of the measurement reagent, it is normal to perform calibration regularly, for example, once a month. On the other hand, for a reagent in which a decrease in sensitivity is suppressed, if calibration is performed only at the first time, stable measured values can be obtained over a long period without performing calibration in subsequent measurements, and it is known that non-calibration measurement becomes possible.

[0419] The protease can be included in at least any one of a composition containing a Trinder's reagent, a composition containing 4-aminoantipyrine, and a composition containing a ferrocyanide. The protease may be any protease that can effectively generate glycated amino acids or glycated peptides by decomposing glycated proteins. The protease is, for example, an endopeptidase. Examples of endopeptidases include serine endopeptidases. The protease includes proteases derived from microorganisms such as the genus Bacillus, the genus Streptomyces, the genus Tritirachium, and the genus Aspergillus, as well as genetically recombinant proteases thereof.Examples of proteases derived from the genus Bacillus include Alcalase, Neutrase, Esperase, Savinase (manufactured by Novozymes), Bioprase OP, Bioprase SP-20FG, Bioprase 30L, Bioprase 30G, Bioprase AL-15FG, Bioprase APL-30, Protease CL-15 (manufactured by Nagase ChemteX), Protin SD PC-10CF, Samoase PC-10F, Protin SD-AY10, Protin SD-NY10 (manufactured by Amano Enzyme), Multifect PR6L, Optimase PR40L, Optimase PR40X, Optimase PR40E (manufactured by Danisco Japan), Orientase 22BF, Nucleasin, Orientase 10NL, Orientase 90N (manufactured by HBI), Aloase AP-10, Aloase NP-10, Aloase XA-10 (manufactured by Yakult Pharmaceutical Industry), Subtilisin, Bacterial Proteinase Type XXIV, Protease Type VIII derived from Bacillus licheniformis, Protease Type IX derived from Bacillus polymyxa, Thermolysin Type X derived from Geobacillus Stearothermophilus, Protease Type XV derived from Bacillus polymyxa, Protease Type XXVII derived from Bacillus sp., Protease Type XXXI derived from Bacillus licheniformis (manufactured by Sigma), Thermolysin (manufactured by Fujifilm Wako Pure Chemical), Dispase I, Dispase II (manufactured by Godo Shusei), Neutral Proteinase (manufactured by TOYOBO USA), etc. Examples of proteases derived from the genus Streptomyces include Pronase, Protease Type XIV derived from Streptomyces griseus (manufactured by Sigma), Alkalophilic proteinase (manufactured by TOYOBO USA), Denazyme PMC SOFTER (manufactured by Nagase ChemteX), etc. Examples of proteases derived from the genus Tritirachium include Proteinase K (manufactured by Sigma), etc.Examples of proteases derived from Aspergillus include protease P "Amano" 3SD, protease A "Amano" SD, protease M "Amano" SD (all manufactured by Amano Enzyme Inc.), Sumizyme MP, Sumizyme LPL-G, Sumizyme LP50D, Sumizyme AP (all manufactured by Shin Nippon Chemical Industry Co., Ltd.), Orientase OP, Orientase AY (all manufactured by HBI Co., Ltd.), protease type XIII derived from Aspergillus saitoi, protease type XIX derived from Aspergillus sojae, protease type XXIII derived from Aspergillus melleus, protease derived from Aspergillus oryzae (all manufactured by Sigma), Denazyme AP, Denapsin 2P (all manufactured by Nagase ChemteX Corporation), Pancidase NP-2, Pancidase MP, Pancidase P (all manufactured by Yakult Pharmaceutical Industry Co., Ltd.), and the like. Among these, Alcalase, Bioprase SP-20FG, Protin SD-AY10, Multifect PR6L, Optimase PR40L, Alowase XA-10, subtilisin, pronase, proteinase K, and PR "Amano" K are preferred.

[0420] From another perspective, examples of preferred proteases include those with enzyme numbers EC:3.4 or EC:3.4.21. The protease of EC:3.4.21.62 is also an even more preferred example.

[0421] The stabilizer for protease according to this embodiment can be contained in at least any one of the Trinder reagent-containing partial composition, the 4-aminoantipyrine-containing partial composition, and the ferrocyanide-containing partial composition. The stabilizer for protease according to this embodiment makes the redox potential of ferrocyanide greater than 0.058V when the stabilizer for the protease and ferrocyanide are mixed. The redox potential is the redox potential measured in a reaction system containing the stabilizer for protease and ferrocyanide and not containing glycated protein. The reaction system may further contain N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid and sodium chloride. The redox potential may be measured using a silver / silver chloride electrode as a reference electrode. The reaction system for measuring the redox potential will be described in more detail in Example 1 later.

[0422] The lower limit of the redox potential may be, for example, greater than 0.058V, 0.070V or more, 0.084V or more, or 0.112V or more. The upper limit of the redox potential may be, for example, 0.400V or less, 0.300V or less, 0.250V or less, or 0.235V or less.

[0423] Examples of the stabilizer for protease include, for example, propylene glycol, trimethylene glycol, ethylene glycol, and carboxyphenylboronic acid, and combinations thereof. An example of carboxyphenylboronic acid is 2-carboxyphenylboronic acid. From the viewpoints of effective concentration, solubility, cost, etc., the stabilizer is preferably propylene glycol. In this embodiment, the stabilizer for protease does not contain dimethyl sulfoxide. A stabilizer that does not make the redox potential of ferrocyanide greater than 0.058V when mixed with the stabilizer for protease and ferrocyanide is excluded from the stabilizer for protease according to this embodiment.

[0424] Ferrocyanide may be included in at least any one of the Trinder's reagent-containing partial composition, the 4-aminoantipyrine-containing partial composition, and the ferrocyanide-containing partial composition. Ferrocyanide suppresses the influence of bilirubin contained in the specimen on the measurement of glycated proteins. As an example of ferrocyanide, any compound containing ferrocyanide ions may be used, for example, potassium ferrocyanide (Fe(CN) 6 K 4 ) and sodium ferrocyanide (Fe(CN) 6 Na 4 ).

[0425] In the glycated protein measurement reagent according to the embodiment, when ferrocyanide is included in at least any one of the Trinder's reagent-containing partial composition and the 4-aminoantipyrine-containing partial composition, the protease and the protease stabilizer are included in, for example, either one of the Trinder's reagent-containing partial composition and the 4-aminoantipyrine-containing partial composition. Alternatively, the protease, the protease stabilizer, and ferrocyanide are included in, for example, either one of the Trinder's reagent-containing partial composition and the 4-aminoantipyrine-containing partial composition. Preferably, the protease, the protease stabilizer, and ferrocyanide are included in the 4-aminoantipyrine-containing partial composition.

[0426] In the glycated protein measurement reagent according to the embodiment, when ferrocyanide is included in the ferrocyanide-containing partial composition, the protease and the protease stabilizer are included in, for example, any one of the Trinder's reagent-containing partial composition, the 4-aminoantipyrine-containing partial composition, and the ferrocyanide-containing partial composition. For example, the protease and the protease stabilizer are included in the Trinder's reagent-containing partial composition. Alternatively, the protease and the protease stabilizer are included in the 4-aminoantipyrine-containing partial composition. Or alternatively, the protease and the protease stabilizer are included in the ferrocyanide-containing partial composition.

[0427] The glycated amino acid oxidase contained in at least any one of the Trinder reagent-containing partial composition, the 4-aminoantipyrine-containing partial composition, and the ferricyanide-containing partial composition may be any glycated amino acid oxidase that effectively acts on glycated amino acids or glycated peptides derived from human proteins. Examples thereof include glycated amino acid oxidases derived from the genera Gibberella, Aspergillus, Candida, Penicillium, Fusarium, Acremonium, Debaryomyces, and Corynebacterium, and genetically recombinant glycated amino acid oxidase mutants and the like. More specifically, examples include ketoamine oxidase (KAOD, manufactured by Asahi Kasei Pharma Corporation, described in Clinica Chimica Acta, 2002, 324, p. 61-71), mutant KAOD (KAOD-V, manufactured by Asahi Kasei Pharma Corporation, described in Patent Document 1), and FAOD-E (manufactured by Kikkoman Corporation) and the like.

[0428] Examples of the Trinder's reagent contained in the Trinder's reagent-containing partial composition include, specifically, N-(3-sulfopropyl)aniline (HALPS), N-ethyl-N-(3-sulfopropyl)-3-methylaniline (TOPS), N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethylaniline (MAOS), N-(3-sulfopropyl)-3,5-dimethoxyaniline (HDAPS), N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline (HDAOS), N-ethyl-N-(3-sulfopropyl)-3,5-dimethoxyaniline (DAPS), N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline (DAOS), N-ethyl-N-(3-sulfopropyl)aniline (ALPS), N-ethyl-N-(3-sulfopropyl)-3-methoxyaniline (ADPS), N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methoxyaniline (ADOS), N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline (TOOS), N,N-bis(4-sulfobutyl)-3-methylaniline (TODB), and 3-hydroxy-2,4,6-triiodobenzoic acid (HTIB), and their salts and the like. Among these, TOPS, ALPS, TOOS, and TODB are preferred.

[0429] The color development wavelength when using TOPS is 550 nm. The color development wavelength when using ALPS is 561 nm. The color development wavelength when using TOOS is 555 nm. The color development wavelength when using TODB is 550 nm. However, the wavelength measured by the device for measuring absorbance does not necessarily have to exactly match the color development wavelength. For example, it is possible to measure the color development reaction when using each of TOPS, ALPS, TOOS, and TODB with a device that measures absorbance at a wavelength of 546 nm.

[0430] In addition, when the Trinder reagent and 4-aminoantipyrine are included in the same reagent, they will naturally undergo an oxidative condensation reaction and develop color during storage. Therefore, the Trinder reagent is contained in the Trinder reagent-containing partial composition, and 4-aminoantipyrine is contained in the 4-aminoantipyrine-containing partial composition.

[0431] Conventionally, a non-specific color reaction may occur due to peroxides derived from measurement reagents, specimens, etc., and this absorbance may reduce the measurement accuracy. These non-specific colorations are called reagent blanks. In contrast, peroxidase contained in any of the Trinder reagent-containing partial composition, the 4-aminoantipyrine-containing partial composition, or the ferricyanide-containing partial composition has a function of promoting the oxidative condensation reaction between the Trinder reagent and 4-aminoantipyrine and eliminating hydrogen peroxide to reduce the reagent blank. Peroxidase from any origin can be used, and examples thereof include peroxidase derived from plants such as horseradish, and peroxidase derived from microorganisms such as bacteria and molds. Specifically, Peroxidase from horseradish (manufactured by Sigma), peroxidase, derived from horseradish (manufactured by Fujifilm Wako Pure Chemical Corporation), PO “AMANO” 3 (manufactured by Amano Enzyme Inc.), etc. can be mentioned.

[0432] At least one of the 4-aminoantipyrine-containing partial composition and the ferricyanide-containing partial composition may further contain a chelating agent. It is preferable to use a salt of the chelating agent because pH fluctuations are suppressed.

[0433] Examples of chelating agents include ethylenediaminetetraacetic acid, glycol ether diamine tetraacetic acid, N-(2-hydroxyethyl)iminodiacetic acid, nitrilotris(methylphosphonic acid), nitrilotriacetic acid, trans-1,2-diaminocyclohexane-N,N,N,N-tetraacetic acid, iminodiacetic acid, diethylenetriamine-N,N,N’,N”,N”-pentaacetic acid, 1,3-diamino-2-propanol-N,N,N’,N’-tetraacetic acid, gluconic acid, malic acid, succinic acid, citric acid, salicylic acid, tartaric acid, N-[tris(hydroxymethyl)methyl]glycine, N,N-bis(2-hydroxyethyl)glycine, and salts thereof. Preferred chelating agents are, for example, citric acid and its salts.

[0434] The salts of the chelating agent are not particularly limited as long as they can form salts, and may be either acid addition salts or base addition salts, or may be in the form of zwitterions. Examples of base addition salts include base addition salts with inorganic bases such as sodium, potassium, magnesium, calcium, aluminum, etc., or base addition salts with organic bases such as methylamine, 2-aminoethanol, arginine, lysine, or ornithine, etc. Among these, base addition salts with inorganic bases are preferred. Representative base addition salts include sodium salts.

[0435] Each of the 4-aminoantipyrine-containing partial composition, the Trinder reagent-containing partial composition, and the ferrocyanide-containing partial composition may further contain water, an organic solvent, etc. as a solvent. Examples of organic solvents include alcohol solvents (alcohols having 1 to 18 carbon atoms, specifically, methanol, butanol, ethylene glycol, glycerin, etc.), ketone solvents (acetone, methyl ethyl ketone, etc.), and ether solvents (diethyl ether, ethylene glycol monoalkyl ether, cyclic ethers represented by tetrahydrofuran, etc.).

[0436] In addition, each of the 4-aminoantipyrine-containing partial composition, the Trinder reagent-containing partial composition, and the ferricyanide-containing partial composition may contain a buffer.Examples of buffers include buffers using N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES), 3-[4-(2-hydroxyethyl)-1-piperazinyl]propanesulfonic acid (EPPS), 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES), 2-hydroxy-3-[4-(2-hydroxyethyl)-1-piperazinyl]propanesulfonic acid (HEPPSO), N-(2-acetamido)-2-aminoethanesulfonic acid (ACES), N-(2-acetamido)iminodiacetic acid (ADA), N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES), N,N-bis(2-hydroxyethyl)glycine (Bicine), bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane (Bis-Tris), N-cyclohexyl-3-aminopropanesulfonic acid (CAPS), N-cyclohexyl-2-hydroxy-3-aminopropanesulfonic acid (CAPSO), N-cyclohexyl-2-aminoethanesulfonic acid (CHES), 3-[N,N-bis(2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid (DIPSO), 2-morpholinoethanesulfonic acid (MES), 3-morpholinopropanesulfonic acid (MOPS), 2-hydroxy-3-morpholinopropanesulfonic acid (MOPSO), piperazine-1,4-bis(2-ethanesulfonic acid) (PIPES), piperazine-1,4-bis(2-hydroxy-3-propanesulfonic acid) (POPSO), N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS), 2-hydroxy-N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPSO), N-[tris(hydroxymethyl)methyl]glycine (Tricine), and tris(hydroxymethyl)aminomethane (Tris), etc., or boric acid, ammonia, glycine, carbonic acid, acetic acid, phosphoric acid, diethanolamine, p-phenolsulfonic acid, 2-amino-2-methylpropane-1,3-diol, cacodylic acid, maleic acid, veronal, and 3,3-dimethylglutaric acid, etc.

[0437] In addition, each of the 4-aminoantipyrine-containing partial composition, the Trinder reagent-containing partial composition, and the ferricyanide-containing partial composition may further contain, as other additives, a preservative, an enzyme stabilizer, a surfactant, and other enzymes. Examples of the preservative include sodium azide and ProClin. Any stabilizer that stabilizes the enzyme during storage can be used as the enzyme stabilizer. Examples thereof include sugars such as cyclodextrin, sucrose, mannose, fructose, lactose, galactose, and trehalose, and sugar alcohols such as sorbitol and mannitol. Any of a cationic surfactant, an anionic surfactant, an amphoteric surfactant, and a nonionic surfactant may be used as the surfactant. Examples of nonionic surfactants include polyoxyethylene alkyl ether, polyoxyethylene alkyl phenyl ether, polyoxyethylene polyoxypropylene alkyl ether, sorbitan fatty acid ester, polyoxyethylene sorbitan fatty acid ester, glycerin fatty acid ester, and polyoxyethylene polyoxypropylene block polymer. Examples of amphoteric surfactants include alkyl betaine, betaine acetate, sulfobetaine, and alkylamine oxide. Examples of other enzymes include ascorbic acid oxidase, bilirubin oxidase, catalase, etc., which are used to suppress the influence of ascorbic acid, bilirubin, or endogenous peroxide contained in the specimen on the measured value.

[0438] The concentration of 4-aminoantipyrine in the 4-aminoantipyrine-containing partial composition may be a concentration sufficient to react with the generated hydrogen peroxide. Examples of the lower limit include 0.01 mmol / L or more, preferably 0.1 mmol / L or more, and more preferably 0.5 mmol / L or more. From the perspective of cost, examples of the upper limit include 100 mmol / L or less, preferably 50 mmol / L or less, and more preferably 30 mmol / L or less.

[0439] The concentration of protease in the 4-aminoantipyrine-containing partial composition, the Trinder reagent-containing partial composition, or the ferrocyanide-containing partial composition may be any concentration that can decompose glycated proteins within an appropriate time. As the lower limit, 100 U / mL or more, preferably 1 kU / mL or more, more preferably 10 kU / mL or more is exemplified. Also, from the perspective of cost, as the upper limit, 1000 kU / mL or less, preferably 200 kU / mL or less, more preferably 100 kU / mL or less is exemplified.

[0440] The concentration of the protease stabilizer in the glycated protein measurement reagent according to the embodiment is a concentration that can make the redox potential of ferrocyanide greater than 0.058 V when the protease stabilizer and ferrocyanide are mixed in a reaction system containing the protease stabilizer and ferrocyanide and not containing glycated proteins.

[0441] For example, in the glycated protein measurement reagent according to the embodiment, when the Trinder reagent-containing partial composition contains a protease stabilizer and ferrocyanide, the concentration of the protease stabilizer in the Trinder reagent-containing partial composition is adjusted to be the same as the concentration of the protease stabilizer that can make the redox potential of ferrocyanide greater than 0.058 V when the protease stabilizer and ferrocyanide are mixed in a reaction system containing the protease stabilizer and ferrocyanide and not containing glycated proteins.

[0442] For example, when the 4-aminoantipyrine-containing partial composition contains a protease stabilizer and ferrocyanide, the concentration of the protease stabilizer in the 4-aminoantipyrine-containing partial composition is adjusted to be the same as the concentration of the protease stabilizer that can make the redox potential of ferrocyanide greater than 0.058 V when the protease stabilizer and ferrocyanide are mixed in a reaction system containing the protease stabilizer and ferrocyanide and not containing glycated proteins.

[0443] For example, when the trinder reagent-containing partial composition contains a protease stabilizer and the 4-aminoantipyrine-containing partial composition contains a ferrocyanide, the concentration when the trinder reagent-containing partial composition and the 4-aminoantipyrine-containing partial composition are mixed is adjusted to be the same as the concentration of the protease stabilizer that can make the redox potential of the ferrocyanide when the protease stabilizer and the ferrocyanide are mixed greater than 0.058V in a reaction system containing the protease stabilizer and the ferrocyanide and not containing glycated proteins.

