JP2021007341A - 4-aminoantipyrine-containing partial composition for glycated protein measurement reagent containing catalase and chelating agent, glycated protein-measuring reagent, method for measuring glycated protein, method for stabilizing 4-aminoantipyrine-containing partial composition for glycated protein measurement reagent, and method for preserving 4-aminoantipyrine-containing partial composition for glycated protein measurement reagent - Google Patents

Abstract

To provide a 4-aminoantipyrine-containing partial composition that comprises catalase and used for a stable glycated protein-measuring reagent.SOLUTION: A 4-aminoantipyrine-containing partial composition for glycated protein-measuring reagent comprises (1) 4-aminoantipyrine, ferrocyanide, catalase, dimethyl sulfoxide, and protease; and further comprises (2) at least one chelating agent selected from the group consisting of citric acid and a salt thereof, malic acid and a salt thereof, nitrilotris (methylphosphonic acid) and a salt thereof, oxalic acid and a salt thereof, nitrilotriacetic acid and a salt thereof, and N-(2-Hydroxyethyl) iminodiacetic acid and a salt thereof.SELECTED DRAWING: None

Description

The present invention relates to a test agent and stabilizes a 4-aminoantipyrine-containing partial composition for a glycated protein measuring reagent, a glycated protein measuring reagent, a glycated protein measuring method, and a 4-aminoantipyrine-containing partial composition for a glycated protein measuring reagent. The present invention relates to a method and a method for preserving a 4-aminoantipyrine-containing partial composition for a saccharified protein measuring reagent.

Measurement of glycated protein is important in diagnosing and managing diabetes, and reflects glycated hemoglobin (HbA1c, etc.) that reflects the average blood glucose level of the past 1 to 2 months, and the average blood glucose level of the past 2 weeks. Glycated albumin (GA), fructosamine (FRA), which is a general term for glycated proteins in serum, and the like are routinely measured.

An enzyme method is an example of a highly accurate, inexpensive, and easy method for measuring a glycated protein. For example, in Patent Documents 1 and 2, a protease is allowed to act on a glycated protein, hydrogen peroxide is generated using ketoamine oxidase that acts on the produced glycated amino acid or glycated peptide, and peroxidase, a Trinder reagent, and 4-aminoantipyrine are further added. It is described that glycated proteins in serum can be measured by colorimetric quantification using. Patent Document 3 describes saccharification in serum by allowing a protease to act on a saccharified protein and measuring the amount of oxygen consumed when using ketoamine oxidase acting on the produced saccharified amino acid or saccharified peptide by a color development reaction or an oxygen electrode. It states that proteins can be measured. In Patent Documents 4 and 5, specifically, a protease is allowed to act on glycated hemoglobin and glycated albumin, and hydrogen peroxide is generated by using ketoamine oxidase that acts on the generated glycated amino acid or glycated peptide, and further, peroxidase and a Trinder reagent. , And 4-aminoantipyrine is used for colorimetric quantification to measure glycated hemoglobin and glycated albumin.

Currently, liquid products are the mainstream as measurement reagents for the enzyme method, and a two-reagent system in which the reagent components are divided into two liquid storage partial compositions has become the mainstream in consideration of storage stability. There is. The two liquid storage partial compositions may be referred to as a first reagent and a second reagent, respectively. In the glycated protein measurement reagent, ketoamine oxidase and protease are contained in separate liquid storage partial compositions in order to prevent degradation of ketoamine oxidase by protease, and Trinder reagent and 4-aminoantipyrine are contained in order to prevent natural color development during storage. Is preferably contained in separate liquid storage partial compositions. However, since the storage stability is still insufficient even with the two-reagent system, various techniques for improving the storage stability are provided.

For example, Patent Document 5 improves the stability of ketoamine oxidase by containing sugar alcohol, sucrose, water-soluble magnesium salt, water-soluble calcium salt, sulfide, amino acid, or sarcosin in the measurement reagent, and stores the measurement reagent. It states that stability can be improved. Further, Patent Document 5 describes the stability of protease by containing dimethylsulfoxide, alcohol, water-soluble calcium salt, salt, quaternary ammonium salt, or quaternary ammonium salt-type cationic surfactant in the measurement reagent. It is described that it can be improved and the stability of the measurement reagent can be improved. Patent Document 6 describes a fructosyl peptide by coexisting a polyaminocarboxylic acid-based chelating reagent with one or more reagents selected from ammonium salts, oxycarboxylic acid-based chelating reagents, sugar alcohols, and amino acids. It is described that the storage stability of a liquid measurement reagent containing an oxidase can be improved. Patent Document 7 describes that the stability of ketoamine oxidase and ascorbic acid oxidase can be improved and the storage stability of the measurement reagent can be improved by containing a chelating agent in the measurement reagent. Patent Document 8 describes that the storage stability of the measurement reagent can be improved by increasing the concentration of the buffer or 4-aminoantipyrine.

On the other hand, a non-specific color reaction occurs due to a peroxide derived from a measurement reagent or a sample, and the absorbance due to this non-specific color reaction may reduce the measurement accuracy. This non-specific color development is called a reagent blank. In order to eliminate the endogenous peroxide in the measurement reagent and reduce the reagent blank, it is necessary to contain peroxidase or catalase in the reagent, but conventionally, a compound (chelating agent) that coordinates with the iron ion of catalase. However, it is believed that a chelating agent cannot be added to the measurement reagent containing catalase because it inhibits the activity of catalase. For example, Non-Patent Document 1 describes that Clariant's Cartan RCF liq suppresses the decomposition of hydrogen peroxide by catalase by blocking the protoheme in catalase by chelating action. In addition, Non-Patent Document 2 describes that ascorbic acid and ferrous iron form a stable complex, and Non-Patent Document 3 and Non-Patent Document 4 describe that ascorbic acid inhibits catalase. It is described.

Japanese Unexamined Patent Publication No. 6-46846 Japanese Unexamined Patent Publication No. 5-192193 Japanese Unexamined Patent Publication No. 2-195900 International Publication No. 1997/13872 International Publication No. 2002/061119 Japanese Unexamined Patent Publication No. 2006-325547 Japanese Unexamined Patent Publication No. 2004-129531 Japanese Unexamined Patent Publication No. 2005-298632

Yoshitsu et al., "Efficiency of DIP Hydrogen Peroxide Bleaching with Catalase Inhibitors", Paper-Paper Technical Cooperative Magazine, 2010, Vol. Murata et al., "Bactericidal action and mechanism of action of ferrous iron", Saga University, 93: 141-155 (2008) Ohara et al., "Study on the function of catalyst in the decomposition reaction of hydrogen peroxide solution", Kanagawa Prefectural Education Center Research Collection 7: 109-114.1988 The Japanese Biochemical Society, Biochemical Data Book [II], Reduced Edition, 5th Print, June 1, 1989, Tokyo Kagaku Dojin

The present invention contains a 4-aminoantipyrine-containing partial composition for a stable glycated protein measuring reagent containing catalase, a glycated protein measuring reagent, a glycated protein measuring method, and a 4-aminoantipyrine-containing moiety for a glycated protein measuring reagent. Part of the task is to provide a method for stabilizing the composition and a method for preserving the 4-aminoantipyrine-containing partial composition for the saccharified protein measuring reagent.

As a result of diligent research to solve the above problems, the present inventor has increased the activity of catalase by incorporating a specific chelating agent into a 4-aminoantipyrine-containing partial composition for a reagent for measuring glycated protein containing catalase. We have found that the measurement reagent can be stabilized while maintaining it, and have completed the present invention. That is, the present invention includes the following.

[1] A 4-aminoantipyrine-containing partial composition for a saccharified protein measuring reagent, which contains (1) 4-aminoantipyrine, ferrocyanide, catalase, dimethylsulfoxide, and protease, and (2) citric acid. And its salts, malic acid and its salts, nitrilotriacetic acid (methylphosphonic acid) and its salts, oxalic acid and its salts, nitrilotriacetic acid and its salts, and N- (2-hydroxyethyl) iminodiacetic acid and its salts. A 4-aminoantipyrine-containing partial composition for a glycated protein measuring reagent further comprising at least one chelating agent selected from the group.

[2] A liquid 4-aminoantipyrine-containing partial composition for the reagent for measuring glycated protein according to [1].

[3] (1) 4-aminoantipyrine, ferrocyanide, catalase, dimethylsulfoxide, and protease, and (2) citric acid and its salt, malic acid and its salt, nitrilotriacetic acid (methylphosphonic acid) and its salt, Shu An acid and a salt thereof, a nitrilotriacetic acid and a salt thereof, and at least one chelating agent selected from the group consisting of N- (2-hydroxyethyl) iminodiacetic acid and a salt thereof are premixed. The 4-aminoantipyrine-containing partial composition for the glycated protein measuring reagent according to 1] or [2].

[4] The packaged 4-aminoantipyrine-containing partial composition for the glycated protein measuring reagent according to any one of [1] to [3].

[5] The 4-aminoantipyrine-containing partial composition for the glycated protein measuring reagent according to any one of [1] to [4], wherein the chelating agent is citric acid or a salt thereof.

[6] The 4-aminoantipyrine-containing partial composition for the glycated protein measuring reagent according to any one of [1] to [4], wherein the chelating agent is malic acid or a salt thereof.

[7] The glycated protein according to any one of [1] to [4], wherein the chelating agent is nitrilotris (methylphosphonic acid) or a salt thereof, or N- (2-hydroxyethyl) iminodiacetic acid or a salt thereof. 4-Aminoantipyrine-containing partial composition for measurement reagents.

[8] The 4-aminoantipyrine-containing partial composition for the glycated protein measuring reagent according to any one of [1] to [7], wherein the chelating agent is a sodium salt.

[9] The 4-aminoantipyrine-containing partial composition for the glycated protein measuring reagent according to any one of [1] to [8], wherein the ferrocyanide is potassium ferrocyanide.

[10] The 4-aminoantipyrine-containing partial composition for the glycated protein measuring reagent according to any one of [1] to [9], wherein the catalase is derived from Micrococcus.

[11] 4 for the glycated protein measuring reagent according to any one of [1] to [10], wherein the concentration of catalase in the 4-aminoantipyrine-containing partial composition is 0.1 U / mL or more and 10000 U / mL or less. -Aminoantipyrine-containing partial composition.

[12] The glycated protein measuring reagent according to any one of [1] to [11], wherein the concentration of the chelating agent in the 4-aminoantipyrine-containing partial composition is 0.1 μmol / L or more and 1000 mmol / L or less. 4-Aminoantipyrine-containing partial composition.

[13] The 4-aminoantipyrine-containing partial composition for the glycated protein measuring reagent according to [12], wherein the concentration of the chelating agent in the 4-aminoantipyrine-containing partial composition is 5 μmol / L or more.

[14] The 4-aminoantipyrine-containing partial composition for the glycated protein measuring reagent according to [12], wherein the concentration of the chelating agent in the 4-aminoantipyrine-containing partial composition is 10 μmol / L or more.

[15] The 4-aminoantipyrine-containing partial composition for the glycated protein measuring reagent according to [12], wherein the concentration of the chelating agent in the 4-aminoantipyrine-containing partial composition is 50 μmol / L or more.

[16] The 4-aminoantipyrine-containing partial composition for the glycated protein measuring reagent according to [12], wherein the concentration of the chelating agent in the 4-aminoantipyrine-containing partial composition is 500 μmol / L or more.

[17] The 4-aminoantipyrine-containing partial composition for the glycated protein measuring reagent according to [12], wherein the concentration of the chelating agent in the 4-aminoantipyrine-containing partial composition is 1 mmol / L or more.

[18] The 4-aminoantipyrine-containing partial composition for the glycated protein measuring reagent according to [12], wherein the concentration of the chelating agent in the 4-aminoantipyrine-containing partial composition is 100 mmol / L or less.

[19] The 4-aminoantipyrine-containing partial composition for the glycated protein measuring reagent according to any one of [1] to [18], which does not contain glycated amino acid oxidase, peroxidase, and Trinder reagent.

[20] The 4-aminoantipyrine-containing partial composition for the glycated protein measuring reagent according to any one of [1] to [19], which is stored at 2 ° C. or higher and 10 ° C. or lower.

[21] The 4-aminoantipyrine-containing partial composition for the glycated protein measuring reagent according to any one of [1] to [19], which can be stored at 2 ° C. or higher and 10 ° C. or lower for at least one month.

[22] The 4-aminoantipyrine-containing partial composition for the glycated protein measuring reagent according to any one of [1] to [21], wherein the glycated protein is glycated albumin.

[23] Measurement of glycated protein comprising the 4-aminoantipyrine-containing partial composition according to any one of [1] to [22] and a Trinder reagent-containing partial composition containing a glycated amino acid oxidase, a peroxidase, and a Trinder reagent. reagent.

[24] By combining 4-aminoantipyrine and the Trinder reagent, a color-developing reaction occurs in the presence of the glycated protein, and the concentration calculated from the absorbance corresponding to the color-developing reaction is 0.1 g / dL or more and 7.5 g. The glycated protein measuring reagent according to [23], which can be used for detecting a glycated protein of / dL or less.

[25] When the 4-aminoantipyrine-containing partial composition is stored at 30 ° C. for 2 weeks and then used in combination with the Trinder reagent-containing partial composition, the residual measurement sensitivity of the glycated protein is the 4-aminoantipyrine-containing partial composition. The glycated protein measuring reagent according to [23] or [24], which is 88% or more and 100% or less as compared with the case where the product is stored at 2 ° C. for 2 weeks.

[26] When the 4-aminoantipyrine-containing partial composition is stored at 2 ° C. or higher and 10 ° C. or lower for 1 year and then used in combination with the Trinder reagent-containing partial composition, the residual measurement sensitivity of the glycated protein is 4-amino. The saccharified protein measuring reagent according to [23] or [24], which is 76% or more and 100% or less as compared with the case where the antipyrine-containing partial composition is stored at 2 ° C. for 1 month.

[27] When the 4-aminoantipyrine-containing partial composition is stored at 2 ° C. or higher and 10 ° C. or lower for 18 months and then used in combination with the Trinder reagent-containing partial composition, the residual measurement sensitivity of the glycated protein is 4-. The saccharified protein measuring reagent according to [23] or [24], wherein the aminoantipyrine-containing partial composition is 64% or more and 100% or less as compared with the case where the partial composition is stored at 2 ° C. for 1 month.

[28] When the 4-aminoantipyrine-containing partial composition is stored at 2 ° C. or higher and 10 ° C. or lower for 2 years and then used in combination with the Trinder reagent-containing partial composition, the residual measurement sensitivity of the glycated protein is 4-amino. The saccharified protein measuring reagent according to [23] or [24], wherein the antipyrine-containing partial composition is 52% or more and 100% or less as compared with the case where the partial composition is stored at 2 ° C. for 1 month.

[29] The glycated protein measuring reagent according to [23] or [24], wherein the Trinder reagent-containing partial composition is liquid.

[30] The glycated protein measuring reagent according to any one of [23] to [29], wherein the glycated amino acid oxidase, peroxidase, and the Trinder reagent are mixed in advance in the Trinder reagent-containing partial composition.

