JP4854503B2 - Method for measuring stable hemoglobin A1c - Google Patents

Description

The present invention relates to a method for measuring hemoglobin capable of measuring hemoglobin, particularly stable hemoglobin A1c, which is a diagnostic index of diabetes, in a short time and with high accuracy.

Hemoglobin (Hb), particularly hemoglobin A1c (hereinafter referred to as HbA1c), which is a kind of glycated hemoglobin, reflects the average sugar concentration in the blood during the past 1 to 2 months. It is widely used as a test item for grasping blood glucose control status of patients with diabetes.
Conventionally, HPLC methods, immunization methods, electrophoresis methods and the like have been used as methods for measuring HbA1c. Among them, the HPLC method widely used in the clinical laboratory field can measure in 1 to 2 minutes per sample, and the CV value of the simultaneous reproducibility test is about 1.0%. It has been realized. This level of performance is required as a measurement method for grasping the blood glucose control state of a diabetic patient.

On the other hand, the electrophoresis method is a technique capable of producing an inexpensive and small system such as microchip electrophoresis because the apparatus configuration is simple, and clinical examination of high-precision measurement technology of HbA1c in electrophoresis method. Application to can expect a very beneficial effect in terms of cost.
Although the measurement of Hbs by electrophoresis has been used for a long time as a method for separating abnormal Hb having an amino acid sequence different from the usual, separation of HbA1c is very difficult. It took more than a minute. As described above, the electrophoresis method has a problem in terms of measurement time and measurement accuracy when applied to the clinical laboratory field, so far, it has hardly been applied to diabetes diagnosis.

On the other hand, the capillary electrophoresis method that appeared around 1990 generally enables high separation and high accuracy measurement. For example, Patent Document 1 discloses a method for separating HbA1c by capillary electrophoresis. Is disclosed.
However, when the method disclosed in Patent Document 1 is used, the problem that the measurement time is long is not solved, and in addition, the pH of the buffer solution used is as high as 9 to 12, and Hb may be denatured. Due to its nature, this method has been difficult to apply to clinical tests.

Patent Document 2 discloses a method in which a cationic polymer is dynamically coated on the inner surface of a capillary by passing a cationic polymer through the capillary and a buffer containing sulfated polysaccharide is used in capillary electrophoresis. Is disclosed. According to this method, measurement can be performed in about 10 minutes, and measurement can be performed in a shorter time compared to gel electrophoresis.

However, when diagnosing diabetes, stable HbA1c, which is an index of diabetes, must be separated from HbA1c to eliminate the influence of modified Hb such as unstable HbA1c, carbamylated Hb, and acetylated Hb. However, the electropherograms obtained by the methods disclosed in Patent Documents 1 and 2 have insufficient separation performance and measurement accuracy, and it is difficult to separate stable HbA1c within the technical scope described in these methods. there were.
Special table Hei 09-510792 JP 09-105739 A

An object of the present invention is to provide a method for measuring hemoglobin capable of measuring hemoglobins, particularly stable hemoglobin A1c, which is a diagnostic index of diabetes, in a short time with high accuracy.

The present invention relates to a method for measuring stable hemoglobin A1c using capillary electrophoresis or microchip electrophoresis, wherein an electrophoresis path whose inner surface is coated with an ionic polymer, It is a measuring method of stable hemoglobin A1c using a buffer solution containing a chaotropic compound filled inside .
The present invention is described in detail below.

As a result of intensive studies, the present inventors have conducted electrophoresis using a migration path coated with an ionic polymer on the inner surface and a buffer containing a chaotropic compound in the measurement of hemoglobins by electrophoresis. As a result, it has been found that stable HbA1c, which has conventionally been difficult to separate and measure, can be measured with high accuracy in a short time, and the present invention has been completed.

The electrophoresis method used in the method for measuring hemoglobin of the present invention is not particularly limited, and examples thereof include capillary electrophoresis and microchip electrophoresis.