[0444] For example, when the 4-aminoantipyrine-containing partial composition contains a protease stabilizer and the trinder reagent-containing partial composition contains a ferrocyanide, the concentration when the 4-aminoantipyrine-containing partial composition and the trinder reagent-containing partial composition are mixed is adjusted to be the same as the concentration of the protease stabilizer that can make the redox potential of the ferrocyanide when the protease stabilizer and the ferrocyanide are mixed greater than 0.058V in a reaction system containing the protease stabilizer and the ferrocyanide and not containing glycated proteins.

[0445] For example, when the 4-aminoantipyrine-containing partial composition or the trinder reagent-containing partial composition contains a protease stabilizer and the ferrocyanide is contained in the ferrocyanide-containing partial composition, the concentration of the protease stabilizer when either one of the trinder reagent-containing partial composition or the 4-aminoantipyrine-containing partial composition containing the protease stabilizer is mixed with the ferrocyanide-containing partial composition is adjusted to be the same as the concentration of the protease stabilizer that can make the redox potential of the ferrocyanide when the protease stabilizer and the ferrocyanide are mixed greater than 0.058V in a reaction system containing the protease stabilizer and the ferrocyanide and not containing glycated proteins.

[0446] For example, when the ferrocyanide-containing partial composition contains a ferrocyanide and a stabilizer for protease, the concentration of the stabilizer for protease in the ferrocyanide-containing partial composition is adjusted to be the same as the concentration of the stabilizer for protease that can make the redox potential of the ferrocyanide greater than 0.058V when the stabilizer for protease and the ferrocyanide are mixed in a reaction system containing no glycated protein.

[0447] When the 4-aminoantipyrine-containing partial composition or the Trinder reagent-containing partial composition contains a stabilizer for protease and the stabilizer for protease is propylene glycol, the lower limit of the concentration of propylene glycol in the 4-aminoantipyrine-containing partial composition or the Trinder reagent-containing partial composition is, for example, 7.5 wt / vol% or more, 10 wt / vol% or more, 15 wt / vol% or more, 20 wt / vol% or more, 22.5 wt / vol% or more, 25 wt / vol% or more, 27.5 wt / vol% or more, 30 wt / vol% or more, 35 wt / vol% or more, or 37.5 wt / vol% or more. Also, the upper limit of the concentration of propylene glycol in the 4-aminoantipyrine-containing partial composition or the Trinder reagent-containing partial composition is, for example, 80 wt / vol% or less, 62.5 wt / vol% or less, 60 wt / vol% or less, 57.5 wt / vol% or less, 55 wt / vol% or less, 52.5 wt / vol% or less, or 50 wt / vol% or less.

[0448] When the 4 - aminoantipyrine - containing partial composition or the Trinder's reagent - containing partial composition contains a protease stabilizer and the protease stabilizer is trimethylene glycol, the lower limit of the concentration of trimethylene glycol in the 4 - aminoantipyrine - containing partial composition or the Trinder's reagent - containing partial composition is, for example, 40 wt / vol% or more, 42.5 wt / vol% or more, 45 wt / vol% or more, 47.5 wt / vol% or more, 50 wt / vol% or more, 52.5 wt / vol% or more, 55 wt / vol% or more, 57.5 wt / vol% or more, 60 wt / vol% or more, or 62.5 wt / vol% or more. The upper limit of the concentration of trimethylene glycol in the 4 - aminoantipyrine - containing partial composition or the Trinder's reagent - containing partial composition is, for example, 80 wt / vol% or less, 75 wt / vol% or less, or 70 wt / vol% or less.

[0449] When the 4 - aminoantipyrine - containing partial composition or the Trinder's reagent - containing partial composition contains a protease stabilizer and the protease stabilizer is ethylene glycol, the lower limit of the concentration of ethylene glycol in the 4 - aminoantipyrine - containing partial composition or the Trinder's reagent - containing partial composition is, for example, 40 wt / vol% or more, 42.5 wt / vol% or more, 45 wt / vol% or more, 47.5 wt / vol% or more, 50 wt / vol% or more, 52.5 wt / vol% or more, 55 wt / vol% or more, 57.5 wt / vol% or more, 60 wt / vol% or more, or 62.5 wt / vol% or more. The upper limit of the concentration of ethylene glycol in the 4 - aminoantipyrine - containing partial composition or the Trinder's reagent - containing partial composition is, for example, 80 wt / vol% or less, 75 wt / vol% or less, or 70 wt / vol% or less.

[0450] When the 4-aminoantipyrine-containing partial composition or the Trinder reagent-containing partial composition contains a protease stabilizer and the protease stabilizer is 2-carboxyphenylboronic acid, the lower limit of the concentration of 2-carboxyphenylboronic acid in the 4-aminoantipyrine-containing partial composition or the Trinder reagent-containing partial composition is, for example, 0.2 wt / vol% or more, 0.45 wt / vol% or more, 0.5 wt / vol% or more, 0.55 wt / vol% or more, 0.6 wt / vol% or more, 0.75 wt / vol% or more. The upper limit of the concentration of 2-carboxyphenylboronic acid in the 4-aminoantipyrine-containing partial composition or the Trinder reagent-containing partial composition is, for example, 10 wt / vol% or less, 7.5 wt / vol% or less, 5 wt / vol% or less.

[0451] When the ferrocyanide-containing partial composition contains a protease stabilizer and the protease stabilizer is propylene glycol, the lower limit of the concentration of propylene glycol in the ferrocyanide-containing partial composition is, for example, 7.5 wt / vol% or more, 10 wt / vol% or more, 15 wt / vol% or more, 20 wt / vol% or more, 22.5 wt / vol% or more, 25 wt / vol% or more, 27.5 wt / vol% or more, 30 wt / vol% or more, 35 wt / vol% or more, or 37.5 wt / vol% or more. Also, the upper limit of the concentration of propylene glycol in the ferrocyanide-containing partial composition is, for example, 80 wt / vol% or less, 62.5 wt / vol% or less, 60 wt / vol% or less, 57.5 wt / vol% or less, 55 wt / vol% or less, 52.5 wt / vol% or less, or 50 wt / vol% or less.

[0452] When the ferrocyanide-containing partial composition contains a protease stabilizer and the protease stabilizer is trimethylene glycol, the lower limit of the concentration of trimethylene glycol in the ferrocyanide-containing partial composition is, for example, 40 wt / vol% or more, 42.5 wt / vol% or more, 45 wt / vol% or more, 47.5 wt / vol% or more, 50 wt / vol% or more, 52.5 wt / vol% or more, 55 wt / vol% or more, 57.5 wt / vol% or more, 60 wt / vol% or more, or 62.5 wt / vol% or more. The upper limit of the concentration of trimethylene glycol in the ferrocyanide-containing partial composition is, for example, 80 wt / vol% or less, 75 wt / vol% or less, or 70 wt / vol% or less.

[0453] When the ferrocyanide-containing partial composition contains a protease stabilizer and the protease stabilizer is ethylene glycol, the lower limit of the concentration of ethylene glycol in the ferrocyanide-containing partial composition is, for example, 40 wt / vol% or more, 42.5 wt / vol% or more, 45 wt / vol% or more, 47.5 wt / vol% or more, 50 wt / vol% or more, 52.5 wt / vol% or more, 55 wt / vol% or more, 57.5 wt / vol% or more, 60 wt / vol% or more, or 62.5 wt / vol% or more. The upper limit of the concentration of ethylene glycol in the ferrocyanide-containing partial composition is, for example, 80 wt / vol% or less, 75 wt / vol% or less, or 70 wt / vol% or less.

[0454] When the ferrocyanide-containing partial composition contains a protease stabilizer and the protease stabilizer is 2-carboxyphenylboronic acid, the lower limit of the concentration of 2-carboxyphenylboronic acid in the ferrocyanide-containing partial composition is, for example, 0.2 wt / vol% or more, 0.45 wt / vol% or more, 0.5 wt / vol% or more, 0.55 wt / vol% or more, 0.6 wt / vol% or more, or 0.75 wt / vol% or more. The upper limit of the concentration of 2-carboxyphenylboronic acid in the ferrocyanide-containing partial composition is, for example, 10 wt / vol% or less, 7.5 wt / vol% or less, or 5 wt / vol% or less.

[0455] The concentration of ferricyanide in the Trinder reagent-containing partial composition, 4-aminoantipyrine-containing partial composition, or ferricyanide-containing partial composition only needs to be a concentration sufficient to suppress the influence of bilirubin contained in the specimen on the measurement of glycated proteins. As the lower limit, 0.001 mmol / L or more, preferably 0.01 mmol / L or more, more preferably 0.02 mmol / L or more can be exemplified. Also, from the perspective of cost, as the upper limit, 10 mmol / L or less, preferably 1 mmol / L or less, 0.9 mmol / L or less, 0.8 mmol / L or less, 0.7 mmol / L or less, 0.6 mmol / L or less, more preferably 0.5 mmol / L or less can be exemplified.

[0456] The concentration of glycated amino acid oxidase in the Trinder reagent-containing partial composition, 4-aminoantipyrine-containing partial composition, or ferricyanide-containing partial composition only needs to be a concentration sufficient to react with the generated glycated amino acids. As the lower limit, 0.1 U / mL or more, preferably 1 U / mL or more, more preferably 10 U / mL or more can be exemplified. Also, from the perspective of cost, as the upper limit, 1000 U / mL or less, preferably 200 U / mL or less, more preferably 100 U / mL or less can be exemplified.

[0457] The concentration of the Trinder reagent in the Trinder reagent-containing partial composition only needs to be a concentration sufficient to react with the generated hydrogen peroxide. As the lower limit, 0.01 mmol / L or more, preferably 0.1 mmol / L or more, more preferably 0.5 mmol / L or more can be exemplified. Also, from the perspective of cost, as the upper limit, 100 mmol / L or less, preferably 10 mmol / L or less, more preferably 5 mmol / L or less can be exemplified.

[0458] The concentration of peroxidase in the Trinder reagent-containing partial composition, 4-aminoantipyrine-containing partial composition, or cyanide-containing partial composition only needs to be a concentration sufficient to react with the generated hydrogen peroxide. As the lower limit, 0.01 U / mL or more, preferably 0.1 U / mL or more, and more preferably 1 U / mL or more are exemplified. Also, from the perspective of cost, as the upper limit, 500 U / mL or less, preferably 100 U / mL or less, and more preferably 50 U / mL or less are exemplified.

[0459] As the lower limit of the concentration of the chelating agent in the 4-aminoantipyrine-containing partial composition or cyanide-containing partial composition, 5 μmol / L or more, preferably 10 μmol / L or more, 50 μmol / L or more, 75 μmol / L or more, 1 mmol / L or more, and more preferably 1.25 mmol / L or more are exemplified. Also, for example, as the upper limit of the concentration of the chelating agent in the 4-aminoantipyrine-containing partial composition or cyanide-containing partial composition, 1000 mmol / L or less, preferably 100 mmol / L or less, 25 mmol / L or less, 15 mmol / L or less, 10 mmol / L or less, and more preferably 5 mmol / L or less are exemplified.

[0460] The Trinder reagent-containing partial composition may be liquid and may be stored until mixed with the 4-aminoantipyrine-containing partial composition. In the Trinder reagent-containing partial composition, the contained substances are mixed. The 4-aminoantipyrine-containing partial composition may be liquid and may be stored until mixed with the Trinder reagent-containing partial composition. In the 4-aminoantipyrine-containing partial composition, the contained substances are mixed. The 4-aminoantipyrine-containing partial composition and the Trinder reagent-containing partial composition may be separately packaged.

[0461] When the glycated protein measurement reagent according to the embodiment contains a ferricyanide-containing partial composition, the ferricyanide-containing partial composition may be in a liquid state and may be stored until it is mixed with the Trinder reagent-containing partial composition and the 4-aminoantipyrine-containing partial composition. In the ferricyanide-containing partial composition, the contained substances are combined. The ferricyanide-containing partial composition, the 4-aminoantipyrine-containing partial composition, and the Trinder reagent-containing partial composition may each be separately packaged.

[0462] The lower limit of the pH of the 4-aminoantipyrine-containing partial composition and / or the ferricyanide-containing partial composition is, for example, 5 or more, 5.5 or more, 6 or more, 6.5 or more, or 7 or more. The pH of the 4-aminoantipyrine-containing partial composition and / or the ferricyanide-containing partial composition may be, for example, greater than 7. The upper limit of the pH of the 4-aminoantipyrine-containing partial composition and / or the ferricyanide-containing partial composition is, for example, 10 or less, 9.5 or less, or 9 or less.

[0463] A glycated protein measurement reagent that does not contain a protease stabilizer may have problems with storage stability. In contrast, the glycated protein measurement reagent according to the embodiment contains a protease stabilizer, so it has good storage stability over a long period. Therefore, it is possible to accurately measure glycated proteins over a long period.

[0464] The glycated protein measurement reagent according to the embodiment can be stably stored, for example, during a practical period required for distribution. For example, the glycated protein measurement reagent according to the embodiment can be stored for at least 1 month. Examples of the lower limit of the storage period of the glycated protein measurement reagent according to the embodiment include 1 day or more, 1 month or more, 6 months, or 1 year or more. Examples of the upper limit of the storage period of the glycated protein measurement reagent according to the embodiment include 5 years or less, 2 years or less, 1 year and 6 months or less, 1 year and 3 months or less, 6 months or less, or 7 days or less.

[0465] As the lower limit of the storage temperature of the glycated protein measurement reagent according to the embodiment, 2°C or higher, 4°C or higher, or 5°C or higher is exemplified. Further, as the upper limit of the storage temperature of the glycated protein measurement reagent according to the embodiment, 10°C or lower, 8°C or lower, or 5°C or lower is exemplified.

[0466] In the glycated protein measurement reagent according to the embodiment, when ferrocyanide is contained in at least one of the Trinder reagent-containing partial composition and the 4-aminoantipyrine-containing partial composition, the Trinder reagent-containing partial composition or the 4-aminoantipyrine-containing partial composition stored at 5°C for 14 days, and the 4-aminoantipyrine-containing partial composition or the Trinder reagent-containing partial composition stored at 30°C for 14 days, when used in combination, the ratio of the measurement sensitivity of glycated protein to that when the Trinder reagent-containing partial composition or the 4-aminoantipyrine-containing partial composition stored at 5°C for 28 days and the 4-aminoantipyrine-containing partial composition or the Trinder reagent-containing partial composition stored at 30°C for 28 days are used in combination is referred to as the sensitivity retention rate. As the lower limit of the sensitivity retention rate, 80% or higher, 85% or higher, 88% or higher, 90% or higher, 91% or higher, 92% or higher, 93% or higher, 94% or higher, or 95% or higher is exemplified. As the upper limit, 120% or lower, 110% or lower, or 105% or lower is exemplified.

[0467] In the glycated protein measurement reagent according to the embodiment, when ferrocyanide is contained in the 4-aminoantipyrine-containing partial composition, the Trinder reagent-containing partial composition stored at 5°C for 14 days and the 4-aminoantipyrine-containing partial composition stored at 30°C for 14 days are combined and used. The ratio of the measurement sensitivity of glycated protein when used in combination with the Trinder reagent-containing partial composition stored at 5°C for 28 days and the 4-aminoantipyrine-containing partial composition stored at 30°C for 28 days is referred to as the sensitivity retention rate. Examples of the lower limit of the sensitivity retention rate include 80% or more, 85% or more, 88% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, or 95% or more. Examples of the upper limit include 120% or less, 110% or less, or 105% or less.

[0468] In the glycated protein measurement reagent according to the embodiment, when ferrocyanide is contained in the Trinder reagent-containing partial composition, the 4-aminoantipyrine-containing partial composition stored at 5°C for 14 days and the Trinder reagent-containing partial composition stored at 30°C for 14 days are combined and used. The ratio of the measurement sensitivity of glycated protein when used in combination with the 4-aminoantipyrine-containing partial composition stored at 5°C for 28 days and the Trinder reagent-containing partial composition stored at 30°C for 28 days is referred to as the sensitivity retention rate. Examples of the lower limit of the sensitivity retention rate include 80% or more, 85% or more, 88% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, or 95% or more. Examples of the upper limit include 120% or less, 110% or less, or 105% or less.

[0469] In the glycated protein measurement reagent according to the embodiment, when ferricyanide is contained in the ferricyanide-containing partial composition, the Trinder reagent-containing partial composition stored at 5°C for 14 days or the 4-aminoantipyrine-containing partial composition, and the 4-aminoantipyrine-containing partial composition stored at 30°C for 14 days or the Trinder reagent-containing partial composition and the ferricyanide-containing partial composition, when used in combination, the ratio of the measurement sensitivity of glycated protein to that when the Trinder reagent-containing partial composition stored at 5°C for 28 days or the 4-aminoantipyrine-containing partial composition, and the 4-aminoantipyrine-containing partial composition stored at 30°C for 28 days or the Trinder reagent-containing partial composition and the ferricyanide-containing partial composition are used in combination is referred to as the sensitivity retention rate. Examples of the lower limit of the sensitivity retention rate include 80% or more, 85% or more, 88% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, or 95% or more. Examples of the upper limit include 120% or less, 110% or less, or 105% or less.

[0470] In the glycated protein measurement reagent according to the embodiment, when ferricyanide is contained in the ferricyanide-containing partial composition, the ratio of the measurement sensitivity of glycated protein to that when the Trinder reagent-containing partial composition stored at 5°C for 14 days is combined with the 4-aminoantipyrine-containing partial composition and the ferricyanide-containing partial composition stored at 30°C for 14 days, and the Trinder reagent-containing partial composition stored at 5°C for 28 days is combined with the 4-aminoantipyrine-containing partial composition and the ferricyanide-containing partial composition stored at 30°C for 28 days is referred to as the sensitivity retention rate. Examples of the lower limit of the sensitivity retention rate include 80% or more, 85% or more, 88% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, or 95% or more. Examples of the upper limit include 120% or less, 110% or less, or 105% or less.

[0471] In the glycated protein measurement reagent according to the embodiment, when the glycated protein measurement reagent includes a Trinder reagent-containing partial composition and a 4-aminoantipyrine-containing partial composition, the glycated protein measurement sensitivity when the Trinder reagent-containing partial composition stored at 5°C for 28 days and the 4-aminoantipyrine-containing partial composition are used in combination is compared with the glycated protein measurement sensitivity when the Trinder reagent-containing partial composition stored at 5°C for 1 year and the 4-aminoantipyrine-containing partial composition are used in combination. The ratio is called the long-term sensitivity retention rate. Alternatively, when the glycated protein measurement reagent includes a Trinder reagent-containing partial composition, a 4-aminoantipyrine-containing partial composition, and a ferricyanide-containing partial composition, the glycated protein measurement sensitivity when the Trinder reagent-containing partial composition stored at 5°C for 28 days, the 4-aminoantipyrine-containing partial composition, and the ferricyanide-containing partial composition are used in combination is compared with the glycated protein measurement sensitivity when the Trinder reagent-containing partial composition stored at 5°C for 1 year, the 4-aminoantipyrine-containing partial composition, and the ferricyanide-containing partial composition are used in combination. The ratio is called the long-term sensitivity retention rate. Examples of the lower limit of the long-term sensitivity retention rate include 80% or more, 85% or more, 88% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, or 95% or more. Examples of the upper limit include 120% or less, 110% or less, or 105% or less.

