JP4330534B2 - Glycated protein measurement method - Google Patents

Description

  The present invention relates to a method for measuring glycated protein and hemoglobin A1c, which are used as a marker for diagnosis of diabetes in the field of clinical examination. More specifically, the present invention relates to a method for quantifying glycated protein and hemoglobin A1c based on antagonistic inhibition of fructosylamine oxidation reaction by fructosylamine oxidation catalyst by glycated protein and hemoglobin A1c. Furthermore, the present invention relates to a glycated protein, a hemoglobin A1c measurement kit and a sensor constructed based on the method of the present invention.

The amino group of the protein main chain and the side chain is non-enzymatically linked to the reducing end of a reducing sugar such as glucose to produce an Amadori compound, that is, a glycated protein. In blood, it is known that hemoglobin is saccharified to produce glycated hemoglobin (glycohemoglobin; HbA1c). Since the abundance ratio of HbA1c to hemoglobin is higher in diabetic patients than in healthy individuals, and the blood concentration of HbA1c reflects the blood glucose level in the past several weeks, the blood concentration of HbA1c depends on the diagnosis of diabetes and the blood glucose level of the diabetic patient. As an index of control, it is extremely important in clinical trials.
So far, enzymes that act on Amadori compounds, fructosylamine oxidase, have been isolated from various strains, and hydrolyzed products of glycated proteins such as glycated albumin and HbA1c have been analyzed using these enzymes. (JP-A-61-268178, JP-A-61-2280297, JP-A-3-155780, JP-A-5-192193, JP-A-7-289253, JP-A-8-154672, Agric. Biol. Chem., 53 (1), 103-110, 1989, Agric.Biol.Chem., 55 (2), 333-338, 1991, J. Biol.Chem., 269 (44), 27297-27302, 1994, Appl.Environ.Microbiol., 61 (12), 4487-4489, 995, Biosci. Biotech. Biochem., 59 (3), 487-491, 1995, J. Biol. Chem., 270 (1), 218-224, 1995, J. Biol. Chem., 271 (51), 32803-32809, 1996, J. Biol. Chem., 272 (6), 3437-3443, 1997).
In addition, various synthetic polymers having a catalytic action to oxidize fructosylamine have been synthesized, and it is suggested that hydrolyzed products of glycated proteins such as glycated albumin and HbA1c can be analyzed using these catalysts. (WO01 / 90735 WO02 / 22698), Anal. Chim. Acta 435, 151-156, 2001).
However, the fructosylamine oxidation catalysts reported so far cannot oxidize glycated proteins that have not been hydrolyzed as substrates. For this reason, the glycated protein was measured by first hydrolyzing the glycated protein and oxidizing the resulting low molecular weight glycated peptide or fructosylamine using a fructosylamine oxidation catalyst. Therefore, in the said technical field, the method of measuring without decomposing | disassembling glycated protein was desired.
An object of the present invention is to provide a novel method for measuring a glycated protein, in which the concentration of glycated protein and hemoglobin A1c is measured without hydrolysis. More specifically, the present invention provides a method for measuring a glycated protein, particularly hemoglobin A1c (HbA1c) or glycated albumin, which is used in fields such as clinical tests, and a measuring reagent and sensor constructed based on the method. Objective.

