JP2596321B2 - Measurement method of glycated 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 It is known that glycated hemoglobin , particularly A _1C, has an increased blood concentration in diabetic patients, and the measurement of glycated hemoglobin is used in combination with the measurement of blood sugar level to diagnose and progress diabetes. It is used for measurement.

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 have mixed a sample which may contain glycated hemoglobin with unsensitized latex, and subsequently reacted with or simultaneously with an anti-glycated hemoglobin antibody. As a result, it has been found that a step requiring a long time as in the past can be eliminated, and therefore, the measurement of glycated hemoglobin can be performed quickly, and the present invention has been completed.

That is, the present invention comprises mixing a sample which may contain glycated hemoglobin with unsensitized latex,
Subsequently or simultaneously, a method for measuring glycated hemoglobin is provided, comprising reacting an anti-glycated hemoglobin antibody and measuring aggregation of the latex to which the glycated hemoglobin is adsorbed via the anti-glycated hemoglobin antibody. Is sometimes referred to as the first invention).

In the method of the present invention, the glycated hemoglobin of the sample easily binds to the unsensitized latex, so that aggregation occurs only by mixing the anti-glycated hemoglobin antibody. Therefore, in the individual measuring glycated hemoglobin in the sample by simply mixing the latex of the sample and an anti-glycated hemoglobin antibody and naive is adsorbed to the latex and cause antigen-antibody reaction with an anti-glycated hemoglobin antibody, agglutination Can be awakened. This method is 1/10 to 1 in comparison with the conventional process of directly attaching glycated hemoglobin to a solid phase.
Since it can be performed in a time period of / 100, a significant time reduction can be achieved as compared with the conventional method.

Furthermore, the present inventors have found that glycated hemoglobin can be measured by agglutinating the mixture of a specimen and unsensitized latex, reacting the mixture with particles bound with an anti-glycated hemoglobin antibody, and forming the resulting mixture.

That is, the present invention has been obtained after reacting an anti-glycated hemoglobin antibody bound to particles with a sample which may contain glycated hemoglobin and an unsensitized latex to cause an immunoagglutination reaction. There is provided a method for measuring glycated hemoglobin, which comprises measuring the glycated hemoglobin by aggregation (hereinafter, this method may be referred to as a second invention).

According to the method of the second invention, glycated hemoglobin can be measured in a much shorter time than in the conventional method.

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

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, by the method described in detail in the following examples,
It succeeded in producing a monoclonal antibody that specifically reacts with glycated hemoglobin and does not substantially react with normal hemoglobin (monoclonal antibody 3F1).
0, FERM BP-4311). The method of the present invention (first)
In the method of the present invention and the method of the second invention (hereinafter, simply referred to as "the present invention" means the first and the second invention), this monoclonal antibody can be preferably used.

As for the use of unsensitized latex, it is preferable to use a commercially available latex as it is, or to use a latex washed several times with a Tris-succinate buffer, a phosphate buffer or an acetate buffer. Can be. The binding of the anti-glycated hemoglobin antibody to the particles in the method of the second invention is generally performed by a physical adsorption method.
That is, the antibody diluted to 1 to 50 μg / ml is mixed so that the particle concentration becomes about 0.1 to 10% (w / v),
Stir at 4 ° C. to room temperature. This can be washed sufficiently by centrifugation and finally stored in a 1% BSA solution for use.

The particles used in the method of the present invention are latex particles having a diameter of 0.05 to 5 μm, preferably 0.1 to 2 μm, which are unused and dispersed in an ordinary buffer. Examples of the latex include a styrene polymer, a styrene-butadiene copolymer, a styrene-acrylate copolymer, and a styrene-divinylbenzene copolymer.

The mixing of the latex with the specimen and the anti-glycated hemoglobin antibody is carried out at a temperature between 4 ° C. and 40 ° C., and the aggregation is spectroscopically observed immediately after the mixing.

By using particles of 0.05 to 5 μm, the specimen, the anti-human glycated hemoglobin antibody and the particles are mixed on, for example, a black slide glass, and the presence or absence of particles that aggregate and precipitate is observed. Glycated hemoglobin in it can be detected. It is also possible to quantify glycated hemoglobin by measuring absorbance. In this case, the particle size is preferably small, but if the blank can be removed, the change in absorbance can be measured even with a particle size of about 1 μm. Further, by using a latex in which an unsensitized latex and an anti-human glycated hemoglobin antibody are bound, glycated hemoglobin in a sample can be detected with higher sensitivity and speed.

The coagulation of the latex, in addition to be able to verify the aggregation visually, when using latex particles of 0.05 to 1.0 [mu] m, it is possible to check the measured aggregation of the scattered light by spectrometry. Measurement wavelength is 400
It is preferable to use nm to 800 nm or infrared light. Thereby, glycated hemoglobin in the specimen can be detected. Also in this case, as the monoclonal antibody specific to glycated hemoglobin, it is preferable to use the monoclonal antibody 3F10 specific to human glycated hemoglobin _A1C , which will be described in detail in the following Examples.

