JP4912867B2 - Method for measuring hemoglobins - 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 that it has hardly been applied to diabetes diagnosis at present.

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 making a diagnosis of diabetes, among HbA1c, stable HbA1c that is an index of diabetes must be separated 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.

On the other hand, for example, Patent Document 3 discloses a method called electrochromatography in which electrophoresis is performed by filling a capillary for liquid chromatography into a capillary. According to this method, when a measurement substance that is difficult to separate by a simple electrophoresis method is separated, the interaction between the sample and the packing material is used for separation by two actions of electrophoresis and chromatography. It becomes possible to do.
However, in the method using electrochromatography disclosed in Patent Document 3, proteins such as hemoglobins are non-specifically adsorbed on the migration path, so that it is difficult to separate the components of hemoglobins. In particular, in the measurement of hemoglobins that require high-resolution separation within a short time, it is difficult to separate stable HbA1c within the condition setting range of the disclosed technology related to electrochromatography including Patent Document 3. Thus, the separation performance and measurement accuracy at a level capable of managing the blood glucose level of diabetic patients were not obtained.
Japanese Translation of National Publication No. 09-510792 Japanese Patent Laid-Open No. 09-105739 JP 2006-159148 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 is a method for measuring hemoglobin using a capillary electrophoresis method or a microchip electrophoresis method, the inner surface of which is coated with a hydrophilic polymer, and the inside is made of particles that are insoluble in a buffer solution. This is a method for measuring hemoglobin using a migration path filled with an ion exchanger.
The present invention is described in detail below.

As a result of intensive studies, the present inventors have conducted stable electrophoresis, which has been difficult to measure conventionally by performing electrophoresis using a migration path whose inner surface is coated with a hydrophilic polymer and which is filled with an ion exchanger. With respect to the mold HbA1c, it has been found that high-precision measurement can be performed in a short time at a low cost, 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 hemoglobin measurement method of the present invention, the inside of the capillary 14 that is the migration path is filled with an ion exchanger.
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 a hydrophilic polymer and filled with an ion exchanger.
By using such a migration path, it is possible to measure hemoglobins, particularly stable HbA1c, which has conventionally been difficult to separate, with high accuracy in a short time.

The electrophoresis path refers to a site (from an injected site to a detected site) where a 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 means from the part where the sample in the microchip groove in the type electrophoresis apparatus is injected to the part 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.

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.

The inner surface of the migration path used in the present invention is coated with a hydrophilic polymer.
The coating layer formed on the inner surface of the migration path may be a single layer or a multilayer made of the same or different materials.

By coating with a hydrophilic polymer in this way, hydrophilicity is imparted to the inner surface of the migration path, and nonspecific adsorption of each component such as protein and lipid in human blood that is a sample that passes through the interior of the migration path. It is possible to sufficiently separate and measure hemoglobin as a measurement target. If the hydrophilicity is insufficient, each component in the sample may cause nonspecific adsorption on the inner surface of the migration path, which may adversely affect the separation of each component.
In this specification, the hydrophilic polymer refers to a polymer having a hydrophilic functional group.

Although it does not specifically limit as said hydrophilic polymer, For example, an ionic or nonionic polymer is mentioned. Specifically, for example, nonionic polymers such as polyethylene glycol and polyvinyl alcohol; 2-hydroxyethyl (meth) acrylate (acrylate or methacrylate is also referred to as (meth) acrylate hereinafter), 2-hydroxypropyl (meth) acrylate, Glycerol (meth) acrylate; nonionic polymer obtained by polymerizing nonionic monomers such as glycidyl (meth) acrylate, polyethylene glycol (meth) acrylate, polypropylene glycol (meth) acrylate; starch, dextran, agarose, mannan, etc. Nonionic polysaccharides and the like.

The ionic polymer is not particularly limited, and examples thereof include ionic polysaccharides containing a cationic group such as N-substituted cellulose such as chitin, chitosan, aminocellulose, N-methylaminocellulose; dextran sulfate, chondroitin sulfate, heparin. Ionic polysaccharides containing an anionic group such as heparan, fucoidan, alginic acid and pectic acid; proteins such as albumins, lactoferrin and skim milk; and salts or derivatives of these substances.

The other ionic polymer is not particularly limited, and examples thereof include cationic group-containing polymers such as polyethyleneimine, polybrene, poly (2-diethylaminoethyl (meth) acrylate), and copolymers containing these polymers; Anionic group-containing polymers such as (meth) acrylic acid, poly (2-acrylamido-2-methylpropanesulfonic acid), phosphoric acid group-containing (meth) acrylic acid ester polymer, sulfonic acid group-containing (meth) acrylic acid polymer , And copolymers containing these polymers.