[31] The glycated protein measuring reagent according to any one of [23] to [30], wherein the Trinder reagent-containing partial composition is packaged.

[32] The glycated protein measuring reagent according to any one of [23] to [31], wherein the Trinder reagent is N, N-bis (4-sulfobutyl) -3-methylaniline, 2 sodium.

[33] The reagent for measuring a glycated protein according to any one of [23] to [32], wherein the glycated protein is glycated albumin.

[34] Contacting the sample with the Trinder reagent-containing partial composition and the 4-aminoantipyrine-containing partial composition, and
To detect color development that depends on the glycated protein present in the sample,
Including
The Trinder reagent-containing partial composition comprises a glycated amino acid oxidase, a peroxidase, and a Trinder reagent.
The 4-aminoantipyrine-containing partial composition comprises (1) 4-aminoantipyrine, ferrocyanide, catalase, dimethylsulfoxide, and protease, and (2) citric acid and its salt, malic acid and its salt, nitrilotriacetic acid. At least one chelating agent selected from the group consisting of su (methylphosphonic acid) and its salt, citric acid and its salt, nitrilotriacetic acid and its salt, and N- (2-hydroxyethyl) iminodiacetic acid and its salt. Including,
Method for measuring glycated protein.

[35] The method for measuring a glycated protein according to [34], wherein the 4-aminoantipyrine-containing partial composition is liquid.

[36] The method for measuring a glycated protein according to [34] or [35], which is carried out after storing the liquid 4-aminoantipyrine-containing partial composition at 2 ° C. or higher and 10 ° C. or lower for at least one month.

[37] In the 4-aminoantipyrine-containing partial composition, (1) 4-aminoantipyrine, ferrocyanide, catalase, dimethylsulfoxide, and protease, and (2) citric acid and its salt, malic acid and its salt, nitrilotriacetic acid. With at least one chelating agent selected from the group consisting of su (methylphosphonic acid) and its salt, oxalic acid and its salt, nitrilotriacetic acid and its salt, and N- (2-hydroxyethyl) iminodiacetic acid and its salt. , Is premixed, according to any one of [34] to [36], wherein the glycated protein is measured.

[38] The method for measuring a glycated protein according to any one of [34] to [37], wherein the 4-aminoantipyrine-containing partial composition is packaged.

[39] The method for measuring a glycated protein according to any one of [34] to [38], wherein the chelating agent is citric acid or a salt thereof.

[40] The method for measuring a glycated protein according to any one of [34] to [38], wherein the chelating agent is malic acid or a salt thereof.

[41] The glycated protein according to any one of [34] to [38], wherein the chelating agent is nitrilotris (methylphosphonic acid) or a salt thereof, or N- (2-hydroxyethyl) iminodiacetic acid or a salt thereof. Measurement method.

[42] The method for measuring a glycated protein according to any one of [34] to [41], wherein the chelating agent is a sodium salt.

[43] The method for measuring a glycated protein according to any one of [34] to [42], wherein the ferrocyanide is potassium ferrocyanide.

[44] The method for measuring a glycated protein according to any one of [34] to [43], wherein the catalase is derived from Micrococcus.

[45] The method for measuring a glycated protein according to any one of [34] to [44], wherein the concentration of catalase in the 4-aminoantipyrine-containing partial composition is 0.1 U / mL or more and 10000 U / mL or less.

[46] The method for measuring a glycated protein according to any one of [34] to [45], wherein the concentration of the chelating agent in the 4-aminoantipyrine-containing partial composition is 0.1 μmol / L or more and 1000 mmol / L or less.

[47] The method for measuring a glycated protein according to [46], wherein the concentration of the chelating agent in the 4-aminoantipyrine-containing partial composition is 5 μmol / L or more.

[48] The method for measuring a glycated protein according to [46], wherein the concentration of the chelating agent in the 4-aminoantipyrine-containing partial composition is 10 μmol / L or more.

[49] The method for measuring a glycated protein according to [46], wherein the concentration of the chelating agent in the 4-aminoantipyrine-containing partial composition is 50 μmol / L or more.

[50] The method for measuring a glycated protein according to [46], wherein the concentration of the chelating agent in the 4-aminoantipyrine-containing partial composition is 500 μmol / L or more.

[51] The method for measuring a glycated protein according to [46], wherein the concentration of the chelating agent in the 4-aminoantipyrine-containing partial composition is 1 mmol / L or more.

[52] The method for measuring a glycated protein according to [46], wherein the concentration of the chelating agent in the 4-aminoantipyrine-containing partial composition is 100 mmol / L or less.

[53] The method for measuring a glycated protein according to any one of [34] to [52], wherein a glycated protein having a concentration of 0.1 g / dL or more and 7.5 g / dL or less can be detected.

[54] One of [34] to [53], wherein the absorbance of the sample added with the Trinder reagent-containing partial composition and the 4-aminoantipyrine-containing partial composition is measured depending on the concentration of the glycated protein. The method for measuring a glycated protein according to the above.

[55] By combining 4-aminoantipyrine and the Trinder reagent, a color-developing reaction occurs in the presence of the glycated protein, and the concentration calculated from the absorbance corresponding to the color-developing reaction is 0.1 g / dL or more and 7.5 g. The method for measuring a glycated protein according to any one of [34] to [54], which can detect a glycated protein of / dL or less.

[56] Any of [34] to [55], wherein the sample is brought into contact with the Trinder reagent-containing partial composition, and then the sample and the Trinder reagent-containing partial composition are brought into contact with the 4-aminoantipyrine-containing partial composition. Method for measuring glycated protein described in Crab.

[57] The method for measuring a glycated protein according to any one of [34] to [56], wherein the glycated amino acid oxidase, peroxidase, and the Trinder reagent are premixed in the Trinder reagent-containing partial composition.

[58] The method for measuring a glycated protein according to any one of [34] to [57], wherein the Trinder reagent-containing partial composition is liquid.

[59] The method for measuring a glycated protein according to any one of [34] to [58], wherein the Trinder reagent-containing partial composition is packaged.

[60] The method for measuring a glycated protein according to any one of [34] to [59], wherein the Trinder reagent is N, N-bis (4-sulfobutyl) -3-methylaniline, 2 sodium.

[61] The method for measuring a glycated protein according to any one of [34] to [60], wherein the glycated protein is glycated albumin.

[62] A method for stabilizing a 4-aminoantipyrine-containing partial composition for a glycated protein measuring reagent.
In the 4-aminoantipyrine-containing partial composition for the saccharified protein measurement reagent, (1) 4-aminoantipyrine, ferrocyanide, catalase, dimethylsulfoxide, and protease, and (2) citric acid and its salt, malic acid and its above. At least one selected from the group consisting of salts, nitrilotriacetic acid (methylphosphonic acid) and salts thereof, oxalic acid and salts thereof, nitrilotriacetic acid and salts thereof, and N- (2-hydroxyethyl) iminodiacetic acid and salts thereof. To contain a chelating agent,
A method for stabilizing a 4-aminoantipyrine-containing partial composition for a glycated protein measuring reagent.

[63] The method for stabilizing a 4-aminoantipyrine-containing partial composition for a glycated protein measuring reagent according to [62], wherein the 4-aminoantipyrine-containing partial composition is liquid.

[64] The method for stabilizing a 4-aminoantipyrine-containing partial composition for a glycated protein measuring reagent according to [62] or [63], further comprising packaging a 4-aminoantipyrine-containing partial composition.

[65] The method for stabilizing a 4-aminoantipyrine-containing partial composition for a glycated protein measuring reagent according to any one of [62] to [64], wherein the chelating agent is citric acid or a salt thereof.

[66] The method for stabilizing a 4-aminoantipyrine-containing partial composition for a glycated protein measuring reagent according to any one of [62] to [64], wherein the chelating agent is malic acid or a salt thereof.

[67] The glycated protein according to any one of [62] to [64], wherein the chelating agent is nitrilotris (methylphosphonic acid) or a salt thereof, or N- (2-hydroxyethyl) iminodiacetic acid or a salt thereof. A method for stabilizing a 4-aminoantipyrine-containing partial composition for a measurement reagent.

[68] The method for stabilizing a 4-aminoantipyrine-containing partial composition for a glycated protein measuring reagent according to any one of [62] to [67], wherein the chelating agent is a sodium salt.

[69] The method for stabilizing a 4-aminoantipyrine-containing partial composition for a glycated protein measuring reagent according to any one of [62] to [68], wherein the ferrocyanide is potassium ferrocyanide.

[70] The method for stabilizing a 4-aminoantipyrine-containing partial composition for a glycated protein measuring reagent according to any one of [62] to [69], wherein the catalase is derived from Micrococcus.

[71] The 4-for glycated protein measuring reagent according to any one of [62] to [70], wherein the concentration of catalase in the 4-aminoantipyrine-containing partial composition is 0.1 U / mL or more and 10000 U / mL or less. A method for stabilizing a partial composition containing aminoantipyrine.

[72] 4 for the glycated protein measuring reagent according to any one of [62] to [71], wherein the concentration of the chelating agent in the 4-aminoantipyrine-containing partial composition is 0.1 μmol / L or more and 1000 mmol / L or less. -A method for stabilizing a partial composition containing aminoantipyrine.

[73] The method for stabilizing a 4-aminoantipyrine-containing partial composition for a glycated protein measuring reagent according to [72], wherein the concentration of the chelating agent in the 4-aminoantipyrine-containing partial composition is 5 μmol / L or more.

[74] The method for stabilizing a 4-aminoantipyrine-containing partial composition for a glycated protein measuring reagent according to [72], wherein the concentration of the chelating agent in the 4-aminoantipyrine-containing partial composition is 10 μmol / L or more.

[75] The method for stabilizing a 4-aminoantipyrine-containing partial composition for a glycated protein measuring reagent according to [72], wherein the concentration of the chelating agent in the 4-aminoantipyrine-containing partial composition is 50 μmol / L or more.

[76] The method for stabilizing a 4-aminoantipyrine-containing partial composition for a glycated protein measuring reagent according to [72], wherein the concentration of the chelating agent in the 4-aminoantipyrine-containing partial composition is 500 μmol / L or more.

[77] The method for stabilizing a 4-aminoantipyrine-containing partial composition for a glycated protein measuring reagent according to [72], wherein the concentration of the chelating agent in the 4-aminoantipyrine-containing partial composition is 1 mmol / L or more.

[78] The method for stabilizing a 4-aminoantipyrine-containing partial composition for a glycated protein measuring reagent according to [72], wherein the concentration of the chelating agent in the 4-aminoantipyrine-containing partial composition is 100 mmol / L or less.

[79] The 4-aminoantipyrine for the glycated protein measuring reagent according to any one of [62] to [78], wherein the 4-aminoantipyrine-containing partial composition does not contain a glycated amino acid oxidase, a peroxidase, and a Trinder reagent. A method for stabilizing the contained partial composition.

[80] The 4-aminoantipyrine-containing partial composition for the glycated protein measuring reagent according to any one of [62] to [79], wherein the 4-aminoantipyrine-containing partial composition is stored at 2 ° C. or higher and 10 ° C. or lower. Stabilization method.

[81] 4-Amino for the glycated protein measuring reagent according to any one of [62] to [79], wherein the 4-aminoantipyrine-containing partial composition can be stored at 2 ° C. or higher and 10 ° C. or lower for at least one month. A method for stabilizing an antipyrine-containing partial composition.

[82] The description in any of [62] to [81], wherein the 4-aminoantipyrine-containing partial composition can be used in combination with a Trinder reagent-containing partial composition containing a glycated amino acid oxidase, a peroxidase, and a Trinder reagent. A method for stabilizing a 4-aminoantipyrine-containing partial composition for a reagent for measuring glycated protein.

[83] By combining 4-aminoantipyrine and the Trinder reagent, a color-developing reaction occurs in the presence of the glycated protein, and the concentration calculated from the absorbance corresponding to the color-developing reaction is 0.1 g / dL or more and 7.5 g. The 4-aminoantipyrine-containing partial composition for the glycated protein measuring reagent according to [82], wherein the 4-aminoantipyrine-containing partial composition for the glycated protein measuring reagent can be used for the detection of the glycated protein of / dL or less. Stabilization method.

[84] When the 4-aminoantipyrine-containing partial composition is stored at 30 ° C. for 2 weeks and then used in combination with the Trinder reagent-containing partial composition, the residual measurement sensitivity of the glycated protein is the 4-aminoantipyrine-containing partial composition. The method for stabilizing a 4-aminoantipyrine-containing partial composition for a glycated protein measuring reagent according to [82] or [83], which is 90% or more as compared with the case where the product is stored at 2 ° C. for 2 weeks.

[85] When the 4-aminoantipyrine-containing partial composition is stored at 2 ° C. or higher and 10 ° C. or lower for 1 year and then used in combination with the Trinder reagent-containing partial composition, the residual measurement sensitivity of the glycated protein is 4-amino. The 4-aminoantipyrine-containing reagent for the glycated protein measuring reagent according to [82] or [83], which is 76% or more and 100% or less as compared with the case where the antipyrine-containing partial composition is stored at 2 ° C. for 1 month. Method for stabilizing the composition.

[86] When the 4-aminoantipyrine-containing partial composition is stored at 2 ° C. or higher and 10 ° C. or lower for 18 months and then used in combination with the Trinder reagent-containing partial composition, the residual measurement sensitivity of the glycated protein is 4-. Containing 4-aminoantipyrine for the glycated protein measuring reagent according to [82] or [83], which is 64% or more and 100% or less as compared with the case where the aminoantipyrine-containing partial composition is stored at 2 ° C. for 1 month. A method for stabilizing a partial composition.

[87] When the 4-aminoantipyrine-containing partial composition is stored at 2 ° C. or higher and 10 ° C. or lower for 2 years and then used in combination with the Trinder reagent-containing partial composition, the residual measurement sensitivity of the glycated protein is 4-amino. The 4-aminoantipyrine-containing reagent for the glycated protein measuring reagent according to [82] or [83], which is 52% or more and 100% or less as compared with the case where the antipyrine-containing partial composition is stored at 2 ° C. for 1 month. Method for stabilizing the composition.

[88] The method for stabilizing a 4-aminoantipyrine-containing partial composition for a glycated protein measuring reagent according to any one of [82] to [87], wherein the Trinder reagent-containing partial composition is liquid.

[89] The method for stabilizing a 4-aminoantipyrine-containing partial composition for a glycated protein measuring reagent according to any one of [82] to [88], wherein the Trinder reagent-containing partial composition is packaged.

[90] Containing 4-aminoantipyrine for the glycated protein measuring reagent according to any one of [82] to [89], wherein the Trinder reagent is N, N-bis (4-sulfobutyl) -3-methylaniline disodium. A method for stabilizing a partial composition.

[91] The method for stabilizing a 4-aminoantipyrine-containing partial composition for a glycated protein measuring reagent according to any one of [62] to [90], wherein the glycated protein is glycated albumin.

