METHOD FOR REDUCING MEASUREMENT ERROR

Abstract

An object of the present invention is to provide a method for measuring an object to be measured in a specimen by an enzymatic method, the measurement method being able to suppress the positive influence of peroxide derived from the specimen. More specifically, an object of the present invention is to provide a measurement method and a measurement reagent that can suppress elevation in value regardless of whether or not the specimen is a catalase-free specimen. Provided is a measurement method that can accurately quantify hydrogen peroxide derived from an object to be measured, without influence derived from a specimen, by contacting the specimen with an enzyme in the presence of at least one compound selected from the group consisting of a compound represented by the following general formula (I), a benzimidazole derivative having an electron-donating substituent at position 2, and histidine, wherein R1 and R2 are the same or different and each represent hydrogen, a linear or branched alkyl group having 1 to 6 carbon atoms and optionally having a substituent, an aryl group optionally having a substituent, or an alkyloxy group having 1 to 6 carbon atoms.

##STR00001##

Claims

1. A method for measuring a component to be measured by quantifying hydrogen peroxide produced through the reaction of the component to be measured in a specimen with an enzyme, the measurement method comprising: (A) producing hydrogen peroxide by contacting the specimen with the enzyme in the presence of at least one compound selected from the group consisting of a compound represented by the following general formula (I), a benzimidazole derivative having an electron-donating substituent at position 2, and histidine; (B) reacting the produced hydrogen peroxide with a coloring agent in the presence of peroxidase; and (C) detecting color change: ##STR00010## wherein R1 and R2 are the same or different and each represent hydrogen, a linear or branched alkyl group having 1 to 6 carbon atoms and optionally having a substituent, an aryl group optionally having a substituent, or an alkyloxy group having 1 to 6 carbon atoms.

2. The measurement method according to claim 1, wherein each of R1 and R2 in formula (I) is hydrogen, a linear alkyl group having 1 to 4 carbon atoms, a phenyl group or an alkyloxy group having 1 to 4 carbon atoms.

3. The measurement method according to claim 1, wherein either one of R1 and R2 in formula (I) is hydrogen or a methyl group, and the other is any selected from the group consisting of hydrogen, a methyl group, an ethyl group, a phenyl group, a methoxy group and an ethoxy group.

4. The measurement method according to claim 1, wherein the compound represented by formula (I) is phenylglyoxal, glyoxal, diacetyl, 2,3-pentanedione, methyl pyruvate or ethyl pyruvate.

5. The measurement method according to claim 1, wherein the benzimidazole derivative having an electron-donating substituent at position 2 is 2-aminobenzimidazole.

6. The measurement method according to claim 1, wherein the component to be measured is HbA1c.

7. A measurement reagent comprising an enzyme that acts on a component to be measured to produce hydrogen peroxide, a coloring agent, peroxidase and at least one compound selected from the group consisting of a compound represented by the following general formula (I), a benzimidazole derivative having an electron-donating substituent at position 2, and histidine: ##STR00011## wherein R1 and R2 are the same or different and each represent hydrogen, a linear or branched alkyl group having 1 to 6 carbon atoms and optionally having a substituent, an aryl group optionally having a substituent, or an alkyloxy group having 1 to 6 carbon atoms.

8. The measurement reagent according to claim 7, wherein each of R1 and R2 in formula (I) is hydrogen, a linear alkyl group having 1 to 4 carbon atoms, a phenyl group or an alkyloxy group having 1 to 4 carbon atoms.

9. The measurement reagent according to claim 7, wherein either one of R1 and R2 in formula (I) is hydrogen or a methyl group, and the other is any selected from the group consisting of hydrogen, a methyl group, an ethyl group, a phenyl group, a methoxy group and an ethoxy group.

10. The measurement reagent according to claim 7, wherein the compound represented by formula (I) is phenylglyoxal, glyoxal, diacetyl, 2,3-pentanedione, methyl pyruvate or ethyl pyruvate.

11. The measurement reagent according to claim 7, wherein the benzimidazole derivative having an electron-donating substituent at position 2 is 2-aminobenzimidazole.

