JPH0556799A - Reagent for detecting saccharified protein - Google Patents

Abstract

PURPOSE:To obtain the title detection reagent consisting of a boronic acid derivative containing a dihydroxyboryl group bonded to an enzyme such as peroxidase or alkali phosphatase used as a labeled material and capable of readily measuring in high sensitivity by a general purpose method. CONSTITUTION:One or more kinds of enzymes such as peroxidase or alkali phosphatase and a boronic acid derivative such as m-aminophenylboronic acid having a dihydroxyboryl group are dissolved in 0.5M boric acid buffer solution (pH 9.0) and both substances are bonded using glutaric aldehyde, etc., and reduced using sodium borohydride to stabilize the bond and then absorbed into an affinity chromatography using an absorbent consisting of a saccharified albumin-immobilized agarose gel and eluted with 2M acetic acid buffer solution (pH 3.0) and the eluted material is put in a cellulose tube, etc., and dialyzed to 0.25M ammonium acetate (pH 9.0) to provide the objective detection reagent for saccharified protein expressed by the formula.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a reagent for detecting a glycated protein.

[0002]

2. Description of the Related Art A sugar boric acid complex is formed by adding borate to an aqueous solution of a compound having a hydroxyl group adjacent to the cis position among polyoxy compounds such as sugar, sugar alcohol and derivatives thereof [chemical Encyclopedia 6, page 363, Kyoritsu Shuppan, October 26, 1981, issue 26].

Boron acid affinity chromatography using boronic acid which is a derivative of boron hydride is widely used as a separation / purification means applying the above-mentioned principle. More specifically, the boronic acid residue binds to a hydroxide ion under a weak alkaline condition to form a boronic acid anion (pK value 8.66), which forms a stable ester with a compound having a cis-diol group. Form. And pH
When the value is reduced to 6 or less, the boronic acid anion decreases, and the ester with the cis-diol group dissociates with the decrease. FIG. 6 shows the reaction pattern between this change in charge and the cis-diol group. Regarding the application of boronic acid affinity chromatography, examples of ribonucleotide and enzyme substrate purification are reviewed [Protein Nucleic Acid Enzymes, Supplement No. 22, 72 to 75].
Page, published March 1980]. In recent years, boronic acid affinity chromatography has been applied to diabetes-related substances such as glycated hemoglobin [glycohemoglobin, a type of glycated protein (Glycated Protein)].
An example applied to the measurement of
8,879-884,1989].

On the other hand, a reagent for detecting a glycated protein applying the same principle has been developed. For example, a boronic acid derivative bound with a fluorescent substance as a label has been reported as a reagent for detecting glycated albumin [Clinica Chimica Acta, 149, 13, 1985]. In this report, the target to be detected is glycated albumin, and the boronic acid derivative bound with a fluorescent substance as a label is dansylphenylboronic acid. By applying the fact that the fluorescence wavelength changes from 530 nm to 490 nm due to the binding of glycated albumin and dansyl phenylboronic acid, dansyl phenylboronic acid is reported to be useful for measuring the glycation ratio of albumin in human serum. is doing.

Further, there is a report that a boronic acid derivative having a fluorescent substance as a label is used in an immunological assay for glycated hemoglobin [US Pat. No. 4,861,728]. In this report,
The target to be detected is glycated hemoglobin, and the boronic acid derivative bound with a fluorescent substance as a label is an m-aminophenylboronic acid derivative bound with phycoerythrin. Then, m in which glycated hemoglobin and phycoerythrin are bound
-After reacting with an aminophenylboronic acid derivative, contact with a resin piece to which an antibody against hemoglobin is bound, capture hemoglobin on the resin piece, and measure the total amount of hemoglobin by the absorption wavelength of hemoglobin and the amount of glycated hemoglobin by fluorescence measurement. The glycation ratio of hemoglobin is measured.

The boronic acid derivative to which the fluorescent substance shown in the above reported example is bound as a label is very useful as a specific detection reagent for glycated proteins.

However, it is known that fluorescent substances are strongly influenced by temperature, concentration of fluorescent substances, liquid properties, and coexisting substances [fluorescence / phosphorescence analysis method, Keizo Hiraki / Taiji Nishikawa, 19
It was considered that a boronic acid derivative bound with a fluorescent substance as a label could also be used as a detection reagent in some cases, since it is considered that the boronic acid derivative bound with a fluorescent substance as a label is also affected by the above. In addition, since the fluorescence measurement method is not as general as the colorimetric measurement method and the measurement equipment is not widely used, it is considered that the use of the boronic acid derivative bound with a fluorescent substance as a label is limited. ..

