JPH04157366A - Measuring method for saccharified specific protein and reagent therefor - Google Patents

Abstract

PURPOSE:To enable measurement excellent in accuracy and repeatability without problems such as dissociation between a solid phase antibody and saccharified specific protein with good reduction by using reduction reagent of pH 10 or higher. CONSTITUTION:After a solid phase with antibody to specific protein coupled and sample containing saccharified specific protein are reacted with each other, saccharide ingredients dissociated from the solid phase are removed at the time of or after separation between the solid phase and a liquid phase. Then a solid phase in which saccharified parts of saccharified specific protein coupled to the solid phase is reduced by using reducing agent and indicated anti-reduction saccharide protein are reacted with each other. Then after the separation between the solid phase and the liquid phase, either phase is used to measure an indicator amount, from which content of the saccharified specific protein or saccharification ratio is calculated. At this time reducing reagent of pH 10 or higher which does not lose reducing ability but is stabilized even if it is left for a long time is used as the reducing reagent. In this case coupling between the antibody coupled to the solid phase and saccharified protein is not dissociated but a saccharified part can be securely reduced.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of the Invention The present invention relates to a method for measuring specific glycated proteins, and reagents and kits used therefor.

[Prior art] In recent years, the relationship between non-enzymatically glycated proteins and diseases such as diabetes has been studied in detail, and it has been found that the glycation rate of glycated proteins in the blood, such as albumin and hemoglobin, reflects past blood sugar conditions. It turns out that albumin,
Measuring the glycation rate of blood proteins such as hemoglobin is becoming important in diagnosing diabetes.

Conventionally, various methods for measuring glycated proteins in blood have been studied, but a simple and excellent method that can be put to practical use has not yet been found.

reported that glycated proteins can be measured by using antibodies that have specific affinity for glutinol-lysine residues (Japanese Unexamined Patent Publication No. 62-254055). (Japanese Patent Application Laid-open No. Sho E4-IE9ET).

[Problems to be Solved by the Invention] The method previously reported by the present inventor (hereinafter referred to as the conventional method) is a method that is expected to be put into practical use as a simple method that can process a large number of samples in a short time. However, the stability of the reducing reagent used in the reduction reaction of glycated proteins was poor (and the reduction reaction had to be carried out immediately after the reduction reaction was performed).

That is, the reducing reagent used in the conventional method is a borohydride salt such as sodium borohydride at pH 8.2 to 8.
．． Dissolved in the buffer solution of 3.

It loses its reducing power in less than 2 minutes after preparation.

Therefore, conventional methods that use such unstable reagents require great care in measurement, and in some cases, the desired reduction reaction cannot be completed, resulting in variations in measured values. Sometimes it happened.

Accordingly, one object of the present invention is to provide a new method that improves the reduction step of the conventional method, and to provide reagents for use in the method.

[Means for Solving the Problems] The present inventor has conducted various studies in order to find a method to solve the drawbacks of the above-mentioned conventional methods. By setting the value to 10 or more, the reducing power will not disappear even if left for a long time.
∎ Although it was previously believed that antigen-antibody reactions would not proceed well unless immunoassays were performed under neutral pH conditions, Even if the reduction reaction of the glycated protein is carried out using a reducing reagent with a pH of 10 or more, the bond between the antibody bound to the solid phase and the glycated protein does not dissociate, and the glycated portion of the glycated protein can be reliably reduced, and the subsequent They discovered that the antigen-antibody reaction also proceeds smoothly, and completed the present invention.

