MXPA06010540A - Method of measuring glycoprotein - Google Patents

Abstract

It is intended to provide a method of measuring a glycoprotein, a glycopeptide or a glycoamino acid at a high accuracy while regulating the digesting effect of a protease, and a measurement reagent to be used in this measuring method. A method of measuring a glycoprotein, a glycopeptide or a glycoamino acid which comprises treating a glycoprotein-containing sample with a protease, treating the glycopeptide or the glycoamino acid thus liberated with a corresponding oxidase, and measuring the thus formed hydrogen peroxide by using peroxidase and an reagent undergoing color-development when oxidized, characterized in that the liquid reaction mixture before the treatment with the oxidase is regulated to pH 1 to 5;and a reagent for measuring the same.

Description

METHOD FOR MEASURING A GLYCED PROTEIN TECHNICAL FIELD This invention relates to a method for measuring a glycated protein, a glycated peptide, or a glycated amino acid in a sample, and a reagent used in said measurement of the glycated protein.

TECHNICAL BACKGROUND A glycated protein is a protein generated by nonenzymatic glycation of a protein, mainly an Amadori compound generated by the formation of a Schiff base through the union of the aldehyde group of the sugar and the amino group of the protein and the subsequent rearrangement of Amadori . Glycated proteins are found in a wide variety of locations in the living body and between said glycated proteins, the concentration of the glycated proteins present in the blood depends on the concentration of individual sugars such as glucose dissolved in blood. Examples of the glycated protein include those which have an amino-glycosylated group at the amino terminus (eg, glycated hemoglobin) and those where the e-amino group of the lysine within the protein has been glycated (e.g. , glycated albumin). Since the concentration of glycated albumin in the blood, and the ratio of the glycated hemoglobin to the non-glycated hemoglobin in the erythrocyte reflex, an average level of blood glucose in the preceding period of a certain length, said concentration and ratio are used as a index to diagnose diabetes, control the disease condition, and evaluate the therapeutic effects. A typical method for measuring the glycated protein is one that uses an enzyme (see, for example, patent documents 1 and 2). This enzymatic method comprises the step of pretreatment wherein a protease is reacted with the glycated protein in the sample to release the glycated peptide or glycated amino acid that serves as a substrate in the subsequent step; the step wherein the glycated peptide-specific enzyme or a glycated amino acid-specific enzyme (e.g., oxidase) is reacted with the substrate released for generation of a detectable substance (e.g., hydrogen peroxide); and the step of detecting the detectable substance. By measuring the glycated protein by an enzymatic method, the protease has the role of producing the substrate of the color reaction. Therefore, a sufficient amount of the protease is required to supply a sufficient amount of the substrate in a predetermined period. However, it is not only the glycated protein that is broken down by the protease, and other enzymes required for the test (eg, oxidase) are also decomposed simultaneously with the glycated protein, and therefore, the presence of the protease a high concentration results in a deficient pressure of the test of the glycated protein that is the objective of the measurement. Meanwhile, hydrogen peroxide is generally measured using a Trinder reagent which develops heat by oxidative condensation between a coupler such as a 4-aminotypyrine (4-AA) or 3-methyl-2-benzothiazolinonehydrazone (MBTH) and a phenol, aniline, or toluidine chromogen in the presence of peroxidase (POD); or a leuco dye that develops directly color in the presence of PDO. Exemplary known leuco dyes include triphenylmethane leuco dyes having improved water solubility (see patent document 3), and said dye is useful in the measurement of high sensitivity. [Patent Document 1] JP-A-5-192193 [Patent Document 2] JP-A-2001 -95598 [Patent Document 3] JP-A-3-206896 BRIEF DESCRIPTION OF THE INVENTION Problems to be solved by the invention In view of the situation as described above, an object of the present invention is to provide a method for measuring a glycated protein, a glycated peptide, or a glycated amino acid wherein the proteolytic activity of the protease it is controlled to perform a high accuracy of the measurement. Another objective of the present invention is to provide a reagent that is used in said measurement.

Means for solving the problems The inventors of the present invention elaborated an intensive study in view of said situation and found that, by enzymatically measuring the glycated protein, the proteolytic activity of the protease can be controlled to allow the measurement of the glycated protein and the like to a higher accuracy by adjusting the reaction solution before reacting with the glycated peptide specific enzyme or the glycated amino acid specific enzyme at a pH of 1 to 5. The present invention has been completed on the basis of said findings. Also, this invention provides a method for measuring a glycated protein, a glycated peptide, a glycated amino acid comprising the steps of treating a sample containing the glycated protein with a protease to release a glycated peptide or a glycated amino acid, by reacting the peptide glycated liberated or glycated amino acid with corresponding oxidase for generation of hydrogen peroxide; and measuring the resulting hydrogen peroxide with peroxidase and oxidizable color development reagent; wherein the reaction solution prior to the reaction with the oxidase is adjusted to a pH of 1 to 5. This invention also provides a reagent for measuring a glycated protein, a glycated peptide, or a glycated amino acid that at least contains 1) a oxidase that reacts with the glycated peptide or the glycated amino acid to produce hydrogen peroxide, 2) a solution for adjusting the reaction solution to a pH of 1 to 5, and 3) peroxidase.

