JPH11196897A - Determination of glycosylated protein and determination apparatus - Google Patents

Abstract

PROBLEM TO BE SOLVED: To determine a glycosylated protein clinically useful as an important index for diagnosis of symptoms and control of symptom of diabetes mellitus in high accuracy by using a protease immobilized column in measuring glycosylated protein in a sample. SOLUTION: A glycosylated protein which is clinically extremely useful as an important index of diagnosis of symptom for diabetes mellitus and control of symptom therefor is measured by using a protease immobilizing column 4 and a fructosylamino acid oxidase-immobilized column 5. A sample is passed from a solution vessel 1 through a transfer pipe and made to flow into a pump 2 and passed through an injection pulp 3 and fragmentized by the protease- immobilized column 4 and the glycosylated protein is decomposed by the fructosylamino acid oxidase-immobilized column 5 and the generated hydrogen peroxide is decomposed by a peroxidase immobilized column 6 and a coloring agent is colored by the decomposed material and the coloring a measured by a detector 8 to determine glycosylated protein.

Description

DETAILED DESCRIPTION OF THE INVENTION

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method and an apparatus for measuring the concentration of a glycated protein present in a sample using an enzyme, specifically, using fructosyl amino acid oxidase.

[0002]

2. Description of the Related Art A glycated protein is a substance produced by non-enzymatically and irreversibly binding an amino group of an amino acid constituting a protein and an aldehyde group of a reducing sugar such as aldose. Since the enzymatic and irreversible bond is called Amadori rearrangement, it is also called an Amadori compound. The production rate of glycated protein depends on the concentration of protein and reducing sugar, the contact time, and the temperature.The larger the amount of protein and reducing sugar, the longer the contact time between them, the lower the temperature at which protein denaturation does not occur. The higher the value, the faster the production rate of glycated protein and the greater the production amount. Further, in a living body, since the concentration of a glycated protein varies depending on the half-life of the protein to be glycated, various information can be obtained by measuring the concentration of the glycated protein. For example, an Amadori compound in which hemoglobin in blood is saccharified is called glycated hemoglobin, an Amadori compound in which albumin is saccharified is called glycated albumin, and the ability to reduce saccharides in blood is called fructosamine. These blood concentrations each reflect the average blood glucose level in the living body over the past certain period, and the measured values can be important indicators for diagnosing and managing the symptoms of diabetes. The establishment is extremely useful clinically.

[0003] Conventional methods for measuring glycated proteins include:
Isoelectric focusing fractionation electrophoresis, cation exchange chromatography, spectrophotometry, colorimetry, HPLC, mini column method, affinity chromatography, etc. (Inspection and Technology, Vol. 14, No. 11, page 1155) (1986)
), And various glycated protein measurements have been used clinically.

[0004] However, these methods have problems such as detection sensitivity, operability, and long sample processing times, and are not necessarily satisfactory methods and must be practically solved. Ideally, there is a need for a method and apparatus that have high detection sensitivity and can easily and accurately measure a large number of samples in a short time.

[0005] As a method for solving these problems, an antiserum or monoclonal antibody having a specific affinity for a glucitol lysine residue, which is one of the saccharification sites of a glycated protein, is obtained, and a radioisotope is obtained. Measurement of glycated protein by labeled immunoassay (RIA) has been reported (J. Clin. Invest., 72, 1427 (1983), clinicopathology No. 33).
Supplementary Volume (1985), p. 226, Diabetes, Vol. 29, No. 581
P. (1986), Clinica. Chimica. Acta., 153, 293 (1986),
Diabetes, 28, 695 (1985)).

[0006] The immunological assay uses an anti-reduced glycated protein antibody against an Amadori compound of glycated protein, and is an excellent method for assaying glycated protein. Specificity and operability remain, such as the necessity of separation into protein and hemoglobin, the necessity of separately performing the total amount of protein, and the necessity of pretreatment for separating sugar and protein before the reduction reaction ( Diabetes Volume 34 Supplement, Page 96 (198
6 years)).

[0007] As an immunological measurement method which has solved the above-mentioned problems, labeled boron is separated by B / F separation by selectively capturing only a specific protein in a sample with an immobilized antibody that recognizes only the specific protein. There is a method in which B / F separation is performed by adding an acid derivative and binding to a saccharification site, and the remaining labeled substance is measured to determine the saccharification ratio of a specific protein (Japanese Patent Application Laid-Open No. 5-87809). Since the F separation must be performed twice, the operation is very complicated. As a result, a long time is required for the measurement, so that it is very difficult to process a large number of samples, and furthermore, it is affected by non-specific reactions. There is a problem that high-precision measurement cannot be performed.