That is, the present invention includes (a) a step of reacting a solid phase bound with an antibody against a specific protein with a sample containing a glycated specific protein; (b) a step of reacting a sample containing a glycated specific protein during or after separation of the solid phase and the liquid phase; Step (c) of reducing the glycated moiety of the specific glycated protein bound to the solid phase using a reducing reagent (d) [Recognizing anti-reduced glycated protein antibody and step (c) above]
(e) A step of reacting the solid phase obtained in the step (e) After separating the solid phase and the liquid phase, measuring the amount of label in either phase and calculating the content of the specific glycated protein or the glycation rate from that value. In the method for measuring glycated specific proteins consisting of the above (
The present invention relates to a method for measuring a specific glycated protein, characterized in that the reduction reaction in step c) is carried out using a reducing reagent having a pH of 10 or more.

The present invention also relates to a reducing reagent used in (c) of the method of the present invention, in which a boron hydride salt is dissolved and the pH thereof is adjusted to 10 or more.

Furthermore, the present invention relates to a measurement kit for carrying out the above-described method of the present invention.

The method of the present invention will be described in detail below.

In the present invention, the term "1 length" is used in the following meanings.

The term "specific protein" refers to a single type of protein whose glycated protein content or glycation rate is to be measured among various proteins present in a sample. Specifically, such specific proteins include albumin, hemoglobin, and low density riboprotein (LDL).
) etc. are exemplified.

[Glycated specific protein] means a glycosylated specific protein.

"Glycation ratio" means the abundance ratio of the specific glycated protein to the specific protein.

"Anti-reduced glycated protein antibody" means an antibody or an active fragment thereof that has specific affinity for reduced glycated protein. Specifically, it is an antibody that specifically recognizes one or more of the characteristic partial structures of reduced glycated proteins, such as glucitolouridine, glucitolouvaline, and mannitol-lysine, which are produced by reducing glycated proteins.

The solid phase used in the method of the present invention is one in which an antibody against a specific protein of interest or an active fragment thereof is bound to a carrier, and can be prepared by a conventional method. That is, as the material of the carrier, commonly used materials can be used, for example,
Polysaccharides (e.g. cellulose, dextran, starch, dextrin, hydroxyethylcellulose,
activated cyanogen halides of p-aminophenoxyhydroxypropyldextran, agarose, Sephadex, etc. (see Japanese Patent Publication No. 45-38543),
Sodium metaperiodate activated products of the above polysaccharides (see JP-A-58-758), aminoethyl or aminopropylated products of cellulose or derivatives thereof (see JP-A-59-42452), cellulose or derivatives thereof mercapto compound of
-2303 No. 1), shrimp chlorohydrin-p-aminophenol treated product of the above polysaccharide (Japanese Patent Application Laid-open No. 1983-
158994), diazotized products of polysaccharide derivatives containing aromatic amino groups (by treatment with dilute hydrochloric acid and sodium nitrite), cellulose carbonate derivatives, cellulose acetate, natural fibers such as natural fibers (cotton, hemp, wool, etc.) Molecular derivative carriers, such as ethylene, propylene, vinyl chloride, vinyl acetate, vinyl propionate, acrylic acid, acrylic esters, methacrylic acid, methacrylic esters, styrene, methylstyrene, butadiene, isoprene, acrylamide, acrylonitrile, methacrylonitrile, etc. amino groups, hydroxyl groups, carboxyl groups, sulfone groups by polymeric or copolymer carriers or by means known thereto.

Examples include those into which reactive functional groups such as thiol groups, azide groups, and isocyano groups are introduced.

The shape of the carrier is tube-shaped, test plate-shaped,
Examples include beads, disks, spheres, sticks, and latex.

The antibody bound to the carrier is an antibody that specifically reacts with the specific protein to be measured, and the reactivity of the antibody does not vary depending on whether the specific protein is glycosylated or not. The antibody may be either a monoclonal antibody or a polyclonal antibody, and may be used as an antibody molecule itself or as an active fragment thereof (e.g., F(ab')a, Fab', Fab, etc.). Good too.

The antibody can be prepared by conventional methods4.