The hydrophilic polymer may be used as a mixture of plural kinds or as a copolymer with other monomers.
A preferred lower limit of the weight average molecular weight of the hydrophilic 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 hydrophilic polymer on the migration path is not particularly limited, and a conventionally known method can be used. For example, by passing a solution containing the hydrophilic polymer into the migration path. Dynamic coating method for coating; Immobilization coating method in which the hydrophilic polymer is brought into contact with the inner surface of the migration path and physically adsorbed and immobilized using hydrophobic or electrostatic interaction, etc. Examples thereof include an immobilization coating method in which the inner surface and the hydrophilic polymer are bonded and fixed by a covalent bond via a functional group of each substance or other substances. In the case of using the above-described immobilizing coating method, the coating layer does not peel off through a heating step, a drying step, and the like, and therefore, repeated measurement is possible. Specifically, the immobilization coating method includes, for example, passing a solution containing the hydrophilic polymer through the migration path, injecting air into the migration path, and then drying the solution, followed by drying. And the like.

Among the coating methods, the immobilization coating method is particularly 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 hydrophilic polymer in the migration path, another type of layer may be formed between the inner surface of the migration path and the layer made of the hydrophilic polymer. When the different kind of layer is formed, the layer made of the hydrophilic polymer only needs to be formed on the innermost surface of the migration path.

When the hydrophilic polymer is coated on the migration path, the hydrophilic polymer is preferably supplied to the coating as a solution containing the hydrophilic polymer, depending on the type and the coating method.
As a concentration of the hydrophilic 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 hydrophilic polymer cannot be formed uniformly and may be peeled off during the measurement of hemoglobin, which may cause a decrease in reproducibility.

In addition to the hydrophilic polymer coating on the inner surface, the migration path is filled with an ion exchanger. In this specification, an ion exchanger refers to an insoluble carrier having an ion exchange group.

The ion exchanger may be filled in the entire inside of the migration path, or may be filled only in a part of the inside of the migration path.
The hemoglobins in the sample are introduced into the migration path filled with the ion exchanger, and then separated into each component by contacting with the ion exchanger while moving by electrophoresis. Therefore, each separated component can be detected by the detector.

The ion exchanger is not particularly limited, and examples thereof include insoluble polymers having ion-exchange functional groups.
The ion exchange functional group is not particularly limited, and examples thereof include a cation exchange group such as a carboxyl group, a phosphate group, and a sulfonate group; and an anion exchange group such as an amino group.

The skeleton of the polymer having an ion-exchange functional group is not particularly limited, and examples thereof include organic polymers such as organic synthetic polymers and polysaccharides; inorganic polymers such as silica and ceramics, and the like. It is done.
These ion exchangers may be particles that are insoluble at the time of electrophoresis, and may be crosslinked or non-crosslinked.
These ion exchangers may be a polymer obtained by polymerizing a monomer having the ion-exchange functional group. After the polymer is prepared, the ion-exchange functional group is introduced. It may be a thing.
In the method for measuring hemoglobin of the present invention, since blood is used as a sample, it is necessary to suppress non-specific adsorption of components in the sample to the ion exchanger. Therefore, these ion exchangers have hydrophilic properties. It is preferable to have. Specifically, for example, an ion exchanger made of a hydrophilic material, or an ion exchanger made of a hydrophilized polysaccharide, an organic synthetic polymer, silica or the like is preferable, and a hydrophilic organic synthetic polymer is more preferable. preferable.

The 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 thereof and derivatives thereof, and insolubilized products such as cationic group-containing polysaccharides such as salts thereof. In addition, sulfonic acid group-containing polysaccharides such as chondroitin sulfate, dextran sulfate, heparin, heparan, fucoidan, and salts thereof; carboxyl group-containing polysaccharides such as alginic acid and pectic acid; and salts thereof; cellulose, dextran, agarose, Examples thereof include neutral polysaccharides such as mannan and starch, an anionic group-introduced product thereof, and insolubilized products such as known anionic group-containing polysaccharides such as salts thereof.
You may use the said polysaccharide which has the said ion exchange property, and neutral polysaccharides, such as starch, dextran, agarose, a mannan, as a mixture or a conjugate | bonded material.