[0008]

In view of these drawbacks, it is an object of the present invention to provide a reagent for detecting a glycated protein which can be used for general purposes.

[0009]

[Means for Solving the Problems] The inventors of the present invention have conducted eager researches to detect glycated proteins in a versatile manner, and when the detection reagent of the present invention labeled with a boronic acid derivative with an enzyme was glycated. As a reagent for detecting proteins, they have found that they can be used not only for colorimetric measurement methods but also for fluorescence measurement methods and luminescence measurement methods, and arrived at the present invention.

That is, the present invention comprises (1) detection of a glycated protein, which comprises a boronic acid derivative containing a dihydroxyboryl group to which one or more enzymes selected from peroxidase or alkaline phosphatase are bound as a label. reagent.

(2) The reagent for detecting a glycosylated protein according to the above item (1), wherein the boronic acid derivative containing a dihydroxyboryl group is an aminophenylboronic acid derivative. It is about.

The reagent for detecting a glycated protein, which comprises a boronic acid derivative to which the enzyme of the present invention is bound as a label, is an enzyme immunoassay (EIA) used for measuring trace substances.
As in the case of the enzyme-labeled antibody described in (1) above, the catalytic activity of the enzyme labeled in the boronic acid derivative is used, and thus the colorimetric measurement method can be performed with high sensitivity. Further, by selecting an enzyme substrate, it is possible to perform detection by a fluorescence measurement method or a luminescence measurement method.
Therefore, the degree of freedom of measurement is widened.

The present invention is described in detail below, but the present invention is not limited to the substances and substance groups listed below.

As the boronic acid derivative, a boronic acid derivative containing a dihydroxyboryl group such as 3-hydroxyphenylboronic acid or 4-aminophenylboronic acid can be synthesized and used, and commercially available m-aminophenylboron is used. Acids are preferably used.

Further, by introducing a suitable functional group or spacer into the amino group of m-aminophenylboronic acid,
It can also be used as an aminophenylboronic acid derivative. For example, s-acetylmercaptosuccinic anhydride can be reacted to introduce a thiol group for use. That is, a thiol group can be introduced by suspending m-aminophenylboronic acid and anhydrous s-acetylmercaptosuccinic acid in dehydrated pyridine and leaving the suspension at room temperature for 12 hours.

Similarly, N- (m-dihydroxyborylphenyl) succinamic acid [N- (m-Dihydrox) in which a carboxyl group is introduced by reacting with succinic anhydride is introduced.
yborylphenyl) succinamic acid] can be synthesized and used.

Further, as the spacer, an aminophenylboronic acid derivative into which an amino acid, a protein, an aliphatic diamine, an aliphatic monoaminomonocarboxylic acid or the like is introduced can be used. That is, the spacer having an amino group is, for example, glutaraldehyde or the like, and the spacer having a carboxyl group is a water-soluble carbodiimide or the like.
It can be introduced into aminophenylboronic acid.

Boronic acid derivatives containing a dihydroxyboryl group other than m-aminophenylboronic acid can also be used by introducing a functional group or a spacer.

To bond the boronic acid derivative and the enzyme,
Examples of the cross-linking reagent include glutaraldehyde, periodate, N-succinimidyl maleimide compound, water-soluble carbodiimide [1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride, 1-cyclohexyl-3- (2-morpholinoethyl) carbodiimide
Meso-p-toluenesulfonate] and the like can be used.

For details on the method of using the cross-linking reagent and the method of introducing the functional group and the spacer, see "Enzyme Immunoassay".
[Third Edition, pp. 75-151, Ikusho Shoin, May 15, 1987] and "Experimental and Applied Affinity Chromatography" [Ichiro Chibata, Tetsuya Tosa, Yushi Matsuo, pp. 30-109, Kodansha Scientific Fick, 19
The publicly known method described in, for example, the 4th printing of February 20, 1984] can be referred to and applied.

For the measurement of the boronic acid derivative to which an enzyme is bound as a labeled substance, a known measuring method can be used depending on the kind of the enzyme labeling the boronic acid.

