JP2543630B2 - Measuring method of glycation ratio of specific protein - Google Patents

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a method for measuring the glycation ratio of a specific protein.

[0002]

2. Description of the Related Art Since complications occurring in the late stage of diabetes decisively influence the prognosis of a patient, prevention of the progress thereof is the most important issue in the treatment of diabetes.

It has been reported that a protein in blood of a diabetic patient undergoes non-enzymatic glycation, and the amount of non-enzymatic glycated protein in blood is increased as compared with a healthy person, and the relationship between the non-enzymatic glycated protein and a disease is detailed. Being researched. (Biochem.Bi
ophys.Res.Commun., 67,103, (1975), J.Biol.Chem., 254,7
02 (1979), Proc.Natl.Acad.Sci.USA, 78,2393 (1982), Diab
etes, 31,283 (1982), Proc.Natl.Acad.Sci.USA, 75,2918 (1
978), J. Clinical Pahtology, 37,841 (1984), Annals of C
linical Biochemistry, 21, 2 (1984)).

[0004] As an index of blood glucose control, the fasting blood glucose reflecting the current glucose metabolism state, as well as the past 1
Non-enzymatic glycated hemoglobin, which has a good correlation with the blood glucose level for two months, is clinically useful and is widely adopted as an index for treating diabetic patients. However, there are problems that fasting blood glucose has a large diurnal variation, and that non-enzymatic glycated hemoglobin requires a long period of time to confirm the therapeutic effect.

For the purpose of knowing the therapeutic effect earlier, fructosamine, which is a reducing ability of blood non-enzymatic glycated protein reflecting blood glucose status in the past 1-2 weeks, has been measured and used for diabetes treatment. However, the effect of coexisting substances, such as bilirubin and reducing substances such as L-ascorbic acid, remains a problem (clinical test equipment,
Reagent, Vol. 13, No. 1, p. 87 (1990)). Similarly, non-enzymatic glycated albumin attracts attention as an indicator of blood glucose control in a short period of time, and research for developing a simple assay method thereof has been actively conducted.

Furthermore, non-enzymatic glycated proteins include Schiff base type (unstable type) and Amadori transfer product (stable type) in which the Schiff base is stabilized by Amadori transfer (Clinical test No. 33, No. 8, (Page 893 (1989)), it is becoming important to measure only Amadori metastases that are not affected by changes in blood glucose level for a short period of time and that well reflect the blood glucose level during a certain past period.

[0007] Conventional methods for measuring glycated proteins include:
In addition to isoelectric focusing, cation exchange chromatography, spectrophotometry, colorimetric method, etc., HPLC method,
The mini-column method, affinity chromatography and the like can be mentioned (see Diabetologia, 31, 627-631 (1988), Inspection and Technology, Vol. 14, No. 11, page 1155 (1986)),
It is widely used clinically for measuring the amount of glycated protein in a sample (test liquid).

However, it is difficult to say that these methods simultaneously satisfy the points of measurement sensitivity, operability, sample processing time, and the like, and there are some points that must be improved in practical use. Therefore, it has been demanded to establish a method capable of easily and highly accurately measuring a large number of samples in a short time.

As a method for solving these problems, application of an immunological assay method is considered, and an antiserum having a specific affinity for a glycitol lysine residue, which is one of the glycosylation sites of glycosylated proteins, It has been reported that a glycated protein can be measured by obtaining a monoclonal antibody and radiolabeling immunoassay (RIA) (J. Clin. Invest., 72,
1427 (1983), Clinical Pathology Vol. 33, Supplement (1985) 226.
P. Diabetes Vol. 29, p. 581 (1986), Clinica.Ch
imica.Acta., 153,293 (1986), Diabetes Vol. 28, No. 6
95 (1985)).

All of the above-mentioned immunological assay methods use an anti-reducing glycated protein antibody against the Amadori transfer product of the glycated protein, which is an excellent method for glycated protein measurement, but is clinically measured. There are problems that it is necessary to perform specific separation of highly significant specific glycated proteins and to measure the total amount thereof separately, and pretreatment to separate sugar and protein before the reduction reaction is required (Diabetes Vol. 34). See Supplement 96, 1986).

