JP2001004623A - Immunoassay for age-hemoglobin - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a measurement method for AGE(advanced glycation end product)-hemoglobin allowing rapid and easy measurement of AGE-hemoglobin. SOLUTION: This immunological measurement method for AGE-hemoglobin is as follows: AGE-hemoglobin derived from a specimen is sensitized to unsensitized latex, the obtained AGE-hemoglobin-sensitized latex is reacted with an AGE-hemoglobin antibody, then the turbidity of generated aggregates is measured. Otherwise, first latex sensitized with the AGE-hemoglobin derived from the specimen is reacted with second latex sensitized with the AGE- hemoglobin antibody, then the turbidity of generated aggregates is measured.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for immunologically measuring an advanced glycated product of hemoglobin, that is, an AGE-modified hemoglobin by utilizing an agglutination reaction.

[0002]

2. Description of the Related Art In recent years, AGEs (end glycation end products) have become increasingly serious in diabetic complications (Brownlee, M., et al.
al, 1988, N.M. Engl. J. Med. , 31
8: 1315-1321) and aging (Araki, N., et al.).
et al, 1992, J. Am. Biol. Chem. , 2
67: 10211-10214), chronic kidney disease (Miy)
ata, T .; , Et al, 1996, J. Am. Am. So
c. Nephrol. , 7: 1198-1206), Alzheimer's disease (Smith, MA, et al,
1994, Proc. Natl. Acad. Sci. U
SA, 91: 5710). In particular, it has been found that AGE-containing hemoglobin containing carboxymethylated hemoglobin (hereinafter sometimes referred to as “CM-containing hemoglobin”) is associated with the development of diabetic complications (Japanese Patent Laid-Open No. 10-1998).
185917, Makita, Z .; , Et a
1, 1992, Science, 258: 651-65.
From 3), it is a useful index for diagnosis, treatment and prevention of diabetic complications or evaluation of its efficacy.

Conventionally, the measurement of AGE-containing hemoglobin including CM-containing hemoglobin in blood is mainly performed by ELISA (enzyme-labeled immunoassay) (Japanese Patent Application Laid-Open No. 10-504640) and dot blotting. . However, in the ELISA method, for example,
As described in JP-504640A, it is necessary to perform a pretreatment of the specimen with a surfactant, a protein denaturant, or the like,
There is a problem that the operation is complicated and takes time. Also, E
Both the LISA method and the dot blotting method require complicated and time-consuming operations, so it has been difficult to quickly measure a large amount of a sample.

[0004]

SUMMARY OF THE INVENTION An object of the present invention is to provide a method for measuring AGE hemoglobin, which can quickly and easily measure AGE hemoglobin. Other objects and advantages of the present invention will become apparent from the following description.

[0005]

Means for Solving the Problems The inventors of the present invention have found that if AGE-modified hemoglobin derived from a subject is immobilized on unsensitized latex, it can be used in a subject without pretreatment with a surfactant or a protein denaturant. Can be measured. In addition, AGE-modified hemoglobin derived from a test substance is immobilized on unsensitized latex, followed by reaction with an antibody that specifically recognizes AGE-modified hemoglobin, and the turbidity is measured. The inventors have found that AGE-modified hemoglobin in a sample can be measured, and have completed the present invention.

That is, according to the present invention, the above-mentioned objects and advantages of the present invention are as follows. First, (1) sensitizing unsensitized latex to AGE-modified hemoglobin derived from a subject;
(2) reacting the obtained AGE-modified hemoglobin-sensitized latex with an antibody that specifically recognizes the AGE-modified hemoglobin, and then (3) measuring the turbidity of the formed aggregate. This is achieved by an immunoassay for hemoglobin.