That is, colorimetric measurement was carried out by labeling with peroxidase, o-phenylenediamine, 3,3 ',
5,5'-tetramethylbenzidine, 4-chloro-1-
When alkaline phosphatase is labeled using naphthol or the like as a substrate, 4-nitrophenyl phosphate, 5-bromo-4-chloro-3-indole phosphate or the like can be used as a substrate.

Fluorescence measurement is carried out when peroxidase is labeled, 4-hydroxyphenylacetic acid, 3- (4-hydroxyphenyl) propionic acid and the like are used as substrates, and when alkaline phosphatase is labeled, 4-methylumbelliferyl phosphate and the like. Can be used as a substrate. Luminescence can be measured with a luminol-hydrogen peroxide system or the like when peroxidase is labeled. The details can be applied by referring to the above-mentioned "Enzyme Immunoassay", pages 56 to 73.

[0024]

As described above, according to the present invention, since a boronic acid derivative containing a dihydroxyboryl group to which one or more enzymes selected from peroxidase or alkaline phosphatase are bound as a labeling substance is used as a detection reagent, glycated protein can be generally detected. It is possible to provide a detection reagent which can be easily detected by a conventional colorimetric measurement method and which can be detected by a fluorescence measurement method or a luminescence measurement method by selecting an enzyme substrate.

[0025]

EXAMPLES The present invention will now be described in more detail with reference to examples, but the present invention is not limited thereto.

[Example-1] Measurement of glycation ratio of human serum albumin Preparation of adsorbent bound with glycated albumin 50 mg of human serum albumin (manufactured by Sigma), 750 mg of D-glucose and 4 mg of sodium borohydride
Was dissolved in 1.5 ml of 10 mM (M is mol / l) phosphate buffer pH 8.0 and kept at 37 ° C. for 10 days.

This albumin was designated as "Sephadex G-2.
5 "(Pharmacia gel chromatography carrier), eluent 0.25M ammonium acetate buffer pH
After desalting by gel filtration chromatography of 9.0, m-
Affinity chromatography using an adsorbent in which aminophenylboronic acid (manufactured by Aldrich) was bound to "Sepharose 4B" (chromatography carrier manufactured by Pharmacia) activated with bromocyan was used as an eluent 0.25M ammonium acetate buffer. Solution pH 9.0, 2M acetate buffer pH 3.0 was performed, and albumin eluted with eluent 0.25M ammonium acetate buffer pH 9.0 was deglycated albumin fraction, 2M acetate buffer pH 3.0.
The albumin eluted in 1. was separated and purified as a saccharified albumin fraction. The adsorbent bound with glycated albumin is
The glycated albumin fraction adjusted to pH 8.0 with 1 N sodium hydroxide was added to "Sepharose 4B" activated with bromocyan, and left standing at 4 ° C for 24 hours.

Preparation of peroxidase-labeled phenylboronic acid derivative 4 mg of horseradish-derived peroxidase (manufactured by Sigma)
And 16 mg of m-aminophenylboronic acid in 2 ml of 0.
It was dissolved in 5M borate buffer pH 9.0, 5 μl of glutaraldehyde (25 w / w%) was added, and the mixture was reacted at room temperature for 48 hours. Furthermore, sodium borohydride, 2m
g was added and reacted at room temperature for 3 hours.

"Sephadex G-25", eluate 0.
After desalting by gel filtration chromatography with 25 M ammonium acetate buffer pH 9.0, affinity chromatography was performed with an adsorbent bound to glycated albumin as an eluent 0.25 M ammonium acetate buffer pH 9.0, 2 M acetic acid buffer pH 3. 0, 2M acetate buffer pH
The peroxidase-labeled phenylboronic acid derivative eluting at 3.0 was separated and purified (Fig. 4).

The peroxidase-labeled phenylboronic acid derivative thus eluted was adjusted to pH 8.5 by adding tris (hydroxymethyl) aminomethane, and then put into a dialysis tube (cellulose tube) to prepare a 0.25 M ammonium acetate buffer solution at pH 9 It was dialyzed against 0.0.

Of the peaks in the chromatogram of FIG. 4,
The peak on the left is the peroxidase that has not bound to the boronic acid derivative, and the peak on the right is the peak of the peroxidase-labeled phenylboronic acid derivative of the present invention.
This is supported by the fact that, when the peroxidase itself is subjected to the affinity chromatography described above, the peroxidase is not retained by the adsorbent and is eluted (FIG. 5).