The present inventor has found that a glycated protein can antagonistically inhibit fructosylamine oxidation reaction by a fructosylamine oxidation catalyst, and has completed the present invention. That is, the present invention is a method for measuring a glycated protein in a sample, wherein the sample is brought into contact with a fructosylamine oxidation catalyst in the presence of a certain amount of fructosylamine, and the fructosylamine oxidized by the catalyst is used. By measuring the amount of glycated protein, the method comprises measuring the amount of the glycated protein. According to the present invention, it is possible to measure a glycated protein using hydrolysis inhibition of fructosylamine oxidation catalyst as an index without hydrolyzing the glycated protein.
In the present specification, the “fructosylamine oxidation catalyst” refers to a catalyst that catalyzes the oxidation reaction of fructosylamine and that the reaction is inhibited in a concentration-dependent manner in the presence of a glycated protein. Examples of the fructosylamine oxidation catalyst include fructosylamine oxidase and a polymer that catalyzes the oxidation of fructosylamine in the presence of an electron acceptor, such as an imidazole group-containing polymer.
In a preferred embodiment, the present invention provides a method for measuring a glycated protein, wherein the fructosylamine oxidation catalyst described above is a molecule having an imidazole group. In a preferred embodiment, the present invention provides a method for measuring a glycated protein, wherein the fructosylamine oxidation catalyst described above is a fructosylamine oxidase. More preferably, the fructosylamine oxidation catalyst is selective for fructosyl valine.
Preferably, in the method of the present invention, the step of measuring the amount of fructosylamine is carried out by spectroscopically measuring the amount of electron acceptor that has been reduced as a result of oxidation of fructosylamine. Preferably, in the method of the present invention, the step of measuring the amount of fructosylamine is performed by electrochemically measuring the amount of electron acceptor reduced with the oxidation of fructosylamine using an electrode. Done.
Furthermore, the present invention provides a method for measuring a glycated protein, characterized in that the glycated protein described above is hemoglobin A1c.
The present invention also provides a glycated protein measurement kit based on the principle described above. Preferably, the kit includes a fructosylamine oxidation catalyst and fructosylamine. The present invention also provides a hemoglobin A1c measurement kit based on the principle described above.
Furthermore, the present invention provides a sensor for measuring glycated protein based on the principle described above. The present invention also provides a sensor for measuring hemoglobin A1c based on the principle described above.