12. The measurement reagent according to claim 7, wherein the component to be measured is HbA1c, and the enzyme that acts on a component to be measured to produce hydrogen peroxide is protease and fructosyl peptide oxidase.

13. A measurement reagent kit comprising an enzyme that acts on a component to be measured to produce hydrogen peroxide, a coloring agent, peroxidase and at least one compound, the kit comprising: a first reagent and a second reagent wherein the first reagent comprises at least one compound selected from the group consisting of a compound represented by the following general formula (I), a benzimidazole derivative having an electron-donating substituent at position 2, and histidine; and the second reagent comprises the enzyme: ##STR00012## wherein R1 and R2 are the same or different and each represent hydrogen, a linear or branched alkyl group having 1 to 6 carbon atoms and optionally having a substituent, an aryl group optionally having a substituent, or an alkyloxy group having 1 to 6 carbon atoms.

14. A method for reducing a measurement error in a method for measuring a component to be measured by circumventing the influence of hydrogen peroxide derived from a component other than the component to be measured contained in a specimen, and quantifying hydrogen peroxide derived from the component to be measured by an enzymatic method, comprising: contacting the specimen with at least one compound selected from the group consisting of a compound represented by the following general formula (I), a benzimidazole derivative having an electron-donating substituent at position 2, and histidine: ##STR00013## wherein R1 and R2 are the same or different and each represent hydrogen, a linear or branched alkyl group having 1 to 6 carbon atoms and optionally having a substituent, an aryl group optionally having a substituent, or an alkyloxy group having 1 to 6 carbon atoms.

Description

EXAMPLES

[0103] Hereinafter, the present invention will be described in detail with reference to Examples. However, the present invention is not limited by the following Examples.

[Reference Example 1] Preparation of Catalase-Free Model Specimen

[0104] Blood A or B collected from each of two healthy persons using an EDTA blood collection tube was centrifuged to obtain blood cells A or B. An aqueous solution of sodium azide known to inhibit catalase activity was added to the blood cells to prepare catalase-free model specimen A or B.

[Example 1] Study Using Catalase-Free Model Specimen

[0105] 1. Measurement Reagent and Measurement Sample:

[0106] <Protease-Containing Substrate Reagent (R1)>

[0107] 50 mM MES pH 6.0

[0108] 1.0% Emal 20C (Kao Corp.)

[0109] Protin PC10F (Daiwa Kasei Industry Co., Ltd.)

[0110] DA-67 (sodium 10-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino)phenothiazine, FUJIFILM Wako Pure Chemical Corp.)

[0111] Additives described in Tables 1 to 4

[0112] The manufacturers and distributors of the additives described in Tables 1 to 4 represent the following.

[0113] TCI: Tokyo Chemical Industry Co., Ltd.

[0114] Kishida: Kishida Chemical Co., Ltd.

[0115] Fuji: FUJIFILM Wako Pure Chemical Corp.

[0116] <Coloring Reagent (R2)>

[0117] 50 mM citric acid pH 6.0

[0118] Fructosyl peptide oxidase (Kikkoman Corp.)

[0119] Peroxidase (Toyobo Co., Ltd.)

[0120] <Measurement Sample> [0121] (1) Norudia® N HbA1c calibrators (Sekisui Medical Co., Ltd.) 1 and 2 [0122] (2) Catalase-free model specimens A and B prepared in Reference Example 1

[0123] 2. Operation

[0124] The absorbance of HbA1c (5) in each measurement sample was measured according to parameters given below using an automatic analyzer JCA-9130 (JEOL Ltd.) and a combination of reagent R1 and reagent R2 containing various additives shown in Tables 1 to 4. Time setting described below is a setting in which R2 is added approximately 5 minutes after the start of first reaction by the addition of R1.