Example

[0472] Next, examples of the present invention will be described, but it goes without saying that the present invention is not limited to the following examples at all.

[0473] For the measurement of absorbance in the examples, a 7170 type Hitachi automatic analyzer (manufactured by Hitachi High-Technologies Corporation) was used. In the apparatus, the main wavelength was set to 546 nm and the sub-wavelength was set to 700 nm. The absorbance Abs of the sample was calculated using the following formula (1), where the absorbance at a wavelength of 546 nm was Abs 546 and the absorbance at a wavelength of 700 nm was Abs 700 . Abs = Abs 546 - Abs700 (1)

[0474] [Reference Example 1: Generation mechanism of reagent blank] (Preparation of liquid partial composition containing Trinder reagent) The following reagent components were dissolved in purified water so as to have the described concentrations, and the pH was adjusted to 7.6 with sodium hydroxide to prepare a liquid partial composition containing a Trinder reagent. 100 mmol / L N-Tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (manufactured by Dojindo Laboratories) 2 mmol / L N,N-Bis(4-sulfobutyl)-3-methylaniline, disodium salt (manufactured by Dojindo Laboratories)

[0475] (Preparation of liquid partial composition containing 4-aminoantipyrine and cyanide) The following reagent components were dissolved in purified water so as to have the described concentrations, and the pH was adjusted to 7.5 with sodium hydroxide to prepare a liquid partial composition containing 4-aminoantipyrine and cyanide. 200 mmol / L 3,3-Dimethylglutaric acid (manufactured by Tokyo Chemical Industry Co., Ltd.) 5 mmol / L 4-Aminoantipyrine (manufactured by Actic Co., Ltd.) Stabilizer with the concentration described later Cyanide or a mixture of cyanide and ferricyanide with the concentration described later

[0476] (Stabilizer added to the liquid partial composition containing 4-aminoantipyrine and cyanide) 30 wt / vol% Dimethyl sulfoxide (manufactured by Nacalai Tesque, Inc.) 30 wt / vol% Ethanol (manufactured by Kanto Chemical Co., Inc.) 30 wt / vol% Ethylene glycol (manufactured by Fujifilm Wako Pure Chemical Corporation) 30 wt / vol% Propylene glycol (manufactured by Fujifilm Wako Pure Chemical Corporation)

[0477] (Ferrocyanide or a mixture of ferrocyanide and ferricyanide added to a liquid partial composition containing 4-aminoantipyrine and ferrocyanide) 0.1 mmol / L potassium ferrocyanide (manufactured by Kokusan Chemical Co., Ltd.) 0.09 mmol / L potassium ferrocyanide (manufactured by Kokusan Chemical Co., Ltd.) and 0.01 mmol / L potassium ferricyanide (manufactured by Fujifilm Wako Pure Chemical Corporation) 0.07 mmol / L potassium ferrocyanide (manufactured by Kokusan Chemical Co., Ltd.) and 0.03 mmol / L potassium ferricyanide (manufactured by Fujifilm Wako Pure Chemical Corporation)

[0478] (Sample) The following samples were prepared. Purified water

[0479] (Measurement procedure) 4.5 μL of purified water was added to 180 μL of the Trinder reagent-containing liquid partial composition, and after reacting at 37°C for 5 minutes, 45 μL of the 4-aminoantipyrine and ferrocyanide-containing liquid partial composition was added and the reaction was continued at 37°C for another 5 minutes. The change in absorbance from the absorbance Abs1 at 5 minutes after adding purified water to the Trinder reagent-containing liquid partial composition to the absorbance Abs2 at 5 minutes after adding the 4-aminoantipyrine and ferrocyanide-containing liquid partial composition to the mixture of the Trinder reagent-containing liquid partial composition and purified water was measured. The absorbance change A C is given by the following formula (2). A C =(Abs2) - (Abs1) × 180 / 225 (2) Here, 180 represents the volume of the Trinder reagent-containing liquid partial composition, and 225 represents the total volume of the Trinder reagent-containing liquid partial composition and the 4-aminoantipyrine and ferrocyanide-containing liquid partial composition.

[0480] When purified water was used as the sample, the absorbance change was taken as the reagent blank Acb, and the results are shown in Table 1.

Table 1

[0481] As is clear from Table 1, the reagent blank tended to increase when ferricyanide was added. Therefore, it was suggested that ferricyanide contributed to the generation of the reagent blank. Also, when dimethyl sulfoxide was added as a stabilizer, a reagent blank of 1 mAbs or more occurred even with ferrocyanide, suggesting that dimethyl sulfoxide might change ferrocyanide to ferricyanide, which causes the reagent blank.

[0482] [Reference Example 2: Reagent Blank When Reagent Arrangement Is Changed] (Preparation of Trinder Reagent-Containing Liquid Partial Composition (1)) The following reagent components were dissolved in purified water to the described concentrations and adjusted to pH 7.6 with sodium hydroxide to prepare the Trinder reagent-containing liquid partial composition (1). 100 mmol / L N-Tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (manufactured by Dojindo Laboratories) 2 mmol / L N,N-Bis(4-sulfobutyl)-3-methylaniline, disodium salt (manufactured by Dojindo Laboratories)

[0483] (Preparation of Trinder Reagent and Ferrocyanide-Containing Liquid Partial Composition (1)) The following reagent components were dissolved in purified water to the described concentrations and adjusted to pH 7.6 with sodium hydroxide to prepare the Trinder reagent and ferrocyanide-containing liquid partial composition (1). 100 mmol / L N-Tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (manufactured by Dojindo Laboratories) 2 mmol / L N,N-Bis(4-sulfobutyl)-3-methylaniline, disodium salt (manufactured by Dojindo Laboratories) 0.025 mmol / L Potassium Ferricyanide (manufactured by Kokusan Chemical Co., Ltd.)

[0484] (Preparation of 4-Aminoantipyrine-Containing Liquid Partial Composition (1)) The following reagent components were dissolved in purified water to the described concentrations and adjusted to pH 7.6 with sodium hydroxide to prepare a 4-aminoantipyrine-containing liquid partial composition (1). 100 mmol / L N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (manufactured by Dojindo Laboratories) 1.25 mmol / L 4-aminoantipyrine (manufactured by Actic Co., Ltd.)

[0485] (Preparation of a liquid partial composition (1) containing 4-aminoantipyrine and ferricyanide) The following reagent components were dissolved in purified water to the described concentrations and adjusted to pH 7.6 with sodium hydroxide to prepare a liquid partial composition (1) containing 4-aminoantipyrine and ferricyanide. 100 mmol / L N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (manufactured by Dojindo Laboratories) 1.25 mmol / L 4-aminoantipyrine (manufactured by Actic Co., Ltd.) 0.025 mmol / L potassium ferricyanide (manufactured by Kokusan Chemical Co., Ltd.)

[0486] (Preparation of a liquid partial composition (2) containing 4-aminoantipyrine and ferricyanide) The following reagent components were dissolved in purified water to the described concentrations and adjusted to pH 7.5 with sodium hydroxide to prepare a liquid partial composition (2) containing 4-aminoantipyrine and ferricyanide. 200 mmol / L 3,3-dimethylglutaric acid (manufactured by Tokyo Chemical Industry Co., Ltd.) 5 mmol / L 4-aminoantipyrine (manufactured by Actic Co., Ltd.) 0.1 mmol / L potassium ferricyanide (manufactured by Kokusan Chemical Co., Ltd.) A stabilizer at the concentration described later

[0487] (Stabilizer added to the liquid partial composition (2) containing 4-aminoantipyrine and ferricyanide) 30 wt / vol% dimethyl sulfoxide (manufactured by Nacalai Tesque, Inc.) 30 wt / vol% Propylene Glycol (manufactured by FUJIFILM Wako Pure Chemical Corporation)

[0488] (Preparation of Liquid Partial Composition (2) Containing 4-Aminoantipyrine) The following reagent components were dissolved in purified water to the described concentrations and adjusted to pH 7.5 with sodium hydroxide to prepare the liquid partial composition (2) containing 4-aminoantipyrine. 200 mmol / L 3,3-Dimethylglutaric Acid (manufactured by Tokyo Chemical Industry Co., Ltd.) 5 mmol / L 4-Aminoantipyrine (manufactured by Actic Co., Ltd.) Stabilizer at the concentration described later

[0489] (Stabilizer Added to Liquid Partial Composition (2) Containing 4-Aminoantipyrine) 30 wt / vol% Propylene Glycol (manufactured by FUJIFILM Wako Pure Chemical Corporation)

[0490] (Preparation of Liquid Partial Composition (2) Containing Trinder Reagent and Ferrocyanide) The following reagent components were dissolved in purified water to the described concentrations and adjusted to pH 7.5 with sodium hydroxide to prepare the liquid partial composition (2) containing Trinder reagent and ferrocyanide. 200 mmol / L 3,3-Dimethylglutaric Acid (manufactured by Tokyo Chemical Industry Co., Ltd.) 8 mmol / L N,N-Bis(4-Sulfobutyl)-3-Methylaniline, Disodium Salt (manufactured by Dojindo Laboratories) 0.1 mmol / L Potassium Ferrocyanide (manufactured by Kokusan Chemical Co., Ltd.) Stabilizer at the concentration described later

[0491] (Stabilizer Added to Liquid Partial Composition (2) Containing Trinder Reagent and Ferrocyanide) 30 wt / vol% Dimethyl Sulfoxide (manufactured by Nacalai Tesque, Inc.) 30 wt / vol% Propylene Glycol (manufactured by FUJIFILM Wako Pure Chemical Corporation)

[0492] (Preparation of Liquid Partial Composition (2) Containing Trinder Reagent) The following reagent components were dissolved in purified water to the described concentrations and adjusted to pH 7.5 with sodium hydroxide to prepare a Trinder reagent-containing liquid partial composition (2). 200 mmol / L 3,3-dimethylglutaric acid (manufactured by Tokyo Chemical Industry Co., Ltd.) 8 mmol / L N,N-bis(4-sulfobutyl)-3-methylaniline, disodium salt (manufactured by Dojindo Laboratories) A stabilizer at the concentration described later

[0493] (Stabilizer added to the Trinder reagent-containing liquid partial composition (2)) 30 wt / vol% propylene glycol (manufactured by FUJIFILM Wako Pure Chemical Corporation)

[0494] (Sample) The following samples were prepared. Purified water

[0495] (Measurement procedure) 4.5 μL of purified water was added to 180 μL of the Trinder reagent-containing liquid partial composition (1), and after reacting at 37°C for 5 minutes, 45 μL of the 4-aminoantipyrine and ferricyanide-containing liquid partial composition (2) was added and the reaction was continued at 37°C for another 5 minutes. The change in absorbance from the absorbance Abs1 of the mixture of the Trinder reagent-containing liquid partial composition (1) and purified water 5 minutes after adding purified water to the absorbance Abs2 of the mixture of the Trinder reagent-containing liquid partial composition (1) and purified water and the 4-aminoantipyrine and ferricyanide-containing liquid partial composition (2) 5 minutes after addition was measured. The absorbance change A C is given by the following formula (3). A C =(Abs2) - (Abs1)×180 / 225 (3) Here, 180 means the volume of the Trinder reagent-containing liquid partial composition (1), and 225 means the total volume of the Trinder reagent-containing liquid partial composition (1) and the 4-aminoantipyrine and ferricyanide-containing liquid partial composition (2).

[0496] 180 μL of the Trinder reagent and the ferrocyanide-containing liquid partial composition (1) was added with 4.5 μL of purified water and reacted at 37°C for 5 minutes. Then, 45 μL of the 4-aminoantipyrine-containing liquid partial composition (2) was added and reacted at 37°C for another 5 minutes. The change in absorbance from the absorbance Abs1 at 5 minutes after adding purified water to the Trinder reagent and the ferrocyanide-containing liquid partial composition (1) to the absorbance Abs2 at 5 minutes after adding the 4-aminoantipyrine-containing liquid partial composition (2) to the mixture of the Trinder reagent and the ferrocyanide-containing liquid partial composition (1) and purified water was measured. The absorbance change A C is given by the following formula (4). A C =(Abs2) - (Abs1) × 180 / 225 (4) Here, 180 means the volume of the Trinder reagent and the ferrocyanide-containing liquid partial composition (1), and 225 means the total volume of the Trinder reagent and the ferrocyanide-containing liquid partial composition (1) and the 4-aminoantipyrine-containing liquid partial composition (2).

[0497] 180 μL of the 4-aminoantipyrine-containing liquid partial composition (1) was added with 4.5 μL of purified water and reacted at 37°C for 5 minutes. Then, 45 μL of the Trinder reagent and the ferrocyanide-containing liquid partial composition (2) was added and reacted at 37°C for another 5 minutes. The change in absorbance from the absorbance Abs1 at 5 minutes after adding purified water to the 4-aminoantipyrine-containing liquid partial composition (1) to the absorbance Abs2 at 5 minutes after adding the Trinder reagent and the ferrocyanide-containing liquid partial composition (2) to the mixture of the 4-aminoantipyrine-containing liquid partial composition (1) and purified water was measured. The absorbance change A C is given by the following formula (5). A C =(Abs2) - (Abs1) × 180 / 225 (5) Here, 180 means the volume of the 4-aminoantipyrine-containing liquid partial composition (1), and 225 means the total volume of the 4-aminoantipyrine-containing liquid partial composition (1) and the Trinder reagent and the ferrocyanide-containing liquid partial composition (2).

[0498] To 180 μL of the liquid partial composition (1) containing 4-aminoantipyrine and ferricyanide, 4.5 μL of purified water was added, and after reacting at 37 °C for 5 minutes, 45 μL of the liquid partial composition (2) containing the Trinder reagent was added and further reacted at 37 °C for 5 minutes. After adding purified water to the liquid partial composition (1) containing 4-aminoantipyrine and ferricyanide, the absorbance change from the absorbance Abs1 after 5 minutes to the absorbance Abs2 after 5 minutes when the liquid partial composition (2) containing the Trinder reagent was added to the mixed solution of the liquid partial composition (1) containing 4-aminoantipyrine and ferricyanide and purified water was measured. The absorbance change A C is given by the following formula (6). A C =(Abs2) - (Abs1) × 180 / 225 (6) Here, 180 means the liquid volume of the liquid partial composition (1) containing 4-aminoantipyrine and ferricyanide, and 225 means the total liquid volume of the liquid partial composition (1) containing 4-aminoantipyrine and ferricyanide and the liquid partial composition (2) containing the Trinder reagent.

[0499] To 180 μL of the liquid partial composition (2) containing 4-aminoantipyrine and ferricyanide, 4.5 μL of purified water was added, and after reacting at 37 °C for 5 minutes, 45 μL of the liquid partial composition (1) containing the Trinder reagent was added and further reacted at 37 °C for 5 minutes. After adding purified water to the liquid partial composition (2) containing 4-aminoantipyrine and ferricyanide, the absorbance change from the absorbance Abs1 after 5 minutes to the absorbance Abs2 after 5 minutes when the liquid partial composition (1) containing the Trinder reagent was added to the mixed solution of the liquid partial composition (2) containing 4-aminoantipyrine and ferricyanide and purified water was measured. The absorbance change A C is given by the following formula (7). A C =(Abs2) - (Abs1) × 180 / 225 (7) Here, 180 means the liquid volume of the liquid partial composition (2) containing 4-aminoantipyrine and ferricyanide, and 225 means the total liquid volume of the liquid partial composition (2) containing 4-aminoantipyrine and ferricyanide and the liquid partial composition (1) containing the Trinder reagent.

[0500] To 180 μL of the Trinder reagent and the liquid partial composition (2) containing ferrocyanide, 4.5 μL of purified water was added, and after reacting at 37°C for 5 minutes, 45 μL of the liquid partial composition (1) containing 4-aminoantipyrine was added and the reaction was continued at 37°C for another 5 minutes. To the Trinder reagent and the liquid partial composition (2) containing ferrocyanide, purified water was added, and the change in absorbance from the absorbance Abs1 after 5 minutes to the absorbance Abs2 after 5 minutes when the liquid partial composition (1) containing 4-aminoantipyrine was added to the mixture of the Trinder reagent and the liquid partial composition (2) and purified water was measured. The change in absorbance A C is given by the following formula (8). A C =(Abs2) - (Abs1)×180 / 225 (8) Here, 180 means the volume of the Trinder reagent and the liquid partial composition (2) containing ferrocyanide, and 225 means the total volume of the Trinder reagent and the liquid partial composition (2) and the liquid partial composition (1) containing 4-aminoantipyrine.

[0501] When purified water was used as the sample, the change in absorbance was used as the reagent blank Acb, and the results are shown in Table 2.

Table 2

[0502] As shown in Table 2, in both cases where the liquid partial composition that reacts with the sample first contains the Trinder reagent and where the liquid partial composition that reacts with the sample first contains 4-aminoantipyrine, the reagent blank became larger when dimethyl sulfoxide was used as the stabilizer than when propylene glycol was used as the stabilizer. Also, it became clear that the suppressing effect on the reagent blank does not greatly depend on the order of addition of the reagents or the combination method with ferrocyanide.

