JP2596322B2 - Method for measuring the ratio of glycated hemoglobin to total hemoglobin - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for measuring glycated hemoglobin.

[0002]

2. Description of the Related Art Glycated hemoglobin , especially glycated hemoglobin
Emissions A _1C is known that the blood concentration increases in diabetic patients, measurement of glycated hemoglobin is used for diagnosis and measurement progress of diabetes in combination with the measurement of blood glucose level.

Conventionally, glycated hemoglobin is measured by directly binding glycated hemoglobin in a sample (red blood cell extract) to a solid phase, washing, reacting a labeled anti-glycated hemoglobin antibody, washing, and measuring the label. It is performed by. However, this method requires a pretreatment (eg, overnight incubation at 4 ° C.) for directly binding glycated hemoglobin in each sample to a solid phase,
This is time consuming and it is not possible to measure large quantities of specimens quickly.

[0004]

SUMMARY OF THE INVENTION Accordingly, an object of the present invention is to provide a method for measuring glycated hemoglobin, which can easily and quickly measure glycated hemoglobin.

[0005]

Means for Solving the Problems As a result of intensive studies, the inventors of the present invention reacted a sample with an anti-hemoglobin antibody previously bound to a solid phase, and further reacted with an anti-glycated hemoglobin antibody to obtain a sample. The present inventors have found that the time-consuming step of binding glycated hemoglobin in the sample to the solid phase can be eliminated, and that the measurement of glycated hemoglobin can be carried out quickly, thus completing the present invention.

That is, the present invention comprises reacting an anti-hemoglobin antibody bound to a solid phase with a sample that may contain glycated hemoglobin, and then reacting the anti-glycated hemoglobin antibody with the glycated hemoglobin and the anti-hemoglobin. Measuring the amount of anti-glycated hemoglobin antibody bound to the solid phase via the antibody, the sugar to total hemoglobin
The present invention provides a method for measuring the ratio of activated hemoglobin .

In the method of the present invention, if a solid phase to which an anti-hemoglobin antibody is bound is prepared in advance in a large amount, in each measurement, the antigen-antibody is bound between the sample and the anti-hemoglobin antibody bound to the solid phase. Antihemo by reacting
Hemoglobin and glycated hemoglobin in the sample via globin antibody
Globin can be bound to the solid phase at the same ratio as in the sample.
Reacts with anti-glycated hemoglobin antibody
It is possible to measure the percentage of glycated hemoglobin in robin
Wear. In addition, glycated hemoglobin is mediated through this anti-hemoglobin antibody.
The method of solidifying globin can be performed in 1/10 or less of the time of the step of directly binding glycated hemoglobin to the solid phase, so that a significant time reduction can be achieved as compared with the conventional method.

[0008]

[0009]

[0010]

[0011]

[0012]

Hereinafter, the present invention will be described in more detail.

The solid phase used in the method of the first invention may be any of those used in conventional immunoassays. For example, wells of a plastic microplate can be preferably used.

[0015] Anti-hemoglobin antibodies are well known in the art, and may be polyclonal or monoclonal. The binding of the anti-hemoglobin antibody to the solid phase is performed, for example, by adding an antibody solution having a concentration of about 1 μg / ml to the solid phase and adding the antibody solution at 4 ° C.
At room temperature overnight. After binding, blocking is performed with a protein such as BSA according to a conventional method to block nonspecific adsorption sites of the protein. When measuring human glycated hemoglobin, it is preferable to bind an anti-human hemoglobin antibody to a solid phase.

Next, the anti-hemoglobin antibody bound to the solid phase is reacted with the sample, and the glycated hemoglobin in the sample is reacted by an antigen-antibody reaction via the anti-hemoglobin antibody bound to the solid phase. Combine to phase. The specimen may be a red blood cell extract or whole blood. This antigen-antibody reaction can be performed, for example, at room temperature for about 2 hours.