Further, according to the present invention, the above-mentioned object and advantages of the present invention are as follows: (1) a first latex sensitized with an AGE-modified hemoglobin derived from a subject;
Immunoreactivity of AGE-activated hemoglobin, which is reacted with a second latex sensitized with an antibody that specifically recognizes activated hemoglobin, and then (2) measuring the turbidity of the formed aggregate. Achieved by law.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in more detail. Latex used in the present invention is a latex used as a normal immunoassay carrier, for example, a synthetic resin such as styrene, propylene, acrylamide, and acrylonitrile, or a reactive functional group introduced therein by a method known per se. It is not particularly limited as long as it is a thing. The latex surface preferably has at least one functional group selected from the group consisting of a hydroxyl group, a carboxyl group, and an epoxy group. The amount of the functional group introduced to the latex surface is not particularly limited, but may be 10 ^-5 mol / g of latex.
It is preferable that 1 or more have been introduced. If the amount is less than this, the effect of introducing a functional group is hardly seen. It is noteworthy that the latex into which these functional groups are introduced has a property that non-specific aggregation is unlikely to occur in the reaction between the latex sensitized with AGE-modified hemoglobin and the antibody that specifically recognizes AGE-modified hemoglobin. .

Although the shape of the latex is not particularly limited,
Spherical ones are preferably used because the latex can be easily produced. The average particle diameter of the spherical latex that sensitizes AGE-modified hemoglobin is 0.05 to 0.05 from the viewpoints of easy aggregation after the antigen-antibody reaction, measurement of the degree of aggregation (turbidity), and easy determination of the degree of aggregation. It is preferably 0.5 μm.

In the present invention, AGE-linked hemoglobin is associated with at least one of the three characteristic phenomena (fluorescence, browning, and intra / intermolecular cross-linking) observed in the late stage reaction of the Maillard reaction. Refers to hemoglobin to which a reaction product has been added, and / or CM-modified hemoglobin. As a reaction product accompanied by at least one of the three characteristic phenomena, for example, pentosidinated hemoglobin,
Imidazolone hemoglobin, cross-phosphorylated hemoglobin and the like. Examples of CM-modified hemoglobin include hemoglobin in which the N-terminal α-amino group is carboxymethylated, and hemoglobin in which the ε-amino group of the lysine side chain is carboxymethylated. Of these, N
Hemoglobin in which the terminal α-amino group is carboxymethylated is a marker for diabetic complications since the blood concentration in patients with diabetic complications is significantly higher than that in healthy subjects. It is preferably used as

The sensitization of unaged latex with AGE-modified hemoglobin derived from a subject is generally carried out at 4 to 50 ° C. by a physical adsorption method. Usually, for latex, AG
A sample carrying about 0.001 to 10% by weight of a subject-derived hemoglobin that may contain E-modified hemoglobin is used as AGE-modified hemoglobin-sensitized latex.
And subjected to a reaction with an antibody that specifically recognizes modified hemoglobin. The sensitization of the AGE-modified hemoglobin to the unsensitized latex can be usually carried out in a buffer used for the antigen-antibody reaction, and the salt concentration of the buffer is preferably about 10 to 50 mM. If the salt concentration is higher than this, aggregation of latex or aggregation of latexes via hemoglobin during sensitization tends to occur. If the salt concentration is lower than this, the buffer capacity is weak, which is not preferable. The pH at the time of sensitization of AGE-modified hemoglobin to unsensitized latex is preferably 7.5 to 10.0. When the pH is lower than this, aggregation of the latex via hemoglobin is liable to occur, and when the pH is higher than this, the denaturation of hemoglobin tends to be remarkable.

The latex sensitized with the AGE-modified hemoglobin is subjected to a reaction with an antibody that specifically recognizes the AGE-modified hemoglobin. The antibody that specifically recognizes the AGE-linked hemoglobin (hereinafter sometimes referred to as “AGE-linked hemoglobin antibody”) may be a polyclonal antibody or a monoclonal antibody. Is preferred. Furthermore, it is preferable that the monoclonal antibody be derived from a mouse in terms of ease of preparation. Known methods can be applied to the preparation of the monoclonal antibody.