[0503] [Example 1: Measurement of the Redox Potential of Ferrocyanide in the Presence of a Stabilizer] (Preparation of Ferrocyanide-Containing Liquid Composition) The following reagent components were dissolved in purified water to the described concentrations and adjusted to pH 7.5 with sodium hydroxide to prepare a ferrocyanide-containing liquid composition. 100 mmol / L N-Tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (manufactured by Dojindo Laboratories) 1 mmol / L Potassium Ferrocyanide (manufactured by Kokusan Chemical Co., Ltd.) 150 mmol / L Sodium Chloride (manufactured by FUJIFILM Wako Pure Chemical Corporation) Stabilizer at the concentration described later

[0504] (Stabilizer Added to Ferrocyanide-Containing Liquid Composition) 6 wt / vol% Propylene Glycol (manufactured by FUJIFILM Wako Pure Chemical Corporation) 7.5 wt / vol% Propylene Glycol (manufactured by FUJIFILM Wako Pure Chemical Corporation) 10 wt / vol% Propylene Glycol (manufactured by FUJIFILM Wako Pure Chemical Corporation) 30 wt / vol% Propylene Glycol (manufactured by FUJIFILM Wako Pure Chemical Corporation) 37.5 wt / vol% Propylene Glycol (manufactured by FUJIFILM Wako Pure Chemical Corporation) 40 wt / vol% Propylene Glycol (manufactured by FUJIFILM Wako Pure Chemical Corporation) 50 wt / vol% Propylene Glycol (manufactured by FUJIFILM Wako Pure Chemical Corporation) 62.5 wt / vol% Propylene Glycol (manufactured by FUJIFILM Wako Pure Chemical Corporation) 30 wt / vol% Trimethylene Glycol (manufactured by FUJIFILM Wako Pure Chemical Corporation) 37.5 wt / vol% Trimethylene Glycol (manufactured by FUJIFILM Wako Pure Chemical Corporation) 50 wt / vol% Trimethylene Glycol (manufactured by FUJIFILM Wako Pure Chemical Corporation) 62.5 wt / vol% Trimethylene Glycol (manufactured by FUJIFILM Wako Pure Chemical Corporation) 30 wt / vol% Ethylene Glycol (manufactured by FUJIFILM Wako Pure Chemical Corporation) 37.5 wt / vol% ethylene glycol (manufactured by Fujifilm Wako Pure Chemical Corporation) 50 wt / vol% ethylene glycol (manufactured by Fujifilm Wako Pure Chemical Corporation) 62.5 wt / vol% ethylene glycol (manufactured by Fujifilm Wako Pure Chemical Corporation) 37.5 wt / vol% ethanol (manufactured by Fujifilm Wako Pure Chemical Corporation) 37.5 wt / vol% 1-propanol (manufactured by Fujifilm Wako Pure Chemical Corporation) 37.5 wt / vol% glycerin (manufactured by Fujifilm Wako Pure Chemical Corporation) 0.6 wt / vol% 2-carboxyphenylboronic acid (manufactured by Tokyo Chemical Industry Co., Ltd.) 0.75 wt / vol% 2-carboxyphenylboronic acid (manufactured by Tokyo Chemical Industry Co., Ltd.) 1 wt / vol% 2-carboxyphenylboronic acid (manufactured by Tokyo Chemical Industry Co., Ltd.) 1 wt / vol% lauryltrimethylammonium chloride (manufactured by Fujifilm Wako Pure Chemical Corporation) 1 wt / vol% benzyltriethylammonium chloride (manufactured by Fujifilm Wako Pure Chemical Corporation) 1 wt / vol% benzyltributylammonium chloride (manufactured by Fujifilm Wako Pure Chemical Corporation) 1 wt / vol% calcium chloride (manufactured by Fujifilm Wako Pure Chemical Corporation) 1 wt / vol% boric acid (manufactured by Fujifilm Wako Pure Chemical Corporation) 150 mmol / L sodium chloride (manufactured by Fujifilm Wako Pure Chemical Corporation) (total 300 mmol / L sodium chloride together with the above 150 mmol / L sodium chloride) 30 wt / vol% dimethyl sulfoxide (manufactured by Nacalai Tesque, Inc.) 37.5 wt / vol% dimethyl sulfoxide (manufactured by Nacalai Tesque, Inc.) 50 wt / vol% dimethyl sulfoxide (manufactured by Nacalai Tesque, Inc.) 62.5 wt / vol% dimethyl sulfoxide (manufactured by Nacalai Tesque, Inc.)

[0505] (Measurement procedure) 10 mL of the ferrocyanide-containing liquid composition was added to a cell for voltammetry (BAS), and cyclic voltammetry was performed using the following electrodes and apparatus. Working electrode: GCE glassy carbon electrode, outer diameter 6 mm × inner diameter 3 mm (BAS) Counter electrode: Pt counter electrode, 23 cm (coiled), length 23 cm × electrode part diameter 0.5 mm (BAS) Reference electrode: RE-1B aqueous reference electrode (Ag / AgCl) (BAS) Apparatus: AUTOMATIC POLARIZATION SYSTEM HZ-7000 (Hokuto Denko)

[0506] During cyclic voltammetry measurement, the potential applied to the working electrode was changed with respect to time. The sweep rate was set to 0.1 V / second. With the initial potential as the natural potential, the potential was swept in the positive direction up to the first set potential. Subsequently, a sweep was performed to fold back the electrode potential from the first set potential to the second set potential in the negative direction. Further, a sweep was performed to fold back the electrode potential from the second set potential to the first set potential in the positive direction. The first set potential and the second set potential were 0.5 V and -0.5 V, respectively. Also, the cyclic voltammetry measurement was carried out at 25°C.

[0507] The redox potential (E´0) was calculated as the average value [(Ered + Eox) / 2] of the potential value (Eox) when the oxidation current showed a peak and the potential value (Ered) when the reduction current showed a peak. The measurement results are shown in Table 3.

Table 3-1

Table 3-2

[0508] As is clear from Table 3, the redox potential of ferrocyanide varies depending on the type and concentration of the stabilizer, and it was found that dimethyl sulfoxide, propylene glycol, trimethylene glycol, and ethylene glycol decrease the redox potential in a concentration-dependent manner. On the other hand, the redox potential hardly changed with 2-carboxyphenylboronic acid. In particular, dimethyl sulfoxide was found to significantly decrease the redox potential compared to propylene glycol, trimethylene glycol, and ethylene glycol. Generally, when the redox potential decreases, it becomes easier to be oxidized, so it was suggested that ferricyanide is more likely to be generated by mixing ferrocyanide with a specific stabilizer.

[0509] [Example 2: Changes in reagent blanks and stability of analyte sensitivity depending on the type of protease stabilizer] (Preparation of liquid partial composition containing Trinder reagent) The following reagent components were dissolved in purified water to the described concentrations and adjusted to pH 7.6 with sodium hydroxide to prepare a liquid partial composition containing Trinder reagent. 100 mmol / L N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (manufactured by Dojindo Laboratories) 0.05 wt / vol% sodium azide (manufactured by Merck) 1 mmol / L sodium ethylenediaminetetraacetate (manufactured by Kokusan Chemical Co., Ltd.) 6 wt / vol% D(-)-sorbitol (manufactured by FUJIFILM Wako Pure Chemical Corporation) 2 mmol / L N,N-bis(4-sulfobutyl)-3-methylaniline, disodium salt (manufactured by Dojindo Laboratories) 10 U / mL peroxidase (manufactured by Amano Enzyme Inc.) 30 U / mL ketoamine oxidase (manufactured by Asahi Kasei Pharma Corporation)

[0510] (Two-reagent system: Preparation of liquid partial composition containing 4-aminoantipyrine and ferrocyanide) The following reagent components were dissolved in purified water to the described concentrations, adjusted to pH 7.5 with sodium hydroxide, and a liquid partial composition containing 4-aminoantipyrine and ferricyanide with a stabilizer was prepared. 200 mmol / L 3,3-dimethylglutaric acid (manufactured by Tokyo Chemical Industry Co., Ltd.) 0.05 wt / vol% ProClin300 (manufactured by Sigma-Aldrich) 5 mmol / L 4-aminoantipyrine (manufactured by Actichem Co., Ltd.) 0.1 mmol / L potassium ferricyanide (manufactured by Kokusan Chemical Co., Ltd.) 500 U / mL catalase (manufactured by Nagase ChemteX Corporation) 40 kU / mL protease for glycated albumin (obtained according to the method described in Japanese Patent No. 4565807, manufactured by Asahi Kasei Pharma Corporation) A stabilizer at the concentration described later

[0511] (Two-reagent system: stabilizer contained in the liquid partial composition containing 4-aminoantipyrine and ferricyanide) 30 wt / vol% propylene glycol (manufactured by FUJIFILM Wako Pure Chemical Corporation) 37.5 wt / vol% propylene glycol (manufactured by FUJIFILM Wako Pure Chemical Corporation) 50 wt / vol% trimethylene glycol (manufactured by FUJIFILM Wako Pure Chemical Corporation) 62.5 wt / vol% trimethylene glycol (manufactured by FUJIFILM Wako Pure Chemical Corporation) 50 wt / vol% ethylene glycol (manufactured by FUJIFILM Wako Pure Chemical Corporation) 62.5 wt / vol% ethylene glycol (manufactured by FUJIFILM Wako Pure Chemical Corporation) 0.6 wt / vol% 2-carboxyphenylboronic acid (manufactured by Tokyo Chemical Industry Co., Ltd.) 0.75 wt / vol% 2-carboxyphenylboronic acid (manufactured by Tokyo Chemical Industry Co., Ltd.) 30 wt / vol% dimethyl sulfoxide (manufactured by Nacalai Tesque, Inc.) 37.5 wt / vol% dimethyl sulfoxide (manufactured by Nacalai Tesque, Inc.)

[0512] (3 - Reagent System: Preparation of 4 - Aminoantipyrine - Containing Liquid Partial Composition (1)) The following reagent components were dissolved in purified water to the described concentrations and adjusted to pH 7.5 with sodium hydroxide to prepare a 4 - aminoantipyrine - containing liquid partial composition (1) containing a stabilizer. 250 mmol / L 3,3 - Dimethylglutaric acid (manufactured by Tokyo Chemical Industry Co., Ltd.) 0.05 wt / vol% ProClin300 (manufactured by Sigma - Aldrich Co., LLC) 6.25 mmol / L 4 - Aminoantipyrine (manufactured by Actech Co., Ltd.) 625 U / mL Catalase (manufactured by Nagase ChemteX Corporation) 50 kU / mL Protease for Glycated Albumin (obtained according to the method described in Japanese Patent No. 4565807, manufactured by Asahi Kasei Pharma Corporation.) Stabilizer at the concentration described later

[0513] (3 - Reagent System: Stabilizers Contained in 4 - Aminoantipyrine - Containing Liquid Partial Composition (1)) 37.5 wt / vol% Propylene Glycol (manufactured by FUJIFILM Wako Pure Chemical Corporation) 50 wt / vol% Propylene Glycol (manufactured by FUJIFILM Wako Pure Chemical Corporation) 62.5 wt / vol% Trimethylene Glycol (manufactured by FUJIFILM Wako Pure Chemical Corporation) 62.5 wt / vol% Ethylene Glycol (manufactured by FUJIFILM Wako Pure Chemical Corporation) 0.75 wt / vol% 2 - Carboxyphenylboronic Acid (manufactured by Tokyo Chemical Industry Co., Ltd.) 37.5 wt / vol% Dimethyl Sulfoxide (manufactured by Nacalai Tesque, Inc.)

[0514] (3 - Reagent System: Preparation of Ferrocyanide - Containing Liquid Partial Composition (1)) The following reagent components were dissolved in purified water to the described concentrations and adjusted to pH 7.0 with sodium hydroxide to prepare a ferrocyanide - containing liquid partial composition (1). 100 mmol / L 3,3 - Dimethylglutaric acid (manufactured by Tokyo Chemical Industry Co., Ltd.) 0.05 wt / vol% ProClin300 (manufactured by Sigma-Aldrich) 0.5 mmol / L potassium ferricyanide (manufactured by Kokusan Chemical Co., Ltd.)

[0515] (3-reagent system: Preparation of liquid partial composition (2) containing 4-aminoantipyrine) The following reagent components were dissolved in purified water to the described concentrations, adjusted to pH 7.0 with sodium hydroxide, and liquid partial composition (2) containing 4-aminoantipyrine was prepared. 100 mmol / L 3,3-dimethylglutaric acid (manufactured by Tokyo Chemical Industry Co., Ltd.) 0.05 wt / vol% ProClin300 (manufactured by Sigma-Aldrich) 25 mmol / L 4-aminoantipyrine (manufactured by Actic Co., Ltd.)

[0516] (3-reagent system: Preparation of liquid partial composition (2) containing ferricyanide) The following reagent components were dissolved in purified water to the described concentrations, adjusted to pH 7.5 with sodium hydroxide, and liquid partial composition (2) containing ferricyanide with a stabilizer was prepared. 250 mmol / L 3,3-dimethylglutaric acid (manufactured by Tokyo Chemical Industry Co., Ltd.) 0.05 wt / vol% ProClin300 (manufactured by Sigma-Aldrich) 0.125 mmol / L potassium ferricyanide (manufactured by Kokusan Chemical Co., Ltd.) 625 U / mL catalase (manufactured by Nagase ChemteX Corporation) 50 kU / mL protease for glycated albumin (manufactured by Asahi Kasei Pharma Corporation, obtained according to the method described in Japanese Patent No. 4565807.) Stabilizer at the concentration described later

[0517] (3-reagent system: Stabilizer contained in liquid partial composition (2) containing ferricyanide) 37.5 wt / vol% propylene glycol (manufactured by FUJIFILM Wako Pure Chemical Corporation) 37.5 wt / vol% dimethyl sulfoxide (manufactured by Nacalai Tesque, Inc.)

[0518] (Sample Specimen) The following sample specimens were prepared. Control Serum L (manufactured by Asahi Kasei Pharma Corporation) Control Serum H (manufactured by Asahi Kasei Pharma Corporation)

[0519] (Reagent Storage) The prepared liquid partial composition containing Trinder reagent was stored at 5°C for 14 days or 28 days. Also, the partial composition containing 4-aminoantipyrine and ferricyanide, the liquid partial composition containing 4-aminoantipyrine, and the liquid partial composition containing ferricyanide were stored at 30°C for 14 days or 28 days.

[0520] (Measurement Preparation) The liquid partial composition (1) containing 4-aminoantipyrine and the liquid partial composition (1) containing ferricyanide in the three-reagent system were mixed at a volume ratio of 4:1 to prepare the liquid partial composition (1) containing 4-aminoantipyrine and ferricyanide for measurement. The liquid partial composition (2) containing 4-aminoantipyrine and the liquid partial composition (2) containing ferricyanide in the three-reagent system were mixed at a volume ratio of 1:4 to prepare the liquid partial composition (2) containing 4-aminoantipyrine and ferricyanide for measurement.

[0521] (Measurement Procedure) 4.5 μL of the sample specimen was added to 180 μL of the liquid partial composition containing Trinder reagent, and after reacting at 37°C for 5 minutes, 45 μL of the liquid partial composition containing 4-aminoantipyrine and ferricyanide was added and further reacted at 37°C for 5 minutes. The absorbance change from the absorbance Abs1 at 5 minutes after adding the sample specimen to the liquid partial composition containing Trinder reagent to the absorbance Abs2 at 5 minutes after adding the liquid partial composition containing 4-aminoantipyrine and ferricyanide to the mixed solution of the liquid partial composition containing Trinder reagent and the sample specimen was measured. The absorbance change A C is given by the following formula (9). A C =(Abs2)-(Abs1)×180 / 225 (9) Here, 180 means the liquid volume of the liquid partial composition containing the Trinder reagent, and 225 means the total liquid volume of the liquid partial composition containing the Trinder reagent and the liquid partial composition containing 4 - aminoantipyrine and ferricyanide.

[0522] The absorbance change Acs measured using the sample specimen, and the absorbance change measured using purified water instead of the sample specimen was defined as the reagent blank Acb. As shown in the following formula (10), the value obtained by subtracting the absorbance change Acb of the reagent blank from the absorbance change Acs measured using the sample specimen was calculated as the sensitivity ΔAbs. ΔAbs = Acs - Acb (10)

[0523] The sensitivity after storing the partial composition for 14 days was defined as ΔAbs14, and the sensitivity after storing the partial composition for 28 days was defined as ΔAbs28. As shown in the following formula (11), the sensitivity retention rate was calculated as the ratio of ΔAbs28 to ΔAbs14. Together with the results of the reagent blank Acb after storing the partial composition for 14 days, the sensitivity retention rate is shown in Table 4. Sensitivity retention rate (%) = ΔAbs28 ÷ ΔAbs14 × 100 (11)

[0524] Also, the relationship between the reagent blank after storing the partial composition for 14 days and the redox potential of ferricyanide is shown in Table 5 and Figure 1. For the redox potential of ferricyanide, the redox potential value at the concentration when the stabilizer and ferricyanide were mixed was used. For example, when the stabilizer and ferricyanide are contained in the same partial composition (the liquid partial composition containing 4 - aminoantipyrine and ferricyanide in the 2 - reagent system, and the liquid partial composition containing ferricyanide in the 3 - reagent system (2)), the redox potential value at the stabilizer concentration in the partial composition was used. Also, when the stabilizer and ferricyanide are contained in different partial compositions (the liquid partial composition containing 4 - aminoantipyrine (1) and the liquid partial composition containing ferricyanide (1) in the 3 - reagent system), the redox potential value at the stabilizer concentration when the liquid partial composition containing 4 - aminoantipyrine (1) and the liquid partial composition containing ferricyanide (1) were mixed was used. [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4] [Table 5-1] [Table 5-2] [Table 5-3]

[0525] As is clear from Table 4, Table 5, and FIG. 1, in both the two-reagent system and the three-reagent system, the effect of suppressing the reagent blank is remarkable with a stabilizer that gives a redox potential higher than the redox potential of ferrocyanide (0.058 V) when mixed with ferrocyanide. Also, in any of the stabilizers, it was shown that the residual rate of the analyte sensitivity improves by changing from the two-reagent system to the three-reagent system. Furthermore, it was shown that the higher the stabilizer concentration, the higher the analyte sensitivity stabilization effect.