Next, after washing, the glycated hemoglobin bound to the solid phase via the anti-hemoglobin antibody and the anti-glycated hemoglobin antibody are subjected to an antigen-antibody reaction. The anti- glycated hemoglobin antibody may be a polyclonal antibody or a monoclonal antibody, but is preferably a monoclonal antibody in order to increase the measurement accuracy. The present inventors have succeeded in producing a monoclonal antibody that specifically reacts with glycated hemoglobin and does not substantially react with normal hemoglobin by the method described in detail in the Examples below (monoclonal antibody 3F10). , FERM BP-431
1). In the method of the present invention, this monoclonal antibody can be preferably used. This antigen-antibody reaction can also be performed under the same conditions as described above.

Next, after washing, the anti-glycated hemoglobin antibody bound to the solid phase via the glycated hemoglobin and the anti-hemoglobin antibody is measured. This can be performed by various methods conventionally known in the field of immunoassay. For example, it can be performed by labeling an anti-glycated hemoglobin antibody with an enzyme, a fluorescent dye, a radioactive substance, or the like, and measuring the label. Alternatively, it can be performed by binding biotin to an anti-glycated hemoglobin antibody, reacting the biotin with labeled avidin, and measuring the label. Furthermore, it can be performed by further reacting a labeled antibody that specifically reacts with the anti-glycated hemoglobin antibody, and measuring the label.

[0019]

[0020]

[0021]

[0022]

[0023]

[0024]

EXAMPLE 1 (1) Preparation human glycated hemoglobin A _1C of monoclonal antibodies against human glycated hemoglobin A _1C well dispersed in Freund's complete adjuvant Bal in this 100μl
b / c mice were immunized four times every two weeks. The immune mice were excised sacrificed after spleen to obtain 10 ⁶ splenocytes than this. It was fused with mouse myeloma cells in the presence of PEG and cultured. The supernatant of the proliferated cells is collected and ELI
The presence or absence of an anti-human glycated hemoglobin antibody was examined by the SA method. Cells positive for the antibody were tested by the limiting dilution method to confirm cells producing an anti-human glycated hemoglobin antibody.

The cells thus obtained were cultured in large quantities as anti-human glycated hemoglobin mouse monoclonal antibody-producing cells (hybridoma cells) and injected into the peritoneal cavity of mice. Two weeks later, ascites was collected every three days to obtain the desired anti-human glycated hemoglobin monoclonal antibody. This anti-human glycated hemoglobin monoclonal antibody was
And hybridoma producing 3F10
Deposited with JICA on June 10, 2016. This deposit was converted to an international deposit on May 25, 1993, and its accession number is FERM BP-4311. 3F10 is I
gG was confirmed.

(2) Measurement of human glycated hemoglobin A _1c by ELISA method Anti-human hemoglobin antibody (rabbit antibody) 10 μg / ml
50 μl each into Nunc 96-well ELISA plate
And left at 4 ° C. overnight. 2% of this plate
The plate was washed 5 times with BSA / 0.02% Tween 20 / phosphate-buffered saline, and finally 200 µl of this buffer was added and stored at 4 ° C. The supernatant of this plate was removed, and 150 μl of a sample containing human glycated hemoglobin was added dropwise.
Left for hours. The plate is placed in 0.02% Tween2
Washed four times with physiological saline containing 0.

Next, a peroxidase-labeled anti-human glycated hemoglobin-specific mouse monoclonal antibody (3F10)
After adding 150 μl (500 ng / ml) and leaving the mixture at room temperature for 2 hours, it was again washed four times with a physiological saline solution containing 0.02% Tween 20, and then 0.1% 2,2′-azobis (3
-Ethylbenzothiazoline-6-sulfonic acid (ABT
200 μl of H ₂ O ₂ solution (0.005%) containing S)
After the addition, and the mixture was left at room temperature for 30 minutes, the color development was measured by colorimetry. Colorimetry was performed by measuring the absorbance at 415 nm and 492 nm. The results are shown in Table 1 below.

[0028]

[Table 1]

Example 2, Comparative Example 1 Using the anti- human glycated hemoglobin monoclonal antibody prepared in Example 1 and a commercially available anti-human glycated hemoglobin monoclonal antibody, glycated human hemoglobin in a sample containing various concentrations of glycated human hemoglobin was determined. Quantified. The quantification was performed as follows. The anti-human glycated hemoglobin monoclonal antibody prepared in Example 1 or a commercially available anti-human glycated hemoglobin monoclonal antibody (Chemicon, USA) was sensitized to a 96-well microplate. Then, peroxidase (POD) -labeled glycated hemoglobin antigen was added. At this time, the amount of addition (dilution ratio) was varied and added. Next, the reaction was carried out at room temperature for 2 hours, and PBS-Twe
Washed with en. 100 μl of ABTS / H ₂ O ₂ (substrate) was added and reacted at room temperature for 1 hour. 50 μl of the reaction stop solution was added, and the absorbance at a wavelength of 415 nm was measured.