Affinity chromatography of phenylboronic acid, which is less susceptible to small fluctuations in temperature, pH or ionic strength, is an excellent method for separating glycated proteins, and is fixed by Amicon Corporation and Pierce Chemical Company. Kits have been developed for clinical use which include modified phenylboronic acid (UK Patent Publication No. 2,024,829 and US Pat. No. 4,269,605). However, these methods also have problems that a relatively large amount of sample is required and that a protein amount measurement operation lacking specificity is involved.

Further, a method in which a glycated protein is separated by immobilized phenylboronic acid and a labeled antibody having a specific affinity for a specific protein is used (JP 62-100660 A).
No.) has been reported, but there are practical problems such as being affected by changes in the ratio of each type of glycated protein and the need to separately measure the total amount of specific proteins in order to know the glycation ratio. There is.

A method of quantifying glycated albumin from changes 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. This method is, in principle, that the detection reagent reacts with an unspecified glycated protein, that the total amount of the specific protein must be separately measured, and that the specificity of the protein measuring method for the specific protein is low. It is not suitable as a method for measuring the saccharification rate.

As a method for solving these problems, Japanese Patent Laid-Open No.
64-16964 discloses that a specific protein is separated by a solid-phased antibody, and the amount of the specific glycated protein is determined by a monoclonal antibody that specifically recognizes reduced glycated moieties such as glycitol lysine and glycitol valine residues. , Or a method for measuring the glycation ratio of a specific protein has been reported. This method is an excellent method in that the glycation ratio can be measured without measuring the total amount of a specific protein, but the reproducibility and calibration curve linearity are poor, and the recognition glycation site is only a part of the glycation site. There are problems such as not being present and requiring a measurement time of 5 hours or more.

Further, 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, and the total amount of hemoglobin is measured by colorimetry, and further glycated hemoglobin is measured. A method for measuring the saccharification ratio of hemoglobin by measurement by fluorescence measurement has been reported (US Pat. No. 4,861,728). However, it is not an economical assay because it requires a boronic acid derivative labeled with a fluorescent dye that is more than the total amount of glycated hemoglobin in the sample, which is present in a large excess relative to the amount of antibody immobilized on the carrier. There are problems in operability, convenience, and versatility in that two measurements, colorimetric measurement and fluorescence measurement, are required to measure the ratio.

[0016]

In view of these drawbacks of the conventional methods, the present invention provides an immunological method and an affinity adsorption method capable of accurately measuring the glycation ratio of a specific protein in a short time with a simpler operation. The purpose is to provide a measurement method that combines methods.

[0017]

[Means for Solving the Problems] As a result of intensive studies conducted by the present inventors to easily measure the glycation ratio of a specific protein, first, it has a specific affinity for the specific protein to be measured. An antibody or an active fragment of an antibody (the part containing the antigen recognition site of the antibody) is immobilized on a solid carrier and the insolubilized solid phase is allowed to react with a sample that is the test liquid to capture a specific protein, and then the liquid phase (sample)
And the solid phase are separated, and a labeled boronic acid derivative having an affinity with the cis-diol group existing at the glycosylation site of the specific protein captured on the solid phase is reacted,
The present inventors have found that the glycation ratio of a specific protein in a sample can be easily measured by binding a label to a glycation site and measuring the amount of the label bound or not bound to a solid phase.

That is, in the present invention, a sample is brought into contact with a solid phase in which an antibody or an active fragment of the antibody having a specific affinity for a specific protein is immobilized on a carrier to selectively bind the specific protein to the solid phase. After capturing on the phase, the solid phase is separated from the solid phase, and then the labeled boronic acid derivative having an affinity for the glycosylation site of a protein is contacted with the separated solid phase. The method is characterized in that a labeling substance is bound to the glycation site of a specific protein captured by, and then the solid phase and the liquid phase of this reaction mixture are separated, and the amount of the separated solid phase or liquid phase labeling substance is measured. The present invention relates to a method for measuring the glycation ratio of a specific protein.

The present invention also provides a method for measuring the saccharification ratio of a specific protein as described above, wherein the boronic acid derivative is a boronic acid derivative containing a dihydroxyboryl group.

The present invention further provides a method for measuring the glycation ratio of a specific protein as described above, which is one kind selected from urine, blood, plasma and serum from which the above sample was collected.