[0008] Affinity chromatography of phenylboronic acid, which is not easily affected by fluctuations in pH or ionic strength in the measurement reaction system, is an excellent method for separating glycated proteins, and is available from Amicon Corporation and Pierce Chemical Company. Has developed a kit for clinical use containing immobilized phenylboronic acid (UK Patent Application Publication No. 2,024,829 and US Pat. No. 4,269,605). However, the measurement cannot be performed with high accuracy because a relatively large amount of sample is required for the measurement and the bond between glycated protein and phenylboronic acid is affected by the temperature.

A method of separating a glycated protein with immobilized phenylboronic acid and using a labeled antibody that recognizes only a specific protein (JP-A-62-100660) has been reported. It is necessary to separately measure the total amount of the specific protein, and there remains a problem that high-precision measurement cannot be performed because the bond between glycated protein and phenylboronic acid is affected by temperature.

A method of quantifying glycated albumin from a change in fluorescence wavelength using a fluorescently labeled boronic acid derivative as a reagent for detecting glycated albumin (Clin. Chem. Acta, 149, 13 (1985))
) Has been reported. In principle, this method requires that the detection reagent reacts with an unspecified glycated protein and that the total amount of the specified protein be separately measured. Not a good way to do that.

As a method for solving these problems, Japanese Patent Application Laid-Open
64-16964 discloses that a specific protein is separated by an immobilized antibody, and the amount of specific glycated protein is determined by a monoclonal antibody that specifically recognizes a reduced glycated portion such as glycitol lysine or glycitol valine residue. Or methods for measuring the saccharification ratio of specific proteins. This method is excellent in that the saccharification ratio is measured without having to measure the total amount of the specific protein,
There are problems such as poor reproducibility and linearity of the calibration curve, that the recognized glycation site is only a part of the glycation site, and that the measurement time requires 5 hours or more.

A boronic acid derivative labeled with a fluorescent dye is reacted with hemoglobin in a sample, hemoglobin is captured by an antibody immobilized on a carrier, the total amount of hemoglobin is measured by colorimetry, and glycated hemoglobin is measured by fluorescence. And a method for measuring the saccharification ratio of hemoglobin has been reported (US Pat. No. 4,861,728). However, the point that a boronic acid derivative labeled with a fluorescent dye that is equal to or more than the total amount of glycated hemoglobin in the sample that is present in a large excess with respect to the amount of the antibody immobilized on the carrier is required, is not an economical measurement method, There are problems in operability, simplicity, and versatility in that two measurements of colorimetry and fluorescence are required to measure the saccharification ratio.

As a method for measuring glycated protein other than the methods described above, there is a measurement method using an enzyme, and the principle of measurement is as shown in the following reaction formula: R ¹ -CO-CH ₂ -NH-R ² + O ₂ + H ₂ O → R ¹ —CO—CHO + R ² —NH ₂ + H ₂ O ₂ (wherein R ¹ represents an aldose residue and R ² represents an amino acid, protein or peptide residue) Glycated protein The glycated protein concentration can be determined by reacting oxidoreductase with the oxidized reductase and measuring the amount of oxygen consumed or the amount of product (eg, hydrogen peroxide) produced (JP-B 5-33997, JP-B-6-33997). -65300, JP-A-2-195900, JP-A3-1
No. 55780, JP-A-4-4874, JP-A-5-192193, JP-A-6-46846, JP-A-7-289253, JP-A-8-154672, JP-A-8-336386 Gazette). further,
Methods for measuring glycated proteins for the diagnosis of diabetes are also known (JP-A-2-95899, JP-A-2-195900, JP-A-5-192193 (EP0526150A), -Four
No. 6846 (EP 0 576 838 A), JP-A-7-289253, JP-A-8-154672, JP-A-8-336386.