[92] A method for preserving a 4-aminoantipyrine-containing partial composition for a glycated protein measuring reagent.
(1) Containing 4-aminoantipyrine, ferrocyanide, catalase, dimethylsulfoxide, and protease, and (2) citric acid and its salt, malic acid and its salt, nitrilotriacetic acid (methylphosphonic acid) and its salt, shu 4 for glycated protein measurement reagents further comprising at least one chelating agent selected from the group consisting of acids and salts thereof, nitrilotriacetic acid and salts thereof, and N- (2-hydroxyethyl) iminodiacetic acid and salts thereof. -Preparing a partial composition containing aminoantipyrine and
Preserving 4-aminoantipyrine-containing partial compositions and
A method for storing a 4-aminoantipyrine-containing partial composition for a glycated protein measuring reagent.

[93] The method for preserving the 4-aminoantipyrine-containing partial composition for a glycated protein measuring reagent according to [92], wherein the 4-aminoantipyrine-containing partial composition for the glycated protein measuring reagent is liquid.

[94] Preservation of 4-aminoantipyrine-containing partial composition for glycated protein measuring reagent according to [92] or [93], wherein the 4-aminoantipyrine-containing partial composition for glycated protein measuring reagent is packaged and stored. Method.

[95] The method for preserving a 4-aminoantipyrine-containing partial composition for a glycated protein measuring reagent according to any one of [92] to [94], wherein the chelating agent is citric acid or a salt thereof.

[96] The method for preserving a 4-aminoantipyrine-containing partial composition for a glycated protein measuring reagent according to any one of [92] to [94], wherein the chelating agent is malic acid or a salt thereof.

[97] The glycated protein according to any one of [92] to [94], wherein the chelating agent is nitrilotris (methylphosphonic acid) or a salt thereof, or N- (2-hydroxyethyl) iminodiacetic acid or a salt thereof. A method for storing a 4-aminoantipyrine-containing partial composition for a measurement reagent.

[98] The method for preserving a 4-aminoantipyrine-containing partial composition for a glycated protein measuring reagent according to any one of [92] to [97], wherein the chelating agent is a sodium salt.

[99] The method for preserving a 4-aminoantipyrine-containing partial composition for a glycated protein measuring reagent according to any one of [92] to [98], wherein the ferrocyanide is potassium ferrocyanide.

[100] The method for preserving a 4-aminoantipyrine-containing partial composition for a glycated protein measuring reagent according to any one of [92] to [99], wherein the catalase is derived from Micrococcus.

[101] 4-The glycated protein measuring reagent according to any one of [92] to [100], wherein the concentration of catalase in the 4-aminoantipyrine-containing partial composition is 0.1 U / mL or more and 10000 U / mL or less. A method for preserving a partial composition containing aminoantipyrine.

[102] 4 for the glycated protein measuring reagent according to any one of [92] to [101], wherein the concentration of the chelating agent in the 4-aminoantipyrine-containing partial composition is 0.1 μmol / L or more and 1000 mmol / L or less. -A method for preserving a partial composition containing aminoantipyrine.

[103] The method for preserving a 4-aminoantipyrine-containing partial composition for a glycated protein measuring reagent according to [102], wherein the concentration of the chelating agent in the 4-aminoantipyrine-containing partial composition is 5 μmol / L or more.

[104] The method for preserving a 4-aminoantipyrine-containing partial composition for a glycated protein measuring reagent according to [102], wherein the concentration of the chelating agent in the 4-aminoantipyrine-containing partial composition is 10 μmol / L or more.

[105] The method for preserving a 4-aminoantipyrine-containing partial composition for a glycated protein measuring reagent according to [102], wherein the concentration of the chelating agent in the 4-aminoantipyrine-containing partial composition is 50 μmol / L or more.

[106] The method for preserving a 4-aminoantipyrine-containing partial composition for a glycated protein measuring reagent according to [102], wherein the concentration of the chelating agent in the 4-aminoantipyrine-containing partial composition is 500 μmol / L or more.

[107] The method for preserving a 4-aminoantipyrine-containing partial composition for a glycated protein measuring reagent according to [102], wherein the concentration of the chelating agent in the 4-aminoantipyrine-containing partial composition is 1 mmol / L or more.

[108] The method for preserving a 4-aminoantipyrine-containing partial composition for a glycated protein measuring reagent according to [102], wherein the concentration of the chelating agent in the 4-aminoantipyrine-containing partial composition is 100 mmol / L or less.

[109] The glycated protein measuring reagent according to any one of [92] to [108], wherein the 4-aminoantipyrine-containing partial composition for the glycated protein measuring reagent does not contain the glycated amino acid oxidase, peroxidase, and Trinder reagent. 4-Amino Antipyrin-Containing Subcomposition for Preservation.

[110] The 4-aminoantipyrine-containing partial composition for the glycated protein measuring reagent according to any one of [92] to [109], wherein the 4-aminoantipyrine-containing partial composition is stored at 2 ° C. or higher and 10 ° C. or lower. Preservation method.

[111] Containing 4-aminoantipyrine for the glycated protein measuring reagent according to any one of [92] to [109], wherein the 4-aminoantipyrine-containing partial composition is stored at 2 ° C. or higher and 10 ° C. or lower for at least one month. How to store the partial composition.

[112] Any of [92] to [111], wherein the conserved 4-aminoantipyrine-containing partial composition can be used in combination with a Trinder reagent-containing partial composition containing a glycated amino acid oxidase, a peroxidase, and a Trinder reagent. A method for preserving a 4-aminoantipyrine-containing partial composition for a glycated protein measuring reagent according to the above.

[113] By combining 4-aminoantipyrine and the Trinder reagent, a color-developing reaction occurs in the presence of the glycated protein, and the concentration calculated from the absorbance corresponding to the color-developing reaction is 0.1 g / dL or more and 7.5 g. The method for preserving a 4-aminoantipyrine-containing partial composition for a saccharified protein measuring reagent according to [112], wherein a conserved 4-aminoantipyrine-containing partial composition can be used for detecting a glycated protein of / dL or less. ..

[114] When the 4-aminoantipyrine-containing partial composition is stored at 30 ° C. for 2 weeks and then used in combination with the Trinder reagent-containing partial composition, the residual measurement sensitivity of the glycated protein is the 4-aminoantipyrine-containing partial composition. The method for preserving a 4-aminoantipyrine-containing partial composition for a glycated protein measuring reagent according to [112] or [113], which is 90% or more as compared with the case where the product is stored at 2 ° C. for 2 weeks.

[115] When the 4-aminoantipyrine-containing partial composition is stored at 2 ° C. or higher and 10 ° C. or lower for 1 year and then used in combination with the Trinder reagent-containing partial composition, the residual measurement sensitivity of the glycated protein is 4-amino. The 4-aminoantipyrine-containing reagent for the glycated protein measuring reagent according to [112] or [113], which is 76% or more and 100% or less as compared with the case where the antipyrine-containing partial composition is stored at 2 ° C. for 1 month. How to store the composition.

[116] When the 4-aminoantipyrine-containing partial composition is stored at 2 ° C. or higher and 10 ° C. or lower for 18 months and then used in combination with the Trinder reagent-containing partial composition, the residual measurement sensitivity of the saccharified protein is 4-. Containing 4-aminoantipyrine for the saccharified protein measuring reagent according to [112] or [113], which is 64% or more and 100% or less as compared with the case where the aminoantipyrine-containing partial composition is stored at 2 ° C. for 1 month. How to store a partial composition.

[117] When the 4-aminoantipyrine-containing partial composition is stored at 2 ° C. or higher and 10 ° C. or lower for 2 years and then used in combination with the Trinder reagent-containing partial composition, the residual measurement sensitivity of the glycated protein is 4-amino. The 4-aminoantipyrine-containing reagent for the glycated protein measuring reagent according to [112] or [113], which is 52% or more and 100% or less as compared with the case where the antipyrine-containing partial composition is stored at 2 ° C. for 1 month. How to store the composition.

[118] The method for preserving a 4-aminoantipyrine-containing partial composition for a glycated protein measuring reagent according to any one of [112] to [117], wherein the Trinder reagent-containing partial composition is liquid.

[119] The method for preserving a 4-aminoantipyrine-containing partial composition for a glycated protein measuring reagent according to any one of [112] to [118], wherein the Trinder reagent-containing partial composition is packaged.

[120] Containing 4-aminoantipyrine for the glycated protein measuring reagent according to any one of [112] to [119], wherein the Trinder reagent is N, N-bis (4-sulfobutyl) -3-methylaniline disodium. How to store the partial composition.

[121] The method for preserving a 4-aminoantipyrine-containing partial composition for a glycated protein measuring reagent according to any one of [92] to [120], wherein the glycated protein is glycated albumin.

According to the present invention, a partial composition for a stable glycated protein measuring reagent containing catalase, a glycated protein measuring reagent, a glycated protein measuring method, and a 4-aminoantipyrine-containing partial composition for a glycated protein measuring reagent. It is possible to provide a method for stabilizing and a method for preserving a partial composition containing 4-aminoantipyrine for a glycation measuring reagent.

Hereinafter, preferred embodiments of the present invention (hereinafter referred to as “embodiments”) will be described in detail. The embodiments shown below exemplify devices and methods for embodying the technical idea of the present invention, and the technical idea of the present invention specifies combinations of constituent members and the like to the following. It's not a thing. The technical idea of the present invention can be modified in various ways within the scope of claims.

The 4-aminoantipyrine-containing partial composition for the saccharified protein measuring reagent according to the embodiment includes (1) 4-aminoantipyrine, ferrocyanide, catalase, dimethylsulfoxide, and protease, and (2) citric acid and a salt thereof. Selected from the group consisting of malic acid and its salts, nitrilotriacetic acid (methylphosphonic acid) and its salts, oxalic acid and its salts, nitrilotriacetic acid and its salts, and N- (2-hydroxyethyl) iminodiacetic acid and its salts. Includes at least one chelating agent.

In the present disclosure, the reagent is a reagent for measuring an object to be measured, for example, an aggregate of partial compositions. The reagent can detect the measurement target by itself. The partial composition is a composition that constitutes a part of a reagent. The partial composition cannot measure the measurement target by itself.

The glycated protein is, for example, glycated albumin (GA), glycated hemoglobin (HbA1c, etc.).

When measuring a glycated protein such as glycated albumin, the 4-aminoantipyrine-containing partial composition for the glycated protein measuring reagent according to the embodiment can be used in combination with the Trinder reagent-containing partial composition. The Trinder reagent-containing partial composition may include a glycated amino acid oxidase, a Trinder reagent, and a peroxidase. The 4-aminoantipyrine-containing partial composition according to the embodiment preferably does not contain glycated amino acid oxidase, Trinder reagent, and peroxidase.

The 4-aminoantipyrine-containing partial composition according to the embodiment may be liquid or may be stored until mixed with the Trinder reagent-containing partial composition. The Trinder reagent-containing partial composition may be liquid or may be stored until mixed with the 4-aminoantipyrine-containing partial composition according to the embodiment. The contents of the 4-aminoantipyrine-containing partial composition according to the embodiment may be mixed in advance. The inclusions of the Trinder reagent-containing partial composition may be premixed. The 4-aminoantipyrine-containing partial composition and the Trinder reagent-containing partial composition according to the embodiment may be packaged, respectively.

When the 4-aminoantipyrine-containing partial composition according to the embodiment, the Trinder reagent-containing partial composition, and a glycated protein such as albumin to which glucose contained in the sample to be measured (sample sample) is bound are brought into contact with each other, glycation occurs. Proteins are broken down by proteases into glycated amino acids or glycated peptides. The saccharified amino acid or saccharified peptide is decomposed into amino acids or peptides and glucosone by glycated amino acid oxidase. At this time, the saccharified amino acid oxidase reacts with water and oxygen to generate hydrogen peroxide. The Trinder reagent and 4-aminoantipyrine undergo an oxidative condensation reaction in the presence of hydrogen peroxide and peroxidase to develop color. For example, when N, N-bis (4-sulfobutyl) -3-methylaniline, or 2 sodium (TODB) is used as the Trinder reagent, a blue-purple color reaction occurs. The bluish-purple coloration is detected by measuring the absorbance at a wavelength of 546 nm. The concentration of glycated protein contained in the sample is quantified based on the absorbance at a wavelength of 546 nm.

When measuring the concentration of a glycated protein, it is known that it is preferable to first add the Trinder reagent-containing partial composition to the sample sample because free glycated amino acids or glycated peptides mixed in the sample sample are eliminated. In the usual method of using the partial composition according to the embodiment, the Trinder reagent-containing partial composition is first added to the sample sample, and the sample sample and the Trinder reagent-containing partial composition are added for several minutes, for example, about 5 to 10 minutes. The glycated protein can be measured by leaving the mixture to stand, then adding the 4-aminoantipyrine-containing partial composition according to the embodiment to the mixture, and measuring the degree of color development. However, in some cases, the Trinder reagent-containing partial composition and the 4-aminoantipyrine-containing partial composition may be added and mixed at the same time when added to the sample, or the Trinder reagent-containing partial composition and 4-amino may be added and mixed at the same time. The antipyrine-containing partial composition may be added in advance and used immediately, or the 4-aminoantipyrine-containing partial composition may be added to the sample sample first, and then the Trinder reagent-containing partial composition may be added.

In order to quantify a glycated protein, it is common to measure and calibrate one or more calibration substances (calibrators) having a known glycated protein concentration. A person skilled in the art can carry out the method for creating a calibrator and the calibration method based on publicly known information. For example, the method will be appropriately carried out with reference to the descriptions in International Publication No. 2001/094618 and JP-A-2005-261383. be able to. Further, in order to correct the fluctuation of the measured value due to the decrease in sensitivity during storage of the measuring reagent, it is usual to perform calibration periodically, for example, once a month.