Furthermore, in the present invention, the labeled boronic acid derivative is a radioisotope, an enzyme, a coenzyme, a fluorescent dye,
The present invention relates to the method for measuring the saccharification ratio of a specific protein as described above, which is a boronic acid derivative labeled with either a chemiluminescent substance or a specific binding protein.

According to this measuring method, since the boronic acid derivative mainly captures the Amadori transfer product (stable form) of the glycated protein, it is necessary to remove the Schiff base type (unstable) glycation product by pretreatment of the sample. Not (Clin.Chem., 28,10,2
088-2094 (1982)).

As described above, according to the present invention, first, a specific protein for specifically separating only a specific protein of interest (specifically, albumin, hemoglobin, etc.) from a sample that is a test liquid is used. A specific antibody or a specific protein or an active fragment of the antibody is captured on a solid phase on which a specific antibody is immobilized on a carrier, and then the specific protein is glycated A labeled boronic acid derivative (a boronic acid derivative with a radioisotope, an enzyme, or another labeled substance) is used to measure the.
Therefore, the Amadori transfer product (the Schiff base type glycation product of the glycated protein becomes stable due to Amadori transfer) can be efficiently measured without preparing an antibody against the glycated protein.

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

The antibody used in the present invention is not particularly limited in its origin, and may be obtained by immunizing a mammal or the like (eg, mouse, rat, rabbit, etc.) with a specific protein or a purified product thereof as an antigen and immunizing it. The obtained antiserum, ascites fluid or the like is used as it is, or is purified as a polyclonal antibody by a conventionally known method such as salting out method, gel filtration method, ion exchange chromatography, electrophoresis method, affinity chromatography and the like. You can Alternatively, a monoclonal antibody prepared by obtaining hybrid cells (hybridomas) from antibody-producing cells (spleen cells, lymph node cells, etc.) of mammals sensitized with an antigen and myeloma cells, or conventionally known salting out methods, various chromatographies, etc. A monoclonal antibody purified by chromatography can be used.

These antibodies may be antibody molecules themselves, or Fab and Fa obtained by enzymatic treatment of these antibodies.
An active fragment of an antibody such as b'or F (ab ') 2 (a portion containing the antigen recognition site of the antibody) may be used.

The shape of the solid phase carrier may be appropriately selected according to the purpose of use, and examples thereof include beads, test plates, tubes, disks, spheres, sticks, and latex. it can. Further, as the material thereof, those used as a carrier for a usual enzyme immunoassay (EIA), for example, glass, polysaccharides (cellulose, dextran, starch, dextrin) or derivatives thereof, silica gel, porous ceramics, metal oxides Materials, various synthetic resins (eg propylene, vinyl chloride, vinyl acetate, vinyl propionate, acrylic acid,
Acrylic acid ester, methacrylic acid, methacrylic acid ester, styrene, methylstyrene, butadiene, isoprene, acrylamide, acrylonitrile, methacrylonitrile, or other polymer or copolymer carrier), or a sulfo group or an amino group by known means. And the like into which a reactive functional group is introduced.

As a method for immobilizing an antibody on a carrier, the same methods as those for immobilized enzymes such as physical adsorption method, covalent bond method and cross-linking method may be applied. For example, “Immobilized enzyme” edited by Ichiro Chibata. (Published by Kodansha, Ltd. on March 20, 1975)
Pages 9 to 75 can be applied.

The boronic acid derivative may have a dihydroxyboryl group in structure, and examples thereof include (1-amino-2-phenylethyl) boronic acid, [3-[(aminocarbonyl) amino] phenyl] boronic acid, (3-Aminophenyl) boronic acid, [3- (hydroxy) phenyl] boronic acid hydrochloride, [2- (hydroxyamino) phenyl] boronic acid, [4- (hydroxyamino) phenyl] boronic acid hydrochloride, etc. Can be used. Particularly, a boronic acid derivative having an amino group is preferably used.