A simplified model of the peroxidase-labeled phenylboronic acid derivative molecule obtained by the above-mentioned preparation method is shown in FIG.

Measurement of Glycation Ratio of Human Serum Albumin By mixing non-glycated albumin and glycated albumin, the human serum albumin concentration is the albumin concentration in normal human serum 3
g / dl, the ratio of glycated albumin is 10, 20, 40,
Samples were prepared that were 60, 80, 100%.

20 μl of this sample was diluted to 100 with physiological saline.
Diluted twice to prepare a diluted sample.

Dispense 500 μl of the diluted sample into a test tube,
Warm for 5 minutes in a 7 ° C water bath. Subsequently, an anti-human serum albumin antibody-immobilized carrier was placed in a test tube and reacted for 5 minutes to capture glycated albumin and non-glycated albumin on the carrier. After separating the carrier and the liquid phase, the carrier was washed with physiological saline, and a peroxidase-labeled phenylboronic acid derivative solution [2 U / ml (U represents a unit which is a unit of enzyme activity), 0.25 M acetic acid. 400 μl of ammonium buffer solution pH 9.0 was added and reacted at 37 ° C. for 30 minutes. The carrier was washed with 0.25 M ammonium acetate buffer (pH 9.0) and then heated to 37 ° C to 1 mg / m 2.
l o-phenylenediamine solution (0.1 M phosphate citrate buffer pH 6.0, containing 5 mM hydrogen peroxide) 500
μl was added, and the mixture was reacted at 37 ° C. for 10 minutes. After 10 minutes, the reaction was stopped with 2.0 ml of 1N sulfuric acid, and the absorbance at 492 nm was measured.

The result of the measurement is shown in FIG. As shown in FIG. 3, a calibration curve having excellent linearity could be created between the ratio of glycated albumin [glycation ratio] and the absorbance (ΔA492 nm) obtained by subtracting the sample blank. The time required for measurement is about 1 hour, and the required sample volume is 20 μl.
It became possible to measure the saccharification rate of human serum albumin with a small amount of sample.

Since the o-phenylenediamine solution added as a substrate thus develops color depending on the amount of peroxidase bound, the saccharification ratio can be detected accurately.

FIG. 2 is a simplified model diagram showing a state in which the peroxidase-labeled phenylboronic acid derivative of the present invention is bound to the cis-diol group of glycated albumin captured on the anti-human serum albumin-immobilized carrier.

[Example-2] Measurement of glycation ratio of human serum albumin by fluorometric method According to Example-1, the ratio of glycated albumin was 0,50,
A 100% sample was prepared, the measurement operation was performed, and the substrate of the peroxidase-labeled phenylboronic acid derivative was measured according to the above-mentioned "enzyme immunoassay", pages 61 to 62.
Fluorescence was measured as-(4-hydroxyphenyl) propionic acid.

As a result of the measurement, it was possible to prepare a calibration curve having excellent linearity between the ratio of glycated albumin and the fluorescence intensity as in Example-1.

As described above, 3- (4-hydroxyphenyl) propionic acid used as a substrate emits fluorescence in accordance with the binding amount of peroxidase, so that the saccharification ratio can be detected with high sensitivity.

Example 3 Detection of Glycated Albumin Bound to Nitrocellulose Membrane Binding of Nitrocellulose Membrane to Glycated Albumin A nitrocellulose membrane (manufactured by Toyo Roshi Kaisha, Ltd.) was fixed on a dot plate (manufactured by Toyo Ruigami). , 0.01 to 100 μg of glycated albumin is gradually added to the holes of each dot plate, and the mixture is allowed to stand at room temperature for 15 minutes and then sucked,
Glycated albumin was adsorbed on a nitrocellulose membrane. Then, the nitrocellulose membrane having glycated albumin adsorbed thereon was dipped in a bovine serum albumin 1 g / dl solution (0.25 M ammonium acetate buffer solution pH 9.0) and allowed to stand at room temperature for 30 minutes.