The protease contained in the 4-aminoantipyrine-containing partial composition for the glycated protein measuring reagent according to the embodiment may be any protease that decomposes a glycated protein derived from a human protein to effectively produce a glycated amino acid or a glycated peptide. .. The protease is, for example, an endopeptidase. Examples of endopeptidases include serine endopeptidases. Proteases are preferably microorganism-derived proteases such as the genus Bacillus, the genus Streptomyces, the genus Tritirachium, and the genus Aspergillus. Examples of Bacillus-derived proteases include alcalase, nutrase, esperase, sabinase (above, manufactured by Novozymes), bioprase OP, bioprase SP-20FG, bioprase 30L, bioprase 30G, bioprase AL-15FG, bioprase APL-30, protease CL. -15 (above, manufactured by Nagase ChemteX), Protease SD PC-10CF, Samoase PC-10F, Protease SD-AY10, Protin SD-NY10 (above, manufactured by Amano Enzyme), Multifect PR6L, Optimase PR40L, Optimase Optimase PR40E (above, manufactured by Danisco Japan), Orientase 22BF, Nucresin, Orientase 10NL, Orientase 90N (above, manufactured by HBI), Aloase AP-10, Aloase NP-10, Aloase XA-10 (above, Yakult) (Manufactured by Yakuhin Kogyo Co., Ltd.), Subtilicin, Bacterial proteinase type XXIV, Protease Bacillus licheniformis-derived type VIII, Protease Bacillus polymyxa-derived type IX, Thermolysin Geobacillus Stearothermofilus-derived type X, Protease Derived type XXVII, Protease Bacillus licheniformis Derived type XXXI (above, manufactured by Sigma), Thermolysin (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), Dispase I, Dispase II (above, manufactured by Joint Shusei Co., Ltd.), Neutral Proteinase (manufactured by TOYOBO) ) Etc. can be mentioned. Examples of proteases derived from the genus Streptomyces include pronase, protease Streptomyces griseus-derived type XIV (manufactured by Sigma), Alkalopicular proteinase (manufactured by TOYOBO USA), Denateam PMC SOFTER (manufactured by Nagasechemtex), etc. .. Examples of proteases derived from the genus Tritilatium include Proteinase K (manufactured by Sigma). Examples of proteases derived from Aspergillus include Protease P "Amano" 3SD, Protease A "Amano" SD, Protease M "Amano" SD (above, manufactured by Amano Enzyme), Sumiteam MP, Sumiteam LPL-G, Sumiteam LP50D, Sumiteam. AP (above, manufactured by Shin Nihon Kagaku Kogyo Co., Ltd.), Orientase OP, Orientase AY (above, manufactured by HBI), Protease Aspergillus Saitoi-derived type XIII, Protease Aspergillus sojae-derived type XIX, Protease Aspergillus Xeleus-derived type XIX, Protease Aspergillus Examples include oryzae-derived (above, manufactured by Sigma), Denateam AP, denapsin 2P (above, manufactured by Nagase Chemtex), Punchpase NP-2, Punchdase MP, Punchdase P (above, manufactured by Yakult Pharmaceutical Co., Ltd.) and the like. Of these, alcalase, bioprase SP-20FG, protein SD-AY10, multifect PR6L, Optimase PR40L, alloase XA-10, subtilisin, pronase, proteinase K, and PR "Amano" K are preferable.

From another point of view, the protease contained in the 4-aminoantipyrine-containing partial composition for the glycated protein measuring reagent according to the embodiment includes, for example, the enzyme number EC: 3.4 or EC: 3.4.21. Proteases are preferred examples. Further, a protease of EC: 3.4.21.62 is also mentioned as a more preferable example.

Dimethyl sulfoxide contained in the 4-aminoantipyrine-containing partial composition for the glycated protein measuring reagent according to the embodiment stabilizes the protease.

The glycated amino acid oxidase contained in the Trinder reagent-containing partial composition may be a glycated amino acid derived from a human protein or a glycated amino acid oxidase that effectively acts on the glycated peptide, and examples thereof include Gibbella and Aspergillus. ), Candida, Penicillium, Fusarium, Acremonium, Debaryomyces, and Corynebacterium from the genus Corynebacterium Examples thereof include recombinant glycated amino acid oxidase variants. More specifically, ketoamine oxidase (KAOD, manufactured by Asahi Kasei Pharma Co., Ltd., Clinica Chimica Acta, 2002, 324, p.61-71), mutant KAOD (KAOD-V, manufactured by Asahi Kasei Pharma Co., Ltd., patent document). 1), FAOD-E (manufactured by Kikkoman Co., Ltd.) and the like.

Specific examples of the Trinder reagent contained in the Trinder reagent-containing partial composition include N- (3-sulfopropyl) aniline sodium monohydrate (HALPS) and N-ethyl-N- (3-sulfopropyl). -3-Methylaniline sodium monohydrate (TOPS), N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline sodium monohydrate (MAOS), N- (3) -Sulfopropyl) -3,5-dimethoxyaniline sodium monohydrate (HPAPS), N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline sodium (HDAOS), N-ethyl-N- (3-Sulfopropyl) -3,5-dimethoxyaniline sodium monohydrate (DAPS), N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline sodium (DAOS), N-ethyl-N- (3-sulfopropyl) sodium aniline (ALPS), N-ethyl-N- (3-sulfopropyl) -3-methoxyaniline sodium monohydrate (ADPS), N-ethyl-N- (2-Hydroxy-3-sulfopropyl) -3-methoxyaniline sodium dihydrate (ADOS), N-ethyl-N- (2-hydroxy-3-sulfopropyl) -m-toluidine (TOOS), N, Examples thereof include N-bis (4-sulfobutyl) -3-methylaniline disodium (TODB), 3-hydroxy-2,4,6-triiodobenzoic acid (HTIB) and the like. Of these, TOPS, ALPS, TOOS, and TODB are preferable.

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

When the Trinder reagent and 4-aminoantipyrine are included in the same reagent, they naturally undergo an oxidative condensation reaction to develop color during storage. Therefore, the Trinder reagent is contained in the Trinder reagent-containing partial composition, and 4-aminoantipyrine is used. Is contained in the 4-aminoantipyrine-containing partial composition according to the embodiment.

The peroxidase contained in the Trinder reagent-containing partial composition has a function of promoting the oxidative condensation reaction between the Trinder reagent and 4-aminoantipyrine, eliminating peroxides, and reducing reagent blanks. Therefore, by including peroxidase in the measurement reagent, it is possible to suppress the non-specific color reaction due to the peroxide derived from the measurement reagent or the sample, and contribute to the improvement of the measurement accuracy. Any source of peroxidase can be used, and examples thereof include peroxidase derived from plants such as horseradish, and peroxidase derived from microorganisms such as bacteria and mold. Specific examples include Peroxidase from horseradish (manufactured by Sigma), peroxidase, horseradish derived (manufactured by Wako Junyaku Co., Ltd.), PO "AMANO" 3 (manufactured by Amano Enzyme), Peroxidase (manufactured by Toyobo Enzyme), and the like. ..

Catalase contained in the 4-aminoantipyrine-containing partial composition for the glycated protein measuring reagent according to the embodiment stabilizes the 4-aminoantipyrine-containing partial composition according to the embodiment. Catalase also has the function of eliminating peroxides that may be present in the 4-aminoantipyrine-containing partial composition and reducing reagent blanks. Catalase can be of any origin, for example Aspergillus.
Examples include bacterial catalases such as niger, Micrococcus, and Rhodopseudomonas, and animal catalases such as bovine liver and human erythrocytes. Specifically, Catalase from Aspergillus niger (manufactured by Sigma), Catalase from human erythrocytes (manufactured by Sigma), catalase, derived from bovine liver (manufactured by Wako Junyaku Co., Ltd.), catalase (manufactured by Nagase ChemteX), etc. Be done. Catalase is preferably derived from microorganisms, more preferably from Micrococcus.

It can also be expressed as catalase contained in the 4-aminoantipyrine-containing partial composition for the glycated protein measuring reagent according to the embodiment, and catalase having an enzyme number of EC: 1.11.1.6.

The ferrocyanide contained in the 4-aminoantipyrine-containing partial composition for the glycated protein measuring reagent according to the embodiment suppresses the influence of bilirubin contained in the sample on the measurement of the glycated protein. Examples of the ferrocyanide may be any compound containing a ferrocyanide ion, such as potassium ferrocyanide (Fe (CN) ₆ K ₄ ) and sodium ferrocyanide (Fe (CN) ₆ Na ₄ ). Can be mentioned.

Citric acid and its salt, malic acid and its salt, nitrilotriacetic acid (methylphosphonic acid) and its salt, oxalic acid and its salt, which are contained in the 4-aminoantipyrine-containing partial composition for the saccharified protein measuring reagent according to the embodiment. At least one chelating agent selected from the group consisting of nitrilotriacetic acid and its salt, and N- (2-hydroxyethyl) iminodiacetic acid and its salt suppresses the decrease in sensitivity of the glycated protein measuring reagent. It is preferable to use a salt of the above compound because pH fluctuation is suppressed.

The type of the chelating agent salt is not particularly limited as long as it forms a salt, and may be either an acid addition salt or a base addition salt, or may be in the form of zwitterion. Examples of the base addition salt include a base addition salt with an inorganic base such as sodium, potassium, magnesium, calcium and aluminum, or a base addition with an organic base such as methylamine, 2-aminoethanol, arginine, lysine or ornithine. Includes salt and the like. Of these, a base addition salt with an inorganic base is preferable. A sodium salt is exemplified as a typical base addition salt.

The concentration of the chelating agent may be a concentration sufficient to stabilize the 4-aminoantipyrine-containing partial composition for the glycated protein measuring reagent according to the embodiment, and the lower limit is usually 0.1 μmol / L or more. , Or 1 μmol / L or more, preferably 5 μmol / L or more, or 10 μmol / L or more, more preferably 50 μmol / L or more, or 100 μmol / L or more, particularly preferably 500 μmol / L or more, or 1 mmol / L or more. To. Further, in some cases, it is preferably 2 mmol / L or more, 5 mmol / L or more, and further preferably 10 mmol / L or more. Further, since the degree of the stabilizing effect may be saturated, the upper limit can be determined from the viewpoint of cost and the like. For example, the upper limit is usually 1000 mmol / L or less, preferably 100 mmol / L or less, more preferably 100 mmol / L or less. Examples include 50 mmol / L or less.

Each of the 4-aminoantipyrine-containing partial composition and the Trinder reagent-containing partial composition according to the embodiment may further contain water, an organic solvent and the like as the 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 the like. Examples thereof include ether solvents and the like (tetratetra, diethyl ether, ethylene glycol monoalkyl ether, cyclic ether and the like).

In addition, each of the 4-aminoantipyrine-containing partial composition and the Trinder reagent-containing partial composition according to the embodiment may contain a buffer. Examples of buffers are 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-Acetamide) -2-aminoethanesulfonic acid (ACES), N- (2-acetamide) 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), piperazin-1 , 4-bis (2-ethanesulfonic acid (PIPES), piperazin-1,4-bis (2-hydroxy-3-propanesulfonic acid) (POPSO), N-tris (hydroxymethyl) methyl-3-aminopropanesulfon Acid (TAPS), 2-Hydroxy-N-Tris (Hydroxymethyl) Methyl-3-aminopropanesulfonic Acid (TAPSO), N- [Tris (Hydroxymethyl) Methyl] Glycine (Tricine), and Trishydroxymethylaminomethane ( A buffer using Tris), etc., or boric acid, ammonia, glycine, carbonic acid, acetic acid, phosphoric acid, diethanolamine, p-phenolsulfonic acid, 2-amino-2-methylpropan-1,3-diol, cacodylate , Maleic acid, veronal, 3,3-dimethylglutaric acid and the like.

In addition, each of the 4-aminoantipyrine-containing partial composition and the Trinder reagent-containing partial composition according to the embodiment further contains preservatives, enzyme stabilizers, surfactants, and other enzymes as other additives. It may be included. Examples of preservatives include sodium azide, ProClin and the like. As the stabilizer of the enzyme, any stabilizer that stabilizes the enzyme during storage can be used. For example, sugars such as cyclodextrin, sucrose, mannose, fructose, lactose, galactose and trehalose, etc. Examples thereof include sugar alcohols such as sorbitol and mannitol. As the surfactant, any of a cationic surfactant, an anionic surfactant, an amphoteric surfactant, and a nonionic surfactant may be used, for example, as an example of the nonionic surfactant. Polyoxyethylene alkyl ether, polyoxyethylene alkyl phenyl ether, polyoxyethylene polyoxypropylene alkyl ether, sorbitan fatty acid ester, polyoxyethylene sorbitan fatty acid ester, glycerin fatty acid ester, polyoxyethylene polyoxypropylene block polymer, etc. Can be mentioned. Examples of amphoteric surfactants include alkyl betaine, betaine acetate, sulfobetaine, and alkylamine oxide. Examples of other enzymes include ascorbic acid oxidase and bilirubin oxidase, which are used to suppress the influence of ascorbic acid and bilirubin contained in the sample on the measured value.

The concentration of the glycated amino acid oxidase contained in the Trinder reagent-containing partial composition may be a concentration sufficient to react with the produced glycated amino acid or glycated peptide, and the lower limit is 0.1 U / mL or more, preferably 1 U. / ML or more, more preferably 10 U / mL or more is exemplified. From the viewpoint of cost, the upper limit is preferably 1000 U / mL or less, preferably 200 U / mL or less, and more preferably 100 U / mL or less. The concentration of peroxidase may be a concentration sufficient to react with the generated hydrogen peroxide, and the lower limit is 0.01 U / mL or more, preferably 0.1 U / mL or more, and more preferably 1 U / mL or more. Is exemplified. From the viewpoint of cost, the upper limit is exemplified by 500 U / mL or less, preferably 100 U / mL or less, and more preferably 50 U / mL or less. The concentration of the Trinder reagent may be a concentration sufficient to react with the generated hydrogen peroxide, and the lower limit is 0.01 mmol / L or more, preferably 0.1 mmol / L or more, more preferably 0.5 mmol. / L or more is exemplified. Further, as the upper limit, from the viewpoint of cost, 100 mmol / L or less, preferably 10 mmol / L or less, more preferably 5 mmol / L or less is exemplified.

The concentration of 4-aminoantipyrine contained in the 4-aminoantipyrine-containing partial composition for the saccharified protein measuring reagent according to the embodiment may be a concentration sufficient to react with the generated hydrogen peroxide, and the lower limit is set. , 0.01 mmol / L or more, preferably 0.1 mmol / L or more, and more preferably 0.5 mmol / L or more. Further, as the upper limit, from the viewpoint of cost, 100 mmol / L or less, preferably 50 mmol / L or less, more preferably 10 mmol / L or less is exemplified. The concentration of the ferrocyanide may be a concentration sufficient to suppress the influence of bilirubin contained in the sample on the measurement of the glycated protein, and the lower limit is 1 μmol / L or more, preferably 0.01 mmol / L or more. , More preferably 0.02 mmol / L or more. Further, as the upper limit, from the viewpoint of cost, 10 mmol / L or less, preferably 1 mmol / L or less, more preferably 0.5 mmol / L or less is exemplified.

The concentration of dimethyl sulfoxide contained in the 4-aminoantipyrine-containing partial composition for the glycated protein measuring reagent according to the embodiment may be a concentration sufficient to stabilize the protease, and the lower limit is 1% or more. An example is preferably 10% or more, more preferably 20% or more. Further, as the upper limit, 90% or less, preferably 70% or less, more preferably 50% or less is exemplified from the viewpoint of solubility of other components. The concentration of the protease may be a concentration capable of decomposing the glycated protein within an appropriate time, and the lower limit is exemplified by 100 U / mL or more, preferably 1 kU / mL or more, and more preferably 10 kU / mL or more. Further, as the upper limit, from the viewpoint of cost, 1000 kU / mL or less, preferably 200 kU / mL or less, more preferably 100 kU / mL or less is exemplified.

The lower limit of the concentration of citric acid and a salt thereof contained in the 4-aminoantipyrine-containing partial composition for the saccharified protein measuring reagent according to the embodiment is 10 μmol / L or more, preferably 100 μmol / L or more, more preferably 1 mmol / L. L and above are exemplified. Examples of the lower limit of the concentration of malic acid and its salt are 1 mmol / L or more, preferably 5 mmol / L or more, and more preferably 10 mmol / L or more. Examples of the lower limit of nitrilotris (methylphosphonic acid) and a salt thereof are 5 μmol / L or more, preferably 10 μmol / L or more. Examples of the lower limit of the concentration of oxalic acid and its salt are 10 μmol / L or more, preferably 100 μmol / L or more, and more preferably 500 μmol / L or more. Examples of the lower limit of the concentration of nitrilotriacetic acid and its salt are 10 μmol / L or more, preferably 100 μmol / L or more, and more preferably 500 μmol / L or more. Examples of the lower limit of the concentration of N- (2-hydroxyethyl) iminodiacetic acid and a salt thereof are 5 μmol / L or more, preferably 10 μmol / L or more, and more preferably 50 μmol / L or more.