Effect of the Invention According to the present invention, a glycated protein, a glycated peptide, or a glycated amino acid can be measured at a high accuracy by controlling the proteolytic activity of the protease, and due to the convenience of the method, the method of The present invention is very useful in the field of clinical examination.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a view showing the results of the measurement of hemoglobin concentration when Triton X-100 was added to the acid reagent (example 3). Figure 2 is a view showing the results of the measurement of hemoglobin concentration when EMAL 20C was added to the acid reagent (example 3). Figure 3 is a view showing the results of measuring the hemoglobin concentration when a surfactant was not added to the acid reagent (comparative example 3).

Figure 4 is a view showing the correlation between the HbA1 c value of the present invention and the HbAlc value measured by "Rapidia HbA1c" (example 4). Figure 5 is a view showing the correlation between the HbAlc value of the present invention and the HbAlc value measured by "Rapidia HbAlc" (example 4). Figure 6 is a view showing the correlation between the HbAlc value of the present invention and the HbAlc value measured by "Rapidia HbAlc" (example 5). Figure 7 is a view showing the correlation between the value HbAlc of the present invention and the HbAlc value measured by "Rapidia HbAlc" (example 6).

DETAILED DESCRIPTION OF THE INVENTION As described above, the protein glycated in the present invention can be any glycated protein produced by non-enzymatic binding between a protein and an aldose such as glucose. Exemplary glycated proteins of biological sample include glycated albumin and glycated hemoglobin, and use of the present invention facilitates the measurement of, for example, hemoglobulin Ale (HbAlc). Examples of the sample containing glycated protein include biological samples such as whole blood, blood cell, serum, plasma, spinal fluid, sweat, urine, tear fluid, saliva, skin, mucosa, and hair, etc. Glycated proteins are also contained in a wide variety of foods such as juice and sauce, etc. Of these samples, whole blood, blood cell, serum and plasma are preferred, and these samples can be used in a test without further processing, or after a treatment such as filtration or dialysis, and in some cases, after concentrating or extracting the glycated protein to be measured and optionally diluted with water or a buffer solution. In the present invention, the sample containing a glycated protein is allowed to react with a protease to release a glycated peptide (e.g., a fructosyl peptide) or a glycated amino acid (e.g., a fructosyl amino acid). In the case of a biological sample or a food, the fructosyl peptides or fructosyl amino acids generated by the binding of glucose to the peptide or the amino acid formed by proteolics of the glycated protein and the subsequent rearrangement of Amadori are already present in the sample before treatment with the protease, and said fructosyl peptides and fructosyl amino acids are also included in "the fructosyl peptides released or fructosyl amino acids". The protease used is not particularly limited as long as it has proteolytic or peptidolytic activity. However, the protease preferably used is one capable of releasing the fructosyl peptide or the fructosyl amino acid of the glycated protein in a short time and at a high efficiency. In particular, when the glycated protein is HbAlc, the protease preferably used is the fructosyl valil peptide release or fructosyl valil histidine, and the most preferred is the fructosyl valil histidine release. Examples of the protease releasing the fructosyl peptide or the fructosyl amino acid include those derived from a microorganism such as Bacillus sp., Aspergillus sp., Or Streptomyces sp.; an animal; or a plant. The protease can also be one that belongs to the metalloproteinase, neutral protease or basic protease, or that produced by genetic engineering of the genes included in the microorganism. If desired, the protease can also be chemically modified. Exemplary proteases include those readily available as a commercial product for research purposes such as proteinase, trypsin, papain and pronase; and those available as a commercial product for industrial purpose such as neutral protease and Toyozyme NEP (both are products of Toyobo Co., Ltd.), Sumizyme LP, Sumizyme FP, and Sumizyme MP (these three are products of Shin Nihon Chemical Co. , Ltd), Thermoase P, Protin A, and Protin P (these three are products of Daiwa Kasei KK), Actinase AS, Actinase PF, and actinase E (these three are products of Kaken Pharmaceutical Co., Ltd.), and Umamizyme , Protease S "Amano" G, Protease A "Amano" G, and Protease P "Amano" 3G (these four are products of Amano Enzyme Inc.). Said protease can be used alone or in combination of two or more. Among said proteases, the most preferred ones are those derived from Streptomyces griseus since said protease can release fructosyl valyl histidine at a high efficiency when using the protease alone. Examples of the protease derived from Streptomyces griseus include Actinase AS, Actinase AF, and Actinase E (these three products from Kaken Pharmaceutical Co., Ltd.) and Preñase E (product from Calbiochem-Novabiochem or Sigma). Proteases derived from Bacillus sp. Are also preferred. and examples include Protin PC10F (product of Daiwa Kasei K.K), and Toyozyme (product of Toyobo Co., Ltd.). The