FIG. 1 shows an example of a capillary electrophoresis apparatus used in the method for measuring hemoglobin of the present invention. As shown in FIG. 1, the capillary electrophoresis apparatus 11 includes an anode tank 12, a cathode tank 13, a capillary 14, a high voltage power supply 15, a detector 16, and a pair of electrodes 17 and 18. Both ends of the capillary 14 are immersed in the buffer solution in the anode tank 12 and the cathode tank 13, and the inside of the tubular capillary 14 is filled with the buffer solution. The electrodes 17 and 18 are electrically connected to the high voltage power supply 15.
In the method for measuring hemoglobin of the present invention, the buffer solution contains a chaotropic compound. When measuring hemoglobin, a sample is injected from one of the capillaries 14 and a predetermined voltage is applied from the high-voltage power supply 15 so that the target component moving in the capillaries 14 is measured by the detector 16. .

The method for measuring hemoglobin of the present invention uses an electrophoresis path whose inner surface is coated with an ionic polymer. By using such a migration path, it is possible to perform highly accurate measurement in a shorter time with respect to the stable HbA1c.

The electrophoresis path refers to a site (from a site where the sample is injected to a site where the sample is detected) where the sample moves and / or separates when electrophoresis is performed. Specifically, for example, in the case of capillary electrophoresis, it means from the site where the sample in the capillary is injected to the site detected by the detector, and in the case of microchip electrophoresis, the microchip This refers to the region from the portion where the same sample is injected into the microchip groove in the type electrophoresis apparatus to the portion detected by the detector.
The inner surface of the migration path specifically refers to the inner surface of the migration path of the capillary in the case of capillary electrophoresis, for example, and in the microchip electrophoresis apparatus in the case of the microchip electrophoresis method. It refers to the inner surface of the microchip groove.

It does not specifically limit as a raw material which comprises the said migration path, For example, glass, a metal, resin etc. are mentioned.
The migration path may have a solid substance such as filler particles inside.

A preferable lower limit of the length of the migration path is 10 mm, and a preferable upper limit is 300 mm. If it is less than 10 mm, the sample may not be sufficiently separated, and accurate measurement may not be possible. If it exceeds 300 mm, the measurement time may be extended or the peak shape may be deformed in the obtained electropherogram, so that accurate measurement may not be possible.

The preferable lower limit of the diameter of the migration path is 1 μm, and the preferable upper limit is 100 μm. If it is less than 1 μm, the optical path length for detection by the detector is small, and the measurement accuracy may decrease. When it exceeds 100 μm, the peak becomes broad in the electropherogram obtained by diffusing the sample in the migration path, and the measurement accuracy may be lowered.

In this specification, an ionic polymer means a polymer having an ionic functional group in the molecule.
The ionic polymers are roughly classified into anionic polymers and cationic polymers depending on the ionic type.

The anionic polymer is a polymer having an anionic functional group in the molecule.
It does not specifically limit as said anionic polymer, For example, anionic group containing polysaccharide, anionic group containing organic synthetic polymer, etc. are mentioned.
The anionic functional group is not particularly limited, and examples thereof include a carboxyl group, a phosphoric acid group, and a sulfonic acid group.

The anionic group-containing polysaccharide is not particularly limited. For example, sulfonic acid group-containing polysaccharides such as chondroitin sulfate, dextran sulfate, heparin, heparan, fucoidan, and salts thereof; carboxyl groups such as alginic acid and pectinic acid. Polysaccharides and salts thereof; neutral polysaccharides such as cellulose, dextran, agarose, mannan and starch; anionic group-introduced products of derivatives thereof; and known anionic group-containing polysaccharides such as salts thereof Can be mentioned.

The anionic group-containing organic synthetic polymer is not particularly limited. For example, polyacrylic acid, polymethacrylic acid (hereinafter also referred to as (meth) acrylic acid), phosphoric acid group-containing (meth) acrylic acid ester polymer, Examples include sulfonic acid group-containing (meth) acrylic acid polymers, 2- (meth) acrylamide-2-methylpropanesulfonic acid polymers, and known anionic group-containing organic synthetic polymers such as copolymers thereof. It is done.