Parameter:

[HbA1c]

[0125] Analysis method: EPA

[0126] Calculation method: MSTD

[0127] Measurement wavelength (sub/main): HbA1c 805/658

[0128] Main DET.P1-P.m-P.n: HbA1c 0-95-98

[0129] Sub DET.P.p-P.r: HbA1c 44-47

[0130] Reaction time: 10 min

[0131] No specimen dilution

[0132] Amount of reaction specimen (amount of sample): 6.4 μL

[0133] Amount of first reagent (R1): 60 μL, amount of second reagent: 0 μL,

[0134] Amount of third reagent (R2): 20 μL, amount of fourth reagent: 0 μL

[0135] 3. Results

[0136] The relative value (%) of the absorbance of HbA1c (%) measured under each condition to the absorbance obtained under condition 1 (additive absent) was calculated and is shown in Tables 1 to 4. The relative value of the measured absorbance of the calibrator 1 or the calibrator 2 is preferably a value close to 100% to that obtained under condition 1. The relative value of the measured absorbance of the catalase-free model specimen A or B is preferably lower than that obtained under condition 1.

TABLE-US-00001 TABLE 1 Measurement results Relative value of measured Relative value of absorbance to that under measured absorbance to Manufacturer condition 1 (%) that under condition 1 (%) and Concentration Calibrator Calibrator Specimen Specimen Condition Additive distributor (wt %) 1 2 A B 1 Absent — — 100.0 100.0 100.0 100.0 2 Pyruvic acid TCI 0.10% 87.1 70.1 20.1 22.1 3  1.0% 593.2 204.3 97.7 86.2 4 2-Ketoglutaric TCI 0.10% 81.0 57.3 62.0 61.7 5 acid  1.0% 895.6 292.4 164.8 146.8 6 Levulinic acid TCI 0.10% 89.4 81.3 132.1 127.9 7 Phenylglyoxal TCI 0.01% 106.1 100.3 83.5 84.0 8 0.10% 110.4 92.4 21.0 21.6 9 0.20% 109.0 80.6 17.1 17.5 10 Glyoxal TCI 0.10% 101.2 98.6 89.7 89.7 11 Diacetyl TCI 0.01% 103.2 100.5 93.7 92.9 12 0.10% 116.5 100.7 59.7 58.7

TABLE-US-00002 TABLE 2 Measurement results Relative value of measured Relative value of absorbance to that under measured absorbance to condition 1 (%) that under condition 1 (%) Manufacturer Concentration Calibrator Calibrator Specimen Specimen Condition Additive and distributor (wt %) 1 2 A B 1 Absent — — 100.0 100.0 100.0 100.0 13 Imidazole Kishida 0.01% 101.4 98.5 100.3 100.5 14 0.10% 101.9 86.5 92.9 92.1 15 0.20% 54.8 78.2 110.8 109.3 16 Benzimidazole Kishida 0.01% 83.3 101.1 97.9 97.1 17 0.10% 66.4 68.8 76.4 75.2 18 0.20% 63.1 50.5 56.2 55.3 19 2- Fuji 0.01% 106.6 111.1 85.2 86.0 20 Aminobenzimidazole 0.10% 120.0 99.6 55.2 55.3 21 0.20% 81.2 82.5 47.2 47.8

TABLE-US-00003 TABLE 3 Measurement results Relative value of measured Relative value of absorbance to that under measured absorbance to condition 1 (%) that under condition 1 (%) Manufacturer Concentration Calibrator Calibrator Specimen Specimen Condition Additive and distributor (wt %) 1 2 A B 1 Absent — — 100.0 100.0 100.0 100.0 22 Methionine Kishida 0.10% 113.8 105.4 105 106.9 23 Tryptophan TCI 0.10% 104.9 112.6 96.6 96.4 24 L-Histidine Kishida 0.10% 94.5 93.4 91.7 92.0 25 0.20% 86.9 88.3 82.5 83.6