[0526] [Example 3: Effect of Adding Chelating Agent] (Preparation of Liquid Partial Composition Containing Trinder Reagent) The following reagent components were dissolved in purified water to the described concentrations and adjusted to pH 7.6 with sodium hydroxide to prepare a liquid partial composition containing a Trinder reagent. 100 mmol / L N-Tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (manufactured by Dojindo Laboratories) 0.05 wt / vol% Sodium azide (manufactured by Merck) 1 mmol / L Sodium ethylenediaminetetraacetate (manufactured by Kokusan Chemical Co., Ltd.) 6 wt / vol% D(-)-Sorbitol (manufactured by Fujifilm Wako Pure Chemical Corporation) 2 mmol / L N,N-Bis(4-sulfobutyl)-3-methylaniline, disodium salt (manufactured by Dojindo Laboratories) 10 U / mL Peroxidase (manufactured by Amano Enzyme Inc.) 30 U / mL Ketamine Oxidase (manufactured by Asahi Kasei Pharma Corporation)

[0527] (Two-reagent system: Preparation of liquid partial composition containing 4-aminoantipyrine and ferricyanide) The following reagent components were dissolved in purified water to the described concentrations, adjusted to pH 7.5 with sodium hydroxide, and a liquid partial composition containing 4-aminoantipyrine and ferricyanide containing propylene glycol as a stabilizer, a partial composition containing trisodium citrate, and a partial composition not containing trisodium citrate were prepared. 200 mmol / L 3,3-Dimethylglutaric acid (manufactured by Tokyo Chemical Industry Co., Ltd.) 0.05 wt / vol% ProClin300 (manufactured by Sigma-Aldrich) 5 mmol / L 4-Aminoantipyrine (manufactured by Actichem Co., Ltd.) 0.1 mmol / L Potassium Ferricyanide (manufactured by Kokusan Chemical Co., Ltd.) 500 U / mL Catalase (manufactured by Nagase ChemteX Corporation) 40 kU / mL Protease for Glycated Albumin (manufactured by Asahi Kasei Pharma Corporation, obtained according to the method described in Japanese Patent No. 4565807.) 1 mmol / L Trisodium Citrate (manufactured by Fujifilm Wako Pure Chemical Corporation) Stabilizer at the concentration described later

[0528] (Two-reagent system: Stabilizer contained in liquid partial composition containing 4-aminoantipyrine and ferricyanide) 30 wt / vol% Propylene Glycol (manufactured by Fujifilm Wako Pure Chemical Corporation) 37.5 wt / vol% Propylene Glycol (manufactured by Fujifilm Wako Pure Chemical Corporation)

[0529] (3 Reagent System: Preparation of 4-Aminoantipyrine-Containing Liquid Partial Composition (1)) The following reagent components were dissolved in purified water to the described concentrations and adjusted to pH 7.5 with sodium hydroxide to prepare a 4-aminoantipyrine-containing liquid partial composition (1) containing a stabilizer, and a partial composition containing trisodium citrate and a partial composition not containing trisodium citrate were prepared. 250 mmol / L 3,3-Dimethylglutaric acid (manufactured by Tokyo Chemical Industry Co., Ltd.) 0.05 wt / vol% ProClin300 (manufactured by Sigma-Aldrich) 6.25 mmol / L 4-Aminoantipyrine (manufactured by Actic Co., Ltd.) 625 U / mL Catalase (manufactured by Nagase ChemteX Corporation) 50 kU / mL Protease for Glycated Albumin (manufactured by Asahi Kasei Pharma Corporation, obtained according to the method described in Japanese Patent No. 4565807.) 1.25 mmol / L Trisodium Citrate (manufactured by FUJIFILM Wako Pure Chemical Corporation) A stabilizer at the concentration described later

[0530] (3 Reagent System: Stabilizer Contained in 4-Aminoantipyrine-Containing Liquid Partial Composition (1)) 37.5 wt / vol% Propylene Glycol (manufactured by FUJIFILM Wako Pure Chemical Corporation) 62.5 wt / vol% Trimethylene Glycol (manufactured by FUJIFILM Wako Pure Chemical Corporation) 62.5 wt / vol% Ethylene Glycol (manufactured by FUJIFILM Wako Pure Chemical Corporation) 0.75 wt / vol% 2-Carboxyphenylboronic Acid (manufactured by Tokyo Chemical Industry Co., Ltd.)

[0531] (3 Reagent System: Preparation of Ferrocyanide-Containing Liquid Partial Composition (1)) The following reagent components were dissolved in purified water to the described concentrations and adjusted to pH 7.0 with sodium hydroxide to prepare a ferrocyanide-containing liquid partial composition (1), and a partial composition containing trisodium citrate and a partial composition not containing trisodium citrate were prepared. 100 mmol / L 3,3-dimethylglutaric acid (manufactured by Tokyo Chemical Industry Co., Ltd.) 0.05 wt / vol% ProClin300 (manufactured by Sigma-Aldrich Co., LLC) 0.5 mmol / L potassium ferrocyanide (manufactured by Kokusan Chemical Co., Ltd.) 5 mmol / L trisodium citrate (manufactured by FUJIFILM Wako Pure Chemical Corporation)

[0532] (3-reagent system: Preparation of liquid partial composition (2) containing 4-aminoantipyrine) A partial composition containing a 4-aminoantipyrine-containing liquid partial composition (2) was prepared by dissolving the following reagent components in purified water to the described concentrations and adjusting the pH to 7.0 with sodium hydroxide. 100 mmol / L 3,3-dimethylglutaric acid (manufactured by Tokyo Chemical Industry Co., Ltd.) 0.05 wt / vol% ProClin300 (manufactured by Sigma-Aldrich Co., LLC) 25 mmol / L 4-aminoantipyrine (manufactured by Actic Co., Ltd.)

[0533] (3-reagent system: Preparation of ferrocyanide-containing liquid partial composition (2)) A ferrocyanide-containing liquid partial composition (2) containing propylene glycol as a stabilizer was prepared by dissolving the following reagent components in purified water to the described concentrations and adjusting the pH to 7.5 with sodium hydroxide. A partial composition containing trisodium citrate and a partial composition not containing trisodium citrate were prepared. 250 mmol / L 3,3-dimethylglutaric acid (manufactured by Tokyo Chemical Industry Co., Ltd.) 0.05 wt / vol% ProClin300 (manufactured by Sigma-Aldrich Co., LLC) 0.125 mmol / L potassium ferrocyanide (manufactured by Kokusan Chemical Co., Ltd.) 625 U / mL catalase (manufactured by Nagase ChemteX Corporation) 50 kU / mL protease for glycated albumin (manufactured by Asahi Kasei Pharma Corporation, obtained according to the method described in Japanese Patent No. 4565807.) 1.25 mmol / L trisodium citrate (manufactured by FUJIFILM Wako Pure Chemical Corporation) 37.5 wt / vol% propylene glycol (manufactured by Fujifilm Wako Pure Chemical Corporation)

[0534] (sample specimen) The following sample specimens were prepared. Control serum L (manufactured by Asahi Kasei Pharma Corporation) Control serum H (manufactured by Asahi Kasei Pharma Corporation)

[0535] (reagent storage) The prepared liquid partial composition containing Trinder reagent was stored at 5°C for 14 days or 28 days. Also, the liquid partial composition containing 4-aminoantipyrine and ferricyanide, the liquid partial composition containing 4-aminoantipyrine, and the liquid partial composition containing ferricyanide were stored at 30°C for 14 days or 28 days.

[0536] (measurement preparation) The 4-aminoantipyrine-containing liquid partial composition (1) and the ferricyanide-containing liquid partial composition (1) of the three-reagent system were mixed at a volume ratio of 4:1 to prepare the 4-aminoantipyrine and ferricyanide-containing liquid partial composition (1) for measurement. The 4-aminoantipyrine-containing liquid partial composition (2) and the ferricyanide-containing liquid partial composition (2) of the three-reagent system were mixed at a volume ratio of 1:4 to prepare the 4-aminoantipyrine and ferricyanide-containing liquid partial composition (2) for measurement.

[0537] (measurement procedure) 4.5 μL of the sample specimen was added to 180 μL of the liquid partial composition containing Trinder reagent, and after reacting at 37°C for 5 minutes, 45 μL of the liquid partial composition containing 4-aminoantipyrine and ferricyanide was added and reacted at 37°C for another 5 minutes. The change in absorbance from the absorbance Abs1 at 5 minutes after adding the sample specimen to the liquid partial composition containing Trinder reagent to the absorbance Abs2 at 5 minutes after adding the liquid partial composition containing 4-aminoantipyrine and ferricyanide to the mixed solution of the liquid partial composition containing Trinder reagent and the sample specimen was measured. The absorbance change A C is given by the following formula (12). AC = (Abs2) - (Abs1) × 180 / 225 (12) Here, 180 means the liquid volume of the liquid part of the Trinder reagent-containing liquid composition, and 225 means the total liquid volume of the Trinder reagent-containing liquid composition and the liquid composition containing 4-aminoantipyrine and ferricyanide.

[0538] The change in absorbance Acs measured using the sample specimen, the change in absorbance measured using purified water instead of the sample specimen is defined as the reagent blank Acb. As shown in the following formula (13), the value obtained by subtracting the change in absorbance Acb of the reagent blank from the change in absorbance Acs measured using the sample specimen was calculated as the sensitivity ΔAbs. ΔAbs = Acs - Acb (13)

[0539] The sensitivity after storing the partial composition for 14 days is ΔAbs14, and the sensitivity after storing the partial composition for 28 days is ΔAbs28. As shown in the following formula (14), the sensitivity retention rate was calculated as the ratio of ΔAbs14 to ΔAbs28. Combined with the result of the reagent blank Acb after storing the partial composition for 14 days, the sensitivity retention rate is shown in Table 6. Sensitivity retention rate (%) = ΔAbs28 ÷ ΔAbs14 × 100 (14)

Table 6-1

Table 6-2

Table 6-3

Table 6-4

[0540] As is clear from Table 6, when mixed with ferrocyanide, when using a stabilizer that gives a redox potential of ferrocyanide greater than 0.058V, the reagent blank when no chelating agent (trisodium citrate) was added and the reagent blank when a chelating agent (trisodium citrate) was added were almost the same.

[0541] [Example 4: Effects of pH and Chelating Agent Type] (Preparation of Trinder Reagent-Containing Liquid Partial Composition) The following reagent components were dissolved in purified water to the described concentrations and adjusted to pH 7.6 with sodium hydroxide to prepare a Trinder reagent-containing liquid partial composition. 100 mmol / L N-Tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (manufactured by Dojindo Laboratories) 0.05 wt / vol% Sodium azide (manufactured by Merck) 1 mmol / L Sodium ethylenediaminetetraacetate (manufactured by Kokusan Chemical Co., Ltd.) 6 wt / vol% D(-)-Sorbitol (manufactured by FUJIFILM Wako Pure Chemical Corporation) 2 mmol / L N,N-Bis(4-sulfobutyl)-3-methylaniline, disodium salt (manufactured by Dojindo Laboratories) 10 U / mL Peroxidase (manufactured by Amano Enzyme Inc.) 30 U / mL Ketoamine oxidase (manufactured by Asahi Kasei Pharma Corporation)

[0542] (3-Reagent System: Preparation of 4-Aminoantipyrine-Containing Liquid Partial Composition) The following reagent components were dissolved in purified water to the described concentrations and adjusted to pH 7.5 with sodium hydroxide to prepare a 4-aminoantipyrine-containing liquid partial composition containing propylene glycol as a stabilizer. 250 mmol / L 3,3-Dimethylglutaric acid (manufactured by Tokyo Chemical Industry Co., Ltd.) 0.05 wt / vol% ProClin300 (manufactured by Sigma-Aldrich) 6.25 mmol / L 4-Aminoantipyrine (manufactured by Actic Co., Ltd.) 625 U / mL Catalase (manufactured by Nagase ChemteX Corporation) 50 kU / mL Protease for Glycated Albumin (manufactured by Asahi Kasei Pharma Corporation, obtained according to the method described in Japanese Patent No. 4565807.) 50 wt / vol% Propylene Glycol (manufactured by FUJIFILM Wako Pure Chemical Corporation)

[0543] (3 - reagent system: Preparation of Ferrocyanide - containing Liquid Partial Composition (1)) The following reagent components were dissolved in purified water to the described concentrations, and the pH was adjusted with sodium hydroxide to prepare Ferrocyanide - containing Liquid Partial Composition (1) with different pH values. 100 mmol / L 3,3 - Dimethylglutaric Acid (manufactured by Tokyo Chemical Industry Co., Ltd.) 0.05 wt / vol% ProClin300 (manufactured by Sigma - Aldrich Corporation) 0.5 mmol / L Potassium Ferrocyanide (manufactured by Kokusan Chemical Co., Ltd.) Adjusted to the pH of the concentration described later

[0544] (pH of Ferrocyanide - containing Liquid Partial Composition (1)) pH 6.5 pH 7.0 pH 7.5 pH 8.0

[0545] (3 - reagent system: Preparation of Ferrocyanide - containing Liquid Partial Composition (2)) The following reagent components were dissolved in purified water to the described concentrations, and the pH was adjusted to 7.0 with sodium hydroxide to prepare Ferrocyanide - containing Liquid Partial Composition (2) containing a chelating agent. 100 mmol / L 3,3 - Dimethylglutaric Acid (manufactured by Tokyo Chemical Industry Co., Ltd.) 0.05 wt / vol% ProClin300 (manufactured by Sigma - Aldrich Corporation) 0.5 mmol / L Potassium Ferrocyanide (manufactured by Kokusan Chemical Co., Ltd.) Chelating agent of the concentration described later

[0546] (Chelating agent contained in Ferrocyanide - containing Liquid Partial Composition (2)) 5 mmol / L sodium ethylenediaminetetraacetate (manufactured by Kokusan Chemical Co., Ltd., abbreviated as EDTA) 5 mmol / L glycol ether diamine tetraacetic acid (manufactured by Dojindo Laboratories, abbreviated as EGTA) 5 mmol / L N-(2-hydroxyethyl)iminodiacetic acid (manufactured by Dojindo Laboratories, abbreviated as HIDA) 5 mmol / L nitrilotris(methylphosphonic acid), trisodium salt (manufactured by Dojindo Laboratories, abbreviated as NTPO) 5 mmol / L nitrilotriacetic acid (manufactured by Dojindo Laboratories, abbreviated as NTA) 5 mmol / L trans-1,2-diaminocyclohexane-N,N,N,N-tetraacetic acid, monohydrate (manufactured by Dojindo Laboratories, abbreviated as CyDTA) 5 mmol / L iminodiacetic acid (manufactured by Dojindo Laboratories, abbreviated as IDA) 5 mmol / L diethylenetriamine-N,N,N’,N”,N”-pentaacetic acid (manufactured by Dojindo Laboratories, abbreviated as DTPA) 5 mmol / L 1,3-diamino-2-propanol-N,N,N’,N’-tetraacetic acid (manufactured by Dojindo Laboratories, abbreviated as DPTA-OH) 5 mmol / L sodium gluconate (manufactured by FUJIFILM Wako Pure Chemical Corporation) 5 mmol / L L(-)-sodium malate (manufactured by FUJIFILM Wako Pure Chemical Corporation) 5 mmol / L succinic acid (manufactured by Kanto Chemical Co., Inc.) 5 mmol / L trisodium citrate (manufactured by FUJIFILM Wako Pure Chemical Corporation) 5 mmol / L salicylic acid (manufactured by Nacalai Tesque, Inc.) 5 mmol / L (+)-potassium tartrate (manufactured by FUJIFILM Wako Pure Chemical Corporation) 5 mmol / L N-[tris(hydroxymethyl)methyl]glycine (manufactured by Dojindo Laboratories, abbreviated as Tricine) 5 mmol / L N,N-bis(2-hydroxyethyl)glycine (manufactured by Dojindo Laboratories, abbreviated as Bicine)

[0547] (Sample) The following sample specimens were prepared. Control serum L (manufactured by Asahi Kasei Pharma Corporation) Management Serum H (manufactured by Asahi Kasei Pharma Corporation)

[0548] (Reagent Storage) The prepared liquid partial composition containing Trinder reagent was stored at 5°C for 14 days or 28 days. Also, the liquid partial compositions containing 4-aminoantipyrine and the liquid partial compositions (1) and (2) containing ferricyanide were stored at 30°C for 14 days or 28 days.

[0549] (Measurement Preparation) The liquid partial composition containing 4-aminoantipyrine and the liquid partial composition (1) or (2) containing ferricyanide in the 3-reagent system were mixed at a volume ratio of 4:1 to prepare the liquid partial composition containing 4-aminoantipyrine and ferricyanide for measurement.

[0550] (Measurement Procedure) 4.5 μL of the sample specimen was added to 180 μL of the liquid partial composition containing Trinder reagent and reacted at 37°C for 5 minutes. Then, 45 μL of the liquid partial composition containing 4-aminoantipyrine and ferricyanide was added and reacted at 37°C for another 5 minutes. The absorbance change from the absorbance Abs1 at 5 minutes after adding the sample specimen to the liquid partial composition containing Trinder reagent to the absorbance Abs2 at 5 minutes after adding the liquid partial composition containing 4-aminoantipyrine and ferricyanide to the mixed solution of the liquid partial composition containing Trinder reagent and the sample specimen was measured. The absorbance change A C is given by the following formula (15). A C =(Abs2)-(Abs1)×180 / 225 (15) Here, 180 means the liquid volume of the liquid partial composition containing Trinder reagent, and 225 means the total liquid volume of the liquid partial composition containing Trinder reagent and the liquid partial composition containing 4-aminoantipyrine and ferricyanide.

[0551] The absorbance change Acs measured using the sample specimen, with the absorbance change measured using purified water instead of the sample specimen as the reagent blank Acb, and as shown in the following formula (16), the value obtained by subtracting the absorbance change Acb of the reagent blank from the absorbance change Acs measured using the sample specimen was calculated as the sensitivity ΔAbs. ΔAbs = Acs - Acb (16)

[0552] Taking the sensitivity after storing the partial composition for 14 days as ΔAbs14 and the sensitivity after storing the partial composition for 28 days as ΔAbs28, the sensitivity retention rate was calculated as the ratio of ΔAbs14 to ΔAbs28 as shown in the following formula (17). Together with the results of the reagent blank Acb after storing the partial composition for 14 days, the sensitivity retention rate is shown in Table 7 and Figure 2. Sensitivity retention rate (%) = ΔAbs28 ÷ ΔAbs14 × 100 (17)

Table 7-1

Table 7-2

[0553] As is clear from Table 7 and Figure 2, even when the pH of the ferrocyanide-containing liquid partial composition was changed, the change in the reagent blank was small. Also, even when the type of chelating agent added to the ferrocyanide-containing liquid partial composition was changed, the change in the reagent blank was small.

[0554] [Example 5: Effect of pH] (Preparation of Liquid Partial Composition Containing Trinder Reagent) The following reagent components were dissolved in purified water to the described concentrations and adjusted to pH 7.6 with sodium hydroxide to prepare a liquid partial composition containing the Trinder reagent. 100 mmol / L N-Tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (manufactured by Dojindo Laboratories) 0.05 wt / vol% Sodium azide (manufactured by Merck) 1 mmol / L Sodium ethylenediaminetetraacetate (manufactured by Kokusan Chemical Co., Ltd.) 6 wt / vol% D(-)-Sorbitol (manufactured by Fujifilm Wako Pure Chemical Corporation) 2 mmol / L N,N-Bis(4-sulfobutyl)-3-methylaniline, disodium salt (manufactured by Dojindo Laboratories) 10 U / mL Peroxidase (manufactured by Amano Enzyme Inc.) 30 U / mL Ketamine oxidase (manufactured by Asahi Kasei Pharma Corporation)

[0555] (3 reagent system: Preparation of the liquid partial composition (1) containing 4-aminoantipyrine) The following reagent components were dissolved in purified water to the described concentrations and adjusted to pH 7.5 with sodium hydroxide to prepare a liquid partial composition (1) containing 4-aminoantipyrine and a stabilizer, and a partial composition containing trisodium citrate and a partial composition not containing trisodium citrate were prepared. 250 mmol / L 3,3-Dimethylglutaric acid (manufactured by Tokyo Chemical Industry Co., Ltd.) 0.05 wt / vol% ProClin300 (manufactured by Sigma-Aldrich) 6.25 mmol / L 4-Aminoantipyrine (manufactured by Actic Co., Ltd.) 625 U / mL Catalase (manufactured by Nagase ChemteX Corporation) 50 kU / mL Protease for glycated albumin (manufactured by Asahi Kasei Pharma Corporation, obtained according to the method described in Japanese Patent No. 4565807.) 1.25 mmol / L Trisodium citrate (manufactured by Fujifilm Wako Pure Chemical Corporation) Stabilizer at the concentration described below

[0556] (3 reagent system: Stabilizer added to the liquid partial composition (1) containing 4-aminoantipyrine) 37.5 wt / vol% Propylene glycol (manufactured by Fujifilm Wako Pure Chemical Corporation) 62.5 wt / vol% Trimethylene glycol (manufactured by Fujifilm Wako Pure Chemical Corporation) 62.5 wt / vol% Ethylene glycol (manufactured by Fujifilm Wako Pure Chemical Corporation) 0.75 wt / vol% 2-Carboxyphenylboronic acid (manufactured by Tokyo Chemical Industry Co., Ltd.)