As an enzyme that catalyzes the above reaction, fructosyl amino acid oxidase derived from various microorganisms has been provided. Fusarium, Gibberella (G
glycerin protein can be measured using an enzyme derived from a bacterium belonging to the genus Penicillium, Penicillium, or the like. (JP-A-7-289253, JP-A-8-15
No. 4672, JP-A-8-336386. ) Fusarium oxysporum S-1F4 (FERM)
BP-5010) and Gibberella Fujikuroi (IFO NO.6356 and I
FO NO.6605) (Gibberella fujikuroi) produces fructosyl amino acid oxidase (hereinafter referred to as FAO, respectively)
DS and FAOD-G) have strong activity against fructosyl lysine and fructosyl poly lysine, and thus have been found to be particularly useful for measuring glycated albumin among Amadori compounds. (JP-A-7-289253). Therefore, if a method for measuring a glycated protein using these fructosyl amino acid oxidases is established, the method can be applied to a general-purpose automatic analyzer, so that the measurement can be performed at a lower cost and in a shorter time than the conventional method. . Furthermore, it is also possible to accurately determine the glycated protein in a biological component by the specificity of the enzyme, and this is a measurement method that is greatly expected as a screening test in a health checkup or a therapeutic marker for a diabetic patient.

In the measurement of glycated protein using fructosyl amino acid oxidase, the glycated protein must be efficiently bound to the active center of fructosyl amino acid oxidase, and when the glycated protein molecule is large, pretreatment with a protease is required. Become. It is already well known that fructosyl amino acid oxidase increases the enzyme reaction rate by fragmenting a glycated protein into small fragments using a protease. However, the types of proteases are extremely large, and the size of the ideal substrate for the enzyme reaction varies depending on the type of fructosyl amino acid oxidase.Therefore, the ideal combination of protease and fructosyl amino acid oxidase used in the measurement reaction is limited. Come.

[0016] After fragmenting glycated albumin using at least one of Sumizyme MP or Protease XIV as a protease, FAOD-S or FAO-S
A method of measuring the concentration of glycated albumin using DG fructosyl amino acid oxidase has been established.
(Japanese Patent Application No. 9-107106)

The amount of fructosyl amino acid oxidase separated and purified by culturing the above-mentioned bacteria is extremely small, and it takes time and effort to obtain a large amount of the enzyme. When measuring hydrogen peroxide generated by an enzymatic reaction, it is necessary to add fructosyl amino acid oxidase to each sample to be measured. Had the disadvantage that Similarly, for the fragmentation of glycated proteins, it is necessary to add a protease, which has further increased the cost.

Another important problem is that the protease itself added for the fragmentation treatment is saccharified,
This has caused a problem that it reacts with fructosyl amino acid oxidase as a substrate, which has a large effect on the measurement result as an error.

[Problems to be solved by the invention]

After fragmentation treatment is performed by adding protease to the sample containing glycated protein, fructosyl amino acid oxidase is added to determine the amount of hydrogen peroxide generated or the amount of oxygen consumed, thereby obtaining the amount of saccharification or saccharification rate. In the case of the conventional method of measuring glycated protein, the fragmented glycated protein must specifically react with fructosyl amino acid oxidase. That is, only the glycated protein contained in the sample must react with fructosyl amino acid oxidase. If a glycated protein other than the glycated protein contained in the sample is present in the measurement system, for example, if glycated protease or glycated fructosyl amino acid oxidase is present, fructosyl amino acid oxidase reacts at those glycated sites. As a result, the true saccharification amount or saccharification rate contained in the measurement sample cannot be determined, and high-accuracy measurement cannot be performed.

Proteins such as enzymes are industrially mass-produced by screening a gene serving as a template and culturing a recombinant introduced into E. coli or the like. Since glucose is added as a nutrient at the time of culturing this recombinant, the obtained protein is saccharified. Since sugar is indispensable for culturing recombinants, saccharification of protein is unavoidable in an environment where protein and sugar coexist.As a result, protease and fructosyl amino acid oxidase used for measurement of glycated protein are also saccharified. I'm done.

In addition to the case where the saccharified protease acts as a substrate for fructosyl amino acid oxidase and causes an error in the measurement result, the protease added for fragmenting the glycated protein may fragment the protease itself. The effect of the fragmentation of the glycated protease cannot be ignored. The smaller the size of the substrate that fructosyl amino acid oxidase can recognize, the higher the substrate specificity of the enzyme. Therefore, the saccharified protease auto-digests more than the non-fragmented saccharified protease.
It will react with fructosyl amino acid oxidase.

[0022]

In order to measure a glycated protein in a sample, a protease and a fructosyl amino acid oxidase are immobilized on an immobilized carrier, a protease-immobilized column filled with the immobilized carrier, and a fructosyl amino acid oxidase. An immobilization carrier is prepared in advance, and the measurement sample is subjected to fragmentation treatment using a protease-immobilized column, and then hydrogen peroxide is generated using a fructosyl amino acid oxidase-immobilized column, and the saccharification is performed by detecting the hydrogen peroxide. A highly accurate measurement method of glycated protein from which the rate or the amount of glycated protein can be determined was established.