For example, a purified product of a specific protein whose content or percentage of glycated protein is to be measured is emulsified using physiological saline and complete Freud's adjuvant, and this is emulsified in warm-blooded animals (e.g., rabbits, sheep, goats, rats, etc.). mice, guinea pigs, cows, chickens, pigeons, ducks, etc.) are inoculated several times every 1 to 4 weeks.
Antiserum containing the polyclonal antibody of interest can be obtained by collecting blood for up to 14 days and centrifuging it. When purification of a polyclonal antibody is required, it can be purified using conventional antibody purification methods such as salting out method and affinity chromatography method.

Furthermore, monoclonal antibodies can be prepared by known methods such as cell fusion methods. For example, a warm-blooded animal (e.g., rat,
Antibody-producing cells (e.g., tile cells, lymph node cells, peripheral blood lymphocytes, etc.) obtained by intramuscular immunization (guinea pigs, mice, etc.) and available myeloma cells (eg.
P3X83Ag8 (ATCCTIB-9), P3x6
3Ag 8tJ, I (P3Ul) (ATCCCR
L-1597), P3/NSI/1-Ag4-1 (A
TCC'MB-18), 210. RCY, Ag1.2.
3 (ATCC CRL-1831), etc.) in the presence of a cell fusion promoter (eg, polyethylene glycol, Sendai virus, etc.) and under conditions of applying an electric pulse to 1. The obtained hybridomas and unfused cells are selected using a selective medium (for example, hypoxanthine-aminopriten-thymidine (HAT) medium, etc.), and then the target antibody is detected by a known screening method (for example, enzyme immunoassay). Select hybridomas that produce The selected hybridomas are cloned by conventional monocloning methods (for example, limiting dilution method, soft agar method, cell sorter method, etc.),
Monoclonal antibodies can be obtained by growth in conventional liquid culture or intraperitoneally in phlebotomy animals.

When it is necessary to purify the antibody, it can be purified by known methods such as salting-out methods and various chromatography methods.

All of the above-mentioned antibody preparations are established and conventional methods, and for details of the preparation methods, you can refer to books, reviews, reports, etc. (for example, Tatsuo Iwasaki et al., "
Monoclonal antibodies - hybridomas and BLISA-J
(Published by Kodansha Co., Ltd., February 20, 1982), Eu
r, J, Immunol, , fi, pp511-5
19 (1976), Methods in ENZYM
OLOGY Vol, 121 (ls+munoches
ical Techniques Part I(H
hybridomaTechnology and Mo
noclonal^ntibodies) (published by Academic Press), Nature4m, pp269~
270 (+978), Clinical Pathology, Vol. 34, No. 2, No. 1
21-124 (1988), J, ^pp1. Bio
chess, 4.41 (+982), etc.).

The antibody can be bound to the carrier by adsorption method, covalent bonding method, crosslinking method,
Any of the comprehensive laws may be applied.

For details of such immobilization methods, methods for immobilized enzymes can be applied, for example, "Immobilized Enzymes" edited by Chibata, Vol. 9 (March 20, 1975, published by Kodansha Co., Ltd.) You may refer to the collection or reviews on pages 75 to 75.

The types of samples used in the method of the present invention may be those of biological origin that may contain specific proteins, such as urine, blood, serum, cerebrospinal fluid, various organ extracts, etc. can be mentioned.

When measuring the content of glycated specific protein in the sample, all of the fixed amount of immobilized antibody obtained by binding to the carrier should be combined with specific protein content (glycated specific protein and non-glycated specific protein). When measuring the saccharification rate of a specific protein, a fixed amount of solid phase obtained by immobilizing it on a carrier is used. It is important to use antibodies that can contain specific proteins at a concentration higher than that at which the specific proteins (glycosylated specific proteins and non-glycosylated specific proteins) can bind. When measuring the glycation rate of a specific protein by using such a sample, the amount of the specific protein bound to the immobilized antibody is always constant, and the operation of measuring the total amount of the specific protein can be omitted.