The AGE-linked hemoglobin antibody may be used alone, or may be cross-linked at the Fc portion to increase the number of antibody active groups to more than two. The AGE-linked hemoglobin antibody may be cross-linked with an antibody that specifically recognizes the Fc portion of the AGE-linked hemoglobin antibody. Alternatively, an AGE-conjugated hemoglobin antibody sensitized to latex may be used for the following antigen-antibody reaction. For sensitization of the AGE-conjugated hemoglobin antibody to latex, a method known per se is used without any limitation.

The reaction conditions of the AGE-conjugated hemoglobin antibody and the AGE-conjugated hemoglobin-sensitized latex may be any reaction conditions used for ordinary antigen-antibody reactions without any limitation.
The resulting aggregate of the latex may be detected according to a known method. For example, the absorbance is measured by an automatic analyzer for latex turbidimetric determination, and the AGE-modified hemoglobin in the subject can be detected from the turbidity or a change in the absorbance over a certain period of time. Measurement wavelength is 400-80
It is preferred to use 0 nm. Further, by mixing the AGE-conjugated hemoglobin antibody and the AGE-coupled hemoglobin-sensitized latex on a black slide glass and observing the presence or absence of the aggregated latex, the AGE-linked hemoglobin in the subject can be detected.

[0015]

According to the method for measuring AGE-modified hemoglobin of the present invention, the concentration of AGE-modified hemoglobin in a subject can be simply and rapidly measured. In particular, when an automatic analyzer for latex turbidimetric determination is used, AGE hemoglobin in a large amount of a sample can be measured at a time, and the operation is simple.

[0016]

EXAMPLES Hereinafter, the present invention will be described in detail with reference to examples, but the present invention is not limited to these examples.

Example 1 (1) [Preparation of antigen] CM-Hb was produced and purified by the following methods. Hb (SIGMA) was added to a borate buffer (0.1 M,
pH 9.0), adjusted to 1 mg / ml, stirred 200 ml of this solution at room temperature for 2 hours with 180 ml of a 1M glyoxylic acid solution, and further stirred at room temperature for 2 hours.
mg / ml sodium cyanoborohydride solution 20ml
And CM-Hb was produced. Further, after dialysis using a dialysis membrane for 20 hours in a phosphate buffer (3.3 mM, pH 7.4), ultrafiltration was performed to obtain purified CM-Hb.

(2) [Immunization of mice] CM-Hb purified by the above method was placed in a phosphate buffered saline (hereinafter sometimes abbreviated as “PBS”) for 0.2 m.
After dilution to a g / ml, the mixture was mixed with an equal amount of Freund's complete adjuvant, and the first immunization was performed by intraperitoneal administration of 0.2 ml per BALB / c mouse (female, 5 weeks old). Thereafter, three times at two-week intervals, CM-Hb P
A solution obtained by mixing the BS solution with an equal amount of incomplete Freund's adjuvant was intraperitoneally administered to each mouse at 0.2 ml per mouse to perform booster immunization. Two weeks after the last boost, CM-Hb
The final immunization was performed by intravenously administering 0.1 ml of a PBS solution of the above.

(3) [Cell fusion] Three days after the final immunization was performed by the above method, the spleen was excised from the immunized mouse, and the spleen cells were replaced with 10% fetal bovine serum (FC).
The suspension was suspended in RPMI-1640 medium containing S). on the other hand,
Mouse myeloma cells P3U1 were transformed with R containing 10% FCS.
The cells were cultured in PMI-1640 medium, and the cells were collected during the logarithmic growth phase and used for cell fusion. The mouse spleen cells and P3U1 were each washed three times with RPMI-1640 medium without serum, then mixed at a ratio of 5: 1 and mixed at 1,500 rpm for 5 minutes.
The medium was removed by centrifugation for minutes. 0.5 ml of 50% polyethylene glycol 1500 is gradually added to the cell pellet,
Centrifugation was performed at 1,800 rpm for 8 minutes. Next, R without serum
After gradually adding 20 ml of PMI-1640 medium,
The supernatant was removed by centrifugation at 500 rpm for 5 minutes. The precipitated cells were placed in a HAT medium (1 × 10 ⁻⁴ M hypoxanthine, 4
× 10 ^-7 M aminopterin, 1.5 × 10 ^-5 M thymidine, RPMI-1640 medium containing 20% FCS) 50
The resulting suspension was dispensed in 0.1 ml portions to each well of a 96-well microplate. The fused cells are treated with 5% CO ₂ ,
The cells were cultured at 37 ° C. One day after cell fusion, 0.1 was added to each well.
HAT medium was added in ml. After 7 to 10 days, colonies of hybridomas that grew were observed. The total number of wells in which hybridomas had grown was 235 (53 wells).
%)Met.