The protease used herein is a protease capable of fragmenting a glycated protein and reacting with fructosyl amino acid oxidase, such as protease XIV, pronase, proteinase K, subtilisin, protease S, Sumiteam AP, and Sumiteam M.
An enzyme carrier selected from P, carboxypeptidase Y, and protin FA may be used, or a mixture of a plurality of enzymes may be used. Fructosyl amino acid oxidase may be any enzyme as long as it is the enzyme described above. Carriers for immobilizing these proteases or fructosyl amino acid oxidases include, for example, high-molecular-weight polysaccharides such as agarose, cellulose, and dextran and derivatives thereof, porous glass, silica gel, chitosan, polyacrylamide polystyrene resin, ceramic, nylon, and polyvinyl alcohol. , Acrylic and amino acid copolymers can be used.

The method of binding the enzyme to the above-mentioned immobilized carrier is already generally known, such as a physical adsorption method, an ionic bonding method, a cross-linking method, a glutaraldehyde method, a periodic acid method and the like. Depending on the joining method
Any method may be used as long as the immobilized enzyme can maintain high activity and can be immobilized without detaching from the immobilized carrier. Hydrogen peroxide produced as a result of an enzymatic reaction with fructosyl amino acid oxidase can also be measured using a column packed with a peroxidase-immobilized carrier.

The hydrogen peroxide generated by the fructosyl amino acid oxidase-immobilized column is measured by a method known in the art, for example, a coloring method, a method using a hydrogen peroxide electrode. Examples of the color developing system in the colorimetric method of hydrogen peroxide include couplers such as 4-aminoantipyrine (4AA) and 3-methyl-2-benzothiazolinone hydrazone (MBTH) and chromogens such as phenol in the presence of peroxidase. A system that produces a dye by oxidative condensation with a dye can be used. Examples of the chromogen include a phenol derivative, an aniline derivative, a toluidine derivative and the like. For example, N-ethyl-N- (2-hydroxy-3-
Sulfopropyl) -m-toluidine, N, N-dimethylaniline, N, N-diethylaniline, 2,4-dichlorophenol, N-ethyl-N- (2-hydroxy-3-
Sulfopropyl) -3,5-dimethoxyaniline, N-
Ethyl-N- (3-sulfopropyl) -3,5-dimethylaniline (MAPS), N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3,5-dimethylaniline (MAOS) and the like. Can be Also, leuco-type color reagents that are oxidized in the presence of peroxidase to give a color can be used, and such leuco-type color reagents are known to those skilled in the art, and o-dianisidine, o-tolidine,
3,3'-diaminobenzidine, 3,3 ', 5,5'-tetramethylbenzidine, N- (carboxymethylaminocarbonyl) -4,4'-bis (dimethylamino) diphenylamine.Na (DA64), 10- (Carboxymethylaminocarbonyl) -3,7-bis (dimethylamino) phenothiazine.Na (DA67).

The measurement of hydrogen peroxide using a chromogen includes a colorimetric method, a fluorescence method and a chemiluminescence method. Fluorescent methods include compounds that emit fluorescence upon oxidation, such as homovanillic acid, 4-hydroxyphenylacetic acid, tyramine,
Paracresol, diacetylfluorescin derivatives and the like can be used. In the chemiluminescence method, peroxidase, potassium ferricyanide, hemin or the like can be used as a catalyst, and luminol, lucigenin, isoluminol, pyrogallol or the like can be used as a substrate. Further, for the measurement of hydrogen peroxide, a system in which catalase is allowed to act in the presence of an alcohol (eg, methanol) and a aldehyde generated is colored by a Hunch reaction or a condensation reaction with MBTH can also be used. This aldehyde can be conjugated to aldehyde dehydrogenase to measure changes in NAD (NADH). For measurement of glucosone, an aldose reagent such as diphenylamine can be used.