[0557] (3-Reagent system: Preparation of the liquid partial composition (1) containing ferrocyanide) The following reagent components were dissolved in purified water so as to have the described concentrations, and adjusted to pH 7.0 or pH 8.0 to prepare a liquid partial composition containing ferrocyanide. Also, a partial composition containing trisodium citrate and a partial composition not containing trisodium citrate were prepared for the liquid partial composition containing ferrocyanide at pH 8.0. 100 mmol / L 3,3-Dimethylglutaric acid (manufactured by Tokyo Chemical Industry Co., Ltd.) 0.05 wt / vol% ProClin300 (manufactured by Sigma-Aldrich) 0.5 mmol / L Potassium ferrocyanide (manufactured by Kokusan Chemical Co., Ltd.) 5 mmol / L Trisodium citrate (manufactured by Fujifilm Wako Pure Chemical Corporation)

[0558] (3-Reagent system: Preparation of the liquid partial composition (2) containing 4-aminoantipyrine) The following reagent components were dissolved in purified water so as to have the described concentrations, and adjusted to pH 7.0 or pH 8.0 with sodium hydroxide to prepare a liquid partial composition containing 4-aminoantipyrine. Also, a partial composition containing trisodium citrate and a partial composition not containing trisodium citrate were prepared for the liquid partial composition containing 4-aminoantipyrine adjusted to pH 8.0. 100 mmol / L 3,3-Dimethylglutaric acid (manufactured by Tokyo Chemical Industry Co., Ltd.) 0.05 wt / vol% ProClin300 (manufactured by Sigma-Aldrich) 25 mmol / L 4-Aminoantipyrine (manufactured by Actic Co., Ltd.) 25 mmol / L Trisodium citrate (manufactured by Fujifilm Wako Pure Chemical Corporation) (added only when adjusted to pH 8.0)

[0559] (3-Reagent system: Preparation of the liquid partial composition (2) containing ferrocyanide) The following reagent components were dissolved in purified water to the described concentrations, adjusted to pH 7.5 with sodium hydroxide, and a ferrocyanide-containing liquid partial composition containing propylene glycol as a stabilizer was prepared, including a partial composition containing trisodium citrate and a partial composition not containing trisodium citrate. 250 mmol / L 3,3-dimethylglutaric acid (manufactured by Tokyo Chemical Industry Co., Ltd.) 0.05 wt / vol% ProClin300 (manufactured by Sigma-Aldrich) 0.125 mmol / L potassium ferrocyanide (manufactured by Kokusan Chemical Co., Ltd.) 625 U / mL catalase (manufactured by Nagase ChemteX Corporation) 50 kU / mL protease for glycated albumin (manufactured by Asahi Kasei Pharma Corporation, obtained according to the method described in Patent No. 4565807) 1.25 mmol / L trisodium citrate (manufactured by Fujifilm Wako Pure Chemical Corporation) 37.5 wt / vol% propylene glycol (manufactured by Fujifilm Wako Pure Chemical Corporation)

[0560] (Sample Specimen) The following sample specimens were prepared. Control Serum L (manufactured by Asahi Kasei Pharma Corporation) Control Serum H (manufactured by Asahi Kasei Pharma Corporation)

[0561] (Reagent Storage) The prepared Trinder reagent-containing liquid partial composition was stored at 5°C for 14 days or 28 days. Also, the 4-aminoantipyrine-containing liquid partial composition and the ferrocyanide-containing liquid partial composition were stored at 30°C for 14 days or 28 days.

[0562] (Measurement Preparation) The 4-aminoantipyrine-containing liquid partial composition (1) and the ferrocyanide-containing liquid partial composition (1) of the three-reagent system were mixed at a volume ratio of 4:1 to prepare the 4-aminoantipyrine and ferrocyanide-containing liquid partial composition (1) for measurement. The 4-aminoantipyrine-containing liquid partial composition (2) of the reagent system and the ferricyanide-containing liquid partial composition (2) were mixed at a volume ratio of 1:4 to prepare the 4-aminoantipyrine and ferricyanide-containing liquid partial composition (2) used for measurement.

[0563] (Measurement procedure) 4.5 μL of the sample specimen was added to 180 μL of the Trinder reagent-containing liquid partial composition, and after reacting at 37°C for 5 minutes, 45 μL of the 4-aminoantipyrine and ferricyanide-containing liquid partial composition was added and reacted at 37°C for another 5 minutes. The absorbance change from the absorbance Abs1 at 5 minutes after adding the sample specimen to the Trinder reagent-containing liquid partial composition to the absorbance Abs2 at 5 minutes after adding the 4-aminoantipyrine and ferricyanide-containing liquid partial composition to the mixed solution of the Trinder reagent-containing liquid partial composition and the sample specimen was measured. The absorbance change A C is given by the following formula (18). A C =(Abs2)-(Abs1)×180 / 225 (18) Here, 180 means the liquid volume of the Trinder reagent-containing liquid partial composition, and 225 means the total liquid volume of the Trinder reagent-containing liquid partial composition and the 4-aminoantipyrine and ferricyanide-containing liquid partial composition.

[0564] The absorbance change Acs measured using the sample specimen, and the absorbance change measured using purified water instead of the sample specimen was defined as the reagent blank Acb. As shown in the following formula (19), the value obtained by subtracting the absorbance change Acb of the reagent blank from the absorbance change Acs measured using the sample specimen was calculated as the sensitivity ΔAbs. ΔAbs = Acs - Acb (19)

[0565] The sensitivity after storing the partial composition for 14 days was designated as ΔAbs14, and the sensitivity after storing the partial composition for 28 days was designated as ΔAbs28. As shown in the following formula (20), the sensitivity retention rate was calculated as the ratio of ΔAbs14 to ΔAbs28. Combined with the result of the reagent blank Acb after storing the partial composition for 14 days, the sensitivity retention rate is shown in Table 8. Sensitivity residual rate (%) = ΔAbs28 ÷ ΔAbs14×100 (20)

Table 8-1

Table 8-2

Table 8-3

Table 8-4

Table 8-5

[0566] As is clear from Table 8, when using a stabilizer that gives a redox potential of ferricyanide greater than 0.058V when mixed with ferricyanide, even if the pH was increased above 7, the change in the reagent blank was small. Also, when using a stabilizer that gives a redox potential of ferricyanide greater than 0.058V when mixed with ferricyanide, even when a chelating agent was added, the change in the reagent blank was small.

[0567] [Example 6: Change in Reagent Arrangement] (Preparation of Liquid Partial Composition Containing 4-Aminoantipyrine) The following reagent components were dissolved in purified water to the described concentrations and adjusted to pH 7.0 with sodium hydroxide to prepare a liquid partial composition containing 4-aminoantipyrine. 50 mmol / L Tris(hydroxymethyl)aminomethane (manufactured by FUJIFILM Wako Pure Chemical Corporation) 0.05 wt / vol% Sodium Azide (manufactured by Merck) 8 mmol / L 4-Aminoantipyrine (manufactured by Actic Co., Ltd.) 10 kU / mL Protease for Glycated Albumin (manufactured by Asahi Kasei Pharma Corporation, obtained according to the method described in Japanese Patent No. 4565807.) 10 wt / vol% Propylene Glycol (manufactured by FUJIFILM Wako Pure Chemical Corporation)

[0568] (Preparation of Trinder reagent and ferrocyanide-containing liquid partial composition) The following reagent components were dissolved in purified water so as to have the described concentrations, and adjusted to pH 7.5 with sodium hydroxide to prepare a Trinder reagent and a ferrocyanide-containing liquid partial composition. 50 mmol / L Tris(hydroxymethyl)aminomethane (manufactured by Fujifilm Wako Pure Chemical Corporation) 5 wt / vol% D(-)-sorbitol (manufactured by Fujifilm Wako Pure Chemical Corporation) 0.05 wt / vol% sodium azide (manufactured by Merck) 10 mmol / L TOOS (manufactured by Dojindo Laboratories) 5 U / mL peroxidase (manufactured by Amano Enzyme Inc.) 20 U / mL ketoamine oxidase (manufactured by Asahi Kasei Pharma Corporation) 0.08 mmol / L potassium ferrocyanide (manufactured by Kokusan Chemical Co., Ltd.)

[0569] (Sample specimen) The following sample specimens were prepared. Control serum H (manufactured by Asahi Kasei Pharma Corporation)

[0570] (Reagent storage) The prepared Trinder reagent and ferrocyanide-containing liquid partial composition were stored at 30 °C for 14 days or 28 days, and the 4-aminoantipyrine-containing liquid partial composition was stored at 5 °C for 14 days or 28 days.

[0571] (Measurement procedure) 6 μL of the sample specimen was added to 180 μL of the 4-aminoantipyrine-containing liquid partial composition, and after reacting at 37°C for 5 minutes, 60 μL of the Trinder reagent and ferrocyanide-containing liquid partial composition was added and the reaction was continued at 37°C for another 5 minutes. The absorbance change from the absorbance Abs1 5 minutes after adding the sample specimen to the 4-aminoantipyrine-containing liquid partial composition to the absorbance Abs2 5 minutes after adding the Trinder reagent and ferrocyanide-containing liquid partial composition to the mixed solution of the 4-aminoantipyrine-containing liquid partial composition and the sample specimen was measured. The absorbance change A C is given by the following formula (21). A C =(Abs2) - (Abs1)×180 / 240 (21) Here, 180 means the liquid volume of the 4-aminoantipyrine-containing liquid partial composition, and 240 means the total liquid volume of the 4-aminoantipyrine-containing liquid partial composition, the Trinder reagent, and the ferrocyanide-containing liquid partial composition.

[0572] The absorbance change Acs measured using the sample specimen, the absorbance change measured using purified water instead of the sample specimen was defined as the reagent blank Acb, and as shown in the following formula (22), the value obtained by subtracting the absorbance change Acb of the reagent blank from the absorbance change Acs measured using the sample specimen was calculated as the sensitivity ΔAbs. ΔAbs = Acs - Acb (22)

[0573] The sensitivity after storing the partial composition for 14 days was defined as ΔAbs14, and the sensitivity after storing the partial composition for 28 days was defined as ΔAbs28. As shown in the following formula (23), the sensitivity retention rate was calculated as the ratio of ΔAbs28 to ΔAbs14. Together with the results of the reagent blank Acb after storing the partial composition for 14 days, the sensitivity retention rate is shown in Table 9. Sensitivity retention rate (%) = ΔAbs28 ÷ ΔAbs14×100 (23)

Table 9

[0574] As is clear from Table 9, when mixed with ferrocyanide, it was found that a reagent blank was low when using a stabilizer at a concentration that gives a redox potential of ferrocyanide greater than 0.058V.

[0575] [Example 7: Verification of the change in reagent blank due to the type of protease stabilizer and the long-term stability of the detection sensitivity stability] (Preparation of Trinder reagent-containing liquid partial composition) The following reagent components were dissolved in purified water to the described concentrations and adjusted to pH 7.6 with sodium hydroxide to prepare a Trinder reagent-containing liquid partial composition. 100 mmol / L N-Tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (manufactured by Dojindo Laboratories) 0.05 wt / vol% sodium azide (manufactured by Merck) 1 mmol / L sodium ethylenediaminetetraacetate (manufactured by Kokusan Chemical Co., Ltd.) 6 wt / vol% D(-)-sorbitol (manufactured by FUJIFILM Wako Pure Chemical Corporation) 2 mmol / L N,N-bis(4-sulfobutyl)-3-methylaniline, disodium salt (manufactured by Dojindo Laboratories) 10 U / mL peroxidase (manufactured by Amano Enzyme Inc.) 30 U / mL ketoamine oxidase (manufactured by Asahi Kasei Pharma Corporation)

[0576] (2 reagent system: Preparation of 4-aminoantipyrine and ferrocyanide-containing liquid partial composition) The following reagent components were dissolved in purified water to the described concentrations and adjusted to pH 7.5 with sodium hydroxide to prepare a 4-aminoantipyrine and ferrocyanide-containing liquid partial composition containing a stabilizer. 200 mmol / L 3,3-dimethylglutaric acid (manufactured by Tokyo Chemical Industry Co., Ltd.) 0.05 wt / vol% ProClin300 (manufactured by Sigma-Aldrich) 5 mmol / L 4-aminoantipyrine (manufactured by Actic Co., Ltd.) 0.1 mmol / L potassium ferrocyanide (manufactured by Kokusan Chemical Co., Ltd.) 500 U / mL Catalase (manufactured by Nagase ChemteX Corporation) 40 kU / mL Protease for Glycated Albumin (manufactured by Asahi Kasei Pharma Corporation, obtained according to the method described in Japanese Patent No. 4565807) Stabilizer at the concentration described below

[0577] (2 - reagent system: Stabilizer contained in the liquid partial composition containing 4 - aminoantipyrine and ferrocyanide) 30 wt / vol% Propylene Glycol (manufactured by FUJIFILM Wako Pure Chemical Corporation) 37.5 wt / vol% Propylene Glycol (manufactured by FUJIFILM Wako Pure Chemical Corporation) 50 wt / vol% Trimethylene Glycol (manufactured by FUJIFILM Wako Pure Chemical Corporation) 62.5 wt / vol% Trimethylene Glycol (manufactured by FUJIFILM Wako Pure Chemical Corporation) 50 wt / vol% Ethylene Glycol (manufactured by FUJIFILM Wako Pure Chemical Corporation) 62.5 wt / vol% Ethylene Glycol (manufactured by FUJIFILM Wako Pure Chemical Corporation) 0.6 wt / vol% 2 - Carboxyphenylboronic Acid (manufactured by Tokyo Chemical Industry Co., Ltd.) 0.75 wt / vol% 2 - Carboxyphenylboronic Acid (manufactured by Tokyo Chemical Industry Co., Ltd.) 30 wt / vol% Dimethyl Sulfoxide (manufactured by Nacalai Tesque, Inc.) 37.5 wt / vol% Dimethyl Sulfoxide (manufactured by Nacalai Tesque, Inc.)

[0578] (3 - reagent system: Preparation of the liquid partial composition (1) containing 4 - aminoantipyrine) The following reagent components were dissolved in purified water to the described concentrations and adjusted to pH 7.5 with sodium hydroxide to prepare a 4 - aminoantipyrine - containing liquid partial composition (1) containing a stabilizer. 250 mmol / L 3,3 - Dimethylglutaric Acid (manufactured by Tokyo Chemical Industry Co., Ltd.) 0.05 wt / vol% ProClin300 (manufactured by Sigma - Aldrich) 6.25 mmol / L 4 - Aminoantipyrine (manufactured by Actic Co., Ltd.) 625 U / mL Catalase (manufactured by Nagase ChemteX Corporation) 50 kU / mL Protease for Glycated Albumin (manufactured by Asahi Kasei Pharma Corporation, obtained according to the method described in Japanese Patent No. 4565807) Stabilizer at the concentration described below

[0579] (3 - reagent system: Stabilizer contained in the liquid partial composition (1) containing 4 - aminoantipyrine) 37.5 wt / vol% Propylene Glycol (manufactured by FUJIFILM Wako Pure Chemical Corporation) 50 wt / vol% Propylene Glycol (manufactured by FUJIFILM Wako Pure Chemical Corporation) 62.5 wt / vol% Trimethylene Glycol (manufactured by FUJIFILM Wako Pure Chemical Corporation) 62.5 wt / vol% Ethylene Glycol (manufactured by FUJIFILM Wako Pure Chemical Corporation) 0.75 wt / vol% 2 - Carboxyphenylboronic Acid (manufactured by Tokyo Chemical Industry Co., Ltd.) 37.5 wt / vol% Dimethyl Sulfoxide (manufactured by Nacalai Tesque, Inc.)

[0580] (3 - reagent system: Preparation of the liquid partial composition (1) containing ferrocyanide) The following reagent components were dissolved in purified water to the described concentrations and adjusted to pH 7.0 with sodium hydroxide to prepare the liquid partial composition (1) containing ferrocyanide. 100 mmol / L 3,3 - Dimethylglutaric Acid (manufactured by Tokyo Chemical Industry Co., Ltd.) 0.05 wt / vol% ProClin300 (manufactured by Sigma - Aldrich Corporation) 0.5 mmol / L Potassium Ferrocyanide (manufactured by Kokusan Chemical Co., Ltd.)

[0581] (3 - reagent system: Preparation of the liquid partial composition (2) containing 4 - aminoantipyrine) The following reagent components were dissolved in purified water to the described concentrations and adjusted to pH 7.0 with sodium hydroxide to prepare the liquid partial composition (2) containing 4 - aminoantipyrine. 100 mmol / L 3,3 - Dimethylglutaric Acid (manufactured by Tokyo Chemical Industry Co., Ltd.) 0.05 wt / vol% ProClin300 (manufactured by Sigma - Aldrich) 25 mmol / L 4 - aminoantipyrine (manufactured by Actech)

[0582] (3 - reagent system: Preparation of the liquid partial composition (2) containing ferricyanide) The following reagent components were dissolved in purified water to the described concentrations and adjusted to pH 7.5 with sodium hydroxide to prepare the liquid partial composition (2) containing ferricyanide and a stabilizer. 250 mmol / L 3,3 - dimethylglutaric acid (manufactured by Tokyo Chemical Industry) 0.05 wt / vol% ProClin300 (manufactured by Sigma - Aldrich) 0.125 mmol / L potassium ferricyanide (manufactured by Kokusan Chemical) 625 U / mL catalase (manufactured by Nagase ChemteX) 50 kU / mL protease for glycated albumin (obtained according to the method described in Japanese Patent No. 4565807, manufactured by Asahi Kasei Pharma) A stabilizer with the concentration described later

[0583] (3 - reagent system: Stabilizers contained in the liquid partial composition (2) containing ferricyanide) 37.5 wt / vol% propylene glycol (manufactured by Fujifilm Wako Pure Chemical) 37.5 wt / vol% dimethyl sulfoxide (manufactured by Nacalai Tesque)

[0584] (Sample specimens) The following sample specimens were prepared. Control serum L (manufactured by Asahi Kasei Pharma) Control serum H (manufactured by Asahi Kasei Pharma)

[0585] (Reagent storage) The prepared Trinder reagent - containing liquid partial composition, the 4 - aminoantipyrine and ferricyanide - containing partial composition, the 4 - aminoantipyrine - containing liquid partial composition, and the ferricyanide - containing liquid partial composition were stored at 5°C for 28 days or 1 year.