(4) [Screening] The supernatant of the well in which the hybridoma has sufficiently grown is collected,
By performing the ELISA method as follows, CM
Hybridomas producing antibodies against -Hb were selected. CM-Hb obtained by the above method is
diluted to a concentration of μg / ml, and dispensed 100 μl per well into a 96-well EIA microplate.
Incubated overnight at 0 ° C. Remove the CM-Hb solution from the microplate and add 0.05% Tween80.
Containing PBS (hereinafter sometimes abbreviated as "T-PBS")
After washing three times with PBS, PBS containing 1% bovine serum albumin in each well (hereinafter sometimes abbreviated as “B-PBS”)
Was stored at 4 ° C. and used as an antigen-adsorbing plate for the subsequent operations. The antigen-adsorbing plate was washed three times with T-PBS, and 100 μl of the hybridoma medium supernatant was added to each well, followed by incubation at 37 ° C. for 1 hour. Thereafter, the medium supernatant was removed, and the cells were washed three times with T-PBS. Then, 100 μl of the secondary antibody solution was added to each well, and the mixture was incubated at 37 ° C. for 1 hour. As a secondary antibody, peroxidase-labeled anti-mouse immunoglobulin antibody (Kappel) was diluted 500-fold with B-PBS and used. After removing the secondary antibody solution and washing with T-PBS three times, 100 μl of a chromogenic substrate solution was added to each well, and the mixture was incubated at room temperature for 30 minutes. The chromogenic substrate solution was 0.01% hydrogen peroxide, 0.3 mg / ml ABTS,
[2,2'-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium]
Citrate buffer (pH 5.0) was used. After confirming an appropriate amount of color development, 1% SDS was added to each well to stop the reaction.
The absorbance at a wavelength of 410 nm was measured.

(5) [Cloning of hybridoma] A hybridoma that strongly reacts with CM-Hb was selected by screening according to the above method, and cloned by limiting dilution. The hybridoma was diluted with RPMI-1640 medium containing 20% FCS to a concentration of 0.5 cells / 0.1 ml, and 0.1 ml was dispensed into each well of a 96-well microplate. 5% C
The cells were cultured at 37 ° C. in O ₂ . ELISA was performed on the culture supernatant of the well in which the hybridoma had grown in a single colony in the same manner as described above, and a hybridoma producing an antibody against CM-Hb was selected. Among them, 2B3 was obtained as a hybridoma stably producing a monoclonal antibody that reacts most strongly with CM-Hb. The obtained hybridoma 2B3 was deposited as "Hybridoma CM-Hb 2B3" with the National Institute of Bioscience and Human-Technology, National Institute of Advanced Industrial Science and Technology (Deposit No .: Life Science Bacteria No. 16).
No. 632 (FERM P-16632)).

(6) [Immunoglobulin Class of Monoclonal Antibody] The immunoglobulin class of the antibody in the hybridoma culture supernatant was examined using a monoclonal antibody typing kit by ELISA (American Corex). This kit uses a rabbit IgG antibody specific to each class and subclass of mouse immunoglobulin to examine the immunoglobulin class by a method according to the above-described ELISA method. The immunoglobulin class of the monoclonal antibody produced by hybridoma 2B3 is IgG
Met.