FIG. 1 shows a sensor system constructed in the second embodiment.
FIG. 2 shows a decrease in steady-state current value when the HbA1c sample is added to the sensor system constructed in Example 2.
FIG. 3 shows the sample concentration dependence of the decrease in steady-state current when various proteins and HbA1c were added to the sensor shown in Example 2 in the presence of fructosyl valine.
FIG. 4 shows the results of measuring HbA1c using the sensor system created in Example 2.
FIG. 5 shows the results of measuring HbA1c using the sensor system created in Example 5.
DETAILED DESCRIPTION OF THE INVENTION The method for measuring glycated protein of the present invention is based on the discovery that glycated protein can antagonistically inhibit fructosylamine oxidation reaction by fructosylamine oxidation catalyst. That is, when a sample containing glycated protein is brought into contact with a fructosylamine oxidation catalyst in the presence of a certain amount of fructosylamine, the amount of fructosylamine oxidized by the fructosylamine oxidation catalyst depends on the concentration of glycated protein. And change. Therefore, the concentration of glycated protein in the sample can be determined by measuring the amount of oxidized fructosylamine or the oxidation rate.
As the fructosylamine oxidation catalyst, for example, a synthetic polymer having an imidazole group or fructosylamine oxidase can be used, as long as it has a fructosylamine oxidation activity and is subjected to competitive inhibition by a glycated protein. It is not limited. For example, not only imidazole, but also a polymer polymerized using a monomer containing a functional group that functions as a general base catalyst, such as bipyridyl containing pyridine, can be used.
The method of the present invention is particularly useful for measuring HbA1c. Since HbA1c has a fructosyl valine moiety in its molecule and acts antagonistically against the fructosyl amine selective catalyst for fructosyl valine, it is specifically selected according to the method of the present invention. Can be quantified. As such a catalyst, for example, a synthetic polymer having an imidazole group synthesized by allowing fructosyl valine as a template during polymerization, or fructosyl amine oxidase selective for fructosyl valine is used. However, the catalyst is not limited as long as it has a fructosyl valine oxidation activity and undergoes competitive inhibition by HbA1c. For example, not only fructosyl valine but also a catalyst polymer having an imidazole group synthesized using methyl valine, which is an analog, as a template can be used as a catalyst.
The measurement of the glycated protein in the present invention can be easily performed by measuring the amount of the electron acceptor that is reduced by the fructosylamine oxidation catalyst by the oxidation of fructosylamine or the reduction rate. For example, a sample is added to a solution containing a certain amount of fructosylamine oxidation catalyst and fructosylamine, and an electron acceptor, and the amount or rate of reduction of the electron acceptor as the fructosylamine is oxidized is measured. By comparing the amount of electron acceptor in the absence of the sample or the reduction rate, the glycated protein in the sample can be measured. As a method for measuring the electron acceptor, a method of measuring by a spectroscopic method based on a characteristic spectrum of the reduced state of the electron acceptor, or a method of reoxidizing the electron acceptor in the reduced state on the electrode can be obtained. An electrochemical method or the like by measuring current can be used.
When phenazine methosulfate (PMS) and dichlorophenolindophenol (DCIP) are used as electron acceptors and fructosyl valine is used as a substrate, fructosyl valine is oxidized with time, and DCIP is converted via PMS. It is observed that it is reduced and faded. The rate of this DCIP fading is proportional to the concentration of fructosyl valine present. Thus, for example, when HbA1c is present, the oxidation of fructosyl valine is antagonistically inhibited, resulting in a decrease in the rate of fading of DCIP. The concentration of HbA1c can be measured using the rate of decrease in the fading rate as an index. That is, according to the method of the present invention, an unknown concentration of HbA1c can be quantified with a sensitivity of 10 nM or less in the presence of PMS and DCIP as electron acceptors. Further, various artificial electron acceptors may be used. As the electron acceptor, potassium ferricyanide, ferrocene, osmium derivatives and the like can be used.
On the other hand, when fructosylamine oxidase is used as a catalyst, oxygen can be used as the electron acceptor in addition to the electron acceptor. In this case, by using the amount of oxygen decrease / decrease rate or the amount of hydrogen peroxide generated / production rate as an index, inhibition of the enzyme by the glycated protein can be measured to measure the glycated protein concentration.
In another aspect of the assay kit , the present invention features a kit for measuring a glycated protein such as HbA1c or glycated albumin, which includes a measuring method using the principle of the present invention. The glycated protein measurement kit of the present invention contains the fructosylamine oxidation catalyst according to the present invention, fructosylamine and an electron acceptor in an amount sufficient for at least one assay. Typically, the kit comprises a fructosylamine oxidation catalyst and a buffer adjusted to pH 6.0-10, an appropriate mediator, a standard solution of a glycated protein or derivative thereof for the creation of a calibration curve, and directions for use. including. The glycated protein analysis kit according to the present invention can be provided in various forms, for example, as a lyophilized reagent or as a solution in a suitable storage solution.
In another aspect according to the sensor , the present invention features a sensor for measuring a glycated protein such as glycated albumin or HbA1c. The glycated protein sensor of the present invention electrochemically oxidizes a mediator reduced as fructosylamine is oxidized by a fructosylamine oxidation catalyst on an electrode, and measures the current value at this time. In the presence of glycated protein, the oxidation reaction is antagonistically inhibited depending on the concentration of glycated protein, so current values are measured in the presence of various concentrations of glycated protein to create a standard curve. Based on this, the glycated protein concentration in the sample can be determined. A carbon electrode, a gold electrode, a platinum electrode, or the like is used as an electrode, and a fructosylamine oxidation catalyst is immobilized on this electrode. Examples of immobilization methods include a method using a crosslinking reagent, a method of encapsulating in a polymer matrix, a method of coating with a dialysis membrane, a method of using a photocrosslinkable polymer, a conductive polymer, and a redox polymer. May be used.
In accordance with the measurement principle of the present invention, a sensor for measuring HbA1c can be constructed as follows. When amperometric measurement is performed using a carbon electrode, a gold electrode, a platinum electrode, etc., an electrode on which a fructosylamine oxidation catalyst is immobilized is used as a working electrode, and a counter electrode (for example, a platinum electrode) and a reference electrode (for example, (Ag / AgCl electrode) is inserted into a buffer containing a mediator and kept at a constant temperature. A constant voltage is applied to the working electrode, a certain amount of fructosylamine and a sample are added, and the increase in current is measured. As the mediator, potassium ferricyanide, ferrocene, osmium derivatives, phenazine methosulfate, and the like can be used.
Furthermore, as a method for measuring by an amperometric system using a carbon electrode, a gold electrode, a platinum electrode, etc., there is a system using an immobilized electron mediator. That is, using a fructosylamine oxidation catalyst and an electrode in which an electron mediator such as potassium ferricyanide, ferrocene, osmium derivative, or phenazine methosulfate is immobilized on a polymer matrix by adsorption or covalent bonding as a working electrode, a counter electrode (for example, platinum electrode) Along with the reference electrode (eg, Ag / AgCl electrode), it is inserted into a buffer solution and kept at a constant temperature. A constant potential is applied to the working electrode, a certain amount of fructosylamine and a sample are added, and the increase in current is measured.
Regardless of which electrode is used, the concentration of glycated protein in a sample can be determined according to calibration curves prepared using solutions of various concentrations of glycated protein.
The contents of all patents and references explicitly cited herein are hereby incorporated by reference as part of the present specification. In addition, the contents described in the specification and drawings of Japanese Patent Application No. 2002-319040, which is the application on which the priority of the present application is based, are cited herein as part of the present specification.