[0018]

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 Balb in this 100μl
/ C mice were immunized four times every two weeks. After sacrifice of the immunized mouse, the spleen was excised, and 10 ⁶ spleen cells were obtained therefrom. It was fused with mouse myeloma cells in the presence of PEG and cultured. Collect the supernatant of the proliferating cells and perform ELISA
The presence or absence of an anti-human glycated hemoglobin antibody was examined by Method A. 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) Coagulation method using unsensitized latex
Measurement of Human Glycated Hemoglobin 5 μl of each of the five types of hemolysis specimens were separately added to 2 ml of unsensitized polystyrene latex (0.025% by weight, manufactured by Nippon Synthetic Rubber Co., Ltd.) having a diameter of 0.254 μm and mixed.
5 minutes later, 3F10 anti-glycated hemoglobin A _1C mouse lg
Add 50 μg of G and immediately add 37 μg with Hitachi 220 spectrophotometer.
The change in absorbance at a wavelength of 750 nm was measured while stirring at ℃. The results are shown in Table 1 below.

[0021]

[Table 1]

Example 2 Anti-human glycated hemoglobin A _1C monoclonal antibody 3F1
Preparation of 0-bonded latex Polystyrene latex (10% (w / v), diameter 0.05
100 μl of 20 μl containing the anti-human glycated hemoglobin monoclonal A _1C antibody 3F10 prepared in Example 1.
900 μl of mM acetate buffer (3F10 concentration: 40 μg /
ml, pH 6.0) and stirred with an end-over mixer. This was centrifuged (5000 g × 15 minutes), washed four times with physiological saline, and then added to 20 mM containing 1% by weight BSA.
It was suspended (0.5 wt%) in an acetate buffer (pH 5.0) and stored.

Sample 5 μl and unsensitized latex 100 μl
(0.05 μm, 0.25 wt%), 1 ml of the 3F10- bound latex prepared as described above was mixed, and immediately under stirring, the increase in absorbance at a wavelength of 800 nm was examined. Table 2 below shows the ratio (%) of glycated hemoglobin to total hemoglobin in the sample and the change in absorbance per minute.

[0024]

[Table 2]

Example 3 Specificity of 3F10 Antibody for 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.) tex concentration 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.

[0026] As shown in FIG. 1, 3F10 antibody against sensitized latex A ₀ antigen it was hardly react. In contrast, when the 3-4 antibody was added, an agglutination reaction occurred, and an increase in absorbance was observed. Similarly, _A1C antigen
Latex sensitized with 3F10 antibody or 3-4
Equivalent reactivity was shown by the addition of the antibody.

[0027]

According to the method of the present invention, a method for measuring glycated hemoglobin which can easily and quickly measure glycated hemoglobin is provided.

[Brief description of the drawings]

FIG. 1 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.

Continuing on the front page (72) Inventor Aiko Tanaka 2-7-1, Nishishinjuku, Shinjuku-ku, Tokyo Inside Fuji Rebio Co., Ltd. (72) Inventor Tetsuji Tanimoto 2-7-1 Nishishinjuku, Shinjuku-ku, Tokyo Fuji (56) References JP-A-4-145367 (JP, A) JP-A-61-280571 (JP, A)

Claims (8)

(57) [Claims] 1. A reaction between an unsensitized latex and a sample that may contain glycated hemoglobin, a reaction with an anti-glycated hemoglobin antibody, and agglutination of the latex adsorbed with the glycated hemoglobin via the anti-glycated hemoglobin antibody. A method for measuring glycated hemoglobin comprising measuring. 2. An unsensitized latex and saccharified hemoglobin
React with a sample that may contain
Reacting a hemoglobin antibody, and the glycated hemoglobin is
Adsorbed latex mediated by anti-glycated hemoglobin antibody
The method of claim 1, comprising measuring agglutination. 3. An unsensitized latex and saccharified hemoglobin
And anti-glycated hemoglobin antibody
At the same time, and the glycated hemoglobin adsorbed
Measures aggregation of TEX via anti-glycated hemoglobin antibody
The method of claim 1, comprising: 4. The unsensitized latex has a particle size of 0.05 to 5 μm.
any one method according to the claims 1 to m 3. 5. A reaction between an unsensitized latex, a sample that may contain glycated hemoglobin, and an anti-glycated hemoglobin antibody bound to the particles to cause an immunoagglutination reaction. A method for measuring glycated hemoglobin, comprising measuring the glycated hemoglobin. 6. The unsensitized latex has a particle size of 0.05 to 5 μm.
The method according to claim 5, wherein m is m. 7. Latex whose particles have a diameter of 0.05 to 5 μm,
The method according to claim 5, wherein the method is selected from the group consisting of gelatin particles having a diameter of 0.5 to 10 µm and animal erythrocytes. 8. The method according to any one of the anti-glycated hemoglobin antibody claims 1 which is a monoclonal antibody 3F10 7.