(7) [Preparation of Monoclonal Antibody] Hybridoma 2B3 strain was transformed with RPM containing 10% FCS.
The cells were cultured in the 1-1640 medium. An equivalent amount of saturated ammonium sulfate was added to the culture supernatant of the hybridoma, and the precipitate was collected by centrifugation. This precipitate was dissolved in a small amount of 10 mM Tris-HCl buffer (pH 8.5) and dialyzed against the same buffer. After dialysis, centrifuged to remove insolubles,
Loaded on an E-cellulose column. After washing with a buffer solution, elution was performed with a salt concentration gradient, and an IgG fraction was collected. This fraction was subjected to a gel filtration HPLC column (Bio-Rad, Bio-
(Sil TSK250) and chromatography to obtain a purified monoclonal antibody.

(8) [Measurement by latex agglutination method] Whole blood collected from a human is allowed to stand at room temperature for 3 hours to precipitate erythrocytes, and 10 μl of the erythrocytes is lysed by adding 15 ml of purified water to cause hemolysis. did. Latex particles having a particle diameter of 0.129 μm (Fujikura Kasei: Cat. No. 1232) having an epoxy group introduced into the particle surface are diluted with a 20 mM glycine buffer (pH 8.2) to a 0.1% concentration. 8 μl of the above-mentioned specimen is added to 300 μl, and 37
C. for about 5 minutes and left hemoglobin and / or
Alternatively, CM-modified hemoglobin was adsorbed on the latex.

Next, the CM-modified hemoglobin antibody prepared above was added to the latex solution in a 50 mM glycine buffer (pH 8.2) containing 300 mM sodium chloride.
The antibody solution dissolved to 5 μg / ml was added to 100 μl
l Addition and stirring, wavelength 660 from 30 seconds to 200 seconds after
The optical density change in nm was measured. The above operations were performed using an automatic analyzer TBA-30R (Toshiba Medical)
Was used.

The same operation as described above was performed using an automatic analyzer TBA-30R as a standard using a test sample whose CM-hemoglobin value was known, to obtain a standard curve. This standard curve is shown in FIG. From this standard curve, the concentration of CM hemoglobin in the subject whose CM hemoglobin concentration was unknown was determined. As a target method of the latex method, the amount of carboxymethylated valine per 1 mg of hemoglobin was measured by amino acid analysis, and the correlation with the latex method was determined. The result is shown in FIG. As shown in FIG. 2, a good correlation was found between the amino acid analysis method and the latex agglutination method.

Example 2 In place of the CM hemoglobin antibody solution in Example 1, a suspension containing the same latex sensitized with the CM hemoglobin antibody as in Example 1 was used in the same amount as the antibody solution of Example 1. Except for the fact that in the same manner as in Example 1, the same concentration of CM hemoglobin in the subject as that used in Example 1 was used as a standard using a subject having a known CM hemoglobin value as a standard. It was determined from the curve. The value was compared with the CM hemoglobin value of the subject obtained in Example 1 to obtain a correlation. The result is shown in FIG. As shown in FIG. 3, a good correlation was observed between the case where the antibody was not sensitized to the latex and the case where the antibody-sensitized latex was used.

The latex sensitized with the CM-conjugated hemoglobin antibody was prepared as follows. That is, the particle diameter is 0.12.
Polystyrene particles (Latex N-100 for diagnostics, Sekisui Chemical Co., Ltd.) were suspended in 20 mM Tris-HCl buffer at pH 8.0 so that the latex concentration was 1 μm. 1 ml of this particle suspension and CM diluted to 0.1 mg / ml with 20 mM Tris-HCl buffer at pH 8.0
And 1 ml of the hemoglobin antibody. Then, 0.3% bovine serum albumin (SI
2 ml of 20 mM Tris-HCl buffer containing GMA, fraction V) was added, and the mixture was further allowed to stand at 37 ° C. for 2 hours.
Next, 4 ml of a 100 mM Tris-HCl buffer (pH 8.0) containing 100 mM sodium chloride was added to the sediment (CM hemoglobin antibody-sensitized latex) obtained by centrifugation.
The suspension was added, and a suspension containing a latex sensitized with a CM-conjugated hemoglobin antibody was prepared.