[0586] (Measurement Preparation) The 4 - aminoantipyrine - containing liquid partial composition (1) and the ferrocyanide - containing liquid partial composition (1) of the three - reagent system were mixed at a volume ratio of 4:1 to prepare the 4 - aminoantipyrine and ferrocyanide - containing liquid partial composition (1) for measurement. The 4 - aminoantipyrine - containing liquid partial composition (2) and the ferrocyanide - containing liquid partial composition (2) of the three - reagent system were mixed at a volume ratio of 1:4 to prepare the 4 - aminoantipyrine and ferrocyanide - containing liquid partial composition (2) for measurement.

[0587] (Measurement Procedure) 4.5 μL of the sample specimen was added to 180 μL of the Trinder reagent - containing liquid partial composition, and after reacting at 37°C for 5 minutes, 45 μL of the 4 - aminoantipyrine and ferrocyanide - containing liquid partial composition was added and reacted at 37°C for another 5 minutes. The absorbance change from the absorbance Abs1 at 5 minutes after adding the sample specimen to the Trinder reagent - containing liquid partial composition to the absorbance Abs2 at 5 minutes after adding the 4 - aminoantipyrine and ferrocyanide - containing liquid partial composition to the mixture of the Trinder reagent - containing liquid partial composition and the sample specimen was measured. The absorbance change A C is given by the following formula (24). A C =(Abs2)-(Abs1)×180 / 225 (24) Here, 180 means the volume of the Trinder reagent - containing liquid partial composition, and 225 means the total volume of the Trinder reagent - containing liquid partial composition and the 4 - aminoantipyrine and ferrocyanide - containing liquid partial composition.

[0588] The absorbance change Acs measured using the sample specimen, and the absorbance change measured using purified water instead of the sample specimen was taken as the reagent blank Acb. As shown in the following formula (25), the value obtained by subtracting the absorbance change Acb of the reagent blank from the absorbance change Acs measured using the sample specimen was calculated as the sensitivity ΔAbs. ΔAbs = Acs - Acb (25)

[0589] Taking the sensitivity after storing the partial composition for 28 days as ΔAbs28 and the sensitivity after storing the partial composition for 1 year as ΔAbs365, the long-term sensitivity retention rate was calculated as the ratio of ΔAbs365 to ΔAbs28 as shown in the following formula (26). Combining with the results of the reagent blank after storing the partial composition for 1 year, the long-term sensitivity retention rate is shown in Table 10. Long-term sensitivity retention rate (%) = ΔAbs365 ÷ ΔAbs28 × 100 (26)

[0590] In addition, the relationship between the reagent blank after storing the partial composition for 1 year and the redox potential of ferrocyanide is shown in Table 11 and Figure 3. For the redox potential of ferrocyanide, the redox potential value at the concentration when the stabilizer and ferrocyanide are mixed was used. For example, when the stabilizer and ferrocyanide are contained in the same partial composition (the 4-aminoantipyrine and ferrocyanide-containing liquid partial composition of the 2-reagent system, and the ferrocyanide-containing liquid partial composition (2) of the 3-reagent system), the redox potential value at the stabilizer concentration in the partial composition was used. Also, when the stabilizer and ferrocyanide are contained in different partial compositions (the 4-aminoantipyrine-containing liquid partial composition (1) and the ferrocyanide-containing liquid partial composition (1) of the 3-reagent system), the redox potential value at the stabilizer concentration when the 4-aminoantipyrine-containing liquid partial composition (1) and the ferrocyanide-containing liquid partial composition (1) are mixed was used.

Table 10-1

Table 10-2

Table 10-3

Table 10-4

Table 10-5

Table 11-1

[0591] The reagents stored for a long time also showed the same results as in Example 2. That is, as is clear from Tables 10, 11, and Figure 3, in both the two-reagent system and the three-reagent system, the effect of suppressing the reagent blank was shown to appear as a stabilizer that increases the redox potential of ferrocyanide by more than 0.058 V when mixed with ferrocyanide. Also, in any of the stabilizers, it was shown that the residual rate of the detection sensitivity improved by changing from the two-reagent system to the three-reagent system. In the two-reagent system using dimethyl sulfoxide as the stabilizer, the residual rate of sensitivity decreased to 80% or less. Therefore, it was shown that it is more preferable to use a stabilizer other than dimethyl sulfoxide from the viewpoint of the residual rate of sensitivity.

[0592] [Example 8: Verification of the long-term stability of the effect of adding a chelating agent] (Preparation of a liquid partial composition containing a Trinder reagent) The following reagent components were dissolved in purified water to the described concentrations and adjusted to pH 7.6 with sodium hydroxide to prepare a liquid partial composition containing a Trinder reagent. 100 mmol / L N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (manufactured by Dojindo Laboratories) 0.05 wt / vol% sodium azide (manufactured by Merck) 1 mmol / L sodium ethylenediaminetetraacetate (manufactured by Kokusan Chemical Co., Ltd.) 6 wt / vol% D(-)-sorbitol (manufactured by FUJIFILM Wako Pure Chemical Corporation) 2 mmol / L N,N-bis(4-sulfobutyl)-3-methylaniline, disodium salt (manufactured by Dojindo Laboratories) 10 U / mL peroxidase (manufactured by Amano Enzyme Inc.) 30 U / mL Ketamine Oxidase (manufactured by Asahi Kasei Pharma Corporation)

[0593] (Two-reagent system: Preparation of liquid partial composition containing 4-aminoantipyrine and ferricyanide) The following reagent components were dissolved in purified water to the described concentrations, adjusted to pH 7.5 with sodium hydroxide, and a liquid partial composition containing 4-aminoantipyrine and ferricyanide containing propylene glycol as a stabilizer, a partial composition containing trisodium citrate, and a partial composition not containing trisodium citrate were prepared. 200 mmol / L 3,3-dimethylglutaric acid (manufactured by Tokyo Chemical Industry Co., Ltd.) 0.05 wt / vol% ProClin300 (manufactured by Sigma-Aldrich) 5 mmol / L 4-aminoantipyrine (manufactured by Actic Co., Ltd.) 0.1 mmol / L potassium ferricyanide (manufactured by Kokusan Chemical Co., Ltd.) 500 U / mL Catalase (manufactured by Nagase ChemteX Corporation) 40 kU / mL Protease for glycated albumin (manufactured by Asahi Kasei Pharma Corporation, obtained according to the method described in Japanese Patent No. 4565807.) 1 mmol / L trisodium citrate (manufactured by FUJIFILM Wako Pure Chemical Corporation) Stabilizer at the concentration described below

[0594] (Two-reagent system: Stabilizer contained in the liquid partial composition containing 4-aminoantipyrine and ferricyanide) 30 wt / vol% propylene glycol (manufactured by FUJIFILM Wako Pure Chemical Corporation) 37.5 wt / vol% propylene glycol (manufactured by FUJIFILM Wako Pure Chemical Corporation)

[0595] (Three-reagent system: Preparation of liquid partial composition (1) containing 4-aminoantipyrine) Dissolve the following reagent components in purified water to the specified concentrations, adjust the pH to 7.5 with sodium hydroxide, and prepare a liquid partial composition (1) containing 4-aminoantipyrine and a stabilizer, which includes a partial composition containing trisodium citrate and a partial composition not containing trisodium citrate. 250 mmol / L 3,3-dimethylglutaric acid (manufactured by Tokyo Chemical Industry Co., Ltd.) 0.05 wt / vol% ProClin300 (manufactured by Sigma-Aldrich) 6.25 mmol / L 4-aminoantipyrine (manufactured by Actichem Co., Ltd.) 625 U / mL catalase (manufactured by Nagase ChemteX Corporation) 50 kU / mL protease for glycated albumin (manufactured by Asahi Kasei Pharma Corporation, obtained according to the method described in Japanese Patent No. 4565807). 1.25 mmol / L trisodium citrate (manufactured by FUJIFILM Wako Pure Chemical Corporation) A stabilizer at the concentration described below

[0596] (3 reagent system: Stabilizer contained in the liquid partial composition (1) containing 4-aminoantipyrine) 37.5 wt / vol% propylene glycol (manufactured by FUJIFILM Wako Pure Chemical Corporation) 62.5 wt / vol% trimethylene glycol (manufactured by FUJIFILM Wako Pure Chemical Corporation) 62.5 wt / vol% ethylene glycol (manufactured by FUJIFILM Wako Pure Chemical Corporation) 0.75 wt / vol% 2-carboxyphenylboronic acid (manufactured by Tokyo Chemical Industry Co., Ltd.)

[0597] (3 reagent system: Preparation of the liquid partial composition (1) containing ferrocyanide) Dissolve the following reagent components in purified water to the specified concentrations, and adjust the pH to 7.0 with sodium hydroxide to obtain a liquid partial composition (1) containing ferrocyanide, which includes a partial composition containing trisodium citrate and a partial composition not containing trisodium citrate. 100 mmol / L 3,3-dimethylglutaric acid (manufactured by Tokyo Chemical Industry Co., Ltd.) 0.05 wt / vol% ProClin300 (manufactured by Sigma-Aldrich) 0.5 mmol / L potassium ferricyanide (manufactured by Kokusan Chemical Co., Ltd.) 5 mmol / L trisodium citrate (manufactured by FUJIFILM Wako Pure Chemical Corporation)

[0598] (3-reagent system: Preparation of liquid partial composition (2) containing 4-aminoantipyrine) A partial composition containing a 4-aminoantipyrine-containing liquid partial composition (2) was prepared by dissolving the following reagent components in purified water to the described concentrations and adjusting the pH to 7.0 with sodium hydroxide. 100 mmol / L 3,3-dimethylglutaric acid (manufactured by Tokyo Chemical Industry Co., Ltd.) 0.05 wt / vol% ProClin300 (manufactured by Sigma-Aldrich) 25 mmol / L 4-aminoantipyrine (manufactured by Actic Co., Ltd.)

[0599] (3-reagent system: Preparation of ferricyanide-containing liquid partial composition (2)) The following reagent components were dissolved in purified water to the described concentrations, adjusted to pH 7.5 with sodium hydroxide, and a ferricyanide-containing liquid partial composition (2) containing propylene glycol as a stabilizer was prepared, including a partial composition containing trisodium citrate and a partial composition not containing trisodium citrate. 250 mmol / L 3,3-dimethylglutaric acid (manufactured by Tokyo Chemical Industry Co., Ltd.) 0.05 wt / vol% ProClin300 (manufactured by Sigma-Aldrich) 0.125 mmol / L potassium ferricyanide (manufactured by Kokusan Chemical Co., Ltd.) 625 U / mL catalase (manufactured by Nagase ChemteX Corporation) 50 kU / mL protease for glycated albumin (manufactured by Asahi Kasei Pharma Corporation, obtained according to the method described in Japanese Patent No. 4565807.) 1.25 mmol / L trisodium citrate (manufactured by FUJIFILM Wako Pure Chemical Corporation) 37.5 wt / vol% propylene glycol (manufactured by Fujifilm Wako Pure Chemical Corporation)

[0600] (sample specimen) The following sample specimens were prepared. Control serum L (manufactured by Asahi Kasei Pharma Corporation) Control serum H (manufactured by Asahi Kasei Pharma Corporation)

[0601] (reagent storage) The prepared liquid partial composition containing Trinder's reagent, the liquid partial composition containing 4-aminoantipyrine and ferricyanide, the liquid partial composition containing 4-aminoantipyrine, and the liquid partial composition containing ferricyanide were stored at 5°C for 28 days or 1 year.

[0602] (measurement preparation) The liquid partial composition (1) containing 4-aminoantipyrine and the liquid partial composition (1) containing ferricyanide of the three-reagent system were mixed at a volume ratio of 4:1 to prepare the liquid partial composition (1) containing 4-aminoantipyrine and ferricyanide for measurement. The liquid partial composition (2) containing 4-aminoantipyrine and the liquid partial composition (2) containing ferricyanide of the three-reagent system were mixed at a volume ratio of 1:4 to prepare the liquid partial composition (2) containing 4-aminoantipyrine and ferricyanide for measurement.

[0603] (measurement procedure) 4.5 μL of the sample specimen was added to 180 μL of the liquid partial composition containing Trinder's reagent, and after reacting at 37°C for 5 minutes, 45 μL of the liquid partial composition containing 4-aminoantipyrine and ferricyanide was added and further reacted at 37°C for 5 minutes. The absorbance change from the absorbance Abs1 at 5 minutes after adding the sample specimen to the liquid partial composition containing Trinder's reagent to the absorbance Abs2 at 5 minutes after adding the liquid partial composition containing 4-aminoantipyrine and ferricyanide to the mixed solution of the liquid partial composition containing Trinder's reagent and the sample specimen was measured. The absorbance change A C is given by the following formula (27). A C=(Abs2)-(Abs1)×180 / 225 (27) Here, 180 means the liquid volume of the liquid part composition containing the Trinder reagent, and 225 means the total liquid volume of the liquid part composition containing the Trinder reagent and the liquid part composition containing 4 - aminoantipyrine and ferricyanide.

[0604] The absorbance change Acs measured using the sample specimen, the absorbance change measured using purified water instead of the sample specimen is defined as the reagent blank Acb. As shown in the following formula (28), the value obtained by subtracting the absorbance change Acb of the reagent blank from the absorbance change Acs measured using the sample specimen was calculated as the sensitivity ΔAbs. ΔAbs = Acs - Acb (28)

[0605] Regarding the sensitivity after storing the partial composition for 28 days as ΔAbs28 and the sensitivity after storing the partial composition for 1 year as ΔAbs365, as shown in the following formula (29), the long - term sensitivity retention rate was calculated as the ratio of ΔAbs365 to ΔAbs28. Together with the results of the reagent blank after storing the partial composition for 1 year, the long - term sensitivity retention rate is shown in Table 12. Long - term sensitivity retention rate (%) = ΔAbs365 ÷ ΔAbs28×100 (29)

Table 12 - 1

Table 12 - 2

Table 12 - 3

Table 12 - 4

[0606] As is clear from Table 12, when mixed with ferrocyanide, when using a stabilizer that gives a redox potential of ferrocyanide greater than 0.058 V, the reagent blank when no chelating agent (trisodium citrate) was added and the reagent blank when a chelating agent (trisodium citrate) was added were almost the same.

[0607] [Example 9: Verification of the long-term stability of the effects of pH and chelating agent type] (Preparation of the liquid partial composition containing the Trinder reagent) The following reagent components were dissolved in purified water to the described concentrations and adjusted to pH 7.6 with sodium hydroxide to prepare a liquid partial composition containing the Trinder reagent. 100 mmol / L N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (manufactured by Dojindo Laboratories) 0.05 wt / vol% sodium azide (manufactured by Merck) 1 mmol / L sodium ethylenediaminetetraacetate (manufactured by Kokusan Chemical Co., Ltd.) 6 wt / vol% D(-)-sorbitol (manufactured by FUJIFILM Wako Pure Chemical Corporation) 2 mmol / L N,N-bis(4-sulfobutyl)-3-methylaniline, disodium salt (manufactured by Dojindo Laboratories) 10 U / mL peroxidase (manufactured by Amano Enzyme Inc.) 30 U / mL ketoamine oxidase (manufactured by Asahi Kasei Pharma Corporation)

[0608] (3 reagent system: Preparation of the liquid partial composition containing 4-aminoantipyrine) The following reagent components were dissolved in purified water to the described concentrations and adjusted to pH 7.5 with sodium hydroxide to prepare a liquid partial composition containing 4-aminoantipyrine containing propylene glycol as a stabilizer. 250 mmol / L 3,3-dimethylglutaric acid (manufactured by Tokyo Chemical Industry Co., Ltd.) 0.05 wt / vol% ProClin300 (manufactured by Sigma-Aldrich) 6.25 mmol / L 4-aminoantipyrine (manufactured by Actic Co., Ltd.) 625 U / mL Catalase (manufactured by Nagase ChemteX Corporation) 50 kU / mL Protease for glycated albumin (manufactured by Asahi Kasei Pharma Corporation, obtained according to the method described in Japanese Patent No. 4565807.) 50 wt / vol% Propylene glycol (manufactured by FUJIFILM Wako Pure Chemical Corporation)

[0609] (3 - reagent system: Preparation of ferricyanide - containing liquid partial composition (1)) The following reagent components were dissolved in purified water to the described concentrations, and the pH was adjusted with sodium hydroxide to prepare ferricyanide - containing liquid partial compositions (1) with different pH values. 100 mmol / L 3,3 - dimethylglutaric acid (manufactured by Tokyo Chemical Industry Co., Ltd.) 0.05 wt / vol% ProClin300 (manufactured by Sigma - Aldrich) 0.5 mmol / L Potassium ferricyanide (manufactured by Kokusan Chemical Co., Ltd.) Adjusted to the pH of the concentration described later

[0610] (pH of ferricyanide - containing liquid partial composition (1)) pH 6.5 pH 7.0 pH 7.5 pH 8.0

[0611] (3 - reagent system: Preparation of ferricyanide - containing liquid partial composition (2)) The following reagent components were dissolved in purified water to the described concentrations, and the pH was adjusted to 7.0 with sodium hydroxide to prepare a ferricyanide - containing liquid partial composition (2) containing a chelating agent. 100 mmol / L 3,3 - dimethylglutaric acid (manufactured by Tokyo Chemical Industry Co., Ltd.) 0.05 wt / vol% ProClin300 (manufactured by Sigma - Aldrich) 0.5 mmol / L Potassium ferricyanide (manufactured by Kokusan Chemical Co., Ltd.) Chelating agent of the concentration described later

[0612] (Chelating agent contained in ferricyanide - containing liquid partial composition (2)) 5 mmol / L sodium ethylenediaminetetraacetate (manufactured by Kokusan Chemical Co., Ltd., abbreviated as EDTA) 5 mmol / L glycol ether diamine tetraacetic acid (manufactured by Dojindo Laboratories, abbreviated as EGTA) 5 mmol / L N-(2-hydroxyethyl)iminodiacetic acid (manufactured by Dojindo Laboratories, abbreviated as HIDA) 5 mmol / L nitrilotris(methylphosphonic acid), trisodium salt (manufactured by Dojindo Laboratories, abbreviated as NTPO) 5 mmol / L nitrilotriacetic acid (manufactured by Dojindo Laboratories, abbreviated as NTA) 5 mmol / L trans-1,2-diaminocyclohexane-N,N,N,N-tetraacetic acid, monohydrate (manufactured by Dojindo Laboratories, abbreviated as CyDTA) 5 mmol / L iminodiacetic acid (manufactured by Dojindo Laboratories, abbreviated as IDA) 5 mmol / L diethylenetriamine-N,N,N’,N”,N”-pentaacetic acid (manufactured by Dojindo Laboratories, abbreviated as DTPA) 5 mmol / L 1,3-diamino-2-propanol-N,N,N’,N’-tetraacetic acid (manufactured by Dojindo Laboratories, abbreviated as DPTA-OH) 5 mmol / L sodium gluconate (manufactured by FUJIFILM Wako Pure Chemical Corporation) 5 mmol / L L(-)-sodium malate (manufactured by FUJIFILM Wako Pure Chemical Corporation) 5 mmol / L succinic acid (manufactured by Kanto Chemical Co., Inc.) 5 mmol / L trisodium citrate (manufactured by FUJIFILM Wako Pure Chemical Corporation) 5 mmol / L salicylic acid (manufactured by Nacalai Tesque, Inc.) 5 mmol / L (+)-potassium tartrate (manufactured by FUJIFILM Wako Pure Chemical Corporation) 5 mmol / L N-[tris(hydroxymethyl)methyl]glycine (manufactured by Dojindo Laboratories, abbreviated as Tricine) 5 mmol / L N,N-bis(2-hydroxyethyl)glycine (manufactured by Dojindo Laboratories, abbreviated as Bicine)

[0613] (Sample) The following sample specimens were prepared. Control serum L (manufactured by Asahi Kasei Pharma Corporation) Management Serum H (manufactured by Asahi Kasei Pharma Corporation)

[0614] (Reagent storage) The prepared Trinder reagent-containing liquid partial composition, 4-aminoantipyrine-containing liquid partial composition, and ferricyanide-containing liquid partial compositions (1) and (2) were stored at 5°C for 28 days or 1 year.