The concentration ratio of the chelating agent to the ferrocyanide is preferably 0.001 or more, preferably 0.01 or more, and more preferably 0.1 or more as the lower limit from the viewpoint of obtaining the stabilizing action of the glycated protein measuring reagent. To. From the viewpoint of solubility, the upper limit is preferably 10,000 or less, preferably 1000 or less, and more preferably 200 or less.

The concentration of catalase contained in the 4-aminoantipyrine-containing partial composition for the glycated protein measuring reagent according to the embodiment is sufficient to eliminate the endogenous peroxide and stabilize the glycated protein measuring reagent. The concentration may be any amount, and the lower limit is preferably 0.1 U / mL or more, preferably 1 U / mL or more, and more preferably 10 U / mL or more. From the viewpoint of cost, the upper limit is preferably 10,000 U / mL or less, preferably 5000 U / mL or less, and more preferably 1000 U / mL or less.

The activity / concentration ratio (U / mmol) of catalase to 4-aminoantipyrine is preferably 2 or more, preferably 20 or more, and more preferably 50 or more as the lower limit from the viewpoint of obtaining the stabilizing action of the glycated protein measuring reagent. Will be done. From the viewpoint of cost, the upper limit is preferably 20000 or less, preferably 20000 or less, and more preferably 2000 or less.

Since the 4-aminoantipyrine-containing partial composition for the glycated protein measuring reagent according to the embodiment contains a chelating agent, it has good storage stability for a long period of time. Therefore, it is possible to accurately measure the glycated protein over a long period of time. At the time of measuring glycated protein, for example, when the concentration of glycated protein such as glycated albumin is as low as 7.5 g / dL or less, preferably 7 g / dL or less, more preferably 6 g / dL or less, the storage stability by the chelating agent The effect is noticeable. Examples of the lower limit of the concentration of a glycated protein such as glycated albumin that can be measured are 0.1 g / dL or more, preferably 0.15 g / dL or more, and more preferably 0.2 g / dL or more.

Conventionally, it is considered that a chelating agent coordinates the iron ion of catalase and inhibits the activity of catalase. On the other hand, the above-mentioned chelating agent contained in the 4-aminoantipyrine-containing partial composition for the glycated protein measuring reagent according to the embodiment enhances the storage stability of the 4-aminoantipyrine-containing partial composition, and catalase. Does not substantially inhibit the activity of. Therefore, according to the embodiment, it is possible to achieve both reduction of reagent blanks by catalase and improvement of stabilization by a specific chelating agent.

The 4-aminoantipyrine-containing partial composition for the glycated protein measuring reagent according to the embodiment can be stably stored for a practical period required for distribution. For example, the 4-aminoantipyrine-containing partial composition for the glycated protein measuring reagent according to the embodiment can be stored for at least one month. Examples of the lower limit of the storage period of the 4-aminoantipyrine-containing partial composition for the glycated protein measuring reagent according to the embodiment are 1 day or more, 1 month or more, 6 months or more, or 1 year or more. The upper limit of the storage period of the 4-aminoantipyrine-containing partial composition for the glycated protein measuring reagent according to the embodiment is 7 days or less, 6 months or less, 1 year and 3 months or less, and 1 year and 6 months or less. , Or 2 years or less is exemplified. Examples of the lower limit of the storage temperature of the 4-aminoantipyrine-containing partial composition for the glycated protein measuring reagent according to the embodiment are 2 ° C. or higher, 4 ° C. or higher, or 5 ° C. or higher. Further, as the upper limit of the storage temperature of the 4-aminoantipyrine-containing partial composition for the glycated protein measuring reagent according to the embodiment, 10 ° C. or lower, 8 ° C. or lower, or 5 ° C. or lower is exemplified.

With respect to the measurement sensitivity of the glycated protein when the 4-aminoantipyrine-containing partial composition according to the embodiment stored at 2 ° C. for 2 weeks and the Trinder reagent-containing partial composition stored at 2 ° C. for 2 weeks were used in combination. , A glycated protein when a 4-aminoantipyrine-containing partial composition according to an embodiment stored at a predetermined temperature for a predetermined period and a Trinder reagent-containing partial composition stored at 2 ° C. for a predetermined period are used in combination. The ratio of the measurement sensitivities of is called the sensitivity residual rate. In this case, as the sensitivity residual rate when the partial composition according to the embodiment is used, when the predetermined temperature is 30 ° C. and the predetermined period is 2 weeks, the lower limit is 88% or more, 90% or more. Alternatively, 92% or more is exemplified. Examples of the upper limit are 99% or less, 98% or less, or 97% or less.

Alternatively, a saccharified protein when the 4-aminoantipyrine-containing partial composition according to the embodiment stored at 2 ° C. for 1 month and the Trinder reagent-containing partial composition stored at 2 ° C. for 1 month are used in combination. The 4-aminoantipyrine-containing partial composition according to the embodiment stored at a predetermined temperature for a predetermined period and the Trinder reagent-containing partial composition stored at 2 ° C. for a predetermined period were used in combination with respect to the measurement sensitivity of the above. The ratio of the measurement sensitivity of the saccharified protein in the case is called the sensitivity residual rate. Alternatively, a saccharified protein when the 4-aminoantipyrine-containing partial composition according to the embodiment stored at 2 ° C. for 1 month and the Trinder reagent-containing partial composition stored at 5 ° C. for 1 month are used in combination. The 4-aminoantipyrine-containing partial composition according to the embodiment stored at a predetermined temperature for a predetermined period and the Trinder reagent-containing partial composition stored at 5 ° C. for a predetermined period were used in combination with respect to the measurement sensitivity of the above. The ratio of the measurement sensitivity of the saccharified protein in the case is called the sensitivity residual rate. In these cases, the sensitivity residual rate when the partial composition according to the embodiment is used is that when the predetermined temperature is 2 ° C. or higher and 10 ° C. or lower and the predetermined period is one year, the lower limit of the sensitivity residual rate is set. , 76% or more, 80% or more, or 84% or more are exemplified. Examples of the upper limit are 100% or less, 98% or less, or 96% or less. When the predetermined temperature is 2 ° C. or higher and 10 ° C. or lower and the predetermined period is 18 months, the lower limit of the sensitivity residual rate is, for example, 64% or more, 70% or more, or 76% or more. Examples of the upper limit are 100% or less, 97% or less, or 94% or less. When the predetermined temperature is 2 ° C. or higher and 10 ° C. or lower and the predetermined period is 2 years, the lower limit of the sensitivity residual rate is, for example, 52% or more, 60% or more, or 68% or more. Examples of the upper limit are 100% or less, 96% or less, or 94% or less.

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.

Regarding the reagents used in the examples, the left may be used as an abbreviation for the name described on the right below.
Sodium citrate: trisodium citrate dihydrate Sodium malate: L (-)-sodium malate Sodium thiosulfate: sodium thiosulfate pentahydrate HIDA: N- (2-hydroxyethyl) iminodiacetic acid NTPO: Nitrilotriacetic acid trisodium NTA: Nitrilotriacetic acid ADA: N- (2-acetamide) Iminodiacetic acid EDDP: Ethylenediaminedipropionic acid BAPPTA: O, O'-bis (2-aminophenyl) ethylene glycol-N, N, N', N'-tetrapotassium tetraacetate CyDTA: trans-1,2-diaminocyclohexane-N, N, N', N'-tetraacetic acid monohydrate IDA: iminodiacetic acid TPN: N, N, N', N'-tetrakis (2-pyridylmethyl) ethylenediamine DTPA: diethylenetriamine-N, N, N', N'', N''-pentaacetic acid

A 7180 type Hitachi automatic analyzer (manufactured by Hitachi High-Tech Fielding Corporation) was used for the measurement of the absorbance in the examples. 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 -Abs ₇₀₀ (1)

[Example 1: Suppression of decrease in sensitivity in catalase-containing saccharified albumin measuring reagent]
(Partial composition containing Trinder reagent)
The following reagent components were dissolved in purified water to the described concentrations and adjusted to pH 7.6 to prepare a Trinder reagent-containing partial composition.
100 mmol / L N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid (manufactured by Dojin Chemical Co., Ltd.)
0.05% by mass / volume% Sodium azide (Merck)
2 mmol / L N, N-bis (4-sulfobutyl) -3-methylaniline disodium (manufactured by Dojin Chemical Co., Ltd.)
10 U / mL Peroxidase (manufactured by Amano Enzyme)
30 U / mL ketoamine oxidase (manufactured by Asahi Kasei Pharma)

(Partial composition containing 4-aminoantipyrine)
The following reagent components were dissolved in purified water to the described concentrations and adjusted to pH 7.5 to prepare a 4-aminoantipyrine-containing partial composition. In addition to the following reagents, a 4-aminoantipyrine-containing partial composition containing catalase (manufactured by Nagase ChemteX Corporation) having a final concentration of 500 U / mL was also prepared.
200 mmol / L 3,3-dimethylglutaric acid (manufactured by Tokyo Kasei Co., Ltd.)
0.05% by weight / volume Procrine 300 (manufactured by Spellco)
5 mmol / L 4-aminoantipyrine (manufactured by Actec)
30% by weight / volume dimethyl sulfoxide (manufactured by Nacalai Tesque)
0.1 mmol / L Potassium ferrocyanide (manufactured by Domestic Chemical Co., Ltd.)
40 kU / mL Protease for saccharified albumin (manufactured by Asahi Kasei Pharma, obtained according to the method described in Japanese Patent No. 4565807)

(Sample sample)
As sample samples, controlled serum L (manufactured by Asahi Kasei Pharma Co., Ltd.) and controlled serum H (manufactured by Asahi Kasei Pharma Co., Ltd.) were prepared.

(Preservation of reagents)
The prepared Trinder reagent-containing partial composition was stored at 2 ° C. for 2 weeks. In addition, the 4-aminoantipyrine-containing partial composition was stored at 2 ° C. and 30 ° C. for 2 weeks.

(Measurement)
4.5 μL of sample sample was added to 180 μL of the Trinder reagent-containing partial composition incubated at 37 ° C., and the mixture was reacted at 37 ° C. for 5 minutes. Next, 45 μL of the 4-aminoantipyrine-containing partial composition was added to the mixed solution of the Trinder reagent-containing partial composition and the sample sample, and the mixture was further reacted at 37 ° C. for 5 minutes. The absorbance (Abs1) 5 minutes after the sample sample was added to the Trinder reagent-containing partial composition and the absorbance (Abs2) 5 minutes after the 4-aminoantipyrine-containing partial composition was further added were measured. The change in absorbance was calculated according to the following equation (2).
Change in absorbance = (Abs2)-(Abs1) x 180/225 (2)
Here, 180 represents the amount of liquid of the Trinder reagent-containing partial composition. Further, 225 represents the total liquid amount of the Trinder reagent-containing partial composition and the 4-aminoantipyrine-containing partial composition.

The change in absorbance measured using purified water instead of the sample sample is used as the reagent blank, and as shown in equation (3) below, the value obtained by subtracting the change in absorbance of the reagent blank from the change in absorbance measured using the sample sample. Was calculated as the sensitivity ΔAbs.
ΔAbs = (change in absorbance when measuring sample sample)-(change in absorbance when measuring purified water) (3)

A Trinder stored at 2 ° C. for 2 weeks with a sensitivity ΔAbs measured using a Trinder reagent-containing partial composition stored at 2 ° C. for 2 weeks and a 4-aminoantipyrine-containing partial composition stored at 2 ° C. for 2 weeks as 100%. The ratio of the sensitivity ΔAbs measured using the reagent-containing partial composition and the 4-aminoantipyrine-containing partial composition stored at 30 ° C. for 2 weeks was calculated as the residual rate (sensitivity residual rate). The results are shown in Table 1. Immediately after the reagent was prepared, the sensitivity increased, so the reagent stored at 2 ° C. for 2 weeks when the sensitivity became steady was used as the control group.

As shown in Table 1, about 3% when the 4-aminoantipyrine-containing partial composition containing catalase was used as compared with the case where the 4-aminoantipyrine-containing partial composition without catalase was used. The sensitivity residual rate has improved. Therefore, by adding catalase to the 4-aminoantipyrine-containing partial composition, not only the effect of reducing the reagent blank but also a good sensitivity residual rate can be obtained, so that even a measurement reagent stored for a long period of time is accurate. It was shown that measurement was possible.

[Example 2: Suppression of sensitivity decrease by a chelating agent-containing saccharified albumin measuring reagent]
(Partial composition containing Trinder reagent)
The following reagent components were dissolved in purified water to the described concentrations and adjusted to pH 7.6 to prepare a Trinder reagent-containing partial composition.
100 mmol / L N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid (manufactured by Dojin Chemical Co., Ltd.)
0.05% by mass / volume% Sodium azide (Merck)
2 mmol / L N, N-bis (4-sulfobutyl) -3-methylaniline disodium (manufactured by Dojin Chemical Co., Ltd.)
10 U / mL Peroxidase (manufactured by Amano Enzyme)
30 U / mL ketoamine oxidase (manufactured by Asahi Kasei Pharma)

(Partial composition containing 4-aminoantipyrine)
The following reagent components were dissolved in purified water to the described concentrations and adjusted to pH 7.5 to prepare a 4-aminoantipyrine-containing partial composition. In addition to the following reagents, a 4-aminoantipyrine-containing partial composition containing sodium citrate (manufactured by Wako Pure Chemical Industries, Ltd.) having a final concentration of 1.000 mmol / L as a chelating agent was also prepared.
200 mmol / L 3,3-dimethylglutaric acid (manufactured by Tokyo Kasei Co., Ltd.)
0.05% by weight / volume Procrine 300 (manufactured by Spellco)
5 mmol / L 4-aminoantipyrine (manufactured by Actec)
30% by weight / volume dimethyl sulfoxide (manufactured by Nacalai Tesque)
0.1 mmol / L Potassium ferrocyanide (manufactured by Domestic Chemical Co., Ltd.)
500 U / mL catalase (manufactured by Nagase ChemteX)
40 kU / mL Protease for saccharified albumin (manufactured by Asahi Kasei Pharma, obtained according to the method described in Japanese Patent No. 4565807)

(Sample sample)
Using purified water, hydrogen peroxide (manufactured by Kokusai Kagaku Co., Ltd.) of 0.005 mmol / L or more and 0.500 mmol / L or less was prepared as a sample sample.

(Preservation of reagents)
The prepared Trinder reagent-containing partial composition was stored at 2 ° C. for 2 weeks. In addition, the 4-aminoantipyrine-containing partial composition was stored at 2 ° C. and 30 ° C. for 2 weeks.