To bind a boronic acid derivative and a labeled substance to form a labeled boronic acid derivative, for example, glutaraldehyde, periodate, a maleimide compound (N-succinimidyl-2-maleimidoacetate) can be used as a crosslinking reagent. , N-succinimidyl-4-maleimidobutyrate, N-succinimidyl-4- (N-maleimidomethyl) -cyclohexane-1-carboxylate, N-sulfosuccinimidyl-4- (N-maleimidomethyl)-
Cyclohexane-1-carboxylate, etc., dimaleimide compounds (N, N'-oxydimethylenedimaleimide, N, N'-o-phenylenedimaleimide, etc.) and the like can be used.

As the labeling substance for labeling the boronic acid derivative, those commonly used in immunological assay methods can be used. Specifically, radioisotopes, enzymes (for example, β-galactosidase, peroxidase,
Alkaline phosphatase, glucose oxidase, glucose-6-phosphate dehydrogenase etc.), enzyme substrates, fluorescent dyes (eg fluorescein derivatives, rhodamine derivatives, umbelliferone etc.), chemiluminescent substances (eg luminol derivatives, isoluminol derivatives etc.) ,
Coenzymes / prosthetic groups such as biotin and nicotinamide adenine dinucleotide, specific binding proteins (specific binding has the property of binding only to specific compounds,
For example, avidin or the like) that can be relatively easily detected by some method may be used.

Labeling with a radioisotope was conducted in 1983.
February 28, 2014 "Clinical pathology" published by Kanehara Publishing
Special Issue No. 53, pages 24 to 25 (1983) or June 15, 1988, published by the Institute of Medical Sciences "Examination and Technology" Special Issue Vol 16, N
o.7 The method according to pages 581-582 (1988)
For other labeling, published on May 15, 1987, by Eiji Ishikawa, Tadashi Kawakami, Kiyoshi Miyai, edited by "Enzyme Immunoassay," 3rd edition, pages 75-151 or Ichiro Chibata, "Immobilized Enzyme" (Showa era). The method according to pages 9 to 75, etc., published by Kodansha Ltd. on March 20, 1950 can be applied.

As the sample (test liquid) to be measured,
There is no particular limitation as long as it requires measurement of glycated protein, and examples thereof include a sample or protein derived from a living body containing glycated protein such as urine, blood, plasma, serum, etc. and a carbohydrate (for example, glucose or glucose-6- Phosphorus etc.)
And the like. For glycated proteins,
For example, “Clinical Examination” Vol 33, No. 8 pp. 879-899 (1
989).

To measure the glycation ratio of a specific protein in the sample, a fixed amount of an antibody or an active fragment of the antibody having a specific affinity for the specific protein to be measured was immobilized on a carrier. It is important to use a sample that can contain the specific protein at a concentration higher than the concentration at which the specific protein can bind to all the antibodies or active fragments of the antibody on the solid phase.

In order to bind the labeled boronic acid derivative to the glycation site of the glycated protein, it is usually at a pH of 8 to 10.
Ammonium acetate buffer in the range of 9, morpholine hydrochloride buffer, or 2-amino-2-methylpropane-1,3
-It is preferable to use it as a solution such as a diol hydrochloric acid buffer solution.

Hereinafter, in order to facilitate understanding of the present invention,
Taking albumin as an example of the specific protein, the reaction is modeled and shown as a simplified model diagram in FIGS.

FIG. 2 is a molecular model of albumin.
NH ₂ represents the amino group of albumin. FIG. 3 shows glycated albumin (a state in which sugar is bound to the amino group of albumin in FIG. 2 and Amadori is transferred). FIG. 4 shows a state in which the anti-albumin antibody immobilized on the carrier captures albumin without distinguishing between glycated and non-glycated albumin. FIG. 5 shows a state in which the labeled boronic acid derivative is specifically bound only to the glycation site of the glycated albumin captured in FIG. In FIG. 5, * indicates a label.

[0038]

According to the method of the present invention, a certain amount of the antibody or the active fragment of the antibody is immobilized on a carrier, and the antibody or the active fragment of the antibody on the solid phase is obtained at a concentration above the concentration at which the specific protein can bind. By using the sample containing the specific protein, the amount of the specific protein bound to the solid phase becomes constant, so by measuring the amount of the label bound to the glycation site of the specific protein by the labeled boronic acid derivative, The glycation ratio of a specific protein can be measured without separately measuring the total amount of the specific protein. Then, by the method of the present invention, the saccharification ratio of the specific protein in the sample can be more accurately compared with the conventional method,
It is possible to measure easily and quickly in a short time.