The cationic polymer is a polymer having a cationic functional group in the molecule.
The cationic polymer is not particularly limited, and examples thereof include an aminated polysaccharide and an amino group-containing organic synthetic polymer.
The cationic functional group is not particularly limited, and examples thereof include a primary to quaternary amino group.

The aminated polysaccharide is not particularly limited, and examples thereof include chitosan derivatives such as chitin and chitosan and salts thereof; derivatives of N-substituted cellulose such as aminocellulose and N-methylaminocellulose; and salts thereof; dextran. And neutral polysaccharides such as agarose, mannan and starch, and amino group-introduced products and derivatives thereof, and known aminated polysaccharides and the like.

The amino group-containing organic synthetic polymer is not particularly limited. For example, amino group-containing (meth) acrylates and copolymers such as polyethyleneimine, polybrene, poly2-diethylaminoethyl (meth) acrylate, and the like, known amino groups Examples thereof include organic synthetic polymers.

A preferable lower limit of the weight average molecular weight of the ionic polymer is 500. If it is less than 500, it is difficult to sufficiently coat the inner surface of the migration path, and the hemoglobin separation performance may be insufficient.

The method for coating the ionic polymer on the migration path is not particularly limited, and a conventionally known method can be used. For example, a solution containing the ionic polymer is passed through the migration path. A method of dynamic coating by contacting; the ionic polymer is brought into contact with the inner surface of the migration path and physically adsorbed by utilizing a hydrophobic or electrostatic interaction or the like to be immobilized. A method of coating; a method of immobilizing and coating the inner surface of the migration path and the ionic polymer by a covalent bond via a functional group of each substance or another substance. When the immobilization coating method is used, it is possible to immobilize an ionic polymer that is difficult to peel off and can be repeatedly measured through a heating process, a drying process, and the like. As the immobilization coating method, specifically, for example, a solution containing the ionic polymer is passed through the migration path, and the solution is driven out by injecting air into the migration path. The method of repeating drying etc. is mentioned.

Of these, the immobilization coating method is preferable. By using the fixed coating method, once coating is performed, peeling does not occur, and it is not necessary to perform coating again for each measurement. Therefore, the measurement time can be shortened during continuous measurement or the like.

When immobilizing the ionic polymer in the migration path, another kind of layer may be formed between the inner surface of the migration path and the layer made of the ionic polymer. When the different kind of layer is formed, the layer made of the ionic polymer may be formed on the innermost surface of the migration path.

When the ionic polymer is coated on the migration path, the ionic polymer is preferably supplied to the coating as the ionic polymer solution, although depending on the type and the coating method.
As a concentration of the ionic polymer solution, a preferable lower limit is 0.01% and a preferable upper limit is 20%. If it is less than 0.01%, immobilization may be insufficient. If it exceeds 20%, the layer made of the ionic polymer cannot be formed uniformly, and it may be peeled off during the measurement of hemoglobin, which may cause a decrease in reproducibility.

The method for measuring hemoglobin of the present invention uses a buffer solution containing a chaotropic compound.
The buffer solution is a buffer solution that fills the inside of the migration path during electrophoresis, a buffer solution that fills the anode and cathode tanks installed at both ends of the migration path during electrophoresis, and cleans the inside of the migration path. It is used as a buffer solution, a hemolysis dilution solution for dissolving and diluting a sample, and the like.
In the method for measuring hemoglobins of the present invention, all these buffers may contain chaotropic compounds, or only some of the buffers may contain chaotropic compounds.

In this specification, a chaotropic compound is a compound having the property of destroying the interaction between water molecules, weakening the hydrophobic interaction between hydrophobic molecules, and increasing the water solubility of the hydrophobic molecules. Say.

The chaotropic compound is not particularly limited. For example, tribromoacetate ion, trichloroacetate ion, thiocyanate ion, iodide ion, perchlorate ion, dichloroacetate ion, nitrate ion, bromide ion, chloride ion, acetate ion, etc. Anionic chaotropic ions; compounds containing cationic chaotropic ions such as barium ions, calcium ions, lithium ions, cesium ions, potassium ions, magnesium ions, guanidine ions; urea compounds such as urea and thiourea Can be mentioned. Of these, compounds containing thiocyanate ions, perchlorate ions, nitrate ions, guanidine ions, urea compounds, and the like are preferable.