protease as described above is preferably that which has an optimum pH of 5.5 to 10, mainly, that which has a proteolytic activity at a pH of 1 to 5 which is lower than the proteolytic activity at a pH of 5.5 to 10. The Protease activity can be confirmed by a method used casein for the substrate, or by reacting the protease with a glycated peptide or the like, and comparing the samples before and after said reaction by capillary electrophoresis. The conditions used to treat the sample are not particularly limited as long as the glycated peptide or the glycated amino acid can be released at high efficiency in a short time by the action of the protease on the glycated protein. The concentration of the protease used can be suitably selected based on the amount of the glycated protein in the sample and the conditions used in the treatment. However, in a typical embodiment, a protease derived from Streptomyces griseus (e.g., product of Actinase E from Kaken Pharmaceutical Co., Ltd.) is added at a concentration of 0.0001 to 500 mg / mL, and preferably 0.001 to 300 mg. / mL. The pH in the protease treatment is not particularly limited. However, the pH can be adjusted to the optimum pH of the enzyme using a suitable pH adjusting agent such as a pH buffer, for example, at the scale of 5.5 to 10. The pH buffer does not particularly limit, and Exemplary pH buffer solutions include phosphoric acid, phthalic acid, citric acid, Tris, maleic acid, succinic acid, oxalic acid, tartaric acid, acetic acid, boric acid, and good pH buffer. The buffer solution is not particularly limited in its concentration. The concentration, however, preferably is in the range of 0.00001 to 2 mol / L, and more preferably 0.001 to 1 mol / L. the treatment is preferably carried out at a temperature of 10 to 40 ° C. The resulting solution can be used without further processing, or if desired, heated, centrifuged, or diluted as necessary. In the measurement method of the present invention, the solution before reacting with the glycated peptide-specific enzyme or the glycated amino acid-specific enzyme (an oxidase that reacts with the glycated peptide or the glycated amino acid to generate hydrogen peroxide), which is referred to hereafter as "oxidase generating hydrogen peroxide") is adjusted to a pH of 1 to 5 and more preferably, to a scale of 1 to 4. Said pH adjustment on a scale of 1 to 5 It allows controlling the proteolytic activity of the protease to the oxidase that generates hydrogen peroxide. In the present invention, "the reaction solution before reacting with the oxidase generating hydrogen peroxide" means the reaction solution obtained by treating the sample with a protease, or a reaction solution containing both the sample solution before the treatment using the protease as the reaction solution after the treatment. In the latter case, therefore, the pH of the sample solution in addition to the protease treatment should be adjusted to the scale of 1 to 5, and the pH thus adjusted should be maintained during and after the treatment. When the sample solution is adjusted to the scale of 1 to 5 before the proteolytic treatment, the amount of protease or the treatment time may increase. The agent used to adjust the pH is not particularly limited as long as it can comprise an acid pH, an example includes inorganic acids such as hydrochloric acid, sulfuric acid and phosphoric acid; and organic acids such as glycine, phthalic acid, maleic acid, citric acid, succinic acid, oxalic acid, tartaric acid, acetic acid and lactic acid. Inorganic acid and organic acid are not limited to their concentration as long as the acid can reduce the pH of the reaction solution before reacting with the oxidase that generates hydrogen peroxide on a scale of 1 to 5, and the pH of the reaction solution on the scale of 4 to 9 during the reaction of hydrogen peroxide generation. The preferable concentration, however, is in the range of 0.0001 to 1000 mM. A nonionic surfactant or an anionic surfactant each with a polyoxyethylene structure can be added to the reaction solution before reacting with the oxidase generating hydrogen peroxide. The addition of said surfactant to the sample containing glycated protein or the reaction solution after protease treatment can serve as a pre-treatment for the collection of erythrocyte hemoglobin to be used in the reaction, or prevention of turbidity caused by the reactants or the sample. Exemplary nonionic surfactants include polyoxyethylene alkyl ethers, polyoxyethylene alkylphenyl ethers, polyethylene-polyoxypropylene condensates, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene fatty acid esters, and polyoxyethylene polycyclic surfactants, etc., and alkylphenyl ethers are preferred of polyoxyethylene. Exemplary anionic surfactants include polyoxyethylene alkyl ether sulfate, polyoxyethylene alkyl phenyl ether sulphates, polyoxyethylene alkyl ether phosphoric acids, polyoxyethylene alkyl sulfosuccinic acids, polyoxyethylene alkyl ether carboxylates, and polyoxyethylene alkyl ether sulphonates, etc. Preferred are the phosphoric acids of polyoxyethylene alkyl ether, polyoxyethylene alkyl ether sulfates, polyoxyethylene alkylsulfosuccinic acids, and polyethylene alkyl ether sulfate and more preferred are polyoxyethylene alkyl ether sulfates. The surfactant is preferably used in an amount of 0.0001 to 10%, and more preferably 0.001 to 10% in the reaction solution before reacting with the oxidase that generates hydrogen peroxide.