Although it does not specifically limit as said organic synthetic polymer, For example, what is obtained by polymerizing the monomer which has an ion exchange group independently, or copolymerizing with the other hydrophilic monomer which does not have an ion exchange group etc. Can be mentioned. For example, an acrylic polymer obtained by polymerization from an acrylic monomer is preferable.

The monomer having an ion exchange group is not particularly limited, and examples thereof include (meth) acrylic acid having a carboxyl group, 2-acrylamido-2-methylpropanesulfonic acid having a sulfonic acid group, and 2-diethylaminoethyl having an amino group. Examples include those obtained by polymerizing (meth) acrylate and glycidyl (meth) acrylate and then substituting the glycidyl group with a carboxyl group, a sulfonic acid group, or the like. Further, polyethyleneimine having an amino group, insolubilized polybrene, and the like can also be used.

The hydrophilic monomer having no ion exchange group is not particularly limited. For example, 2-hydroxyethyl (meth) acrylate, polyethylene glycol di (meth) acrylate, polymethylolalkane poly (meth) acrylate, (meth) acrylamide, A glycidyl (meth) acrylate etc. are mentioned. Insolubilized materials such as polyvinyl alcohol, polyvinyl pyrrolidone, and polyethylene glycol can also be used.

The shape of the ion exchanger is not particularly limited, and examples thereof include a spherical shape and a crushed shape. Among these, a spherical shape is preferable, and a true sphere or a shape close to a true sphere is more preferable.

The size of the ion exchanger is not particularly limited as long as it can be filled in the migration path, but the preferable lower limit of the diameter is 0.1 μm and the preferable upper limit is 30 μm. If it is less than 0.1 μm, the sample is difficult to move because the gap is too small, and sufficient migration may not be possible. If it exceeds 30 μm, the voids are too large and the mutual adoption of the sample and the ion exchanger becomes insufficient, so that the separation performance may deteriorate.
A more preferable lower limit is 1 μm, and a more preferable upper limit is 20 μm.

In the method for measuring hemoglobin according to the present invention, electrophoresis is carried out using a migration path coated with the inner surface and filled with an ion exchanger, and the migration path is filled with a buffer solution.
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 chaotropic compounds, surfactants, various polymers, and hydrophilic low molecular compounds may be appropriately added to the buffer solution.

Various polymers to be contained in the buffer solution are not particularly limited. For example, a water-soluble polymer having an ionic group is preferable. The water-soluble polymer having an ionic group is a polymer having an ionic functional group in the molecule, and is roughly classified into a polymer having an anionic group and a polymer having a cationic group depending on the ionic type. The Of these, a water-soluble polymer having an anionic group is preferred.

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.

The water-soluble polymer having an anionic group is not particularly limited, and a known polymer can be used. For example, a polysaccharide having an anionic group and a water-soluble organic synthetic polymer having an anionic group can be used. preferable.

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 an acrylic polymer; that is, acrylic acid or methacrylic acid and derivatives thereof. Polymers mainly composed of alcohols and esters are 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.

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 with high accuracy. 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) Preparation of ion exchanger To 2.0 L of 3% aqueous polyvinyl alcohol solution, 300 g of triethylene glycol dimethacrylate (nonionic monomer: manufactured by Shin-Nakamura Chemical Co., Ltd.), diethylaminoethyl methacrylate (monomer having an amino group: Jun Wako) A mixture of 100 g (manufactured by Yakuhin Co., Ltd.) and 1.0 g of benzoyl peroxide (polymerization initiator: manufactured by Nacalai Tesque) was added and heated to 80 ° C. in a nitrogen atmosphere while stirring, and polymerized for 12 hours Went. The obtained polymer was washed and recovered to obtain an ion exchanger having an amino group.

(2) Preparation of migration path A fused silica capillary (inner diameter 100 μm × total length 300 mm: manufactured by GL Science) was passed through 0.2N-NaOH, ion-exchanged water, and 0.5N-HCl in this order to wash the inside of the capillary. Thereafter, a 0.5% polymethacrylic acid aqueous solution (hydrophilic polymer) was passed through for 20 minutes. Thereafter, air was injected into the capillary to drive out the aqueous polyvinyl alcohol solution, and then the product was dried in a dryer at 40 ° C. for 12 hours. Thereafter, a series of steps consisting of injection of an aqueous polyvinyl alcohol solution, injection of air, and drying was repeated 5 times to obtain a capillary coated with polymethacrylic acid. The obtained capillary was filled with the ion exchanger having an amino group obtained in the preparation of the above-mentioned (1) ion exchanger, and set in a capillary electrophoresis apparatus (PAC / E MDQ manufactured by Beckman Coulter). .