TABLE-US-00004 TABLE 4 Measurement Relative value of measured Relative value of absorbance to that under measured absorbance to condition 1 (%) that under condition 1 (%) Manufacturer Concentration Calibrator Calibrator Specimen Specimen Condition Additive and distributor (wt %) 1 2 A B 1 Absent — — 100.0 100.0 100.0 100.0 30 2,3-Pentanedione TCI 0.01% 101.6 100.4 93.8 93.1 31 0.03% 106.8 101.8 76.6 75.4 32 0.05% 110.9 102.6 65.9 63.8 33 1-Phenyl-1,2- TCI 0.01% 103.9 100.8 91.7 90.6 propanedione 34 Methyl pyruvate TCI 0.10% 104.5 98.7 41.4 39.3 35 0.01% 100.4 100.8 87.6 86.1 36 Ethyl pyruvate TCI 0.10% 102.2 99.3 57.1 54.6 37 0.01% 100.7 100.3 92.9 92.0 38 2-Ethyl-1H- TCI 0.01% 101.4 100.6 94.7 94.9 benzimidazole 39 2-Aminoimidazole TCI 0.10% 136.8 120.9 102.5 102.4 40 sulfate 0.01% 104.7 102.6 100.7 99.9

[0137] 3-1. Compound Represented by General Formula (I)

[0138] Under condition 2 where 0.10% of pyruvic acid, α-keto acid described in Patent Literature 2, was added, the relative absorbance values of the catalase-free model specimens were decreased to 20.1 to 22.1%, whereas the relative absorbance values of the calibrators 1 and 2 were also decreased to 70.1 to 87.1%, revealing influence on main reaction. Under condition 3 where 1.0% of pyruvic acid was added, the relative absorbance values of the catalase-free model specimens were decreased to 86.2 to 97.7%, whereas the relative absorbance values of the calibrators were 204.3 to 593.2%, suggesting that abnormality occurred in color reaction. Under conditions 4 and 5 where 2-ketoglutaric acid, an analogous compound of α-keto acid, was added, behavior similar to that obtained with pyruvic acid was exhibited. Under condition 6 where levulinic acid (3-acetylpropionic acid) was added, the relative absorbance values of the calibrators were decreased, whereas the relative absorbance values of the catalase-free model specimens were increased, suggesting that abnormality occurred in color reaction.

[0139] On the other hand, under condition 8 using 0.10% of phenylglyoxal, a compound found in the present application, the relative absorbance values of the catalase-free model specimens were decreased to 21.0 to 21.6%, whereas the relative absorbance values of the calibrators 1 and 2 were kept at 92.4 to 110.4%. Under condition 9 using 0.20% of phenylglyoxal, the relative absorbance values of the catalase-free model specimens were decreased to 17.1 to 17.5%, whereas the relative absorbance values of the calibrators 1 and 2 were kept at 80.6 to 109.0%. Under condition 7 where 0.01% of phenylglyoxal was added, only the absorbance of the catalase-free model specimens was also decreased without decrease in the absorbance of the calibrators. Particularly, the relative absorbance values of the catalase-free model specimens under conditions 8 and 9 were equivalent to those under condition 2, though conditions 8 and 9 were superior to condition 2 in that the relative absorbance values of the calibrators 1 and 2 were maintained.

[0140] Likewise, as for glyoxal (condition 10), diacetyl (conditions 11 and 12), 2,3-pentanedione (conditions 30, 31 and 32), 1-phenyl-1,2-propanedione (condition 33), methyl pyruvate (conditions 34 and 35), and ethyl pyruvate (conditions 36 and 37) having the chemical structure represented by the general formula (I), the relative absorbance values of the catalase-free model specimens were also decreased, whereas the relative absorbance values of the calibrators 1 and 2 were maintained.

[0141] Thus, the compound group having the structure of the general formula (I) was found to be a compound that has the functions, intended by the present invention, of specifically eliminating endogenous peroxide in a specimen, while not influencing main reaction attributed to hydrogen peroxide generated from a substance to be measured.