The reaction between the sample and the immobilized antibody can be carried out in accordance with a normal antigen-antibody reaction, and the reaction temperature can be selected as appropriate from the range of 0 to 50°C and the reaction time from 1 minute to 5 hours. can.

After completion of the reaction, separation of the solid phase and liquid phase can be carried out by appropriately selecting conventional separation methods such as washing, centrifugation, decanting, suction, and filtration, which are frequently used in immunoassays. .

Free sugar components present on the surface phase are removed during or after separation of the surface phase and liquid phase. The removal method is not particularly limited as long as the sugar component can be removed from the solid phase, and examples thereof include washing treatment. The washing treatment may be performed concurrently with the liquid phase separation treatment, or may be performed separately. As the washing solution used for washing, water or a buffer solution can be used, and examples of such buffer solutions include citrate buffer solution, Tris-HCl buffer solution, phosphate buffer solution, phosphate buffered saline solution, etc. Take it by doing it. The degree of washing may be such that coexisting substances that affect the reduction reaction, such as free sugar components, are substantially not present on the solid phase, and this objective can usually be achieved by washing 1 to 3 times. can.

After removing free sugar components on the solid phase, the glycated portion of the specific glycated protein bound to the immobilized antibody is converted into a reduced form using a reducing reagent.

The reducing reagent used in the reduction reaction is a solution of a borohydride salt such as sodium borohydride, lithium borohydride, calcium borohydride, potassium borohydride, zinc borohydride, etc. Examples include those whose liquid properties are adjusted to pHlO or higher. Such a reducing reagent dissolves the borohydride salt in an aqueous medium (e.g., various buffers such as sodium carbonate buffer, alkaline fa solution containing sodium hydroxide, etc.) with a pH of 10 or higher, preferably pH 10 to 14. It can be prepared by Even if the above-mentioned borohydride salt is dissolved in an aqueous medium having a pH below the pH lo, the solution is unstable, and even if this solution is used in a reduction reaction, the above-mentioned problems occur. This will be explained using a test example.

Test Example 1 Method: ■Anti-human serum albumin (HS A) deglycosylated using a boronic acid affinity column (manufactured by Isolab)
Antibody solution (100℃g/1 phosphate buffered saline (P
BS)) were brought into contact with beads (manufactured by Immunochemical) to immobilize the anti-H3A antibody on the beads. Next, a 1% solution of bovine serum albumin (BSA) that had been deglycosylated using a boronic acid affinity column was used to block the portions on the beads to which the antibodies were not bound.

■ Anti-H3A antibody-coupled beads with 1ooμg/ml glycated human serum albumin and 50mM Que' containing 1% BSA;
r [Add 250 μl of 9 buffer (pH 5,0) and
The reaction was carried out at ℃ for 1 hour.

■After the reaction, wash the beads three times with PBS and add 0.1M potassium phosphate buffer containing 1% butanol (pH 8.0).
225 μl and 25 μl of a 10 mM potassium borohydride solution shown in Table 1 below were added and reacted at 25° C. for 10 minutes.

■ Wash the beads three times with PBS, add 8 μg/■ I horseradish bar oxidase-labeled anti-reduced glycated protein antibody and 250 μm of 0.1 M sodium phosphate buffer (pHe, 0) containing 1% BSA at 25°C. The mixture was reacted for 1 hour.

■After washing the beads four times with PBS, add 500 μl of 0.1M citric acid buffer (pH 5,0) containing 0-phenylenediamine (8■/■I) and hydrogen peroxide (0.02%) and incubate at 25°C. I&, 2N-1i reacted for 20 minutes! acid 1
All was added to stop the reaction, and the absorbance at 4θ2 nm was measured.

Results: The results are shown in Figure 1. As is clear from Figure 1,
When reducing reagents dissolved in pH 8 and 9 media were used, the absorbance rapidly decreased. Therefore, it was clearly shown that a reducing reagent in which a boron hydride salt was dissolved in an unvortexed medium at pH 1o was unsuitable for the reduction of the Glycated Protein Award.