When measuring hydrogen peroxide using an electrode,
Any electrode can be used as long as it can exchange electrons with hydrogen peroxide. Platinum, gold, silver and the like are preferable. The measurement can be performed by a method known to those skilled in the art, such as amperometry, potentiometry, and coulometry. In addition, the resulting oxidation or reduction current or the amount of electricity can be measured by interposing an electron mediator in the reaction between the FAOD or the substrate and the electrode. The electron carrier may be a substance known to those skilled in the art, such as a ferrocene derivative or a quinone derivative, or any substance having an electron transfer function that can be generally considered by those skilled in the art.
Further, an electron carrier can be interposed between the electrode and hydrogen peroxide generated by the FAOD reaction, and the resulting oxidation and reduction currents or the amount of electricity can be measured. Also,
After fragmentation of the glycated protein using a protease-immobilized column, the amount of oxygen consumed by the reaction by adding fructosyl amino acid oxidase or the amount of hydrogen peroxide generated by the reaction is detected by the method described above. Or a method in which the amount of hydrogen peroxide is detected by the above-described method using a fructosyl amino acid oxidase-immobilized column after using a protease-immobilized column.

[0028]

BEST MODE FOR CARRYING OUT THE INVENTION After a glycated protein is subjected to a small fragmentation treatment using a protease-immobilized column, hydrogen peroxide is subsequently generated using a fructosyl amino acid oxidase-immobilized column, and the color development of the POD-immobilized column is detected by a detector. A measurement was made. The detailed procedure is shown below. The protease used this time was protease XIV
And fructosyl amino acid oxidase were performed using FAOD-S. (Example 1) A method for producing a carrier immobilized on a column on which protease or fructosyl amino acid oxidase is immobilized is described below. The protease-immobilized column is composed of protease XIV (Sigma) and Sumiteam M as protease.
Chitopearl (BCW-2601) was used as a carrier for immobilizing P (Nippon Chemical Industry) and protease. The preparation of the protease-immobilized carrier was performed as follows. Protease XIV or Sumizyme MP was dissolved in 0.01 M-phosphate buffer, mixed with chitopearl, and stirred at 4 ° C. for 16 hours. After washing and removing the unimmobilized protease XIV with a phosphate buffer, a glutaraldehyde solution was added, and the mixture was stirred at room temperature for 3 hours. Next, the gel was washed using 0.5 M-NaCL-added 0.1 M-Tris-HCl and 0.1 M-Tris-HCl. Protease XIV (Sigma) 100 mg or Sumiteam MP (Shin Nippon Chemical Industry) 100 mg Chitopearl BCW-2601 (Fuji Boseki) 1 g 0.01 M phosphate buffer (pH 7.4) 2.5% glutaraldehyde 8 ml Gel wash 0.1 M Tris- HCl (pH 8.0) +0.5 M-NaCl 0.1 M Tris-HCl (pH 8.0) D. × 3
It was packed in a column of 0 mm size and used as a protease column hereinafter.

Preparation of Fructosyl Amino Acid Oxidase-Immobilized Carrier The fructosyl amino acid oxidase-immobilized column was prepared by purifying FAOD-purified from a culture of Fusarium oxysporum S1F4 as fructosyl amino acid oxidase.
S, and the immobilization carrier is chitopearl (BCW-260
1) was used. The fructosyl amino acid oxidase was prepared as described in JP-A-7-289253. The preparation of the fructosyl amino acid oxidase-immobilized carrier was performed as follows. Fructosyl amino acid oxidase was dissolved in 0.01 M phosphate buffer, mixed with chitopearl, and stirred at 4 ° C. for 16 hours. After the non-immobilized fructosyl amino acid oxidase was removed by washing with a phosphate buffer, a glutaraldehyde solution was added, and the mixture was stirred at room temperature for 3 hours. Next, wash the gel with 0.5M
0.1M Tris-HCl and 0.1M NaCl added.
This was performed using 1M-Tris-HCl. Fructosyl amino acid oxidase FAOD-S 100 mg Chitopearl AL-01 (Fujibo) 1 g 0.01 M phosphate buffer (pH 7.4) 2.5% glutaraldehyde 8 ml Gel wash 0.1 M Tris-HCl (pH 8.0) +0 0.5 M NaCl 0.1 M Tris-HCl (pH 8.0) Fructosyl amino acid oxidase-immobilized carrier gel was 4.6 mm I.D. D. It was packed in a column of × 10 mm size and used as a protease column hereinafter.