(Measurement)
To 120 μL of the Trinder reagent-containing partial composition incubated at 37 ° C., 30 μL of the 4-aminoantipyrine-containing partial composition was added and reacted at 37 ° C. for 5 minutes. Next, 20 μL of the sample sample was added to the mixed solution of the Trinder reagent-containing partial composition and the 4-aminoantipyrine-containing partial composition, and the mixture was reacted at 37 ° C. for 5 minutes. The absorbance (Abs3) 5 minutes after the addition of the 4-aminoantipyrine-containing partial composition to the Trinder reagent-containing partial composition and the absorbance (Abs4) 5 minutes after the addition of the sample sample were measured. The change in absorbance was calculated according to the following equation (4).
Change in absorbance = (Abs4)-(Abs3) x 150/170 (4)
Here, 150 represents the total liquid amount of the Trinder reagent-containing partial composition and the 4-aminoantipyrine-containing partial composition. Further, 170 represents the total liquid amount of the Trinder reagent-containing partial composition, the 4-aminoantipyrine-containing partial composition, and the sample sample.

The change in absorbance measured using purified water instead of the sample sample is used as the reagent blank, and as shown in equation (5) below, the value obtained by subtracting the change in absorbance of the reagent blank from the change in absorbance measured using the sample sample. Was calculated as the sensitivity ΔAbs.
ΔAbs = (change in absorbance when measuring sample sample)-(change in absorbance when measuring purified water) (5)

A Trinder stored at 2 ° C. for 2 weeks with a sensitivity ΔAbs measured using a Trinder reagent-containing partial composition stored at 2 ° C. for 2 weeks and a 4-aminoantipyrine-containing partial composition stored at 2 ° C. for 2 weeks as 100%. The ratio of the sensitivity ΔAbs measured using the reagent-containing partial composition and the 4-aminoantipyrine-containing partial composition stored at 30 ° C. for 2 weeks was calculated as the residual rate (sensitivity residual rate). The results are shown in Table 2. Immediately after the reagent was prepared, the sensitivity increased, so the reagent stored at 2 ° C. for 2 weeks when the sensitivity became steady was used as the control group.

As shown in Table 2, when the 4-aminoantipyrine-containing partial composition without sodium citrate was used, the lower the hydrogen peroxide concentration, the lower the residual sensitivity. This is because, in the case of a measurement reagent having a low absorbance derived from a sample sample or a measurement reagent having a low measurement sensitivity as a whole, the sensitivity residual ratio is greatly affected by the deterioration of the liquid partial composition during storage, and thus, in the long term. It shows that accurate measurement cannot be performed with the stored liquid partial composition.

On the other hand, when the 4-aminoantipyrine-containing partial composition containing sodium citrate was used, the residual sensitivity was high even when the concentration of hydrogen peroxide was 0.2 mmol / L or less. Therefore, by adding sodium citrate to the 4-aminoantipyrine-containing partial composition, a good residual sensitivity can be obtained even when the absorbance derived from the sample sample is as low as 350 mAbs or less. It was shown that accurate measurement can be performed even with measuring reagents stored for a long period of time.

The following measurements were performed to convert the sensitivities listed in Table 2 into glycated albumin concentrations.
(Partial composition containing Trinder reagent)
The following reagent components were dissolved in purified water to the described concentrations and adjusted to pH 7.6 to prepare a Trinder reagent-containing partial composition.
100 mmol / L N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid (manufactured by Dojin Chemical Co., Ltd.)
0.05% by mass / volume% Sodium azide (Merck)
2 mmol / L N, N-bis (4-sulfobutyl) -3-methylaniline disodium (manufactured by Dojin Chemical Co., Ltd.)
10 U / mL Peroxidase (manufactured by Amano Enzyme)
30 U / mL ketoamine oxidase (manufactured by Asahi Kasei Pharma)

(Partial composition containing 4-aminoantipyrine)
The following reagent components were dissolved in purified water to the described concentrations and adjusted to pH 7.5.
200 mmol / L 3,3-dimethylglutaric acid (manufactured by Tokyo Kasei Co., Ltd.)
0.05% by weight / volume Procrine 300 (manufactured by Spellco)
5 mmol / L 4-aminoantipyrine (manufactured by Actec)
30% by weight / volume dimethyl sulfoxide (manufactured by Nacalai Tesque)
0.1 mmol / L Potassium ferrocyanide (manufactured by Domestic Chemical Co., Ltd.)
500 U / mL catalase (manufactured by Nagase ChemteX)
40 kU / mL Protease for saccharified albumin (manufactured by Asahi Kasei Pharma, obtained according to the method described in Japanese Patent No. 4565807)

(Sample sample)
As a sample sample, a highly saccharified albumin sample (high GA sample) concentrated and diluted with physiological saline was prepared. Highly glycated albumin samples are prepared by incubating human serum and glucose at 37 ° C. and the glycated albumin concentration is known.

(Measurement)
4.5 μL of sample sample was added to 180 μL of the Trinder reagent-containing partial composition incubated at 37 ° C., and the mixture was reacted at 37 ° C. for 5 minutes. Next, 45 μL of the 4-aminoantipyrine-containing partial composition was added to the mixed solution of the Trinder reagent-containing partial composition and the sample sample, and the mixture was further reacted at 37 ° C. for 5 minutes. The absorbance (Abs5) 5 minutes after the sample sample was added to the Trinder reagent-containing partial composition and the absorbance (Abs6) 5 minutes after the 4-aminoantipyrine-containing partial composition was further added were measured. The change in absorbance was calculated according to the following equation (6).
Change in absorbance = (Abs6)-(Abs5) x 180/225 (6)
Here, 180 represents the amount of liquid of the Trinder reagent-containing partial composition. Further, 225 represents the total liquid amount of the Trinder reagent-containing partial composition and the 4-aminoantipyrine-containing partial composition.

The change in absorbance measured using purified water instead of the sample sample is used as the reagent blank, and as shown in equation (7) below, the value obtained by subtracting the change in absorbance of the reagent blank from the change in absorbance measured using the sample sample. Was calculated as the sensitivity ΔAbs.
ΔAbs = (change in absorbance when measuring sample sample)-(change in absorbance when measuring purified water) (7)

The glycated albumin concentration of each sample was calculated from a known concentration by the concentration / dilution ratio. The results are shown in Table 3.

From the sensitivity measured in Table 3 and the known glycated albumin concentration, a correlation equation representing the relationship given by the following equation (8) was calculated.
Glycated albumin concentration (g / dL) = ((sensitivity) -1.726) /43.6071 (8)

From 327.02 mAbs, which is the sensitivity corresponding to 0.2 mmol / L of hydrogen peroxide shown in Table 2, and from the above correlation equation, 0.2 mmol / L of hydrogen peroxide corresponds to a saccharified albumin concentration of 7.460 g / dL. Was confirmed. Therefore, by adding sodium citrate to the 4-aminoantipyrine-containing partial composition in Table 2, a good sensitivity residual rate is obtained when the concentration of glycated albumin in the sample sample is as low as 7.5 g / dL or less. Therefore, it was shown that accurate measurement can be performed even with a measurement reagent stored for a long period of time.

[Example 3: Suppression of sensitivity decrease by a chelating agent-containing saccharified albumin measuring reagent]
(Partial composition containing Trinder reagent)
The following reagent components were dissolved in purified water to the described concentrations and adjusted to pH 7.6 to prepare a Trinder reagent-containing partial composition.
100 mmol / L N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid (manufactured by Dojin Chemical Co., Ltd.)
0.05% by mass / volume% Sodium azide (Merck)
2 mmol / L N, N-bis (4-sulfobutyl) -3-methylaniline disodium (manufactured by Dojin Chemical Co., Ltd.)
10 U / mL Peroxidase (manufactured by Amano Enzyme)
30 U / mL ketoamine oxidase (manufactured by Asahi Kasei Pharma)

(Partial composition containing 4-aminoantipyrine)
The following reagent components were dissolved in purified water to the described concentrations and adjusted to pH 7.5 to prepare a 4-aminoantipyrine-containing partial composition. In addition to the following reagents, sodium citrate, sodium thiosulfate, glycylglycine, sodium malate, sodium oxalate (manufactured by Wako Pure Chemical Industries, Ltd.), ADA, which have a final concentration of 1 mmol / L as a chelating agent, Fifteen 4-aminoantipyrine-containing partial compositions containing each of EDDP, BAPTA, CyDTA, HIDA, IDA, TPN, DTPA, NTA, and NTPO (all manufactured by Dojin Chemical Co., Ltd.) were also prepared.
200 mmol / L 3,3-dimethylglutaric acid (manufactured by Tokyo Kasei Co., Ltd.)
0.05% by weight / volume Procrine 300 (manufactured by Spellco)
5 mmol / L 4-aminoantipyrine (manufactured by Actec)
30% by weight / volume dimethyl sulfoxide (manufactured by Nacalai Tesque)
0.1 mmol / L Potassium ferrocyanide (manufactured by Domestic Chemical Co., Ltd.)
500 U / mL catalase (manufactured by Nagase ChemteX)
40 kU / mL Protease for saccharified albumin (manufactured by Asahi Kasei Pharma, obtained according to the method described in Japanese Patent No. 4565807)

(Sample sample)
As sample samples, controlled serum L (manufactured by Asahi Kasei Pharma Co., Ltd.) and controlled serum H (manufactured by Asahi Kasei Pharma Co., Ltd.) were prepared.

(Preservation of reagents)
The prepared Trinder reagent-containing partial composition was stored at 2 ° C. for 2 weeks. In addition, the 4-aminoantipyrine-containing partial composition was stored at 2 ° C. and 30 ° C. for 2 weeks.

(Measurement)
4.5 μL of sample sample was added to 180 μL of the Trinder reagent-containing partial composition incubated at 37 ° C., and the mixture was reacted at 37 ° C. for 5 minutes. Next, 45 μL of the 4-aminoantipyrine-containing partial composition was added to the mixed solution of the Trinder reagent-containing partial composition and the sample sample, and the mixture was further reacted at 37 ° C. for 5 minutes. The absorbance (Abs7) 5 minutes after the sample sample was added to the Trinder reagent-containing partial composition and the absorbance (Abs8) 5 minutes after the 4-aminoantipyrine-containing partial composition was further added were measured. The change in absorbance was calculated according to the following equation (9).
Change in absorbance = (Abs8)-(Abs7) x 180/225 (9)
Here, 180 represents the amount of liquid of the Trinder reagent-containing partial composition. Further, 225 represents the total liquid amount of the Trinder reagent-containing partial composition and the 4-aminoantipyrine-containing partial composition.

The change in absorbance measured using purified water instead of the sample sample is used as the reagent blank, and as shown in equation (10) below, the value obtained by subtracting the change in absorbance of the reagent blank from the change in absorbance measured using the sample sample. Was calculated as the sensitivity ΔAbs.
ΔAbs = (change in absorbance when measuring sample sample)-(change in absorbance when measuring purified water) (10)

A Trinder stored at 2 ° C. for 2 weeks with a sensitivity ΔAbs measured using a Trinder reagent-containing partial composition stored at 2 ° C. for 2 weeks and a 4-aminoantipyrine-containing partial composition stored at 2 ° C. for 2 weeks as 100%. The ratio of the sensitivity ΔAbs measured using the reagent-containing partial composition and the 4-aminoantipyrine-containing partial composition stored at 30 ° C. for 2 weeks was calculated as the residual rate (sensitivity residual rate). The results are shown in Tables 4 to 6. Immediately after the reagent was prepared, the sensitivity increased, so the reagent stored at 2 ° C. for 2 weeks when the sensitivity became steady was used as the control group.

As shown in Tables 4 to 6, the absorbance of the controlled serum L was about 30 mAbs, the absorbance of the controlled serum H was 70 to 80 mAbs, and the absorbance derived from the sample sample was low. Therefore, these sample samples have a concentration of glycated albumin of 7, which is easily affected by the storage stability of the 4-aminoantipyrine-containing partial composition unless a specific chelating agent is added, as described in Example 2. It was a sample sample of .5 g / dL or less.

When the chelating agent-free 4-aminoantipyrine-containing partial composition was stored at 30 ° C. for 2 weeks, the sensitivity residual rate decreased from about 84 to 87%. On the other hand, when the 4-aminoantipyrine-containing partial composition contains sodium citrate, sodium malate, sodium oxalate, HIDA, NTA, or NTPO, the sensitivity residual rate is maintained even when stored at 30 ° C. for 2 weeks. Maintained the 90% level. On the other hand, even when the 4-aminoantipyrine-containing partial composition contained sodium thiosulfate, glycylglycine, ADA, EDDP, BAPTA, CyDTA, IDA, TPN, or DTPA, the residual sensitivity rate was less than 90%. Therefore, by incorporating a specific chelating agent into the 4-aminoantipyrine-containing partial composition, a good sensitivity residual rate can be obtained even when the concentration of glycated albumin in the sample sample is low, and thus a long-term sensitivity residual rate can be obtained. It was shown that accurate measurement can be performed even with the measurement reagent stored in.

[Example 4: Effect of chelating agent concentration on suppression of sensitivity decrease by chelating agent-rich saccharified albumin measuring reagent]
(Partial composition containing Trinder reagent)
The following reagent components were dissolved in purified water to the described concentrations and adjusted to pH 7.6 to prepare a Trinder reagent-containing partial composition.
100 mmol / L N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid (manufactured by Dojin Chemical Co., Ltd.)
0.05% by mass / volume% Sodium azide (Merck)
2 mmol / L N, N-bis (4-sulfobutyl) -3-methylaniline disodium (manufactured by Dojin Chemical Co., Ltd.)
10 U / mL Peroxidase (manufactured by Amano Enzyme)
30 U / mL ketoamine oxidase (manufactured by Asahi Kasei Pharma)

(Partial composition containing 4-aminoantipyrine)
The following reagent components were dissolved in purified water to the described concentrations and adjusted to pH 7.5 to prepare a 4-aminoantipyrine-containing partial composition. In addition to the following reagents, sodium citrate having a final concentration of 1 μmol / L to 10 mmol / L and sodium malate having a final concentration of 100 μmol / L to 100 mmol / L (all manufactured by Wako Pure Chemical Industries, Ltd.) as a chelating agent, 1 μmol / A 4-aminoantipyrine-containing partial composition containing each of L to 5 mmol / L HIDA and 1 μmol / L to 5 mmol / L NTPO (all manufactured by Dojin Chemical Co., Ltd.) was also prepared.
200 mmol / L 3,3-dimethylglutaric acid (manufactured by Tokyo Kasei Co., Ltd.)
0.05% by weight / volume Procrine 300 (manufactured by Spellco)
5 mmol / L 4-aminoantipyrine (manufactured by Actec)
30% by weight / volume dimethyl sulfoxide (manufactured by Nacalai Tesque)
0.1 mmol / L Potassium ferrocyanide (manufactured by Domestic Chemical Co., Ltd.)
500 U / mL catalase (manufactured by Nagase ChemteX)
40 kU / mL Protease for saccharified albumin (manufactured by Asahi Kasei Pharma, obtained according to the method described in Japanese Patent No. 4565807)

(Sample sample)
As sample samples, controlled serum L (manufactured by Asahi Kasei Pharma Co., Ltd.) and controlled serum H (manufactured by Asahi Kasei Pharma Co., Ltd.) were prepared.