To the reaction solution adjusted to pH from 1 to 5, the oxidizable color development reagent can be added together with the peroxidase for color development by reaction with hydrogen peroxide. In the solution having a pH of 1 to 5, the oxidizable color development reagent is highly stable, and no non-specific color development that is otherwise observed gradually is suppressed. The oxidizable color development reagent used can be any color reagent as long as it develops a color upon reaction with hydrogen peroxide, and exemplarily said color reagents include a combination of a coupler such as 4-aminoantipyrine and 3-methyl-2- benxothiazolinonhydrazone with an alinin compound, and a leuco dye. The preferred one is leuco dye. The leuco dye used is not particularly limited, and exemplary leuco dyes include triphenylmethane derivatives, phenothiazine derivatives, and diphenylamine derivatives, etc. Exemplary triphenylmethane derivatives include compounds having high water solubility, such as those described in JP-A-3-206896 and JP-A-6-197795, etc. Exemplary phenothiazine derivatives include compounds such as those described in JP-B2-60-33479, and exemplary diphenylamine derivatives include compounds such as those described in JP-B2-60-33479, JP-A-62-93261 and the like. . Among these, leucomalachite green, leucocristal violet, N- (carboxymethylaminocarbonyl) -4,4'-bis (dimethylamino) -diphenylamine sodium (product DA-64 from Wako Puré Chemical Industries, Ltd.), - (sodium carboxymethylaminocarbonyl) -3,7-bis (dimethylamino) phenothiazine (product DA-67 from Wako Puré Chemical Industries, Ltd.), 10- (N-methylcarbamoyl) -3J-bls (dimethylamino) -10H-phenothiazine ( MCDP product from Dojindo Laboratories), and N, N, N ', N', N ", N" -hexa- (3-sulfopropyl) -4,4 ', 4"-triaminotriphenylmethane (product TPM-PS from Dojindo Laboratories), most preferred are TPM-PS, DA-64, DA-67, and MCDP, and most preferred are TPM-PS and MCDP, although leuco dyes generally have poor shelf stability in a solution, dyes Leuco are stable for a prolonged time in a solution at a pH of 1 to 5. Other leuco dyes that can be used include diaminobenzidine, hydroxyphenyl propionic acid, tetramethylbenzidine, and orthophenylenediamine. The glycated peptide or the glycated amino acid released by the protease or glycated protein treatment can be measured by its reaction with oxidase that produces hydrogen peroxide and the subsequent measurement of hydrogen peroxide. The oxidase that produces hydrogen peroxide is not particularly limited as long as it can metabolize the glycated peptide such as fructosyl peptide or the glycated amino acid such as fructosyl amino acid, and the oxidase can be a derivative of a microorganism, an animal, or a plant. If desired, the protease can also be chemically modified. Exemplary oxidases include fructosyl amino acid oxidase (JP-A-2003-79386 and WO 97/20039), ketamine oxidase (JP-A-5-192193), and fructosyl peptide oxidase (JP-A-2001-95598 and JP-A -2003-235585), and fructosyl peptide oxidase is preferred. Examples of fructosyl peptide oxidase include an enzyme produced by modifying the fructosyl amino acid oxidase produced by Corynebacterium (JP-A-2001-95598), etc., and fructosyl peptide oxidase derived from the templates (JP-A-2003-235585). Most preferred are FPOX-CE and EPOX-EE (these two products from Kikkoman Corporation). The hydrogen peroxide generating oxidase can be used either in the form of a solution or in dry form, can be immobilized or bound to an insoluble carrier, or can be used alone or in combination of two or more. The hydrogen peroxide generating oxidase may be used in an amount of 0.001 to 1000 units / ml, and more preferably 0.1 to 500 units / ml although the amount may vary by the type of enzyme. In the action of oxidase, the pH is adjusted in the range of 4 to 9 when using a pH regulator when taking into consideration an optimum pH of the enzyme. The temperature used for the oxidase action is the temperature commonly used for an enzymatic reaction, and preferably in the range of 10 to 40 ° C. The pH regulator used can be selected from those described above. Although the pH regulator is not limited to its concentration, the concentration is preferably in the range of 0.00001 to 2 mol / l, and more preferably in the range of 0.001 to 1 mol / l. If desired, the oxidase as described above can be used in combination with other enzymes, coenzymes, and the like. Exemplary such enzymes include amino acid metabolizing enzymes that do not use diaphorase or fructosyl valine for the substrate, as well as enzymes such as ascorbate oxidase and bilirubin oxidase which can treat contaminating components in the blood. Exemplary coenzymes include nicotinamide adenine dinucleotide (NAD), nicotinamide adenine dinucleotide reduced (NADH), nicotinamide adenine dinucleotide phosphate (NADP), nicotinamide adenine dinucleotide reduced phosphoric acid (NADPH), thio-NAD, and thio-NADP, etc. The peroxidase preferably used is a derivative of horseradish, and said peroxidase is preferably used in a concentration of 0.01 to 100 units / ml. Hydrogen peroxide can be conveniently measured in a short time by an enzymatic method using a peroxidase and a reagent that develops color by oxidation. The measurement of hydrogen peroxide is typically carried out subsequent to the generation of hydrogen peroxide by the action of the hydrogen peroxide-generating oxidase, and in that case, the solution used for the measurement of hydrogen peroxide is preferably adjusted to a pH from 4 to 9 using the pH buffer as described above. The degree of color development (change in absorbance) can be measured by a spectrophotometer compared to the absorbance of the standard glycated peptide, glycated amino acid, or the like of the known concentration to thereby measure the glycated protein, the glycated peptide, or the amino acid glycated in the sample. The measurement can be carried out by using an automated analyzer commonly used in the art. The reagent for measuring the glycated protein of the present invention contains at least (1) an oxidase that produces hydrogen peroxide by reacting it with a glycated peptide or with a glycated amino acid, (2) a solution for adjusting the reaction solution to a pH from 1 to 5, and (3) peroxidase. The details of each component are as described above. The term "a solution for adjusting the reaction solution at a pH of 1 to 5" of (2) as used herein means the solution having its pH adjusted with the pH adjusting agent described above. The term "reaction solution" means the reaction solution obtained by treating the sample with a protease, and also a reaction solution containing both the sample solution before the treatment using the procedure, and the reaction solution after the treatment. The reagent for measuring the glycated protein of the present invention may also include a protease. Other optional components include an enzyme to process contaminants in the blood; a reaction adjusting agent; a stabilizer; a protein such as albumin, et cetera; a salt such as sodium chloride, potassium chloride, or potassium ferrocyanide, etc .; an amino acid such as lysine, alanite, aspartic acid, or glutamic acid, etc.; a peptide, a polyamino acid, or the like; a tetrazolium salt to prevent the effect of a reducing substance; an antiseptic such as an antibiotic, sodium azide or boric acid; etc.; and a cationic surfactant. The reagent for measuring the glycated protein of the present invention can be provided in the form of a dry product or a gel in addition to the solution, and in addition to the filled form in a glass bottle or a plastic container, the reagent can also be provided by coating it or impregnating it in an insoluble carrier. When the reagent is stored in the form of a solution for a long period, preferably the reagent is stored in a light-resistant container.