[0039]

EXAMPLES In order to facilitate understanding of the present invention, examples will be described below, but the present invention is not limited to these examples.

Example 1 Preparation of Non-Enzymatic Saccharified Albumin and Non-Saccharified Albumin Human serum albumin 50 mg (manufactured by Sigma), D-glucose 50 mg and sodium borohydride 4 mg 10 mM (M indicates mol / l) phosphorus Acid buffer pH
Dissolve in 8.0 and hold at 37 ° C for 10 days. This albumin was used as "Sephadex G-25" (a carrier for gel filtration chromatography manufactured by Pharmacia) and an eluent of 0.
After desalting by gel filtration chromatography with 25 M ammonium acetate buffer pH 9.0, affinity chromatography using an adsorbent to which 3-aminophenylboronic acid (manufactured by Aldrich Co.) was bound was performed with 0.25 M eluent.
Ammonium acetate buffer pH 9.0 and 0.25M acetic acid buffer pH 4.0 were used, and albumin eluted with eluent 0.25M ammonium acetate buffer pH 9.0 was used as a non-glycated albumin fraction to obtain 0.25M acetic acid. Buffer pH
Albumin eluted at 4.0 is separated and purified as a non-enzymatic glycated albumin fraction.

Preparation of peroxidase-labeled phenylboronic acid derivative 4 mg of horseradish-derived peroxidase (manufactured by Sigma)
And 16 mg of 3-aminophenylboronic acid and 0.05% glutaraldehyde are mixed, reacted at room temperature for 48 hours, added with 2 mg of sodium borohydride, and allowed to stand at room temperature for 3 hours. "Sephadex G-25", eluate 0.25M
After desalting by gel filtration chromatography with ammonium acetate buffer pH 9.0, affinity chromatography was performed with the adsorbent to which the non-enzymatic glycated albumin prepared in (4) was eluted as 0.25 M ammonium acetate buffer pH.
9.0, 0.25M acetate buffer pH 4.0,
The peroxidase-labeled phenylboronic acid derivative eluting with 0.25 M acetate buffer (pH 4.0) is separated and purified.

Preparation of anti-human serum albumin antibody-immobilized carrier In order to immobilize the anti-human serum albumin antibody on polystyrene beads and insolubilize it into a solid phase, an anti-human serum albumin antibody solution (100 to 200 μg / ml, 10 mM phosphate buffer) was prepared. Solution pH 7.3) with polystyrene beads (φ6.35)
mm), and allowed to stand overnight at 4 ° C., then immersed in 0.1% bovine serum albumin (BSA) and allowed to stand at 4 ° C. overnight to prepare an anti-human serum albumin antibody-immobilized carrier (solid phase).

Measurement of glycation ratio of human serum albumin Mixing non-glycated albumin and non-enzymatic glycated albumin,
The human serum albumin concentration was set to 3 g / dl, which is the serum albumin concentration in normal humans, and the ratio of glycated albumin was 10,
Samples were prepared which were 20, 40, 60, 80, 100%.

20 μl of this sample was 100
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, the carrier on which the anti-human serum albumin antibody was immobilized was placed in a test tube and reacted for 5 minutes. After separating the carrier and the liquid phase, the carrier is washed with physiological saline, and the peroxidase-labeled phenylboronic acid derivative solution (0.25 M ammonium acetate buffer pH
9.0) 400 μl was added and reacted at 37 ° C. for 30 minutes. Subsequently, 0.25M ammonium acetate buffer p
After washing with H9.0, a 1 mg / ml o-phenylenediamine solution (0.1 M phosphate citrate buffer pH 6.0,
500 μl (containing 5 mM hydrogen peroxide), and
After reacting for 10 minutes, stop the reaction with 2.0 ml of 1N sulfuric acid,
The absorbance at 492 nm was measured.

The results of the measurement are shown in FIG. ΔA on the vertical axis indicates a blank and corrected value. As shown in FIG. 1, a calibration curve can be created between the ratio of glycated protein and the absorbance, and the absorbance increases linearly in proportion to the increase of the percentage of glycated protein. It has been clarified that the measurement method of the present invention enables accurate measurement. Further, since the time required for measurement was about 1 hour and the required sample amount was 20 μl, it became clear that a large amount of sample can be processed in a short time and a small amount of sample is required.