(Preservation of reagents)
The prepared Trinder reagent-containing partial composition was stored at 2 ° C. for 2 weeks. In addition, the 4-aminoantipyrine-containing partial composition was stored at 2 ° C. and 30 ° C. for 2 weeks.

(Measurement)
4.5 μL of sample sample was added to 180 μL of the Trinder reagent-containing partial composition incubated at 37 ° C., and the mixture was reacted at 37 ° C. for 5 minutes. Next, 45 μL of the 4-aminoantipyrine-containing partial composition was added to the mixed solution of the Trinder reagent-containing partial composition and the sample sample, and the mixture was further reacted at 37 ° C. for 5 minutes. The absorbance (Abs7) 5 minutes after the sample sample was added to the Trinder reagent-containing partial composition and the absorbance (Abs8) 5 minutes after the 4-aminoantipyrine-containing partial composition was further added were measured. The change in absorbance was calculated according to the following equation (11).
Change in absorbance = (Abs8)-(Abs7) x 180/225 (11)
Here, 180 represents the amount of liquid of the Trinder reagent-containing partial composition. Further, 225 represents the total liquid amount of the Trinder reagent-containing partial composition and the 4-aminoantipyrine-containing partial composition.

The change in absorbance measured using purified water instead of the sample sample is used as the reagent blank, and as shown in equation (12) below, the value obtained by subtracting the change in absorbance of the reagent blank from the change in absorbance measured using the sample sample. Was calculated as the sensitivity ΔAbs.
ΔAbs = (change in absorbance when measuring sample sample)-(change in absorbance when measuring purified water) (12)

A Trinder stored at 2 ° C. for 2 weeks with a sensitivity ΔAbs measured using a Trinder reagent-containing partial composition stored at 2 ° C. for 2 weeks and a 4-aminoantipyrine-containing partial composition stored at 2 ° C. for 2 weeks as 100%. The ratio of the sensitivity ΔAbs measured using the reagent-containing partial composition and the 4-aminoantipyrine-containing partial composition stored at 30 ° C. for 2 weeks was calculated as the residual rate (sensitivity residual rate). The results are shown in Tables 7 to 10. Immediately after the reagent was prepared, the sensitivity increased, so the reagent stored at 2 ° C. for 2 weeks when the sensitivity became steady was used as the control group.

As shown in Tables 7 to 10, the absorbance of the controlled serum L was about 30 mAbs, the absorbance of the controlled serum H was 70 to 80 mAbs, and the absorbance derived from the sample sample was low. Therefore, these sample samples have a concentration of glycated albumin of 7, which is easily affected by the storage stability of the 4-aminoantipyrine-containing partial composition unless a specific chelating agent is added, as described in Example 2. It was a sample sample of .5 g / dL or less.

When the chelating agent-free 4-aminoantipyrine-containing partial composition was stored at 30 ° C. for 2 weeks, the sensitivity residual rate decreased from about 85 to 87%. On the other hand, when the 4-aminoantipyrine-containing partial composition contains 10 μmol / L or more of sodium citrate, 1 mmol / L or more of sodium malate, 5 μmol / L or more of HIDA, and 5 μmol / L or more of NTPO, Even after storage at 30 ° C. for 2 weeks, the residual sensitivity remained in the 90% range. On the other hand, below the above concentration, the sensitivity residual rate was less than 90%. Therefore, by incorporating a specific chelating agent having a concentration equal to or higher than an appropriate concentration in the 4-aminoantipyrine-containing partial composition, a good sensitivity residual rate can be obtained even when the concentration of glycated albumin is low. It was shown that accurate measurement can be performed even with measurement reagents stored for a long period of time.

[Example 5: Suppression of sensitivity decrease by a chelating agent-containing saccharified albumin measuring reagent]
(Partial composition containing Trinder reagent)
The following reagent components were dissolved in purified water to the described concentrations and adjusted to pH 7.6 to prepare a Trinder reagent-containing partial composition.
100 mmol / L N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid (manufactured by Dojin Chemical Co., Ltd.)
0.05% by mass / volume% Sodium azide (Merck)
2 mmol / L N, N-bis (4-sulfobutyl) -3-methylaniline disodium (manufactured by Dojin Chemical Co., Ltd.)
10 U / mL Peroxidase (manufactured by Amano Enzyme)
30 U / mL ketoamine oxidase (manufactured by Asahi Kasei Pharma)

(Partial composition containing 4-aminoantipyrine)
The following reagent components were dissolved in purified water to the described concentrations and adjusted to pH 7.5 to prepare a 4-aminoantipyrine-containing partial composition. In addition to the following reagents, sodium citrate (manufactured by Wako Pure Chemical Industries, Ltd.) with a final concentration of 1 mmol / L, sodium malate at 10 mmol / L (manufactured by Wako Pure Chemical Industries, Ltd.), 0.1 mmol / L as a chelating agent Four kinds of 4-aminoantipyrine-containing partial compositions containing each of NTPO (manufactured by Dojin Chemical Co., Ltd.) and 0.1 mmol / L HIDA (manufactured by Dojin Chemical Co., Ltd.) were also prepared.
200 mmol / L 3,3-dimethylglutaric acid (manufactured by Tokyo Kasei Co., Ltd.)
0.05% by weight / volume Procrine 300 (manufactured by Spellco)
5 mmol / L 4-aminoantipyrine (manufactured by Actec)
30% by weight / volume dimethyl sulfoxide (manufactured by Nacalai Tesque)
0.1 mmol / L Potassium ferrocyanide (manufactured by Domestic Chemical Co., Ltd.)
500 U / mL catalase (manufactured by Nagase ChemteX)
40 kU / mL Protease for saccharified albumin (manufactured by Asahi Kasei Pharma, obtained according to the method described in Japanese Patent No. 4565807)

(Sample sample)
As sample samples, controlled serum L (manufactured by Asahi Kasei Pharma Co., Ltd.), 6 samples of healthy person serum, controlled serum H (manufactured by Asahi Kasei Pharma Co., Ltd.), and highly saccharified albumin sample (high GA sample) were prepared. Hyperglycated albumin samples were prepared by incubating human serum and glucose at 37 ° C.

(Preservation of reagents)
The prepared Trinder reagent-containing partial composition was stored at either 2 ° C. or 5 ° C. for 12 months. In addition, the 4-aminoantipyrine-containing partial composition was stored at 2 ° C. and 10 ° C. for 12 months, respectively.

(Measurement)
4.5 μL of sample sample was added to 180 μL of the Trinder reagent-containing partial composition incubated at 37 ° C., and the mixture was reacted at 37 ° C. for 5 minutes. Next, 45 μL of the 4-aminoantipyrine-containing partial composition was added to the mixed solution of the Trinder reagent-containing partial composition and the sample sample, and the mixture was further reacted at 37 ° C. for 5 minutes. The absorbance (Abs9) 5 minutes after the sample sample was added to the Trinder reagent-containing partial composition and the absorbance (Abs10) 5 minutes after the 4-aminoantipyrine-containing partial composition was further added were measured. The change in absorbance was calculated according to the following equation (13).
Change in absorbance = (Abs10)-(Abs9) x 180/225 (13)
Here, 180 represents the amount of liquid of the Trinder reagent-containing partial composition. Further, 225 represents the total liquid amount of the Trinder reagent-containing partial composition and the 4-aminoantipyrine-containing partial composition.

The change in absorbance measured using purified water instead of the sample sample is used as the reagent blank, and as shown in equation (14) below, the value obtained by subtracting the change in absorbance of the reagent blank from the change in absorbance measured using the sample sample. Was calculated as the sensitivity ΔAbs.
ΔAbs = (change in absorbance when measuring sample sample)-(change in absorbance when measuring purified water) (14)

The sensitivity ΔAbs measured using a Trinder reagent-containing partial composition stored at 2 ° C. or 5 ° C. for 1 month and a 4-aminoantipyrine-containing partial composition stored at 2 ° C. for 1 month was taken as 100%. Using a Trinder reagent-containing partial composition stored at 2 ° C. or 5 ° C. for 6 months or 12 months and a 4-aminoantipyrine-containing partial composition stored at 2 ° C. or 10 ° C. for 6 months or 12 months. The ratio of the sensitivity ΔAbs measured in the above was calculated as the residual rate (sensitivity residual rate). The results are shown in Tables 11 to 13. Immediately after the reagent was prepared, the sensitivity increased, so the reagent stored at 2 ° C. for 1 month was used as the control group.

As shown in Tables 11 to 13, the absorbance of the controlled serum L and the healthy person serum is about 30 mAbs, the absorbance of the controlled serum H is about 80 mAbs, the absorbance of the hyperglycated albumin sample is about 150 mAbs, and the absorbance derived from the sample sample. Was low. Therefore, these sample samples have a concentration of glycated albumin of 7, which is easily affected by the storage stability of the 4-aminoantipyrine-containing partial composition unless a specific chelating agent is added, as described in Example 2. It was a sample sample of .5 g / dL or less.

Compared with the case of using the 4-aminoantipyrine-containing partial composition without the addition of a chelating agent, the use of the 4-aminoantipyrine-containing partial composition containing sodium citrate, sodium malate, NTPO, or HIDA is saccharified. It was shown that a good sensitivity residual rate can be obtained even when the concentration of albumin is low, and therefore accurate measurement can be performed even with a measurement reagent stored for a long period of time.

[Example 6: Catalase activity in a chelating agent-containing saccharified albumin measuring reagent]
(Catalase solution)
Tris (hydroxymethyl) aminomethane (manufactured by Merck & Co., Inc.) was dissolved in purified water so as to have a concentration of 50 mmol / L, and the pH was adjusted to 7.5 to prepare a catalase solution.

(Substrate solution for measuring catalase activity)
Using a catalase solution, 0.150 mmol / L hydrogen peroxide (manufactured by Kokusan Kagaku Co., Ltd.) was prepared.

(Reagent for measuring catalase activity)
The following reagent components were dissolved in purified water to the described concentrations and adjusted to pH 7.5 to prepare a 4-aminoantipyrine-containing partial composition for measuring catalase activity.
50 mmol / L Tris (hydroxymethyl) aminomethane (manufactured by Merck & Co., Ltd.)
0.1% by mass / volume% Sodium azide (Merck)
0.05% by mass / volume 4-aminoantipyrine (manufactured by Actec)
0.05% by mass / volume% N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methylaniline sodium dihydrate (manufactured by Dojin Chemical Co., Ltd.)
10 U / mL Peroxidase (manufactured by Amano Enzyme)

(4-Aminoantipyrine-containing partial composition for glycated albumin measurement reagent)
The following reagent components were dissolved in purified water to the described concentrations and adjusted to pH 7.5 to prepare a 4-aminoantipyrine-containing partial composition for the saccharified albumin measurement reagent. In addition to the following reagents, a 4-aminoantipyrine-containing partial composition containing sodium azide, which is an activity inhibitor of catalase, in an amount of 0.05% by mass / volume% was also prepared. Further, in addition to the following reagents, six kinds of 4-aminoantipyrine-containing partial compositions containing the chelating agent shown in Table 14 at 1 mmol / L were also prepared.
200 mmol / L 3,3-dimethylglutaric acid (manufactured by Tokyo Kasei Co., Ltd.)
0.05% by weight / volume Procrine 300 (manufactured by Spellco)
5 mmol / L 4-aminoantipyrine (manufactured by Actec)
30% by weight / volume dimethyl sulfoxide (manufactured by Nacalai Tesque)
0.1 mmol / L Potassium ferrocyanide (manufactured by Domestic Chemical Co., Ltd.)
500 U / mL catalase (manufactured by Nagase ChemteX)
40 kU / mL Protease for saccharified albumin (manufactured by Asahi Kasei Pharma, obtained according to the method described in Japanese Patent No. 4565807)

(Sample sample)
The prepared 4-aminoantipyrine-containing partial composition for the saccharified albumin measuring reagent was diluted 250-fold with a catalase solution to prepare a sample sample.

(Measurement)
10 μL of the sample sample was added to 120 μL of the substrate solution for measuring catalase activity incubated at 37 ° C., and the mixture was reacted at 37 ° C. for 5 minutes. Next, 120 μL of a reagent for measuring catalase activity was added to a mixed solution of a substrate solution for measuring catalase activity and a sample sample, and the mixture was further reacted at 37 ° C. for 5 minutes. The absorbance (Abs11) 5 minutes after adding the sample to the substrate solution for measuring catalase activity and the absorbance (Abs12) 5 minutes after adding the reagent for measuring catalase activity were measured and measured as follows (15). ), The change in absorbance was calculated.
Change in absorbance = (Abs12)-(Abs11) x 130/250 (15)
Here, 130 represents the liquid amount of the substrate solution for measuring catalase activity and the sample sample. Further, 250 represents the total amount of the substrate solution for measuring catalase activity, the reagent for measuring catalase activity, and the sample sample.

The change in absorbance measured using a catalase solution instead of the substrate solution for measuring catalase activity is used as a reagent blank, and as shown in equation (16) below, a reagent is used from the change in absorbance measured using a reagent for measuring catalase activity. The value obtained by subtracting the change in the absorbance of the blank was calculated as the blank pulling sensitivity ΔAbs1. ΔAbs1 (blank pulling sensitivity) follows the following formula.
ΔAbs1
= (Change in absorbance measured using a substrate solution for measuring catalase activity)-(Change in absorbance measured using a solution of catalase) (16)

Subsequently, as shown in the following equation (17), the value obtained by subtracting ΔAbs1 measured using the sample sample from ΔAbs1 measured using the catalase solution instead of the sample sample was calculated as the sensitivity ΔAbs2.
ΔAbs2
= (ΔAbs1 measured using a catalase solution)-(ΔAbs1 measured using a sample sample) (17)

Sodium azide as a sample, and saccharified albumin containing sodium azide or a chelating agent, with ΔAbs2 measured using a 4-aminoantipyrine-containing partial composition for a saccharified albumin measuring reagent containing no chelating agent as 100%. The ratio of ΔAbs2 measured using the 4-aminoantipyrine-containing partial composition for the measurement reagent was calculated as the residual ratio (catalase activity residual ratio). The results are shown in Table 14.

表１４に示されるように、糖化アルブミン測定試薬用の４−アミノアンチピリン含有部分組成物として、カタラーゼの活性阻害剤として知られているアジ化ナトリウムを用いると、カタラーゼの活性残存率が５０％を下回った。これに対し、実施例３に記載の保存安定性をもたらすキレート剤であるクエン酸ナトリウム、リンゴ酸ナトリウム、ＮＴＰＯ、ＨＩＤＡ、シュウ酸ナトリウム、又はＮＴＡを含む糖化アルブミン測定試薬用の４−アミノアンチピリン含有部分組成物を用いると、顕著なカタラーゼ活性の阻害は確認されなかった。したがって、糖化アルブミン測定試薬用の４−アミノアンチピリン含有部分組成物に、上記のキレート剤を含有させることで、カタラーゼ活性を阻害することなく、すなわち試薬ブランクやカタラーゼ自体による測定試薬の安定化に影響することなく、保存安定性の良い測定試薬が得られることが示された。

As shown in Table 14, when sodium azide, which is known as an activity inhibitor of catalase, is used as a 4-aminoantipyrine-containing partial composition for a glycated albumin measuring reagent, the residual activity rate of catalase is 50%. It fell below. On the other hand, it contains 4-aminoantipyrine for a saccharified albumin measuring reagent containing sodium citrate, sodium malate, NTPO, HIDA, sodium oxalate, or NTA, which are the chelating agents that provide the storage stability described in Example 3. No significant inhibition of catalase activity was confirmed with the partial composition. Therefore, the inclusion of the above chelating agent in the 4-aminoantipyrine-containing partial composition for the saccharified albumin measuring reagent does not inhibit the catalase activity, that is, affects the stabilization of the measuring reagent by the reagent blank or the catalase itself. It was shown that a measurement reagent with good storage stability can be obtained without any treatment.