Test Example 2 Method: A test was conducted in the same manner as in Test Example 1 using the 10 mM potassium borohydride solution shown in Table 2 below.

Table 2 Results: The results are shown in Figure 2. As is clear from Figure 2, the pH
It was confirmed that a reducing reagent dissolved in a medium with a pH of 10.0 or higher, particularly a pH of 10.5 or higher, is stable and can perform the reduction reaction reliably.

In the reduction reaction, the final degree of borohydride salt in the reaction system is 0.
It can be carried out by adding the above-mentioned reducing reagent to a concentration of 1mM or more, preferably 1 to 100mM, and then reacting at a reaction temperature of 0 to 50°C, preferably 20 to 30'C, for 1 minute to 10 hours. can.

In addition, the pH conditions of the reaction system during the reduction reaction are based on the above-mentioned specific pH conditions.
There is no particular limitation as long as a reducing reagent exhibiting 1 is used, but it is more preferable to maintain the reaction system under conditions exceeding pH 10. There are no particular limitations on the method for maintaining the entire reaction system under pH conditions exceeding pH 10. Examples include methods in which an alkali is added to the reaction system at the start of the reduction reaction, and a buffer solution with a pH exceeding 10 is used as a reaction solvent.

After the above reduction reaction, a labeled anti-reduced glycated protein antibody is added to the reaction solution and reacted.

Anti-reduced glycated protein antibodies can be prepared by appropriately applying known methods that have already been reported (for example, see the method of Daiku et al. mentioned above).

For example, using a reduced glycated protein prepared by reacting protein with glucose or by reducing it with sodium cyanoborohydride during the reaction, as an antigen,
Thereafter, an antibody that specifically reacts with the reduced glycated protein can be prepared according to the known preparation method for polyclonal antibodies or monoclonal antibodies described above.

As a labeling substance for labeling the anti-reduced glycated protein antibody, those frequently used in immunoassay methods can be used. Specifically, 1251, +311
．． 3H1” Crt 0) Radioactive isotope, β-galactosidase, peroxidase, alkaline phosphatase, glucose oxidase, glucose-6
- Enzymes such as phosphate dehydrogenase, fluorescein derivatives (e.g. fluorescein isothionate, fluorescein thiofulbamyl, etc.), rhodamine derivatives (e.g. tetramethylrhodamine B isothiocyanate, etc.), umbelliferole and 1-anilino-8-naphthalene sulfone Fluorescent dyes such as acids, luminol derivatives (e.g. luminol, isoluminol, N
-(6-aminohexyl)-N-ethylisoluminol, etc.), coenzymes such as bition, heme, flavin adenine dinucleotide, and prosthetic groups.

Binding of a labeling substance and an antibody may be performed according to a conventional method. For example, when labeling with a radioactive isotope, the chloramine T method, lactoperoxidase method, glucose okindase method, binding method (Bolton-Hunter method), etc. can be used. When labeling enzymes, glutaraldehyde method, periodic acid method, maleimide method, pyridyl disulfide method, etc. can be used.

The reaction conditions for the labeled anti-reduced glycated protein antibody prepared in this way and the reduced glycated specific protein bound to the immobilized antibody include, for example, a reaction temperature of 0 to 0.
The reaction time at 50°C can be appropriately selected from the range of 1 minute to 5 hours.

After the above reaction, the solid phase and liquid phase are separated by a conventional method, and the amount of label in either phase is measured according to a method commonly used for labeled substances.

A standard curve is created by plotting the relationship between the measured value obtained by measuring a standard solution prepared to have a known glycated specific protein content or glycation ratio by the method of the present invention and the glycated protein content or glycation ratio. do. Based on the standard curve, the glycated specific protein content or the glycated percentage of the specific protein award is calculated with respect to the measured value.

The present invention also includes a kit for carrying out the method of the present invention detailed above. Such a kit includes at least the following reagents.