(3) Blood obtained by collecting heparin from healthy human blood is used as a measurement sample of healthy human blood, and 0.05% Triton X-100 (surfactant: manufactured by Wako Pure Chemical Industries, Ltd.) is included in 70 μL of whole healthy human blood. A solution obtained by adding 200 μL of citrate buffer (pH 6.2) and performing hemolysis dilution was used.
A capillary and an electrode tank at both ends of the capillary are filled with a phosphate buffer (pH 7.6), a sample is injected from one end of the capillary, and electrophoresis is performed by applying a voltage of 20 kV to the buffer at both ends of the capillary. Was measured by capillary electrophoresis of stable HbA1c in the blood of healthy humans.
FIG. 2 is a schematic diagram showing an electropherogram obtained by measurement of blood of a healthy person. In FIG. 2, peak 1 indicates stable HbA1c, and peak 2 indicates HbA0. The stable HbA1c could be separated with an electrophoresis time of about 60 seconds.

(4) Measurement of sample containing modified Hb Glucose is added to whole blood of healthy human blood collected from heparin to 2500 mg / dL, and a sample containing a large amount of unstable HbA1c, which is a type of modified Hb, is artificially prepared. did.
The sample was injected from one of the capillaries, and the stable HbA1c in the modified Hb-containing sample was measured by capillary electrophoresis using the same conditions as in (3) Measurement of healthy human blood.
FIG. 3 is a schematic diagram showing an electropherogram obtained by measuring 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)
(1) Preparation of ion exchanger To 2.0 L of 4% aqueous polyvinyl alcohol solution, 300 g of tetraethylene glycol dimethacrylate (nonionic monomer: Shin-Nakamura Chemical Co., Ltd.), 50 g of tetramethylolethane triacrylate (nonionic monomer: new) Nakamura Chemical Co., Ltd.) and a mixture of 1.0 g of benzoyl peroxide were added, and the temperature was raised to 80 ° C. in a nitrogen atmosphere while stirring to perform polymerization for 1 hour. To this polymerization system, 200 mL of a 50% aqueous solution of 2-acrylamido-2-methylpropanesulfonic acid (monomer having a sulfonic acid group: manufactured by Toagosei Co., Ltd.) was added, and polymerization was further performed for 2 hours. The polymer obtained was washed and recovered to obtain an ion exchanger having a sulfonic acid group.

(2) Preparation of migration path The migration path was prepared in the same manner as in Example 1 except that a 0.5N hydrochloric acid solution containing 0.5% by weight of chitosan (chitosan-100: manufactured by Wako Pure Chemical Industries, Ltd.) was used. Coating was performed. The obtained inner surface-coated capillary was filled with the ion exchanger having a sulfonic acid group obtained by the preparation of the above-mentioned (1) ion exchanger, and then a capillary electrophoresis apparatus (PAC / E MDQ manufactured by Beckman Coulter) Set.

(3) Measurement and modification of healthy human blood by the same method as in Example 1 except that citrate buffer (pH 4.7) was used as a measurement buffer for a sample containing modified Hb. A sample containing Hb was measured.
FIG. 4 is a schematic diagram showing an electropherogram obtained 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 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.

(Example 3)
(1) Preparation of ion exchanger To 2.0 L of 4% aqueous polyvinyl alcohol solution, 300 g of tetraethylene glycol dimethacrylate (nonionic monomer: manufactured by Shin-Nakamura Chemical Co., Ltd.), 50 g of 2-hydroxymethyl methacrylate (nonionic monomer: new) Nakamura Chemical Co., Ltd.), diethylaminoethyl methacrylate (monomer having amino group: Wako Pure Chemical Industries, Ltd.) 100 g and a mixture of 1.0 g of benzoyl peroxide were added, and the temperature was raised to 80 ° C. in a nitrogen atmosphere while stirring. For 10 hours. The obtained polymer was washed and recovered to obtain an ion exchanger having an amino group.

(2) Preparation of migration path An electrophoresis path having a width of 100 μm and a length of 70 mm was formed by forming a cross-shaped groove on a microchip for electrophoresis (50 mm × 100 mm) made of polydimethylsiloxane. The formed migration path was coated with a chitosan solution in the same manner as in Example 2, and further filled with an ion exchanger having an amino group obtained by the preparation of the above (1) ion exchanger.