[Example 7: Suppression of decrease in sensitivity in a chelating agent-containing saccharified albumin measuring reagent containing an additive]
(Partial composition containing Trinder reagent)
The following reagent components were dissolved in purified water to the described concentrations and adjusted to pH 7.6 to prepare a Trinder reagent-containing partial composition.
100 mmol / L N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid (manufactured by Dojin Chemical Co., Ltd.)
0.05% by mass / volume% Sodium azide (Merck)
2 mmol / L N, N-bis (4-sulfobutyl) -3-methylaniline disodium (manufactured by Dojin Chemical Co., Ltd.)
10 U / mL Peroxidase (manufactured by Amano Enzyme)
30 U / mL ketoamine oxidase (manufactured by Asahi Kasei Pharma)

(Partial composition containing 4-aminoantipyrine)
The following reagent components were dissolved in purified water to the described concentrations and adjusted to pH 7.5 to prepare a 4-aminoantipyrine-containing partial composition. In addition to the following reagents, a 4-aminoantipyrine-containing partial composition containing sodium citrate (manufactured by Wako Pure Chemical Industries, Ltd.) having a final concentration of 1 mmol / L, and in addition to the following reagents, a final concentration of 1 mmol A 4-aminoantipyrine-containing partial composition containing / L sodium citrate (manufactured by Wako Pure Chemical Industries, Ltd.) and α-cyclodextrin (manufactured by Wako Pure Chemical Industries, Ltd.) having a final concentration of 1% by mass / volume, and the following. 4-Aminoantipyrine-containing partial composition containing sodium citrate (manufactured by Wako Pure Chemical Industries, Ltd.) with a final concentration of 1 mmol / L and sucrose (manufactured by Wako Pure Chemical Industries, Ltd.) having a final concentration of 1 mass / volume% in addition to the reagents. In addition to the following reagents and the following reagents, sodium citrate (manufactured by Wako Pure Chemical Industries, Ltd.) with a final concentration of 1 mmol / L and nonion P-213 (manufactured by Nichiyu Co., Ltd.) with a final concentration of 0.1 mass / volume% were added. In addition to the 4-aminoantipyrine-containing partial composition contained and the following reagents, sodium citrate (manufactured by Wako Pure Chemical Industries, Ltd.) having a final concentration of 1 mmol / L and Nonion S having a final concentration of 0.1% by mass / volume% A 4-aminoantipyrine-containing partial composition containing -15.4 (manufactured by Nichiyu Co., Ltd.) was prepared.
200 mmol / L 3,3-dimethylglutaric acid (manufactured by Tokyo Kasei Co., Ltd.)
0.05% by weight / volume Procrine 300 (manufactured by Spellco)
5 mmol / L 4-aminoantipyrine (manufactured by Actec)
30% by weight / volume dimethyl sulfoxide (manufactured by Nacalai Tesque)
0.1 mmol / L Potassium ferrocyanide (manufactured by Domestic Chemical Co., Ltd.)
500 U / mL catalase (manufactured by Nagase ChemteX)
40 kU / mL Protease for saccharified albumin (manufactured by Asahi Kasei Pharma, obtained according to the method described in Japanese Patent No. 4565807)

(Sample sample)
As sample samples, controlled serum L (manufactured by Asahi Kasei Pharma Co., Ltd.) and controlled serum H (manufactured by Asahi Kasei Pharma Co., Ltd.) were prepared.

(Preservation of reagents)
The prepared Trinder reagent-containing partial composition was stored at 2 ° C. for 2 weeks. In addition, the 4-aminoantipyrine-containing partial composition was stored at 2 ° C. and 30 ° C. for 2 weeks.

(Measurement)
4.5 μL of sample sample was added to 180 μL of the Trinder reagent-containing partial composition incubated at 37 ° C., and the mixture was reacted at 37 ° C. for 5 minutes. Next, 45 μL of the 4-aminoantipyrine-containing partial composition was added to the mixed solution of the Trinder reagent-containing partial composition and the sample sample, and the mixture was further reacted at 37 ° C. for 5 minutes. The absorbance (Abs13) 5 minutes after the sample sample was added to the Trinder reagent-containing partial composition and the absorbance (Abs14) 5 minutes after the 4-aminoantipyrine-containing partial composition was further added were measured. The change in absorbance was calculated according to the following equation (18).
Change in absorbance = (Abs14)-(Abs13) x 180/225 (18)
Here, 180 represents the amount of liquid of the Trinder reagent-containing partial composition. Further, 225 represents the total liquid amount of the Trinder reagent-containing partial composition and the 4-aminoantipyrine-containing partial composition.

The change in absorbance measured using purified water instead of the sample sample is used as the reagent blank, and as shown in equation (19) below, the value obtained by subtracting the change in absorbance of the reagent blank from the change in absorbance measured using the sample sample. Was calculated as the sensitivity ΔAbs.
ΔAbs = (change in absorbance when measuring sample sample)-(change in absorbance when measuring purified water) (19)

A Trinder stored at 2 ° C. for 2 weeks with a sensitivity ΔAbs measured using a Trinder reagent-containing partial composition stored at 2 ° C. for 2 weeks and a 4-aminoantipyrine-containing partial composition stored at 2 ° C. for 2 weeks as 100%. The ratio of the sensitivity ΔAbs measured using the reagent-containing partial composition and the 4-aminoantipyrine-containing partial composition stored at 30 ° C. for 2 weeks was calculated as the residual rate (sensitivity residual rate). The results are shown in Table 15. Immediately after the reagent was prepared, the sensitivity increased, so the reagent stored at 2 ° C. for 2 weeks when the sensitivity became steady was used as the control group.

As shown in Table 15, the absorbance of the controlled serum L was about 30 mAbs, the absorbance of the controlled serum H was about 80 mAbs, and the absorbance derived from the sample sample was low. Therefore, these sample samples have a concentration of glycated albumin of 7, which is easily affected by the storage stability of the 4-aminoantipyrine-containing partial composition unless a specific chelating agent is added, as described in Example 2. It was a sample sample of .5 g / dL or less.

When the chelating agent-free 4-aminoantipyrine-containing partial composition was stored at 30 ° C. for 2 weeks, the sensitivity decreased to about 90%. On the other hand, when a 4-aminoantipyrine-containing partial composition containing a chelating agent was used, the residual sensitivity was improved to about 95%. Further, even if a saccharide such as α-cyclodextrin or sucrose and a surfactant such as Nonion P-213 or Nonion S-15.4 are further added to the 4-aminoantipyrine-containing partial composition, the same sensitivity residual rate can be obtained. was gotten. Therefore, it was shown that the same storage stability improving effect can be obtained even if the chelating agent-containing measuring reagent additionally contains components such as saccharides and surfactants that are often contained in the enzymatic measuring reagent. ..

Claims (28)

A 4-aminoantipyrine-containing partial composition for a saccharified protein measuring reagent, which comprises (1) 4-aminoantipyrine, ferrocyanide, catalase, dimethylsulfoxide, and protease, and (2) citric acid and a salt thereof. , Malic acid and its salts, nitrilotriacetic acid (methylphosphonic acid) and its salts, oxalic acid and its salts, nitrilotriacetic acid and its salts, and N- (2-hydroxyethyl) iminodiacetic acid and its salts. A 4-aminoantipyrine-containing partial composition for a glycated protein measuring reagent, further comprising at least one chelating agent to be added. The 4-aminoantipyrine-containing partial composition for the glycated protein measuring reagent according to claim 1, which is liquid. The 4-aminoantipyrine-containing partial composition for the glycated protein measuring reagent according to claim 1 or 2, wherein the chelating agent is citric acid or a salt thereof. The 4-aminoantipyrine-containing partial composition for a glycated protein measuring reagent according to any one of claims 1 to 3, wherein the ferrocyanide is potassium ferrocyanide. 4-Amino for the glycated protein measuring reagent according to any one of claims 1 to 4, wherein the concentration of the catalase in the 4-aminoantipyrine-containing partial composition is 0.1 U / mL or more and 10000 U / mL or less. Antipyrine-containing partial composition. 4-The glycated protein measuring reagent according to any one of claims 1 to 5, wherein the concentration of the chelating agent in the 4-aminoantipyrine-containing partial composition is 0.1 μmol / L or more and 1000 mmol / L or less. Aminoantipyrine-containing partial composition. The 4-aminoantipyrine-containing partial composition for the glycated protein measuring reagent according to any one of claims 1 to 6, which is stored at 2 ° C. or higher and 10 ° C. or lower. The 4-aminoantipyrine-containing partial composition for a glycated protein measuring reagent according to any one of claims 1 to 6, which can be stored at 2 ° C. or higher and 10 ° C. or lower for at least one month. The 4-aminoantipyrine-containing partial composition for the glycated protein measuring reagent according to any one of claims 1 to 8, wherein the glycated protein is glycated albumin. The 4-aminoantipyrine-containing partial composition according to any one of claims 1 to 9.
A partial composition containing a Trinder reagent containing a glycated amino acid oxidase, a peroxidase, and a Trinder reagent,
A reagent for measuring glycated protein. By combining the 4-aminoantipyrine and the Trinder reagent, a color-developing reaction occurs in the presence of the glycated protein, and the concentration calculated from the absorbance corresponding to the color-developing reaction is 0.1 g / dL or more and 7.5 g. The glycated protein measuring reagent according to claim 10, which can be used for detecting a glycated protein of / dL or less. The measured sensitivity residual rate of the glycated protein when the 4-aminoantipyrine-containing partial composition is stored at 30 ° C. for 2 weeks and then used in combination with the Trinder reagent-containing partial composition is the 4-aminoantipyrine-containing partial composition. The glycated protein measuring reagent according to claim 10 or 11, which is 88% or more as compared with the case where the product is stored at 2 ° C. for 2 weeks. The reagent for measuring a glycated protein according to any one of claims 10 to 12, wherein the glycated protein is glycated albumin. Contacting the sample with the Trinder reagent-containing partial composition and the 4-aminoantipyrine-containing partial composition, and
To detect color development that depends on the glycated protein present in the sample,
Including
The Trinder reagent-containing partial composition comprises a glycated amino acid oxidase, a peroxidase, and a Trinder reagent.
The 4-aminoantipyrine-containing partial composition comprises (1) 4-aminoantipyrine, ferrocyanide, catalase, dimethylsulfoxide, and protease, and (2) citric acid and its salt, malic acid and its salt, At least one chelating agent selected from the group consisting of nitrilotriacetic acid (methylphosphonic acid) and its salt, citrate and its salt, nitrilotriacetic acid and its salt, and N- (2-hydroxyethyl) iminodiacetic acid and its salt. Including,
Method for measuring glycated protein. The method for measuring a glycated protein according to claim 14, wherein the 4-aminoantipyrine-containing partial composition is in a liquid state. The method for measuring a glycated protein according to claim 15, which is carried out after storing the liquid 4-aminoantipyrine-containing partial composition at 2 ° C. or higher and 10 ° C. or lower for at least one month. The method for measuring a glycated protein according to any one of claims 14 to 16, wherein the chelating agent is citric acid or a salt thereof. The method for measuring a glycated protein according to any one of claims 14 to 17, wherein the ferrocyanide is potassium ferrocyanide. The method for measuring a saccharified protein according to any one of claims 14 to 18, wherein the concentration of the catalase in the 4-aminoantipyrine-containing partial composition is 0.1 U / mL or more and 10000 U / mL or less. The method for measuring a glycated protein according to any one of claims 14 to 19, wherein the concentration of the chelating agent in the 4-aminoantipyrine-containing partial composition is 0.1 μmol / L or more and 1000 mmol / L or less. The method for measuring a glycated protein according to any one of claims 14 to 20, wherein a glycated protein having a concentration of 0.1 g / dL or more and 7.5 g / dL or less can be detected. The present invention according to any one of claims 14 to 21, wherein the absorbance of the sample to which the Trinder reagent-containing partial composition and the 4-aminoantipyrine-containing partial composition are added is measured depending on the concentration of the glycated protein. The method for measuring a glycated protein according to the above. Any of claims 14 to 22, wherein the sample is brought into contact with the Trinder reagent-containing partial composition, and then the mixed solution of the sample and the Trinder reagent-containing partial composition is brought into contact with the 4-aminoantipyrine-containing partial composition. The method for measuring a glycated protein according to item 1. The method for measuring a glycated protein according to any one of claims 14 to 23, wherein the glycated protein is glycated albumin. A method for stabilizing a 4-aminoantipyrine-containing partial composition for a glycated protein measuring reagent.
In the 4-aminoantipyrine-containing partial composition for the saccharified protein measuring reagent, (1) 4-aminoantipyrine, ferrocyanide, catalase, dimethylsulfoxide, and protease, and (2) citric acid and its salt, malic acid and its above. At least one selected from the group consisting of salts, nitrilotriacetic acid (methylphosphonic acid) and salts thereof, oxalic acid and salts thereof, nitrilotriacetic acid and salts thereof, and N- (2-hydroxyethyl) iminodiacetic acid and salts thereof. To contain a chelating agent,
A method for stabilizing a 4-aminoantipyrine-containing partial composition for a glycated protein measuring reagent. A method for preserving a 4-aminoantipyrine-containing partial composition for a glycated protein measuring reagent.
(1) Containing 4-aminoantipyrine, ferrocyanide, catalase, dimethylsulfoxide, and protease, and (2) citric acid and its salt, malic acid and its salt, nitrilotriacetic acid (methylphosphonic acid) and its salt, shu 4 for glycated protein measurement reagents further comprising at least one chelating agent selected from the group consisting of acids and salts thereof, nitrilotriacetic acid and salts thereof, and N- (2-hydroxyethyl) iminodiacetic acid and salts thereof. -Preparing a partial composition containing aminoantipyrine and
Preserving the 4-aminoantipyrine-containing partial composition and
A method for storing a 4-aminoantipyrine-containing partial composition for a glycated protein measuring reagent. The method for preserving a 4-aminoantipyrine-containing partial composition for a glycated protein measuring reagent according to claim 26, wherein the 4-aminoantipyrine-containing partial composition is stored at 2 ° C. or higher and 10 ° C. or lower. The method for storing a 4-aminoantipyrine-containing partial composition for a glycated protein measuring reagent according to claim 26 or 27, wherein the 4-aminoantipyrine-containing partial composition is stored at 2 ° C. or higher and 10 ° C. or lower for at least one month.