EXAMPLES The present invention will now be described in greater detail with reference to the following examples, which in no way limit the scope of the present invention.

EXAMPLE 1 Protease suppression Preparation of samples Samples were prepared by dissolving protease (Actinase E, product of Kaken Pharmaceutical Co., Ltd.) in purified water at a concentration of 0, 1, 5, and 10 mg / ml. (2) Measurement First reagent 3 μM fructosil valine 20 μM TPM-PS (N, N, N ', N ", N" -hexa- (3-sulfopropyl) -4,4', 4"-triaminotriphenylmethane, product of Dojindo Laboratories) 10mM a solution of maleic acid (pH 3) Second reagent 4 units / ml of fructusyl peptide oxidase (EPOX-CE, product of kikkoman Corporation). 20 units / ml POD (product of Toyobo Co., Ltd.) 200mM of a buffer solution of citric acid pH (pH 6) To 20 μL of each sample was added 240 μL of the first reagent, and the mixture was incubated 37 ° C for 5 minutes. After incubation, 80 μL of the second reagent was added and the mixture was incubated at 37 ° C for 5 minutes, and then the absorbance was measured at a wavelength of 600 nm using a Hitachi Model 7150 automatic analyzer. relative values in a condition in which the measured value of the change in absorbance was 100 when the concentration of protease in the sample was 0 mg / ml. The results are shown in table 1.