The amount of the chaotropic compound added is not particularly limited and varies greatly depending on the type, but the preferred lower limit is 0.01% by weight and the preferred upper limit is 30% by weight. If it is less than 0.01% by weight, sufficient hemoglobin separation performance may not be exhibited. If it exceeds 30% by weight, heat is generated during electrophoresis, and peak deformation or the like may occur in the obtained electropherogram. A more preferred lower limit is 0.05% by weight, and a more preferred upper limit is 20% by weight.

The buffer solution is not particularly limited as long as it is a solution containing a conventionally known buffer solution composition having a buffer capacity, and examples thereof include organic acids such as citric acid, succinic acid, tartaric acid, malic acid, and salts thereof. And the like; amino acids such as glycine, taurine and arginine; and solutions containing inorganic acids such as hydrochloric acid, nitric acid, sulfuric acid, phosphoric acid, boric acid and acetic acid, and salts thereof.
Commonly used additives such as surfactants, various polymers, and hydrophilic low molecular weight compounds may be appropriately added to the buffer solution.

The buffer solution preferably further contains a water-soluble polymer having an anionic group. By containing such a polymer, stable HbA1c can be separated and measured more efficiently.

The anionic group is not particularly limited, and examples thereof include conventionally known anionic functional groups such as a carboxyl group, a phosphoric acid group, and a sulfonic acid group. Among these, it is preferable to have a sulfonic acid group.
The water-soluble polymer having an anionic group may have a plurality of the anionic groups in the molecule or may have different types of the anionic groups.

Although the water-soluble polymer which has the said anionic group should just be the state melt | dissolved completely in the said buffer solution at the time of use, the preferable minimum of the solubility with respect to water is 1 g / L. If it is less than 1 g / L, the water-soluble polymer having an anionic group can be used only at a low concentration, so that the effect is hardly exhibited and the measurement accuracy may be insufficient. A more preferred lower limit is 5 g / L.

It does not specifically limit as a water-soluble polymer which has the said anionic group, For example, a well-known polymer can be used. Preferred water-soluble polymers having an anionic group include polysaccharides having an anionic group and water-soluble organic synthetic polymers having an anionic group.

The polysaccharide having an anionic group is not particularly limited, and examples thereof include sulfonic acid group-containing polysaccharides such as chondroitin sulfate, dextran sulfate, heparin, heparan, fucoidan, and salts thereof; carboxyl groups such as alginic acid and pectinic acid. -Containing polysaccharides and salts thereof; neutral polysaccharides such as cellulose, dextran, agarose, mannan and starch; anionic group-introduced products thereof; and polysaccharides having known anionic groups such as salts thereof Etc.

Examples of the organic synthetic polymer having an anionic group include known water-soluble organic synthetic polymers containing an anionic functional group, particularly acrylic polymers; that is, acrylic acid or methacrylic acid and derivatives and esters thereof. A polymer mainly composed of a polymer or the like is preferred.
Specifically, for example, an acrylic polymer obtained by polymerizing a monomer having a carboxyl group such as (meth) acrylic acid and 2- (meth) acryloyloxyethyl succinic acid, ((meth) acryloyloxyethyl) acid phosphate Acrylic polymer obtained by polymerizing a monomer having a phosphate group such as (2- (meth) acryloyloxyethyl) acid phosphate, (3- (meth) acryloyloxypropyl) acid phosphate, 2- (meth) acrylamide Acrylic polymers obtained by polymerizing monomers having a sulfonic acid group such as 2-methylpropanesulfonic acid, (meth) acrylamidepropanesulfonic acid, sulfopropyl (meth) acrylate, (meth) acryloyloxynaphthalenesulfonic acid, etc. Can be mentioned.