FIG. 1 shows the results. As is clear from FIG. 1, when a commercially available anti-human glycated hemoglobin monoclonal antibody was used (Comparative Example 1), it could not cope with an increase in antigen concentration, and almost no response was obtained. When the obtained anti-human glycated hemoglobin monoclonal antibody was used (Example 2), the absorbance increased in proportion to the antigen concentration. Therefore, it was revealed that glycated hemoglobin in a wide concentration range can be measured by using the anti-human glycated hemoglobin monoclonal antibody prepared in Example 1.

[0031]

[0032]

[0033]

[0034]

[0035]

Example 6 Specificity of 3F10 Antibody to A _1C Unsensitized polystyrene latex solution having a diameter of 0.254 μm (Tris-succinate buffer (pH 5.5, 0.2% Triton X-100 (trade name)) the including.) latex concentration of 0.025%) were placed in a cell that regulate 2ml to 37 ° C.. To this, human A _1C antigen (1 mg / ml) or human hemoglobin (A ₀ antigen) (1 mg / ml) obtained by ion-exchange chromatography purification was separately added.
The mixture was heated with stirring at 5 ° C. for 5 minutes. After 5 minutes, the latex solution sensitized with A ₀ antigen 3F10 antibody (8 mg / m
l) was added to the latex solution sensitized with the _A1C antigen by using the 3-4 antibody (anti-human hemoglobin monoclonal antibody, 8 mg /
ml) was added to each, and the change in absorbance at a wavelength of 750 nm was immediately measured with a Hitachi 220 spectrophotometer. One more
After 4 minutes, the 3-4 antibody to the latex solution sensitized with A ₀ antigen, respectively final concentration 3F10 antibody to the latex solution sensitized with A _1C antigen was added to a 8 [mu] g / ml.
The results are shown in FIG.

[0037] As shown in FIG. 2, F310 antibodies against sensitized latex _{A 0} antigen was hardly react. In contrast, when the 3-4 antibody was added, an agglutination reaction occurred, and an increase in absorbance was observed. Similarly, in the latex sensitized with the _A1c antigen, the 3F10 antibody or 3-
The same reactivity was shown by the addition of 4 antibodies.

[Brief description of the drawings]

FIG. 1 shows the anti-human glycated hemoglobin monoclonal antibody prepared in Examples and a commercially available anti-human glycated hemoglobin mono antibody
It is a figure which shows the result of having measured the human glycated hemoglobin in the sample containing various concentrations of human glycated hemoglobin using the clonal antibody.

FIG. 2 shows a 3F10 antibody or 3-4 antibody in a latex particle liquid on which human hemoglobin or human glycated hemoglobin is immobilized.
It is a figure which shows the time-dependent change of the light absorbency at the time of adding an antibody, respectively, and then adding 3-4 antibody or 3F10 antibody, respectively.

 ────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Tetsuji Tanimoto 2-7-1, Nishishinjuku, Shinjuku-ku, Tokyo Inside Fuji Rebio Co., Ltd. (56) References JP-A-61-172064 (JP, A) 61-280571 (JP, A) JP-A-2-8743 (JP, A)

Claims (2)

(57) [Claims] Claims: 1. After reacting an anti-hemoglobin antibody bound to a solid phase with a sample that may contain glycated hemoglobin, reacting the anti-glycated hemoglobin antibody, and reacting the glycated hemoglobin and the anti-hemoglobin antibody Measuring glycated hemoglobin relative to total hemoglobin, comprising measuring anti-glycated hemoglobin antibody bound to the solid phase.
How to measure the percentage . 2. The anti-glycated hemoglobin antibody is monoclonal
2. The method according to claim 1, wherein the antibody is a monoclonal antibody 3F10 .