COMPARATIVE EXAMPLE 1 The procedure of Example 1 was repeated except that the pH of the maleic acid solution in the first reagent was adjusted to 7 using 0.1 N of a sodium hydroxide solution to thereby measure the absorbance.

TABLE 1 The data presented in Table 1 confirm that the proteolysis of FPOX-CE and POD by the protease is suppressed when the first reaction is carried out at a pH of 1 to 5.

EXAMPLE 2 Measurement of glycated hemoglobin Hemolytic reagent 2% EMAL 20C * (Product of Kao Corporation) 1 mg / ml Actinase E (Product of Kaken Pharmaceutical Co. Ltd.) 20 mM Hepes (2- [4- (2-hydroxyethyl) -1-piperazinyl] ethane -sulphonic) buffer solution for pH (pH 8) * EMAL 20C: sodium polyoxyethylene (3) lauryl ether sulfate Acidic reagent 0.1% Triton x-100 20 μM TPM-PS (product of Donjindo Laboratories) 0.5% sodium azide 5mM maleic acid solution (pH 2.8) Enzyme reagent 20 units / ml POD (product of Toyobo Co., Ltd.) 4 units / ml FPOX-CE (product of Kikkoman Corporation) 200mM pH regulating solution of citric acid (pH 6) (1) Preparation of the hemolysed sample Using commercially available equipment "Rapidia HbAlc" (product of Fujirebio Inc.), human blood cells containing HbAlc were used at a known concentration to be used as a sample, and to 10 μL of this sample was added 300 μL of the hemolytic reagent to thereby prepare a hemolyzed sample. (2) Measurement To 20 μL of the hemolysed sample was added 240 μL of the acidic reagent, and this mixture was incubated at 37 ° C for 5 minutes. After measuring the absorbance at a wavelength of 600 nm, 80 μL of the enzyme reagent was added to this solution, and the mixture was allowed to react at 37 ° C for 5 minutes to thereby measure the change in absorbance at a wavelength of 600 nm. The results are shown in table 2.

COMPARATIVE EXAMPLE 2 The procedure of Example 2 was repeated except that the following neutral reagent was used instead of the acidic reagent.

Neutral reagent 0.1% Triton X-100 20 μM TPM-PS (product of Dojindo Laboratories) 0.05% sodium azide 5 M solution of maleic acid (pH 7) TABLE 2 As demonstrated in Table 2, the values measured in Example 2 were higher than those measured in Comparative Example 2, and the correlation coefficient with the known concentration was also higher than that of Comparative Example 2. This indicates that the effect of the protease on other enzymes in the reagent was reduced.

EXAMPLE 3 Measurement of hemoglobin concentration (1) Preparation of the samples To 10 μl of a solution of cells of human blood was added 200 μl of 1% EMAL 20C for hemolysis, and the hemolysed sample was diluted with a solution of 1% EMAL 20C to make 5 solutions in series to use as samples. (2) Measurement To 20 μl of the sample was added 240 μl of the reagent containing 50 mM of a buffer solution of citric acid, and after incubating the mixture at 37 ° C for 5 minutes, the absorbance was measured at a wavelength of 600 nm. The pH regulating solution of citric acid was prepared by adjusting the pH to 3, 4, and 5, respectively, and adding 0.5% Triton X-100 or 1% EMAL 20C as surfactants. The results are shown in figures 1 and 2.

COMPARATIVE EXAMPLE 3 The procedure of Example 3 was repeated except that the surfactant was not used. The results are shown in Figure 3. As shown in Figures 1 to 3, when a surfactant is added to the pH buffer solution of citric acid, the absorbance increases in a manner dependent on the concentration of hemoglobin. On the other hand, in the absence of the surfactant, the absorbance corresponding to the solution in series was not observed due to the turbidity caused by mixing the hemolyzed sample of human blood cells and the acidic reagent.