Preparation of alkaline phosphatase-labeled phenylboronic acid derivative 5 mg of alkaline phosphatase (manufactured by Sigma) and 8 mg of m-aminophenylboronic acid were added to 2 ml of 0.5M.
The solution was dissolved in borate buffer solution pH 9.0, 5 μl of glutaraldehyde (25 w / w%) was added, and the mixture was allowed to stand at 4 ° C. for 1 hour. After that, desalting was performed by gel filtration chromatography similar to the peroxidase-labeled phenylboronic acid derivative of Example 1 to obtain an alkaline phosphatase-labeled phenylboronic acid derivative.

Detection of glycated albumin Alkaline phosphatase-labeled phenylboronic acid derivative solution (0.5 U / ml, 0.25M ammonium acetate buffer pH 9.0, containing 1 g / dl of bovine serum albumin)
A nitrocellulose membrane having glycated albumin adsorbed therein was immersed in the solution and reacted at room temperature for 15 minutes. After washing with 0.25 M ammonium acetate buffer pH 9.0, alkaline phosphatase substrate solution [5-bromo-4-chloro-3-indole phosphate 2.5 mg / 50 μl (N, N-
Dimethylformamide) and nitroblue tetrazolium 5 mg / 15 ml (0.1 M Tris-HCl buffer pH
9.5) was mixed and prepared] and reacted for 30 minutes at room temperature.

As a result of the reaction, 1 μg or more of glycated albumin could be detected in blue.

Example 4 Detection of Glycated Protein Migration Image After Cellulose Acetate Membrane Electrophoresis Cellulose Acetate Membrane Electrophoresis Human globulin and human hemoglobin were analyzed in Example-1.
A glycated protein was prepared according to the method for producing glycated albumin of.
In addition, the same glycated albumin as in Example 1 described above was prepared as a glycated protein. Then, using human serum albumin, human globulin, human hemoglobin and each of the above-mentioned glycated proteins as samples, a cellulose acetate membrane according to "Clinical Test Method Recommendation" [Kanehara Shuppan, pages 414 to 420, issued June 30, 1983]. Electrophoresis was performed.

Preparation of Peroxidase-Labeled Phenylboronic Acid Derivatives Using N- (m-Dihydroxyborylphenyl) succinamic Acid N- (m-dihydroxyborylphenyl) succinamic acid has been described in the literature [Biochemist (Biochemist).
ry), 9, 22, 4396, 1970]. That is,
To 1.37 g of m-aminophenylboronic acid and 1.1 g of succinic anhydride, 5 ml of dehydrated pyridine was added, and the mixture was allowed to stand at room temperature for 12 hours.
Let stand for hours. Next, 5 ml of ion-exchanged water was added, the mixture was allowed to stand at room temperature for 1 hour, and then water was removed under reduced pressure. It was dissolved in 10 ml of deionized water, adjusted to pH 1.0 with hydrochloric acid, and ice-cooled for crystallization. The details were in accordance with the above literature.

Peroxidase (manufactured by Toyobo Co., Ltd.)
20 mg and N- (m-dihydroxyborylphenyl) succinamic acid 10 mg were added to deionized water 2 m.
It was dissolved in 1 and 100 mg of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride was added. 1
The pH was maintained at 5.5 while adding N hydrochloric acid, reacted for 1 hour, and then allowed to stand at 4 ° C. for 12 hours. After that, it was separated and purified in the same manner as the peroxidase-labeled phenylboronic acid derivative of Example-1.

Detection of Proteins and Glycated Proteins Proteins and glycated proteins were detected by the Ponceau 3R staining method in accordance with “Clinical Test Method Recommendation”.

To detect glycated proteins, first, the cellulose acetate membrane after the electrophoresis was immersed in 5% trichloroacetic acid for 2 minutes to immobilize the proteins and the glycated proteins on the cellulose acetate membrane.

Subsequently, the product was washed with a 0.25 M ammonium acetate buffer (pH 9.0), and a peroxidase-labeled phenylboronic acid derivative solution (2.0 U / ml, 0.25
M ammonium acetate buffer (pH 9.0, containing 1 g / dl of bovine serum albumin), reacted at room temperature for 15 minutes, and then washed with 0.25 M ammonium acetate buffer (pH 9.0). After washing, a peroxidase substrate solution (2.
It was immersed in 8 mM 4-chloro-1-naphthol, 20 mM hydrogen peroxide-containing, 20 mM Tris-HCl buffer (pH 7.4) and reacted for 15 minutes.