Example 3 The same procedure as in Example 1 was carried out in the same manner as in Example 1 except that an AGE-conjugated hemoglobin antibody was used in place of the CM-conjugated hemoglobin antibody. The concentration of AGE-bound hemoglobin in the sample was measured. AG in the subject
The E-hemoglobin concentration was converted into the AGE-hemoglobin concentration of the immunogen prepared as described below, and the hemoglobin 1 was converted.
It was expressed as the amount of AGE-bound hemoglobin per mg.
That is, the amount of optical change (absorbance change at a wavelength of 660 nm from 30 seconds to 200 seconds after the addition of the antibody) when measuring AGE-modified hemoglobin is 1000 units (U).
A using the optical change amount of the subject as the immunogen
The value obtained by dividing by the optical change amount of GE-modified hemoglobin and multiplying by 1000 (unit) and dividing by the hemoglobin concentration was defined as the concentration of AGE-modified hemoglobin in the subject.

The obtained AGE-modified hemoglobin value was determined in Example 1.
Was compared with the CM hemoglobin value of the subject obtained in the above, and the correlation was determined. FIG. 4 shows the results. As shown in FIG. 4, CM in the subject measured with the CM-conjugated hemoglobin antibody
A good correlation was found between the conjugated hemoglobin value and the AGE-linked hemoglobin value in the subject measured with the AGE-linked hemoglobin antibody.

The AGE-linked hemoglobin antibody was prepared as follows. (1) Preparation of AGE-modified hemoglobin 0.5 M phosphate buffer 1 containing 0.15 M sodium chloride
0.1 ml of glucose so that human hemoglobin (SIGMA, fraction V) becomes 6 mg / ml.
It dissolved so that it might be set to 7M. 0.22 μl of this solution
Then, the mixture was filtered at 37 ° C. for 2 months. Then, 4 mM with 20 mM phosphate buffer (pH 7.4)
After dialysis at 2 ° C. for 2 days to remove unreacted glucose, it was used as an immunogen for producing an AGE-conjugated hemoglobin antibody.

(2) Preparation of AGE-conjugated hemoglobin antibody Rabbits weighing 2 kg or more were immunized using the above-prepared AGE-conjugated hemoglobin as an antigen in the following manner. 2m
g / ml of the antigen solution 0.5 ml
Was supplemented with 0.5 ml of Freund's complete adjuvant and injected into the rabbit ear vein. Thereafter, every two weeks, 0.25 ml of the 2 mg / ml antigen solution and 0.25 ml of Freund's incomplete adjuvant were boosted. During this period, in order to confirm whether or not AGE-conjugated hemoglobin antibodies were produced, rabbits were partially bled once every two weeks from the outer ear vein. Six weeks later, the production of AGE-linked hemoglobin antibody was determined by enzyme immunoassay (ELIS).
A) Confirmed by method and whole blood was collected.

(3) Preparation of affinity purification column 25 ml of Affigel 15 (BIO-RAD) was
After washing with 1 l of 10 mM acetate buffer (pH 4.5),
62.5 ml of a 10 mg / ml human hemoglobin solution
In addition, the mixture was gently stirred at room temperature. Next, unreacted human hemoglobin was removed by filtration, 30 ml of 1M ethanolamine was added, and the mixture was stirred gently at room temperature to remove unreacted N.
-Hydroxysuccinimide ester was blocked. The support on which the human hemoglobin was immobilized was packed in a column, and washed with ion-exchanged water until the absorbance at 280 nm became zero. Further, the column was equilibrated with 20 mM phosphate buffer (pH 7.4) containing 0.15 M sodium chloride.