Preparation of POD-Immobilized Carrier The POD-immobilized column used was POD (manufactured by Oriental Yeast Co.), and the immobilized carrier was chitopearl (BCW-260).
1) was used. Production of the POD-immobilized carrier was performed as follows. POD was dissolved in a 0.01 M phosphate buffer, chitopearl was mixed, and stirring was continued at 4 ° C for 16 hours. After the non-immobilized POD was removed by washing with a phosphate buffer, a glutaraldehyde solution was added, and the mixture was stirred at room temperature for 3 hours. Next, the gel was washed with 0.5M-NaCl added.
1M-Tris-HCl and 0.1M-Tris-H
Performed using Cl. POD 100 mg Chitopearl AL-01 (Fuji Spinning) 1 g 0.01 M phosphate buffer (pH 7.4) 2.5% glutaraldehyde 8 ml Gel wash 0.1 M Tris-HCl (pH 8.0) + 0.5 M NaCl 1M Tris-HCl (pH 8.0) POD-immobilized carrier gel was 4.6 mmI. D. × 10mm
The column was packed in a size and used as a POD column hereinafter.

FIG. 1 schematically shows an apparatus for measuring a glycated protein. A protease-immobilized column, a fructosyl amino acid oxidase-immobilized column, and a POD-immobilized column were set as shown in the figure to obtain a measuring apparatus. As a solution passing through each column at the time of measurement, a mixture of the following as A solution was used. 1M Tris-HCl buffer (pH 7.5) 2M DTT (manufactured by Wako Pure Chemical Industries) 3mM 4-aminoantipyrine (manufactured by Wako Pure Chemical Industries) 3mM TOOS (manufactured by SIGMA) Pump from solution A container through transfer tube LC-6A (manufactured by Shimadzu Corporation), passes through an injection valve, and passes through a protease-immobilized column, a fructosyl amino acid oxidase-immobilized column, and a POD-immobilized column.
After 20 μl of the measurement sample is injected from the injection valve and fragmented on the protease-immobilized column, the glycated protein is decomposed on the fructosyl amino acid oxidase-immobilized column to generate hydrogen peroxide. Subsequently, the color on the POD-immobilized column was measured using an absorbance meter (SPD-6).
AV manufactured by Shimadzu Corporation) at 555 nm. This time, we measured in-house prepared glycated polylysine as a model substance for Amadori compounds. The preparation was performed by dissolving 100 mg of polylysine (manufactured by Sigma) and 500 mg of D-glucose (manufactured by Nacalai) in 10 ml of distilled water and incubating at 50 ° C. for 1 week. Then, D-glucose not involved in saccharification was removed by gel filtration. . Next, the concentration was measured using a fructosamine “BMY” (Boehringer Mannheim) kit.
It was confirmed to be μmol / l. Using this saccharified polylysine, the results of measurement of the saccharified polylysine solution adjusted to each concentration are shown in FIG. The absorbance at 555 nm depends on the concentration of glycated polylysine,
Linearity was shown.

[0032]

According to the present invention, the concentration of glycated protein in a sample can be measured efficiently, and a highly accurate measurement result can be obtained.

[0033]

[Brief description of the drawings]

FIG. 1 shows a measuring device using an immobilized column.

FIG. 2 shows the results of measurement of glycated polylysine performed using a measurement device.

[0034]

[Explanation of symbols]

 Solution container Pump Injection valve Protease immobilization column Fructosyl amino acid oxidase immobilization column POD immobilization column Oven Detector Waste liquid bottle

Claims (9)

[Claims] 1. A method for measuring a glycated protein, comprising using a protease-immobilized column when measuring a glycated protein in a sample. 2. A method for measuring a glycated protein, wherein a glycated protein in a sample is measured using a protease-immobilized column and a fructosyl amino acid oxidase-immobilized column. 3. The protease-immobilized column used for the measurement of a glycated protein according to claim 1 or 2. 4. A protease-immobilized carrier packed in a protease-immobilized column used for measuring a glycated protein according to claim 3. The fructosyl amino acid oxidase-immobilized column used for the measurement of a glycated protein according to claim 1 or 2. 6. A fructosyl amino acid oxidase-immobilized carrier packed in a fructosyl amino acid oxidase-immobilized column used for the measurement of a glycated protein according to claim 5. 7. The column or immobilized carrier according to claim 3, wherein the protease is at least one selected from the group consisting of protease XIV, Sumizyme MP, pronase, proteinase K, and subtilisin. 8. A measuring device for measuring a glycated protein in a sample using a protease-immobilized column. 9. An apparatus for measuring a glycated protein, wherein when measuring a glycated protein in a sample, at least a protease-immobilized column and a fructosyl amino acid oxidase-immobilized column are used. [0001]