(3) Measurement of healthy human blood and measurement of sample containing modified Hb A citrate buffer solution (pH 4.7) containing 2.0% by weight of chondroitin sulfate was used, and electrophoresis was performed at a voltage of 1000V. Except for the above, the measurement of healthy human blood and the sample containing modified Hb were performed in the same manner as in Example 1.
In the measurement of healthy human blood, an electropherogram similar to that shown in FIG. 4 was obtained. In the measurement of the sample containing the modified Hb, an electropherogram similar to that shown in FIG. 5 was obtained.

(Comparative Example 1)
After washing the inside of the capillary by passing 0.2N-NaOH, ion-exchanged water, and 0.5N-HCl in this order through a capillary made of fused silica (GL Science Co., Ltd .: inner diameter 25 μm × total length 30 cm) The inner surface of the capillary was dynamically coated by passing a malic acid buffer containing 0.5% by weight of BSA (cationic polymer: bovine serum albumin: manufactured by Wako Pure Chemical Industries, Ltd.) for 1 minute. Subsequently, malic acid buffer solution (pH 4.7) containing 0.2% by weight of chondroitin sulfate (manufactured by Wako Pure Chemical Industries, Ltd .: polymer having an anionic group) without filling the capillary with the filler was added to the above-mentioned capillary. Then, the sample containing the modified Hb was measured by the same method 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. 7, peak 1 indicating stable HbA1c overlaps peak 3 indicating modified Hb.

(Evaluation)
The samples obtained in Examples 1 to 3 and Comparative Example 1 were evaluated as follows.

(1) Separation performance test of modified Hb For Examples 1 to 3 and Comparative Example 1, a sample of a healthy human blood and a modified Hb-containing sample prepared based on the same healthy human blood as the healthy human blood sample, Example 1 (4) An unstable HbA1c-containing sample obtained by adding glucose to a healthy human blood prepared in the measurement of a sample containing modified Hb so as to have a concentration of 2000 mg / dL, and another modified Hb-containing sample, That is, an acetylated Hb-containing sample obtained by adding acetaldehyde to healthy human blood to 50 mg / dL, and a carbamylated Hb-containing sample obtained by adding sodium cyanate to 50 mg / dL 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 the obtained modified Hb-containing sample was determined.
The results are shown in Table 1.

In the measurement conditions of Examples 1 to 3, there is almost no difference in the measured value of the stable HbA1c between the modified Hb-containing sample and the healthy human blood sample not containing the 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, under the measurement conditions of Comparative Example 1, it was found that the stable HbA1c value greatly fluctuated because the modified Hbs and the stable HbA1c were not sufficiently separated, and accurate measurement was not possible.

(2) Simultaneous reproducibility test Using the measurement conditions of Examples 1 to 3 and Comparative Example 1, 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).
Further, in Comparative Example 1, when one measurement was completed, 0.2N-NaOH, ion-exchanged water, and 0.5N-HCl were passed in this order to wash the inside of the capillary, and then BSA ( A 0.5 wt% malic acid buffer solution of cationic polymer: bovine serum albumin (manufactured by Wako Pure Chemical Industries, Ltd.) is fed for 1 minute to apply dynamic coating, and then the measurement is repeated by measuring the next sample. A simultaneous reproducibility test was performed.
The results are shown in Table 2.

(3) Durability Test Using the measurement conditions of Examples 1 to 3 and Comparative Example 2, the maximum and minimum values of 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 3, the CV value indicating the degree of variation in the simultaneous reproducibility test 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 at about 0.2%, and it was found that 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 1.2% at the maximum, so that the HbA1c value of diabetic patients can be 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 using the measuring method of hemoglobin of this invention. In Example 1, it is the electropherogram obtained by the measurement of the blood of a healthy person. In Example 1, it is the electropherogram obtained by the measurement of the sample containing modification Hb. In Example 2 and 3, it is the electropherogram obtained by the measurement of the blood of a healthy person. In Example 2 and 3, 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.

Explanation of symbols

1 Peak of stable HbA1c 2 Peak of HbA0 3 Peak of modified Hb (unstable HbA1c)

Claims (2)

A method of measuring hemoglobin using capillary electrophoresis or microchip electrophoresis, wherein an ion exchanger comprising an inner surface coated with a hydrophilic polymer and consisting of particles insoluble in a buffer solution is provided. A method for measuring hemoglobin, which comprises using a packed migration path. Moreover, it filled inside the migration path during electrophoresis, the method for measuring hemoglobin according to claim 1, characterized by using a buffer solution containing a water-soluble polymer having an anionic group.

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