The acrylic polymer may be a copolymer of a (meth) acryl monomer having an anionic group and a (meth) acryl monomer having no anionic group.
The (meth) acrylic monomer having no anionic group is not particularly limited as long as it is a (meth) acrylic monomer copolymerizable with the (meth) acrylic monomer having the anionic group. The (meth) acrylic acid esters having a hydrophilic property are preferably used. Specifically, for example, 2-hydroxyethyl (meth) acrylate, 2-hydroxypropyl (meth) acrylate, glycerol (meth) acrylate; glycidyl (meth) acrylate, polyethylene glycol (meth) acrylate, polypropylene glycol (meth) acrylate Etc.

The addition amount of the (meth) acrylic monomer having no anionic group is not particularly limited as long as the obtained copolymer is water-soluble, but with respect to 100 parts by weight of the (meth) acrylic monomer having the anionic group. The preferred upper limit is 1000 parts by weight. If it exceeds 1000 parts by weight, the resulting copolymer may not be water-soluble.

The preferable lower limit of the content of the water-soluble polymer having an anionic group in the buffer solution is 0.01% by weight, and the preferable upper limit is 10% by weight. If it is less than 0.01% by weight, the effect of adding a water-soluble polymer is hardly exhibited, and separation or the like may be insufficient in the measurement of hemoglobins. If it exceeds 10% by weight, the measurement time may be prolonged or separation may be caused.

In the method for measuring hemoglobin of the present invention, the hemoglobin to be measured is not particularly limited, and examples thereof include conventionally known hemoglobins. Specific examples include HbA1a, HbA1b, HbF, unstable HbA1c, stable HbA1c, HbA0, HbA2; modified hemoglobins such as acetylated Hb and carbamylated Hb; and abnormal hemoglobins such as HbS and HbC. Among these, stable HbA1c can be preferably measured.

ADVANTAGE OF THE INVENTION According to this invention, the measuring method of hemoglobin which can perform the measurement of hemoglobin, especially stable hemoglobin A1c used as a diagnostic index of diabetes with high precision in a short time can be provided. Specifically, while using a simple and low-cost method called electrophoresis, the CV value indicating simultaneous reproducibility is about 1.0% in a short time of several minutes per sample, particularly in the measurement of stable HbA1c. Can be measured with high accuracy. Therefore, it becomes possible to suitably manage the numerical value of HbA1c of diabetic patients by electrophoresis.

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

Example 1
(1) An aqueous solution containing 0.2% by weight of dextran sulfate (manufactured by Wako Pure Chemical Industries, Ltd.), which is a coating ionic polymer for the migration path, was prepared. Next, after 0.2N-NaOH, ion-exchanged water, and 0.5N-HCl are passed through a fused silica capillary (GL Science, Inc .: inner diameter 25 μm × total length 30 cm) in this order, the inside of the capillary is washed. The obtained dextran sulfate aqueous solution was passed through for 20 minutes. Thereafter, air was injected into the capillary to drive out the aqueous dextran sulfate solution, and then the mixture was dried in a dryer at 40 ° C. for 12 hours. Thereafter, a dextran sulfate aqueous solution was injected again, and the inner surface of the capillary was coated by repeating the injection and drying of air five times.
The obtained dextran sulfate-immobilized capillary was set in a capillary electrophoresis apparatus (PAC / E MDQ manufactured by Beckman Coulter).

(2) Preparation of buffer solution Citrate buffer solution (pH 4.8) containing 5.0% by weight of urea (manufactured by Wako Pure Chemical Industries, Ltd.) as a chaotropic compound and 2.0% by weight of dextran sulfate as a water-soluble polymer having an anionic group. ) Was prepared.