Example 2 Measurement of glycation ratio of albumin in serum sample and albumin electrophoretically separated / extracted Separation of albumin by electrophoresis Serum sample was 10 ml of standard control serum "Monnitrol II / X" (manufactured by Dade, USA). D-glucose (5 g) was added and the mixture was kept at 37 ° C for 2 days. A part of the serum sample was subjected to 10 (W / W%) acrylamide gel electrophoresis, and then the gel was immersed in a 30 (W / V%) trichloroacetic acid aqueous solution for about 1 hour to cut out a part corresponding to albumin. The excised gel was placed in a test tube, washed with 0.1 M phosphate buffer (pH 7.3) until neutral, and albumin was extracted by crushing the gel and centrifuging.

Measurement of Saccharification Ratio of Albumin The measurement of the saccharification ratio of albumin in the serum sample and albumin extracted from the electrophoretic gel was carried out according to Example 1. Table 1 shows the measurement results.

[0049]

[Table 1] The saccharification ratios of albumin in serum samples and albumin extracted from gel were almost the same. That is, the glycation ratio of albumin in the serum sample was not affected by the coexisting substance, and could be measured in the same manner as when albumin was isolated by electrophoresis.

Example 3 Measurement of saccharification ratio of human serum albumin using avidin-labeled phenylboronic acid derivative and peroxidase-labeled biotin Preparation of avidin-labeled phenylboronic acid derivative 2 mg of egg white-derived avidin and 8 mg of 3-aminophenylboronic acid 2 ml of 0.5 M borate buffer pH 10.5
Dissolve in 25 (W / W%) glutaraldehyde 1 μl
Was added and left at room temperature for 10 minutes. 10 minutes later, 50
The reaction was stopped by adding 0 μl of 0.25 M Tris-HCl buffer pH 8.5, and the mixture was allowed to stand at room temperature for 1 hour. The avidin-labeled phenylboronic acid derivative was purified by the same method as in Example 1, and then pH was adjusted to 9.0 with 4N-sodium hydroxide.
And

Measurement of saccharification rate of human serum albumin By the same preparation method as in Example 1, the saccharification rate was 0, 50, 1
A sample of human serum albumin having a concentration of 00% was prepared, and in the measurement operation, an avidin-labeled phenylboronic acid derivative was used in place of the peroxidase-labeled phenylboronic acid derivative, and avidin was bound to the glycation site of glycated albumin. The carrier is physiological saline [0.05 (V / V%)
After washing with polyoxyethylene sorbitan monolaurate], a peroxidase-labeled biotin (manufactured by Sigma) solution [0.25 M ammonium acetate buffer pH 9.
0 (2.7 U / ml)] was added, and the mixture was reacted at 37 ° C. for 10 minutes, and then the enzyme activity was measured in the same manner as in Example 1. The measurement results are shown in Table 2.

[0052]

[Table 2] The measured value increased in proportion to the increase in saccharification rate (%).
As described above, the saccharification ratio of human serum albumin can be measured using avidin-labeled phenylboronic acid derivative and peroxidase-labeled biotin by utilizing the specific and selective binding property of avidin, which is a specific binding protein, to biotin. It was possible.

Example 4 Measurement of glycation ratio of human hemoglobin In the same manner as in Example 1, non-enzymatic glycated human hemoglobin and non-glycated human hemoglobin were prepared, separated and purified.
The saccharification ratio was measured in the same manner as in Example 1 except that the carrier for immobilizing the anti-human hemoglobin antibody and the peroxidase-labeled phenylboronic acid derivative solution (2 U / ml) were used in the measurement operation of Example 1. The experimental results are shown in Table 3.

[0054]

[Table 3] As a result of the experiment, it was possible to measure the saccharification ratio of hemoglobin by the same measurement operation as in Example 1 as in the case of albumin.

Example 5 Measurement of glycation ratio of microalbumin in urine Preparation of standard urine sample Urine was deproteinized through an ultrafiltration membrane (manufactured by Toyo Roshi Kaisha, Ltd.). The standard urine sample was prepared by adding the non-enzymatic glycated albumin and the non-glycated albumin obtained in Example 1 to the deproteinized urine.