Reagent 1: A solid phase reagent bound to an antibody that specifically reacts with a specific protein.

Reagent 2: Reducing reagent with a pH of 0 or higher.

Reagent 3゛ Labeled anti-reduced glycated protein antibody reagent.

Note that the reagent 2 consists of a borohydride salt and a solution having a pH of 10 or more, and may be attached to the kit in the form of a ready-to-dissolve type that is prepared at the time of measurement. Furthermore, other reagents may be added to the kit as necessary.

Example 1 Anti-H8A antibody-bound beads were produced in the same manner as in Test Example 1.

lOOμg/mlHS with saccharification rate adjusted to 0-40%
A and 50 mM citrate buffer containing 1% BSA (p
H5,0) 250 μm was added to the above anti-H3A antibody-bound beads and reacted at 25° C. for 1 hour.

After the reaction, wash the beads three times with PBS and add 225μ of 0.1M Tris-HCl buffer (pH 8°5) containing 1% butanol.
1 was added thereto, and 25 μl of a 100 mM potassium borohydride solution shown in Table 3 below was added, followed by a reduction reaction at 25° C. for 20 minutes.

Table 3 After the reduction reaction, wash the beads 3 times with PBS and
Add 1 horseradish bar oxidase IJ-recognizing reduced glycated protein antibody and 250 μm of 0.1 M sodium phosphate buffer (pH 8.0) containing 1% BSA, and
The reaction was carried out at 5°C for 1 hour.

Next, after washing the beads 4 times with PBS, 500 μl of 0.1 M citric acid buffer (pH 5,0) containing 0-phenylenediamine (8 μ/1) and hydrogen peroxide (0°02%) was added for 25 minutes. ℃ for 12 minutes, 2N-sulfuric acid solution nl
ml was added to stop the reaction.

The absorbance of this solution at 429 nm was measured.

The results are shown in FIG. As shown in Figure 3, a reducing reagent dissolved in a medium with a pH of 10.74 can produce a good calibration curve even after several hours of 6 hours, as well as immediately after a+W, whereas with a reducing reagent dissolved in a medium with a pH of 8° The reducing power of the solution dissolved in medium No. 5 disappeared after being left for 6 hours, making it impossible to create a calibration curve. In addition, when serum from diabetic patients and serum from healthy individuals were measured in the same manner as above using the reducing reagent left for 6 hours, the pH was 10,
It was possible to measure the saccharification rate only by the method of the present invention using a reducing reagent dissolved in a medium of 74%.

Example 2 Anti-HSA antibody-bound beads were produced in the same manner as in Test Example 1.

100 μg/ml HS adjusted to a saccharification rate of 0 to 40%
A and 50 mM citrate buffer containing 1% BSA (p
250 μl of H5,0) was added to the anti-H3A antibody-conjugated beads and reacted at 25° C. for 1 hour.

After the reaction, wash the beads three times with PBS and dissolve potassium borohydride in 0.2M Tris-HCl buffer (p88.5 B) or 0.2M sodium carbonate buffer (pH 10,33-11.04). 25 immediately after preparation
250 μl of mM potassium solution (reducing reagent)
was added, and a reduction reaction was carried out at 25°C for 1 hour.

After the reduction reaction, wash the beads 3 times with PBS and add 8μg/-
1 Add horseradish bar okindase drift anti-reduced glycated protein antibody and 250 μl of 0.1 M sodium phosphate solution (pH 8,0) containing 1% BSA,
The reaction was carried out at 5°C for 1 hour.

Once again, the beads were washed four times with PBS (wheat, 0-phenylenediamine (8/1) and hydrogen peroxide (0°02).
%) containing 0.1M citrate buffer (pH 5,0) 500
μl was added and reacted at 25° C. for 12 minutes, and 1 portion of 2N sulfuric acid solution was added to stop the reaction.

The absorbance of this solution at 429 nm was measured.