(3) As a measurement sample of healthy human blood, blood collected from heparin from healthy human blood is used, and 0.05% Triton X-100 (surfactant: manufactured by Wako Pure Chemical Industries, Ltd.) is added to 70 μL of whole healthy human blood. 200 μL of citrate buffer solution (pH 6.0) containing lysate was used and diluted with hemolysis.
A sample is injected from one of the capillaries, subjected to electrophoresis by applying a voltage of 20 kV to the buffer solution at both ends of the capillary, and the change in absorbance at 415 nm visible light is measured, whereby capillary electricity of stable HbA1c in human blood is measured. Measurement by electrophoresis was performed.
FIG. 2 is an electropherogram obtained in Example 1 by measurement of blood of a healthy person. In FIG. 2, peak 1 indicates stable HbA1c, and peak 2 indicates HbA0. In Example 1, stable HbA1c could be separated in an electrophoresis time of about 1.5 minutes.

(4) Measurement of a sample containing modified Hb A sample containing a large amount of unstable HbA1c, which is a type of modified Hb, added to healthy human whole blood used in the measurement of healthy human blood so that glucose is 2500 mg / dL. Was artificially prepared. A sample is injected from one of the capillaries, electrophoresis is performed by applying a voltage of 20 kV to the buffer solution at both ends of the capillary, and the change in absorbance at 415 nm visible light is measured, whereby capillary electricity of stable HbA1c in human blood is measured. Measurement by electrophoresis was performed.
FIG. 3 is an electropherogram obtained in Example 1 by measurement of a sample containing modified Hb.
In FIG. 3, peak 1 shows stable HbA1c, peak 2 shows HbA0, and peak 3 shows modified Hb (unstable HbA1c). As shown in FIG. 3, stable HbA1c and unstable HbA1c, which is a modified Hb, were well separated.

(Example 2)
The inner surface of the capillary was coated by the same method as in Example 1 except that a 0.5% by weight 0.5N hydrochloric acid solution of chitosan (chitosan-100: manufactured by Wako Pure Chemical Industries), which is an ionic polymer, was used. An electrophoresis apparatus was obtained.
As a buffer solution, a malic acid buffer solution (pH 4.8) containing 1.0% by weight of sodium nitrate as a chaotropic compound and 2.0% by weight of chondroitin sulfate as a water-soluble polymer having an anionic group was prepared.
A healthy human blood was measured and a sample containing modified Hb was measured by the same method as in Example 1 except that the obtained electrophoresis apparatus and buffer solution were used. In the measurement of healthy human blood, an electropherogram similar to that shown in FIG. 2 was obtained. In the measurement of the sample containing the modified Hb, an electropherogram similar to that shown in FIG. 3 was obtained.

(Example 3)
The capillary inner surface was coated by the same method as in Example 1 except that a 0.5 wt% aqueous solution of polybrene (manufactured by Wako Pure Chemical Industries, Ltd.), which is an ionic polymer, was used to obtain an electrophoresis apparatus.
As an acrylic monomer having an anionic group, 3.0 g of acrylamide-2-methylpropanesulfonic acid (manufactured by Wako Pure Chemical Industries, Ltd.) and as an acrylic monomer having no anionic group, hydroxyethyl methacrylate (manufactured by Wako Pure Chemical Industries, Ltd.) ) 2.0 g was dissolved in 50 mL of ion exchange water to obtain a solution. To the obtained solution, 0.05 g of potassium persulfate was added, and the temperature was raised to 80 ° C. while stirring in a nitrogen atmosphere to polymerize. After 10 hours of polymerization, the entire contents are transferred to a dialysis tube (manufactured by Sanko Junyaku Co., Ltd .: UC C65-50), dialyzed in ion-exchanged water for 12 hours, and a water-soluble polymer having an acrylic anionic group is obtained. Obtained.
A malic acid buffer solution (pH 4.7) containing 1.0% by weight of sodium perchlorate as a chaotropic compound and 2.0% by weight of the water-soluble polymer having an anionic group described above was prepared.
A healthy human blood was measured and a sample containing modified Hb was measured by the same method as in Example 1 except that the obtained electrophoresis apparatus and buffer solution were used. In the measurement of healthy human blood, an electropherogram similar to that shown in FIG. 2 was obtained. In the measurement of the sample containing the modified Hb, an electropherogram similar to that shown in FIG. 3 was obtained.