[0142] 3-2. Benzimidazole Derivative Having Electron-Donating Substituent at Position 2

[0143] Conditions 13 to 21 and 38 to 40 will be discussed. In the case of adding imidazole, 0.01% (condition 13) caused no change in absorbance for any of the calibrators and the catalase-free model specimens. In the case of adding 0.1 to 0.21 (conditions 14 and 15), the rate of decrease in the relative absorbance values of the calibrators was larger than that of decrease in the relative absorbance values of the catalase-free model specimens, suggesting that these concentrations are unsuitable for measurement. In the case of adding 0.013 to 0.2% of benzimidazole (conditions 16 to 18), all the concentrations decreased the relative absorbance values of the catalase-free model specimens and however, more decreased the relative absorbance values of the calibrators, exhibiting behavior similar to that obtained with imidazole. On the other hand, in the case of adding 0.01% to 0.2% of 2-aminobenzimidazole found in the present application (conditions 19 to 21), all the concentrations significantly decreased the relative absorbance values of the catalase-free model specimen compared with the relative absorbance values of the calibrators. In the case of adding 0.01% of 2-ethyl-1H-benzimidazole (condition 38), the relative absorbance values of the calibrators were also kept at 100.6 to 101.4%, whereas the relative absorbance values of the specimens were decreased. Thus, this compound was found to have the functions intended by the present invention. As for 2-aminoimidazole sulfate (conditions 39 and 40), the relative absorbance values of the specimens were not decreased, suggesting that a benzimidazole skeleton is necessary for a compound having the functions of the present invention.

[0144] Thus, the benzimidazole derivative having an electron-donating substituent at position 2 was found to be a compound that has the functions, intended by the present invention, of specifically eliminating endogenous peroxide in a specimen, while not influencing main reaction attributed to hydrogen peroxide generated from a substance to be measured.

[0145] 3-3. Histidine

[0146] Conditions 22 to 25 will be discussed. Under condition 22 where 0.1% of methionine was added and condition 23 where 0.1% of tryptophan was added, the relative absorbance values of the catalase-free model specimens were rarely changed. On the other hand, under conditions 24 and 25 where L-histidine was added, the relative absorbance values of the catalase-free model specimens were decreased, and the rate of this decrease was larger than that of decrease in the relative absorbance values of the calibrators.

[0147] Thus, histidine among amino acids was found to be a compound having the functions intended by the present invention.

[0148] 4. Assessment Results

[0149] Further, results of assessing the relative values shown in Tables 1 to 4 are shown in Table 5.

[0150] Evaluation criteria were as follows.

[0151] 4-1. Evaluation Criteria for Calibrator [0152] A: Both the relative values of the measured absorbance of the calibrators 1 and 2 are 90 to 110%. [0153] B: Both the relative values of the measured absorbance of the calibrators 1 and 2 are 80 to 120%. [0154] C: One of the relative values of the measured absorbance of the calibrators 1 and 2 is less than 80% or larger than 120%.

[0155] Since the relative values of the measured absorbance of the calibrators 1 and 2 are preferably values close to 100% to that obtained in the absence of an additive, it was concluded that A was most preferred, B was next-preferred, and C was not preferred.

[0156] 4-2. Evaluation Criteria for Specimen

[0157] Under conditions where the calibrator was assessed as A or B, evaluation criteria were as follows. [0158] A: Both the relative values of the specimens A and B are 60% or less. [0159] B: Both the relative values of the specimens A and B are 90% or less. [0160] C: Both the relative values of the specimens A and B are 95% or less. [0161] D: Both the relative values of the specimens A and B are larger than 95% s.

[0162] Since the relative values of the measured absorbance of the catalase-free model specimens A and B are preferably lower than that obtained under condition 1, it was concluded that A was most preferred, B was next-preferred, followed by C, and D did not meet the required performance. Results about a calibrator assessed as “C” were excluded from the specimen evaluation (indicated by “-” in the tables).

[0163] 4-3. Criteria of Comprehensive Evaluation

[0164] Comprehensive evaluation criteria were as follows on the basis of the evaluation of the calibrators and the specimens. [0165] A: Both the calibrators and the specimens are evaluated as A. [0166] B: The calibrators are given A and the specimens are given B, or the calibrators are given B and the specimens are given A or B. [0167] C: The calibrators are given A or B and the specimens are given C. [0168] D: The calibrators are given C or the specimens are given D.