(4) Affinity purification of AGE-conjugated hemoglobin antibody The prepared AGE-conjugated hemoglobin antibody was diluted to 1 mg / ml with a 20 mM phosphate buffer (pH 7.4) containing 0.15 M sodium chloride. To 100
The solution was applied to the affinity purification column so as to be about mg. Next, the above phosphate buffer solution was flowed at a flow rate of 0.5 ml / min until the absorbance at 280 nm became zero.
Antibodies that did not bind to the column were collected as AGE-conjugated hemoglobin antibodies. When the absorbance at 280 nm became 0, the phosphate buffer was replaced with 0.1 M glycine buffer (pH 3.0), and the antibody bound to the column was eluted. The 20 mM solution containing 0.15 M sodium chloride was eluted. The column was equilibrated with a phosphate buffer (pH 7.4), the recovered antibody was applied to the column again, and the antibody not bound to the column was recovered. Then, it was dialyzed against a 50 mM glycine buffer (pH 8.2) and used as an antibody for latex agglutination.

[Brief description of the drawings]

FIG. 1 is a diagram showing a standard curve of CM-modified hemoglobin measured by the latex agglutination method of the present invention.

FIG. 2 is a diagram showing a correlation between measured values obtained by an amino acid analysis method and a latex agglutination method.

FIG. 3 is a diagram showing a correlation between measured values obtained when an unsensitized CM-conjugated hemoglobin antibody is used and when a CM-conjugated hemoglobin antibody-sensitized latex is used.

FIG. 4 is a diagram showing a correlation between a CM-modified hemoglobin value in a subject measured with a CM-conjugated hemoglobin antibody and an AGE-conjugated hemoglobin value in the subject measured with a AGE-conjugated hemoglobin antibody.

 ──────────────────────────────────────────────────の Continued on the front page (72) Inventor Hirofumi Ishida 320-11 Hino, Hino-shi, Tokyo Inside A & T Corporation (72) Inventor Yoshinori Yoshimura 320-11 Hino, Hino-shi, Tokyo Inside A & T Corporation

Claims (12)

[Claims] (1) sensitizing an unsensitized latex with an AGE-conjugated hemoglobin derived from a subject; and (2) specifically recognizing the obtained AGE-linked hemoglobin-sensitized latex with the obtained AGE-linked hemoglobin. An immunoassay for AGE-linked hemoglobin, which comprises reacting with an antibody, and then (3) measuring the turbidity of the formed aggregate. 2. The method according to claim 1, wherein the antibody that specifically recognizes AGE-linked hemoglobin is a mouse-derived monoclonal antibody. 3. The method according to claim 1, wherein the AGE-modified hemoglobin is hemoglobin in which the N-terminal of the α-chain and / or β-chain is carboxymethylated. 4. The latex has a diameter of 0.05 to 0.5 μm.
The method according to claim 1, comprising latex particles of the formula (1). 5. The method according to claim 1, wherein the latex has at least one functional group selected from the group consisting of a hydroxyl group, a carboxyl group and an epoxy group on the surface of the latex particles. 6. The method according to claim 1, wherein the AGE-modified hemoglobin is carboxymethylated hemoglobin. (1) reacting a first latex sensitized with an AGE-conjugated hemoglobin derived from a subject and a second latex sensitized with an antibody that specifically recognizes the AGE-conjugated hemoglobin; 2) An immunoassay for AGE-modified hemoglobin, which comprises measuring the turbidity of the formed aggregate. 8. The method according to claim 7, wherein the antibody that specifically recognizes AGE-linked hemoglobin is a mouse-derived monoclonal antibody. 9. The method according to claim 7, wherein the AGE-modified hemoglobin is hemoglobin in which the N-terminal of the α-chain and / or β-chain is carboxymethylated. 10. The method according to claim 7, wherein at least one of the first latex and the second latex comprises latex particles having a diameter of 0.05 to 0.5 μm. 11. The method according to claim 7, wherein at least one of the first latex and the second latex has at least one functional group selected from the group consisting of a hydroxyl group, a carboxyl group and an epoxy group on the surface of the latex particles. 12. The method according to claim 7, wherein the AGE-modified hemoglobin is carboxymethylated hemoglobin.