Example 4
A cross-shaped migration path was prepared on a polydimethylsiloxane microchip (50 mm × 75 mm × 3 mm), and buffer baths were installed at both ends of the migration path.
The migration path was coated in the same manner as in Example 1 except that chitosan (0.5 wt% 0.5N hydrochloric acid solution) which is an ionic polymer was used.
Microchip electrophoresis was performed at 1000 V using malic acid buffer (pH 4.8) containing 5.0% by weight of urea as a chaotropic compound and 2.0% by weight of chondroitin sulfate as a water-soluble polymer having an anionic group. Except for the above, by the same method as in Example 1, the blood of a healthy person and the sample containing the modified Hb were measured.
FIG. 4 is an electropherogram obtained in Example 3 by measurement of blood of a healthy person. In FIG. 4, peak 1 indicates stable HbA1c, and peak 2 indicates HbA0. FIG. 5 is a schematic diagram showing an electropherogram obtained by measuring a sample containing modified Hb in Example 3. In FIG. 5, peak 1 shows stable HbA1c, peak 2 shows HbA0, and peak 3 shows modified Hb (unstable HbA1c). As shown in FIG. 5, stable HbA1c and unstable HbA1c, which is a modified Hb, were well separated.
In Example 4, stable HbA1c could be separated in an electrophoresis time of about 60 seconds.

(Comparative Example 1)
After the inside of the capillary was washed by passing 0.2 N-NaOH, ion-exchanged water, and 0.5 N-HCl in this order through a fused silica capillary (GL Science, Inc .: inner diameter 25 μm × total length 30 cm), BSA ( The inner surface of the capillary was coated by passing a malic acid buffer solution (pH 4.7) containing 0.5% by weight of ionic polymer: bovine serum albumin: manufactured by Wako Pure Chemical Industries, Ltd. for 1 minute.
Next, malic acid buffer solution (pH 4.7) containing 0.2% by weight of chondroitin sulfate (manufactured by Wako Pure Chemical Industries, Ltd .: water-soluble polymer having an anionic group) is set at both ends of the capillary, Except that the condition was satisfied, the blood of a healthy person and a sample containing modified Hb were measured in the same manner as in Example 1.
FIG. 6 is an electropherogram obtained by measurement of a sample containing modified Hb in Comparative Example 1.
In FIG. 6, peak 1 shows stable HbA1c, peak 2 shows HbA0, and peak 3 shows modified Hb (unstable HbA1c). As shown in FIG. 6, in the obtained electropherogram, peak 1 indicating stable HbA1c and peak 3 indicating modified Hb overlap.

(Comparative Example 2)
A healthy person's blood and a sample containing modified Hb were measured by the same method as in Example 4 except that the chaotropic compound was not used.
FIG. 7 is an electropherogram obtained by measurement of a sample containing modified Hb in Comparative Example 2.
In FIG. 7, peak 1 shows stable HbA1c, peak 2 shows HbA0, and peak 3 shows modified Hb (unstable HbA1c). As shown in FIG. 7, in the obtained electropherogram, peak 1 indicating stable HbA1c and peak 3 indicating modified Hb did not overlap, but separation was insufficient.

(Evaluation)
(1) Separation performance test of modified Hb Containing modified Hb prepared based on healthy human blood sample and the same healthy human blood as the healthy human blood sample for the measurement conditions of Examples 1 to 4 and Comparative Examples 1 and 2 A sample, ie, a sample containing unstable HbA1c obtained by adding glucose to a healthy human blood prepared in Example 1 (4) measurement of a sample containing modified Hb so as to be 2000 mg / dL; A modified Hb-containing sample, that is, an acetylated Hb-containing sample obtained by adding acetaldehyde to a healthy human blood so as to be 50 mg / dL and a sodium cyanate added so as to be 50 mg / dL. Carbamylated Hb-containing samples were prepared, and the stable HbA1c value in each sample was determined.
The stable HbA1c value of each sample was calculated by determining the ratio (%) of the area of the stable HbA1c peak to the area of all hemoglobin peaks.
A value obtained by subtracting the stable HbA1c value of the obtained healthy human blood sample from the stable HbA1c value of each of the obtained modified Hb-containing samples was determined.
The results are shown in Table 1.