[0169] It was concluded in the comprehensive evaluation that A was most preferred, B was next-preferred, followed by C, and D was not effective. Condition 14 was supposed to be given C in the comprehensive evaluation according to the criteria and however, given D in the comprehensive evaluation because the relative values of the specimens A and B were higher than that of the calibrator 2.

[0170] 4-4. Discussion

[0171] From Table 5, phenylglyoxal, methyl pyruvate, and ethyl pyruvate involving comprehensive evaluation A are most preferred compounds. Glyoxal, diacetyl, 2-aminobenzimidazole, histidine, and 2,3-pentanedione involving evaluation B are preferred compounds.

[0172] These results demonstrated that the compound represented by the general formula (I), the benzimidazole derivative having an electron-donating substituent at position 2, or histidine, found in the present invention, specifically eliminates endogenous peroxide in a specimen, while not influencing main reaction attributed to hydrogen peroxide generated from a component to be measured, by the presence thereof in a measurement system in a method for measuring a component to be measured in a specimen by an enzymatic method.

TABLE-US-00005 TABLE 5 Results of evaluating each compound Concentration Calibrator Specimen Comprehensive Condition (wt %) Additive evaluation evaluation evaluation 1 — Absent — — — Compound (I)-related 2 0.10% Pyruvic acid C — D Comparative Example 3  1.0% C — D Comparative Example 4 0.10% 2-Ketoglutaric acid C — D Comparative Example 5  1.0% C — D Comparative Example 6 0.10% Levulinic acid B D D Comparative Example 7 0.01% Phenylglyoxal A B B Example 8 0.10% B A B Example 9 0.20% A A A Example 10 0.10% Glyoxal A B B Example 11 0.01% Diacetyl A C C Example 12 0.10% B A B Example Benzimidazole-related 13 0.01% Imidazole A D D Comparative Example 14 0.10% B C D Comparative Example 15 0.20% C — D Comparative Example 16 0.01% Benzimidazole B D D Comparative Example 17 0.10% C — D Comparative Example 18 0.20% C — D Comparative Example 19 0.01% 2-Aminobenzimidazole B B B Example 20 0.10% B A B Example 21 0.20% B A B Example Histidine-related 22 0.10% Methionine B D D Comparative Example 23 0.10% Tryptophan B D D Comparative Example 24 0.10% L-Histidine B C C Example 25 0.20% B B B Example Compound (I)-related 30 0.01% 2,3-Pentanedione A C C Example 31 0.03% A B B Example 32 0.05% B B B Example 33 0.01% 1-Phenyl-1,2- A C C Example propanedione 34 0.10% Methyl pyruvate A A A Example 35 0.01% A B B Example 36 0.10% Ethyl pyruvate A A A Example 37 0.01% A C C Example Benzimidazole-related 38 0.01% 2-Ethyl-1H- A C C Example benzimidazole 39 0.10% 2-Aminoimidazole C — D Comparative Example 40 0.01% sulfate A D D Comparative Example

[Reference Example 2] Measurement of Healthy Person Specimen and Acatalasia Specimen

[0173] Blood C or D collected from each of two healthy persons using an EDTA blood collection tube, or acatalasia specimen E was centrifuged to obtain blood cells C, D or E.

[0174] The blood cells C, D or E were further evaluated by HPLC (Tosoh G11), a measurement method unsusceptible to the presence or absence of catalase. HbA1c (%) was healthy specimen C: 4.95%, healthy specimen D: 6.50%, and catalase-free specimen E: 6.20%.

[Example 2] Study Using Catalase-Free Specimen

[0175] 1. Measurement Reagent

[0176] <Protease-Containing Substrate Reagent (R1-A)>

[0177] 50 mM MES pH 6.0

[0178] 1.0% Emal 20C (Kao Corp.)

[0179] Protin PC10F (Daiwa Kasei Industry Co., Ltd.)

[0180] DA-67 (sodium 10-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino)phenothiazine, FUJIFILM Wako Pure Chemical Corp.)