  The present invention will be described in more detail with reference to the following examples, but these examples do not limit the scope of the present invention.

  1.6 mmol of 1-vinylimidazole, 0.8 mmol of 4-vinylphenylboronic acid, 2 mmol of ethylene glycol dimethacrylate (EGDMA) as a crosslinking reagent, and 0.06 mmol of 2,2′-azobisisobutyronitrile as a polymerization initiator Then, 0.2 mmol of fructosyl valine was added as a template molecule, 522 μl of methanol and 174 μl of water were added as solvents, and after argon substitution, a polymerization reaction was performed at 45 ° C. for 12 hours. The polymer catalyst obtained here is called BI-MIP. This polymer was ground in a mortar, and a polymer having a particle size of 40 μm was prepared by sieving. Also, BI-CP, which is a control polymer, was synthesized by performing exactly the same operation as above without adding fructosyl valine, which is a template molecule. These polymers were washed twice with methanol: acetic acid (7: 2), twice with acetonitrile: acetic acid (9: 1), once with acetonitrile, and once with methanol.

  20 mg of the polymer catalyst prepared in Example 1 was mixed with 50 mg of the carbon paste, and the carbon paste electrode was filled. This electrode is immersed in a solution of 10 mM phosphate buffered saline (pH 7.4) containing 1 mM 1-methoxyphenazine methosulfate (mPMS), the applied voltage is set to 100 mV (vs. Ag / AgCl), and the substrate is fully charged. Responses were observed when kutosylvaline was added. Furthermore, the sensor system which observes the change of the response value when HbA1c was added here was constructed | assembled (FIG. 1). By adding fructosyl valine, fructosyl valine is oxidized by the polymer catalyst, and mPMS in the solution is reduced accordingly. Since the reductant mPMS is oxidized by the potential applied to the carbon paste electrode, an increase in current is observed. This redox reaction and electrode reaction give a constant steady current value depending on the fructosyl valine concentration in the solution. When a certain amount of HbA1c is dropped here, the steady-state current value decreases (FIG. 2). This is based on the competitive inhibition of the polymer catalyst by HbA1c. Further, when HbA1c is dropped when the steady current value is reached, the steady current value further decreases. That is, depending on the HbA1c concentration, it was shown that the oxidation value of fructosyl valine by the polymer catalyst was inhibited and the current value was lowered.

  For the purpose of investigating the selectivity of the sensor, changes in the sensor response were observed when various proteins were added to the sensor system (FIG. 3). The proteins used as comparison targets are HbA0, bovine serum albumin, and glycated albumin. When any protein was added, the response was reduced to some extent, but this was negligible compared to the decrease when HbA1c was added.