The albumin concentration was set to 5 μg / ml, which is the albumin concentration in urine of healthy subjects. Measurement of glycation ratio of albumin in urine As a measurement sample, 400 μl of the urine sample was placed in a test tube, and
25M ammonium acetate buffer pH 9.0 [2.5 (W
/ V%) sodium chloride, 2.0 (V / V%) containing polyoxyethylene sorbitan monolaurate] 100 μl
Was added and used. The measurement operation was performed in the same manner as in Example 1 except that the antigen-antibody reaction time was changed from 5 minutes to 2 hours and the enzyme reaction time was changed from 10 minutes to 30 minutes in the operation of Example 1. The experimental results are shown in Table 4.

[0057]

[Table 4] Saccharification ratio (%) Sample 1 Sample 225% 50%  ΔA 492nm 0.298 0.529 0.021 0.043  Saccharification rate (%) − − 1.9 3.8 The standard curve shows the standard urine test with saccharification rate of 0, 25 and 50%.
I was able to create it for a fee. In addition, urine samples from healthy subjects
As a result of measuring the saccharification rates of 1 and 2
Was about 2 to 4%.

[0058]

INDUSTRIAL APPLICABILITY As described above, according to the method of the present invention, the glycation ratio of a specific protein in a sample can be measured more accurately, in a shorter time, easily and quickly than in the conventional method.

Further, the method of the present invention is a concentration at which the specific protein can bind to all of the antibodies or the active fragments of the antibody on the solid phase obtained by immobilizing a fixed amount of the antibody or the active fragment of the antibody on the carrier. By using the sample containing the specific protein as described above, the amount of the specific protein captured on the solid phase becomes constant, so the amount of the labeled substance bound to the glycation site of the specific protein by the labeled boronic acid derivative or By measuring the amount of the labeling substance that did not exist, the glycation ratio of the specific protein can be measured without separately measuring the total amount of the specific protein.

In addition, the labeled boronic acid derivative required for the measurement may be present in an amount not less than the amount of the specific protein bound to the solid phase, and an unnecessarily large amount of the labeled boronic acid derivative is required. It does not, and it is also economical.

As described above, according to the present invention,
It is possible to provide a method capable of simply and highly accurately measuring the glycation ratio of a specific protein without any practical problem.

[Brief description of drawings]

FIG. 1 shows a standard curve showing the relationship between the glycation ratio of human serum albumin and the absorbance measured by the amount of labeled substance.

FIG. 2 shows a simplified model diagram of the molecule of albumin.

FIG. 3 shows a simplified model diagram of a glycated albumin molecule.

FIG. 4 shows a simplified model diagram showing a state in which an anti-albumin antibody immobilized on a carrier captures albumin without distinguishing between glycated and non-glycated albumin.

FIG. 5 is a simplified model diagram showing a state in which the labeled boronic acid derivative is specifically bound only to the glycation site of the glycated albumin captured in FIG.

Claims (4)

(57) [Claims] 1. A sample is brought into contact with a solid phase on which an antibody or an active fragment of the antibody having a specific affinity for the specific protein is immobilized on a carrier to selectively bring the specific protein onto the solid phase. After the capture, the sample and the solid phase are separated, and then the labeled boronic acid derivative having an affinity for the glycosylation site of a protein is contacted with the separated solid phase to capture the solid phase. Of the specific protein characterized by binding a label to the glycation site of the specific protein, separating the solid phase and liquid phase of the reaction mixture, and measuring the amount of the separated solid phase or liquid phase label. Method of measuring saccharification rate. 2. The method for measuring the glycation ratio of a specific protein according to claim 1, wherein the boronic acid derivative is a boronic acid derivative containing a dihydroxyboryl group. 3. The method for measuring the glycation ratio of a specific protein according to claim 1, wherein the sample is one kind selected from urine, blood, plasma, and serum. 4. The labeled boronic acid derivative is a boronic acid derivative labeled with any of a radioisotope, an enzyme, a coenzyme, a fluorescent dye, a chemiluminescent substance, or a specific binding protein. 2. The method for measuring the glycation ratio of a specific protein according to 2.