The results are shown in FIG. As shown in FIG. 4, a good standard curve was obtained even under conditions where the pH conditions during the reduction exceeded 1O, confirming that the method was practically applicable.

[Effect] The method of the present invention is characterized by using a reducing reagent with a pH of 10 or higher when reducing the glycated portion of the glycated specific protein award bound to the immobilized antibody using a reducing reagent. Even if an alkaline reducing reagent such as The ratio can be measured with high precision and reproducibility.

Furthermore, by reducing the glycated specific protein bound to the immobilized antibody using the above-mentioned reducing reagent with the specific pH and under conditions exceeding the pH condition during the reduction reaction of 10, it is possible to further reliably reduce the glycated specific protein. It is possible to reduce the glycated portion of the prize, thereby further increasing the accuracy and reproducibility of measurements.

The reducing reagents and kits of the invention are also useful in practicing the methods of the invention.

[Brief explanation of the drawing]

FIGS. 1 and 2 show the results obtained when reducing reagents at various pH values were allowed to stand for a predetermined period of time and then subjected to the method of the present invention. FIG. 3 is a calibration curve prepared using reducing reagents at pH 8.5 and pH 1o, 74 after being left for 0 or 6 hours. FIG. 4 is a calibration curve prepared using reducing reagents at various pH values. Patent Applicant (877) Yamasa Soy Sauce Co., Ltd. No. (1￥J Mouth -... rH>II △-・II″It. ◇-yHzoO-・-fHg 12 Figure ○-・ fH//, 2g △・・・ pHlo, deg ◇ −・・ rl-11F, Pu Otsu-3 Figure 〇 −・ ・ tyH&, ta, direction It'41j7 o
rA)・-11-1'if, t; , radial 11f&76
about △---PH(0,7'l, Rfn&7
0 (k) A --- pH/ρJ9.7jlftf
f/g71 chno*words produced by a fat person

Claims (1)

[Claims] 1) (a) A step of reacting a solid phase bound with an antibody against a specific protein with a sample containing a glycated specific protein (b) A step of reacting the solid phase during or after separation of the solid phase and the liquid phase (c) a step of reducing the glycated moiety of the specific glycated protein bound to the solid phase using a reducing reagent (d) a labeled anti-reduced glycated protein antibody and the above (c)
Step (e) of reacting with the solid phase obtained in the step (e) After separating the solid phase and liquid phase, measuring the amount of label in either phase, and calculating the content of the glycated specific protein or the glycation rate from that value In the method for measuring glycated specific proteins consisting of the above (
c) A method for measuring a specific glycated protein, characterized in that the reduction reaction in the step is carried out using a reducing reagent having a pH of 10 or more. 2) The method for measuring a specific glycated protein according to claim 1, wherein the reducing reagent is one in which a boron hydride salt is dissolved. 3) The method for measuring a specific glycated protein according to claim 1, wherein the reducing reagent exhibits a pH within the range of 10 to 14. 4) The method for measuring a glycated specific protein according to claim 1, wherein the reduction reaction in step (c) is carried out using a reducing reagent with a pH of 10 or higher and under conditions in which the pH exceeds 10. 5) The method for measuring glycated specific protein according to claim 1 (c)
) to dissolve the borohydride salt and p
A reducing reagent with H adjusted to 10 or more. 6) The reducing reagent according to claim 5, which has a pH of 10 to 14. 7) Claim 1 comprising the following reagents as constituent reagents:
Kit for carrying out the described measurement method. Reagent 1: A solid phase reagent bound to an antibody that specifically reacts with a specific protein. Reagent 2: Reducing reagent with a pH of 10 or more. Reagent 3: Labeled anti-reduced glycated protein antibody reagent. 8) The reducing reagent (reagent 2) is a borohydride salt and pH 10.
The kit according to claim 7, which comprises the above-mentioned dissolution solution and is in the form of a ready-to-dissolve type prepared at the time of measurement.