[0615] (Measurement preparation) The 4-aminoantipyrine-containing liquid partial composition and ferricyanide-containing liquid partial composition (1) or (2) of the three-reagent system were mixed at a volume ratio of 4:1 to prepare a 4-aminoantipyrine and ferricyanide-containing liquid partial composition for measurement.

[0616] (Measurement procedure) 4.5 μL of the sample specimen was added to 180 μL of the Trinder reagent-containing liquid partial composition and reacted at 37°C for 5 minutes. Then, 45 μL of the 4-aminoantipyrine and ferricyanide-containing liquid partial composition was added and reacted at 37°C for another 5 minutes. The absorbance change from the absorbance Abs1 at 5 minutes after adding the sample specimen to the Trinder reagent-containing liquid partial composition to the absorbance Abs2 at 5 minutes after adding the 4-aminoantipyrine and ferricyanide-containing liquid partial composition to the mixture of the Trinder reagent-containing liquid partial composition and the sample specimen was measured. The absorbance change A C is given by the following formula (30). A C =(Abs2) - (Abs1)×180 / 225 (30) Here, 180 means the liquid volume of the Trinder reagent-containing liquid partial composition, and 225 means the total liquid volume of the Trinder reagent-containing liquid partial composition and the 4-aminoantipyrine and ferricyanide-containing liquid partial composition.

[0617] The absorbance change Acs measured using the sample specimen, and the absorbance change measured using purified water instead of the sample specimen was defined as the reagent blank Acb. As shown in the following formula (31), the value obtained by subtracting the absorbance change Acb of the reagent blank from the absorbance change Acs measured using the sample specimen was calculated as the sensitivity ΔAbs. ΔAbs = Acs - Acb (31)

[0618] The sensitivity after storing the partial composition for 28 days was defined as ΔAbs28, and the sensitivity after storing the partial composition for 1 year was defined as ΔAbs365. As shown in the following formula (32), the long-term sensitivity survival rate was calculated as the ratio of ΔAbs28 to ΔAbs365. Combining with the results of the reagent blank after storing the partial composition for 1 year, the long-term sensitivity survival rate is shown in Table 13 and Figure 4. Long-term sensitivity survival rate (%) = ΔAbs365 ÷ ΔAbs28 × 100 (32)

Table 13-1

Table 13-2

[0619] As is clear from Table 13 and Figure 4, even when the pH of the ferrocyanide-containing liquid partial composition was changed, the change in the reagent blank was small. Also, even when the type of chelating agent added to the ferrocyanide-containing liquid partial composition was changed, the change in the reagent blank was small.

[0620] [Example 10: Verification of the long-term stability of the effect of pH] (Preparation of the liquid partial composition containing the Trinder reagent) The following reagent components were dissolved in purified water to the described concentrations and adjusted to pH 7.6 with sodium hydroxide to prepare a liquid partial composition containing the Trinder reagent. 100 mmol / L N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (manufactured by Dojindo Laboratories) 0.05 wt / vol% sodium azide (manufactured by Merck) 1 mmol / L sodium ethylenediaminetetraacetate (manufactured by a domestic chemical company) 6 wt / vol% D(-)-sorbitol (manufactured by FUJIFILM Wako Pure Chemical Corporation) 2 mmol / L N,N-bis(4-sulfobutyl)-3-methylaniline, disodium salt (manufactured by Dojindo Laboratories) 10 U / mL peroxidase (manufactured by Amano Enzyme Inc.) 30 U / mL ketoamine oxidase (manufactured by Asahi Kasei Pharma Corporation)

[0621] (3 - reagent system: Preparation of the liquid partial composition (1) containing 4 - aminoantipyrine) The following reagent components were dissolved in purified water to the described concentrations and adjusted to pH 7.5 with sodium hydroxide to prepare a liquid partial composition (1) containing 4 - aminoantipyrine and a stabilizer, and a partial composition containing trisodium citrate and a partial composition not containing trisodium citrate were prepared. 250 mmol / L 3,3 - dimethylglutaric acid (manufactured by Tokyo Chemical Industry Co., Ltd.) 0.05 wt / vol% ProClin300 (manufactured by Sigma - Aldrich) 6.25 mmol / L 4 - aminoantipyrine (manufactured by Actic Co., Ltd.) 625 U / mL catalase (manufactured by Nagase ChemteX Corporation) 50 kU / mL protease for glycated albumin (manufactured by Asahi Kasei Pharma Corporation, obtained according to the method described in Japanese Patent No. 4565807.) 1.25 mmol / L trisodium citrate (manufactured by FUJIFILM Wako Pure Chemical Corporation) Stabilizer at the concentration described later

[0622] (3 - reagent system: Stabilizer contained in the liquid partial composition (1) containing 4 - aminoantipyrine) 37.5 wt / vol% propylene glycol (manufactured by FUJIFILM Wako Pure Chemical Corporation) 62.5 wt / vol% trimethylene glycol (manufactured by FUJIFILM Wako Pure Chemical Corporation) 62.5 wt / vol% ethylene glycol (manufactured by FUJIFILM Wako Pure Chemical Corporation) 0.75 wt / vol% 2 - carboxyphenylboronic acid (manufactured by Tokyo Chemical Industry Co., Ltd.)

[0623] (3 - reagent system: Preparation of the liquid partial composition (1) containing ferrocyanide) The following reagent components were dissolved in purified water to the described concentrations and adjusted to pH 7.0 or pH 8.0 to prepare a ferrocyanide-containing liquid partial composition. Also, a partial composition containing trisodium citrate and a partial composition not containing trisodium citrate were prepared as the ferrocyanide-containing liquid partial composition at pH 8.0. 100 mmol / L 3,3-dimethylglutaric acid (manufactured by Tokyo Chemical Industry Co., Ltd.) 0.05 wt / vol% ProClin300 (manufactured by Sigma-Aldrich) 0.5 mmol / L potassium ferrocyanide (manufactured by Kokusan Chemical Co., Ltd.) 5 mmol / L trisodium citrate (manufactured by Fujifilm Wako Pure Chemical Corporation)

[0624] (3 reagent system: Preparation of 4-aminoantipyrine-containing liquid partial composition (2)) The following reagent components were dissolved in purified water to the described concentrations and adjusted to pH 7.0 or pH 8.0 with sodium hydroxide to prepare a 4-aminoantipyrine-containing liquid partial composition. Also, a partial composition containing trisodium citrate and a partial composition not containing trisodium citrate were prepared as the 4-aminoantipyrine-containing liquid partial composition adjusted to pH 8.0. 100 mmol / L 3,3-dimethylglutaric acid (manufactured by Tokyo Chemical Industry Co., Ltd.) 0.05 wt / vol% ProClin300 (manufactured by Sigma-Aldrich) 25 mmol / L 4-aminoantipyrine (manufactured by Actic Co., Ltd.) 25 mmol / L trisodium citrate (manufactured by Fujifilm Wako Pure Chemical Corporation) (added only when adjusted to pH 8.0)

[0625] (3 reagent system: Preparation of ferrocyanide-containing liquid partial composition (2)) The following reagent components were dissolved in purified water to the described concentrations, adjusted to pH 7.5 with sodium hydroxide, and a ferricyanide-containing liquid partial composition containing propylene glycol as a stabilizer was prepared, comprising a partial composition containing trisodium citrate and a partial composition not containing trisodium citrate. 250 mmol / L 3,3-dimethylglutaric acid (manufactured by Tokyo Chemical Industry Co., Ltd.) 0.05 wt / vol% ProClin300 (manufactured by Sigma-Aldrich) 0.125 mmol / L potassium ferricyanide (manufactured by Kokusan Chemical Co., Ltd.) 625 U / mL catalase (manufactured by Nagase ChemteX Corporation) 50 kU / mL protease for glycated albumin (manufactured by Asahi Kasei Pharma Corporation, obtained according to the method described in Japanese Patent No. 4565807). 1.25 mmol / L trisodium citrate (manufactured by FUJIFILM Wako Pure Chemical Corporation) 37.5 wt / vol% propylene glycol (manufactured by FUJIFILM Wako Pure Chemical Corporation)

[0626] (Sample Specimen) The following sample specimens were prepared. Control Serum L (manufactured by Asahi Kasei Pharma Corporation) Control Serum H (manufactured by Asahi Kasei Pharma Corporation)

[0627] (Reagent Storage) The prepared Trinder reagent-containing liquid partial composition, 4-aminoantipyrine-containing liquid partial composition, and ferricyanide-containing liquid partial composition were stored at 5°C for 28 days or 1 year.

[0628] (Measurement Preparation) The 4-aminoantipyrine-containing liquid partial composition (1) and ferricyanide-containing liquid partial composition (1) of the three-reagent system were mixed at a volume ratio of 4:1 to prepare the 4-aminoantipyrine and ferricyanide-containing liquid partial composition (1) for measurement. The 4 - aminoantipyrine - containing liquid partial composition (2) of the reagent system and the ferrocyanide - containing liquid partial composition (2) were mixed at a volume ratio of 1:4 to prepare the 4 - aminoantipyrine and ferrocyanide - containing liquid partial composition (2) used for measurement.

[0629] (Measurement procedure) 4.5 μL of the sample specimen was added to 180 μL of the Trinder reagent - containing liquid partial composition, and after reacting at 37°C for 5 minutes, 45 μL of the 4 - aminoantipyrine and ferrocyanide - containing liquid partial composition was added and further reacted at 37°C for 5 minutes. The absorbance change from the absorbance Abs1 at 5 minutes after adding the sample specimen to the Trinder reagent - containing liquid partial composition to the absorbance Abs2 at 5 minutes after adding the 4 - aminoantipyrine and ferrocyanide - containing liquid partial composition to the mixed solution of the Trinder reagent - containing liquid partial composition and the sample specimen was measured. The absorbance change A C is given by the following formula (33). A C =(Abs2)-(Abs1)×180 / 225 (33) Here, 180 means the liquid volume of the Trinder reagent - containing liquid partial composition, and 225 means the total liquid volume of the Trinder reagent - containing liquid partial composition and the 4 - aminoantipyrine and ferrocyanide - containing liquid partial composition.

[0630] The absorbance change Acs measured using the sample specimen, the absorbance change measured using purified water instead of the sample specimen was taken as the reagent blank Acb, and as shown in the following formula (34), the value obtained by subtracting the absorbance change Acb of the reagent blank from the absorbance change Acs measured using the sample specimen was calculated as the sensitivity ΔAbs. ΔAbs = Acs - Acb (34)

[0631] The sensitivity after storing the partial composition for 28 days was taken as ΔAbs28, and the sensitivity after storing the partial composition for 1 year was taken as ΔAbs365. As shown in the following formula (35), the long - term sensitivity survival rate was calculated as the ratio of ΔAbs28 to ΔAbs365. Together with the results of the reagent blank after storing the partial composition for 1 year, the long - term sensitivity survival rate is shown in Table 14. Long-term sensitivity retention rate (%) = ΔAbs365 ÷ ΔAbs28×100 (35)

Table 14-1

Table 14-2

Table 14-3

Table 14-4

Table 14-5

[0632] As is clear from Table 14, when using a stabilizer that gives a redox potential of ferrocyanide greater than 0.058V when mixed with ferrocyanide, even if the pH was increased to greater than 7, the change in the reagent blank was small. Also, when using a stabilizer that gives a redox potential of ferrocyanide greater than 0.058V when mixed with ferrocyanide, even if a chelating agent was added, the change in the reagent blank was small.

Claims

1. A method for suppressing a reagent blank of a glycated protein measurement reagent, comprising: preparing a Trinder reagent-containing partial composition containing at least a Trinder reagent; preparing a 4-aminoantipyrine-containing partial composition containing at least 4-aminoantipyrine; including adding a protease and a stabilizer for the protease to either the Trinder reagent-containing partial composition or the 4-aminoantipyrine-containing partial composition; adding a ferrocyanide to either the Trinder reagent-containing partial composition or the 4-aminoantipyrine-containing partial composition; the stabilizer for the protease being a stabilizer that makes the redox potential of the ferrocyanide when mixed with the stabilizer for the protease greater than 0.058 V; the redox potential being preparing a ferrocyanide-containing liquid composition, which contains the stabilizer for the protease, 100 mmol / L of N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid, 1 mmol / L of potassium ferrocyanide, and 150 mmol / L of sodium chloride, does not contain glycated protein, and has a pH adjusted to 7.5 with sodium hydroxide and is an aqueous solution; adding 10 mL of the ferrocyanide-containing liquid composition to a cell for voltammetry, using a glassy carbon electrode with an outer diameter of 6 mm and an inner diameter of 3 mm as the working electrode, using a coiled Pt counter electrode with a length of 23 cm and an electrode part diameter of 0.5 mm as the counter electrode, using an aqueous reference electrode (Ag / AgCl) as the reference electrode, and using an AUTOMATIC POLARIZATION SYSTEM HZ-7000 as the apparatus to measure the redox potential of the ferrocyanide at 25°C by cyclic voltammetry; being the redox potential measured by a method including in the cyclic voltammetry, changing the potential applied to the working electrode with respect to time; in changing the potential applied to the working electrode with respect to time, setting the sweep rate to 0.1 V / second, using the natural potential as the initial potential, sweeping the electrode potential to the positive side up to a first set potential of 0.5 V, then performing a sweep in which the electrode potential is folded back to the negative side from the first set potential to a second set potential of -0.5 V, and further performing a sweep in which the electrode potential is folded back to the positive side from the second set potential to the first set potential; The redox potential calculated as the average value of the potential value when the oxidation current shows a peak and the potential value when the reduction current shows a peak. A method for suppressing the reagent blank of a glycated protein measurement reagent.

2. A method for suppressing the reagent blank of a glycated protein measurement reagent, comprising: Preparing a Trinder reagent-containing partial composition containing at least a Trinder reagent; Preparing a 4-aminoantipyrine-containing partial composition containing at least 4-aminoantipyrine; Preparing a ferrocyanide-containing partial composition containing at least a ferrocyanide; including adding a protease and a stabilizer for the protease to any one of the Trinder reagent-containing partial composition, the 4-aminoantipyrine-containing partial composition, and the ferrocyanide-containing partial composition; the stabilizer for the protease is a stabilizer that makes the redox potential of the ferrocyanide greater than 0.058 V when mixed with the stabilizer for the protease; the redox potential is preparing a ferrocyanide-containing liquid composition, which contains the stabilizer for the protease, 100 mmol / L of N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid, 1 mmol / L of potassium ferrocyanide, and 150 mmol / L of sodium chloride, does not contain glycated protein, and has a pH adjusted to 7.5 with sodium hydroxide and is an aqueous solution; adding 10 mL of the ferrocyanide-containing liquid composition to a cell for voltammetry, using a glassy carbon electrode with an outer diameter of 6 mm and an inner diameter of 3 mm as the working electrode, using a coiled Pt counter electrode with a length of 23 cm and an electrode part diameter of 0.5 mm as the counter electrode, using an aqueous reference electrode (Ag / AgCl) as the reference electrode, and using an AUTOMATIC POLARIZATION SYSTEM HZ-7000 as the apparatus to measure the redox potential of the ferrocyanide at 25 °C by cyclic voltammetry; the redox potential measured by a method including in the cyclic voltammetry, changing the potential applied to the working electrode with respect to time In changing the potential applied to the working electrode with respect to time, the sweep rate is set to 0.1 V / second, the initial potential is the natural potential, the electrode potential is swept to the positive side up to a first set potential of 0.5 V, and then, a sweep is performed in which the electrode potential is folded back to the negative side from the first set potential to a second set potential of -0.5 V, and further, a sweep is performed in which the electrode potential is folded back to the positive side from the second set potential to the first set potential, The redox potential calculated as the average value of the potential value when the oxidation current shows a peak and the potential value when the reduction current shows a peak, A method for suppressing the reagent blank of a glycated protein measurement reagent.

3. A method for suppressing the reagent blank of a glycated protein measurement reagent, preparing a Trinder reagent-containing partial composition containing at least a Trinder reagent, preparing a 4-aminoantipyrine-containing partial composition containing at least 4-aminoantipyrine, including, including a protease and a stabilizer for the protease in either the Trinder reagent-containing partial composition or the 4-aminoantipyrine-containing partial composition, including a ferrocyanide in either the Trinder reagent-containing partial composition or the 4-aminoantipyrine-containing partial composition, wherein the stabilizer for the protease is selected from the group consisting of propylene glycol, trimethylene glycol, ethylene glycol, and carboxyphenylboronic acid, A method for suppressing the reagent blank of a glycated protein measurement reagent.

4. A method for suppressing the reagent blank of a glycated protein measurement reagent, preparing a Trinder reagent-containing partial composition containing at least a Trinder reagent, preparing a 4-aminoantipyrine-containing partial composition containing at least 4-aminoantipyrine, preparing a ferrocyanide-containing partial composition containing at least a ferrocyanide, including, including a protease and a stabilizer for the protease in any one of the Trinder reagent-containing partial composition, the 4-aminoantipyrine-containing partial composition, and the ferrocyanide-containing partial composition, wherein the stabilizer for the protease is selected from the group consisting of propylene glycol, trimethylene glycol, ethylene glycol, and carboxyphenylboronic acid, A method for suppressing the reagent blank of a glycated protein measurement reagent.

5. The method for suppressing a reagent blank of a glycated protein measurement reagent according to any one of claims 1 to 4, wherein the reagent blank is a reagent blank caused by ferricyanide.

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