As a result of the reaction, the migration image of the glycated protein was
It was detected in blue, but non-glycated protein was not detected. On the other hand, in the Ponceau 3R staining method, all electrophoretic images of glycated proteins and non-glycated proteins were detected in red.

[Example-5] Measurement of glycation ratio of human serum albumin by luminescence detection method Preparation of peroxidase-labeled phenylboronic acid derivative using periodoic acid Horseradish-derived peroxidase 4 mg (manufactured by Sigma)
Was dissolved in 1 ml of 0.05 M borate buffer pH 9.0,
0.1 ml of 0.1 M sodium periodate aqueous solution was added, and the mixture was reacted at room temperature for 20 minutes. Further, 6 mg of m-aminophenylboronic acid was dissolved in 0.1 ml of 0.5 M borate buffer solution pH 9.0, added, and reacted at room temperature for 24 hours. Separation and purification of the peroxidase-labeled phenylboronic acid derivative were carried out according to Example-1.

Luminescence detection of glycated albumin According to Example-3, the proportion of glycated albumin was set to 10,2.
After adsorbing 5, 50, 75, 100% of the sample on the nitrocellulose membrane, the peroxidase-labeled phenylboronic acid derivative solution prepared in the same manner as in Example-4 was added to the holes of each dot plate, and Allowed to react for 15 minutes.

0.25M ammonium acetate buffer pH
After washing with 9.0, 1 g / dl solution of bovine serum albumin (0.25M ammonium acetate buffer pH 9.0) was used.
After soaking for 5 minutes, a luminescent reagent solution (1.25 mM luminol, 2.7 mM hydrogen peroxide, 0.136 mM p-iodophenol-containing 0.1 M Tris-HCl buffer pH 8.5)
And allowed to react at room temperature for 1 minute. The nitrocellulose membrane taken out from the luminescent reagent solution was used as a photosensitive film for chemiluminescence detection "Hyperfilm-ECL" (Hy
perfilm-ECL, manufactured by Amersham), and the film was exposed for 1 minute.

"Hyperfilm" is a known method [Molecular cloning, 2nd e
d), Cold Spring Harbor Laboratory (Cold S
pring Harbor Laboratory), page E27, 1989].
And developed according to. As a result of development, the adsorbed dot portion of the sample was strongly exposed to light as the saccharification rate increased, and developed in black.

[0057]

INDUSTRIAL APPLICABILITY As described above, the detection reagent of the present invention enables the detection of glycated protein by a general-purpose colorimetric method. Furthermore, detection by a fluorescence measurement method or a luminescence measurement method is possible by selecting an enzyme substrate.

The reagent also made it possible to specifically detect the migration images of the glycated protein adsorbed on the nitrocellulose membrane and the glycated protein after the electrophoresis on the cellulose acetate membrane.

[Brief description of drawings]

FIG. 1 shows a simplified model diagram of a boronic acid derivative (peroxidase-labeled phenylboronic acid derivative) bound with the enzyme of the present invention as a label when peroxidase and m-aminophenylboronic acid are bound with glutaraldehyde.

FIG. 2 is a simplified model diagram showing a state in which a peroxidase-labeled phenylboronic acid derivative and a glycated albumin captured by an anti-human serum albumin antibody-immobilized carrier are bound.

FIG. 3 shows a standard curve showing the relationship between the ratio of glycated albumin (glycation ratio) and the absorbance.

FIG. 4 shows a chromatogram of a mixed solution of peroxidase and a peroxidase-labeled phenylboronic acid derivative obtained by the method for preparing a peroxidase-labeled phenylboronic acid derivative, which was subjected to affinity chromatography on an adsorbent having glycated albumin bound thereto. ..

FIG. 5 shows a chromatogram obtained by subjecting an adsorbent to which glycated albumin is bound to peroxidase to affinity chromatography.

FIG. 6 is a diagram showing a change in charge of a boronic acid residue due to liquidity and a reaction mode with a cis-diol group.

Claims (2)

[Claims] 1. A reagent for detecting a glycated protein, which comprises a boronic acid derivative containing a dihydroxyboryl group to which one or more enzymes selected from peroxidase or alkaline phosphatase are bound as a label. 2. The reagent for detecting a glycated protein according to claim 1, wherein the boronic acid derivative containing a dihydroxyboryl group is an aminophenylboronic acid derivative.