[0181] <Protease-Containing Substrate Reagent (R1-B)>

[0182] 50 mM MES pH 6.0

[0183] 1.0% Emal 20C (Kao Corp.)

[0184] Protin PC10F (Daiwa Kasei Industry Co., Ltd.)

[0185] DA-67 (sodium 10-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino)phenothiazine, FUJIFILM Wako Pure Chemical Corp.)

[0186] 0.10% phenylglyoxal

[0187] <Coloring Reagent (R2)>

[0188] 50 mM citric acid pH 6.0

[0189] Fructosyl peptide oxidase (Kikkoman Corp.)

[0190] Peroxidase (Toyobo Co., Ltd.)

[0191] 2. Test Sample

[0192] <Calibrator>

[0193] Norudia® N HbA1c calibrators were used.

[0194] <Diluent>

[0195] Purified water was used.

[0196] <Measurement Sample>

[0197] The Normal specimens C and D and the catalase-free specimen E prepared in Reference Example 2 were each diluted 26-fold with purified water and then measured.

[0198] 2. Operation

[0199] Each test sample was measured according to parameters given below using an automatic analyzer JCA-9130 (JEOL Ltd.) and a combination of reagent R1-A and reagent R2 or reagent R1-B and reagent R2, and HbA1c (%) was calculated.

Parameter:

[HbA1c and Hb]

[0200] Analysis method: EPA

[0201] Calculation method: MSTD

[0202] Measurement wavelength (sub/main): HbA1c 805/658, Hb 805/478

[0203] Main DET.Pl-P.m-P.n: HbA1c 0-95-98, Hb 0-44-47

[0204] Sub DET.P.p-P.r: HbA1c 44-47, Hb 0-0

[0205] Reaction time: 10 min

[0206] No specimen dilution

[0207] Amount of reaction specimen (amount of sample): 6.4 μL

[0208] Amount of first reagent (R1): 60 μL, amount of second reagent: 0 μL,

[0209] Amount of third reagent (R2): 20 μL, amount of fourth reagent: 0 μL

[0210] 3. Results

[0211] Results of measurement using R1-A (additive absent) or R1-B (supplemented with phenylglyoxal) as the first reagent are shown in Table 6. In the case of using the R1-A reagent, the healthy specimens C and D were able to be accurately measured, whereas the catalase-free specimen E exhibited drastic increase in value, that is, increase to 15.46%, compared with an HPLC measurement value of 6.20%. On the other hand, in the case of using the R1-B reagent supplemented with 0.10% of phenylglyoxal found in the present invention, the measurement value of the catalase-free specimen was 6.84%, compared with an HPLC value of 6.20% and was thus able to be measured with much higher accuracy than that of the R1-A reagent containing no additive. In the case of using the R1-B reagent, the healthy specimen C exhibited 5.29%, compared with an HPLC value of 4.95%., and the healthy specimen D exhibited 6.65%, compared with an HPLC value of 6.50%. Thus, slight elevation in value was seen. This is presumably a measurement error resulting from use of the calibrators optimized for the R1-A reagent in calibration with the R1-B reagent. Thus, more highly accurate measurement is considered possible by optimizing another calibrator for the R1-B reagent.

TABLE-US-00006 TABLE 6 Measurement value of HbA1c (%) R1-A Supplemented Comparative Additive with Example 2 absent phenylglyoxal Healthy specimen C 4.95 4.97 5.29 Healthy specimen D 6.50 6.48 6.65 Catalase-free specimen E 6.20 15.46 6.84

INDUSTRIAL APPLICABILITY

[0212] The present invention can provide a measurement method and a measurement reagent that can accurately quantify hydrogen peroxide derived from an object to be measured, without the influence of a specimen-derived component other than the object to be measured, in a measurement method based on an enzymatic method. Particularly, the present invention enables more accurate measurement of an object to be measured by an enzymatic method, without the influence of a specimen-derived component other than the object to be measured, regardless of whether or not the specimen is a catalase-free specimen.