  Based on Example 3, HbA1c was measured using as an index the inhibition of the response to fructosyl valine seen in this sensor system. That is, in this sensor system, the response current value to fructosyl valine 0.1 mM in the absence of HbA1c decreases as the concentration of HbA1c to be added increases. The response current value for fructosyl valine 0.1 mM was subtracted from the non-specific decrease in response value observed in the presence of various proteins from the response current value for fructosyl valine 0.1 mM. The response current value for 0.1 mM fructosyl valine observed in the presence of various concentrations of HbA1c is subtracted from this value, and the difference is the response current for 0.1 mM fructosyl valine in the absence of HbA1c. The ratio to the value was defined as the inhibition rate. FIG. 4 shows the results of measurement of HbA1c with the inhibition rate taken on the vertical axis. Thus, this sensor could be detected with a sensitivity of 10 nM without denaturing or hydrolyzing HbA1c.

The ratio of hemoglobin A1c (HbA1c) and hemoglobin Ao (HbAo) in the sample was measured using a sensor system using a platinum electrode as the working electrode, a platinum wire as the counter electrode, and an Ag / AgCl electrode as the reference electrode. The principle of this measurement is based on the fact that HbA1c inhibits fructosyl valine oxidation by fructosylamine oxidase (FAOD) in a concentration-dependent manner.
A mixture of FAOD 20 mU and HbA1c / HbAo (a total of 0.47 μmol as hemoglobin) was added to 600 μl of 50 mM potassium phosphate buffer, incubated at 8 ° C. for 30 minutes, and then 600 mV vs. A potential of Ag / AgCl was applied and incubated at 25 ° C. for 20 minutes. Next, 0.1 mmol of fructosyl valine was added (total amount 650 μl), and the response current value (IC) was measured. The result of plotting the increased current value against the HbA1c concentration is shown in FIG. This measurement was performed by changing various mixing ratios of HbA1c / HbAo in the HbA1c / HbAo mixture, which is the added sample. As is apparent from the figure, the response current value to fructosyl valine decreases as the HbA1c concentration in the sample increases. Using this decrease in response current value as an index, 0-800 nM of HbA1c could be detected using FAOD.

Claims (12)

A method for measuring glycated protein in a sample, wherein the sample is brought into contact with a fructosylamine oxidation catalyst in the presence of a certain amount of fructosylamine, and the amount of fructosylamine oxidized by the catalyst is measured. To measure the amount of the glycated protein. The method for measuring a glycated protein according to claim 1, wherein the fructosylamine oxidation catalyst is a molecule having an imidazole group. The method for measuring a glycated protein according to claim 1, wherein the fructosylamine oxidation catalyst is fructosylamine oxidase. 4. The method for measuring a glycated protein according to claim 1, wherein the fructosylamine oxidation catalyst is selective for fructosyl valine. 5. The method according to claim 1, wherein the step of measuring the amount of fructosylamine is performed by spectroscopically measuring the amount of the electron acceptor reduced with the oxidation of fructosylamine. Method for measuring glycated protein. 5. The method according to claim 1, wherein the step of measuring the amount of fructosylamine is performed by electrochemically measuring the amount of electron acceptor reduced with the oxidation of fructosylamine using an electrode. A method for measuring glycated protein, characterized by the following. 7. The method for measuring a glycated protein according to claim 1, wherein the glycated protein is hemoglobin A1c. A glycated protein measurement kit for carrying out the method according to claim 1. The kit for measuring a glycated protein for carrying out the method according to any one of claims 1 to 7, comprising a fructosylamine oxidation catalyst and fructosylamine. The measurement kit according to claim 8 or 9, wherein the glycated protein is hemoglobin A1c. A sensor for measuring glycated protein for carrying out the method according to claim 1 to 4 and 6. A sensor for measuring hemoglobin A1c for carrying out the method according to claims 1 to 4 and 6.

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