EXAMPLE 4 Measurement of HbAlc concentration Hemolytic reagent 1 2% EMAL 20C (product of Kao Corporation) 1 mg / ml Actinase E (product of Kaken Pharmaceufical Co., Ltd) 20 mM HEPES buffer solution (pH 8) Hemolytic reagent 2 2% EMAL 20C (product of Kao Corporation) 1 mg / ml Actinase E (product of Kaken Pharmaceutical Co., Ltd) 260 μM TPM-PS (product of Dojindo Laboratories) 20 mM HEPES (pH 8) Acidic reagent 1 0.1% Triton X-100 20 μM TPM-PS (product of Dojindo Laboratories) 5 mM maleic acid solution (pH 3) Acidic reagent 2 0.1% Triton X-100 5 mM maleic acid solution (pH 3) Enzymatic reagent 20 units / ml POD (product of Toyobo Co., Ltd.) 3 units / ml FPOX-CE (product of Kikkoman Corporation) 200 mM pH regulating solution of citric acid (pH 6) 1 Preparation of the hemolysed sample To each of the 10 samples of human blood cells were added 300 μL of the hemolytic reagent 1 or the hemolytic reagent 2 to produce the hemolysed sample. In the next measurement, (A) acidic reagent 1 was used when the hemolyzed sample was prepared using the hemolytic reagent 1, and (B) the acidic reagent 2 was used when the hemolyzed sample was prepared using the hemolytic reagent 2, 2 Measurement To 20 μL of the hemolysed sample was added 240 μL of the acidic reagent, and after incubating the mixture at 37 ° C for 5 minutes, the absorbance was measured at a wavelength of 600 nm to determine the dependent value of the concentration of hemoglobin (sample Hb). 80 μL of the enzymatic reagent was added to this reaction solution, and the mixture was allowed to react at 37 ° C for 5 minutes. The change in absorbance at a wavelength of 600 nm was measured, and the value dependent on HbAlc concentration (sample Al) was determined. When using these with the value dependent on the hemoglobin concentration (Hb est) and the HbAlc concentration dependent value (est. A1) obtained using the samples having a known concentration of HbAlc (%), the value was calculated of HbAlc (%) by the following formula: HbAlc (%) = est. HbAl x (est. Hb / est. A1) x (sample A1 / sample Hb) (est. HbAl: HbAlc (%) of the sample having a known concentration of HbAlc). The correlation of the HbAlc value (%) measured with a commercially available immunoassay kit "Rapidia HbAlc" (product of Fujirebio Inc.) (reference example) is shown in figures 4 and 5. As shown in figures 4 and 5, the method of the present invention showed a good correlation with Rapidia HbAlc.

EXAMPLE 5 Measurement of HbAlc Hemolytic reagent 2% EMAL 20C (product of Kao Corporation) 1 mg / ml Actinase E (product of Kaken Pharmaceutical Co., Ltd.) 20mM HEPES (pH 8) Acidic reaction 0.1% Triton X-100 20 μM MCDP (10- (N-methylcarbamoyl) -3,7-bis (dimethylamino) -1 OH-phenothiazine, product of Dojindo Laboratories) 10mM maleic acid solution (pH 3) Enzymatic reagent 20 units / ml POD (product of Toyobo Co., Ltd.) 4 units / ml Fpox-CE (Product of Kikkoman Corporation) 200 mM pH regulating solution of citric acid (pH 6) 1 Preparation of the hemolysed sample To 10 μL of a sample of human blood cells, 300 μL of the hemolytic reagent was added to prepare the hemolysed sample. 2 Measurement To 10 μL of the hemolysed sample was added 240 μL of the acidic reagent, and after incubation of the mixture at 37 ° C for 5 minutes the absorbance at a wavelength of 600 nm was measured to determine the value dependent on the hemoglobin concentration. To the reaction solution was also added 80 μL of the enzyme reagent, and after allowing it to react at 37 ° C for 5 minutes the change in absorbance at a wavelength of 600 nm was measured to determine the value dependent on the HbAlc concentration. The HbAl c (%) value was calculated as in the case of Example 4. The correlation with the HbAl c (%) value measured by "Rapidia HbAlc" (Fujirebio Inc. product) is shown in Figure 6. As shown in FIG. shows in Figure 6, the method of the present invention showed a good correlation with Rapidia HbAlc.

EXAMPLE 6 Measurement of HbAlc concentration Hemolytic reagent 2% EMAL 20C (product of Kao Corporation) 10 mg / ml Proton PC10F (product of Dawin Kasei K.K) 20 mM HEPES (pH 8) Acidic reagent 0.1% Triton X-100 20 μM TPM-PS (product of Dojindo Laboratories) 10 mM maleic acid solution (pH 3) Enzymatic reagent 20 units / ml POD (Product of Toyobo Co., Ltd.) 3 units / ml FPOX-CE (Product of Kikkoman Corporation) 200 mM pH buffer solution of citric acid (pH 6) (1) Preparation of the sample hemolysed To 10 μL of a sample of human blood cells was added 300 μL of the hemolytic reagent to prepare the hemolysed sample. (2) Measurement To 10 μL of the hemolysed sample was added 240 μL of acidic reagent, and after incubating the mixture at 37 ° C for 5 minutes, absorbance was measured at a wavelength of 600 nm to determine the value dependent on the concentration of hemoglobin. To the reaction solution, 80 μL of the reagent was added additionally, and after allowing it to react at 37 ° C for 5 minutes, the change in absorbance at a wavelength of 600 nm was measured to determine the value dependent on the HbAlc concentration. The HbAlc value (%) was calculated as in the case of example 4. The correlation with the HbAlc value (%) measured by "Rapidia HabAlc" (product of Fujirebio Inc.) (reference example) is shown in the figure 7. As shown in Figure 7, the method of the present invention showed a good correlation with Rapidia HbAlc.