In the measurement conditions of Examples 1 to 4, there is almost no difference in the measured value of stable HbA1c between the modified Hb-containing sample and the healthy human blood sample not containing modified Hb, and the measured value difference is 0.2% or less. It was found that stable HbA1c can be measured accurately even in the presence of modified Hbs.
On the other hand, since the modified Hbs and the stable HbA1c could not be separated under the measurement conditions of Comparative Example 1, the stable HbA1c value could not be calculated. In addition, it was found that the stable HbA1c value fluctuates greatly and accurate measurement cannot be performed under the measurement conditions of Comparative Example 2 because the separation of the modified Hb and the stable HbA1c is insufficient.

(2) Simultaneous reproducibility test Using the measurement conditions of Examples 1 to 4 and Comparative Example 2, the CV value of the stable HbA1c value obtained by continuously obtaining the same sample of a healthy human blood sample 10 times was calculated.
The CV value was obtained by calculating (standard deviation / average value).
The results are shown in Table 2.

(3) Durability test Using the measurement conditions of Examples 1 to 4 and Comparative Example 2, the maximum and minimum stable HbA1c values obtained by continuously measuring the same sample of a healthy human blood sample 100 times The difference (R value) from the value was calculated.
The results are shown in Table 2.

As shown in Table 2, in Examples 1 to 4, in the simultaneous reproducibility test, the CV value indicating the degree of variation was around 1%, which was a good result. Further, the variation width at the time of continuous 100 sample measurement in the durability test was also very small, about 0.2%, and it was found that the stable HbA1c can be measured with high accuracy even in continuous measurement of a large number of samples.
On the other hand, in Comparative Example 2, the CV value in the simultaneous reproducibility test is as large as 3% or more, and the variation width in the durability test is about 0.5% at the maximum, so that the HbA1c value of the diabetic patient is managed. It was completely inadequate.

ADVANTAGE OF THE INVENTION According to this invention, the measuring method of hemoglobin which can perform the measurement of hemoglobin, especially stable hemoglobin A1c used as a diagnostic index of diabetes with high precision in a short time can be provided.

It is a schematic diagram which shows an example of the electrophoresis apparatus used for the measuring method of hemoglobin of this invention. In Examples 1, 2, and 3, it is the electropherogram obtained by the measurement of the blood of a healthy person. In Examples 1, 2, and 3, it is the electropherogram obtained by the measurement of the sample containing modification Hb. In Example 4, it is the electropherogram obtained by the measurement of the blood of a healthy person. In Example 4, it is the electropherogram obtained by the measurement of the sample containing modification Hb. In Comparative example 1, it is the electropherogram obtained by the measurement of the sample containing modification Hb. In Comparative Example 2, it is an electropherogram obtained by measuring a sample containing modified Hb.

Explanation of symbols

DESCRIPTION OF SYMBOLS 11 Capillary electrophoresis apparatus 12 Anode tank 13 Cathode tank 14 Capillary 15 High voltage power supply 16 Detector 17 Electrode 18 Electrode 1 Peak of stable HbA1c 2 Peak of HbA0 3 Peak of modified Hb (unstable HbA1c)

Claims (4)

A method for measuring stable hemoglobin A1c by using capillary electrophoresis or microchip electrophoresis , and an inner surface of the electrophoresis path that is coated with an ionic polymer, and the inside of the electrophoresis path is filled during electrophoresis A method for measuring stable hemoglobin A1c, wherein a buffer solution containing a chaotropic compound is used. The method for measuring stable hemoglobin A1c according to claim 1, wherein the buffer further contains a water-soluble polymer having an anionic group. 3. The method for measuring stable hemoglobin A1c according to claim 1, wherein the coating method of the inner surface of the migration path of the ionic polymer is an immobilized coating method . The method for measuring stable hemoglobin A1c according to claim 2 or 3, wherein the anionic group of the water-soluble polymer is a sulfonic acid group.

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