EXAMPLE 7 Stability of TPM-PS (1) TPM-PS was dissolved in the following aqueous solutions so that the resulting concentration of TPM-P] S is 60 μM, and after storing the solution at 37 ° C, absorbance was measured at a wavelength of 600 nm. The absorbance at 0 hours, 2 weeks and 3 weeks is shown in Table 3.

TABLE 3 * PB-K: Potassium phosphate solution As shown in Table 3, the non-specific color development of the TPM-PS was suppressed in the aqueous solution at a pH of 1 to 5.

EXAMPLE 8 Stability of TPM-PS (2) TPM-PS was dissolved in each of the following aqueous solutions at a TPM-PS concentration of 100 μM, and the solution was stored at 25 ° C for 10 days. The absorbance of the solution was then evaluated at a wavelength of 600 nm. The results are shown in table 4.

TABLE 4 As shown in Table 4, the TPMS-PS showed a small change in the absorbance in aqueous solution at pH from 1 to 5 to indicate its stability.

EXAMPLE 9 Stability of MCDP MCDP was dissolved in methanol at 4 mM, and the solution was added to each of the following aqueous solutions containing 0.1% Triton X-100 at a MCDP concentration of 100 μM. The solution was stored at 37 ° C for 24 hours, and the absorbance was measured at a wavelength of 600 nm. The results are shown in table 5.

TABLE 5 As shown in Table 5, the MCDP showed a small change in absorbance in the aqueous solutions at pH from 1 to 5, indicating the suppression of non-specific color development, as well as stability.

Claims (12)

NOVELTY OF THE INVENTION CLAIMS

1. - A method for measuring a glycated protein, a glycated peptide, or a glycated amino acid, comprising the steps of treating a sample containing the glycated protein with a protease to release a glycated peptide or a glycated amino acid; reacting the glycated peptide released or the glycated amino acid with the corresponding oxidase for the generation of hydrogen peroxide; and measuring the resulting hydrogen peroxide with peroxidase and an oxidizable color developing reagent; wherein the reaction solution before the reaction with the oxidase is adjusted to a pH of 1 to 5.

2. The method according to claim 1, further characterized in that the glycated protein is a glycated hemoglobin.

3. The method according to claim 1 or 2, further characterized in that the protease is a protease produced from a microorganism of Bacillus, Aspergillus, or Streptomyces, or a microorganism that is produced by genetically engineering said microorganism; the protease has an optimal pH scale of 5.5 to 10; and the protease is capable of releasing fructosyl vality histidine by itself or using it with another protease.

4. - The method according to any of claims 1 to 3, further characterized in that the protease is a protease produced from a microorganism derived from Streptomyces grisesus-, the protease has an optimum pH of 5.5 to 10; and the protease is capable of releasing fructosyl vality histidine by itself.

5. The method according to any of claims 1 to 4, further characterized in that the reaction solution before the reaction with the oxidase contains an anionic surfactant or a nonionic surfactant each having a structure of polyoexethylene.

6. The method according to any of claims 1 to 5, further characterized in that the reaction solution adjusted to pH 1 to 5 contains an oxidizable color development reagent.

7. The method according to claim 6, further characterized in that the oxidizable color development reagent is a leuco dye selected from leuco dyes of triphenylmethane, leuco dyes of phenothiazine, and dye leuco diphenylamine.

8. The method according to claim 7, further characterized in that the leuco triphenylmethane dye is: N, N, N ', N', N ", N" -hexa- (3-sulfopropyl) -4,4 ' , 4"-triaminotrophenylmethane 9.- The method according to claim 7, further characterized in that the leuco phenothiazine dye is: 10- (carboxymethylaminocarbonyl) -3,7-bis (dimethylamino) phenothiazine or 10- (N-methylcarbamoyl) -3,7-bis (dimethylamino) -10H-phenothiazine 10. The method according to any of claims 1 to 9, further characterized in that the pH adjustment is carried out using at least one member selected from hydrochloric acid, sulfuric acid, phosphoric acid and organic acids 11. The method according to claim 10, further characterized in that the organic acid is glycine, maleic acid or citric acid 12.- A reagent for measuring a glycated protein, a glycated peptide, or a glycated amino acid, comprising at least: (1) a oxidase which reacts with the glycated peptide or with the glycated amino acid to produce hydrogen peroxide; (2) a solution for adjusting the reaction solution to a pH of 1 to 5; and (3) peroxidase.