WO2004029613A1

WO2004029613A1 - Method of measuring glycoprotein

Info

Publication number: WO2004029613A1
Application number: PCT/JP2003/012232
Authority: WO
Inventors: Koji Sode
Original assignee: Koji Sode
Priority date: 2002-09-26
Filing date: 2003-09-25
Publication date: 2004-04-08
Also published as: JPWO2004029613A1; AU2003266612A1; JP4330534B2

Abstract

A method of measuring a glycoprotein such as hemoglobin Alc in a sample. This method is characterized by comprising contacting the sample with a fructosylamine-oxidizing catalyst in the presence of a definite amount of fructosylamine and then quantifying fructosylamine thus oxidized by the catalyst to thereby determine the amount of the glycoprotein. As the fructosylamine-oxidizing catalyst, use can be made of a molecule having an imidazole group, a fructosylamine oxidase, etc.

Description

Specification

Glycoprotein measurement method

The present invention relates to a method for measuring glycated protein and hemoglobin A 1c used as a marker for diagnosing diabetes in the field of clinical examination. More specifically, the present invention provides a method for quantifying glycated protein and hemoglobin A1c based on the fact that glycated protein and hemoglobin A1c antagonistically inhibit the fructosylamine oxidation reaction catalyzed by fructosylamine oxidation. On how to do. Furthermore, the present invention relates to a glycated protein and a kit for measuring hemoglobin A1c and a sensor constructed based on the method of the present invention. Background art

The amino groups of the protein main chain and side chains are non-enzymatically linked to the reducing end of a reducing sugar such as glucose to produce an Amadori compound, ie, a glycated protein. It is known that hemoglobin is glycated in blood to produce glycated hemoglobin (glycohemoglobin; HbAlc). Since the presence of HbAlc in hemoglobin is higher in diabetic patients than in healthy subjects, and the blood concentration of HbAlc reflects blood glucose levels in the past few weeks, the HbAlc blood concentration can be used for diagnosis of diabetes and diabetes. It is extremely important in clinical trials as an indicator of glycemic control in patients.

To date, an enzyme that acts on Amadori compounds, fructosylamine oxidase, has been isolated from various strains. (Japanese Unexamined Patent Publication No. 61-268178, Japanese Unexamined Patent Publication No. 61-280297, Japanese Unexamined Patent Publication No. 3-155780, Japanese Unexamined Patent Publication No. 5-192193, Japanese Unexamined Patent Application Publication No. 7-289253, Japanese Unexamined Patent Application Publication No. Biol. Chem., 53 (1), 103-110, 1989, Agric. Biol. Chem., 55 (2), 333-338, 1991, J. Biol. Chem., 269 (44), 27297-27302. Biol., 1994, Appl. Environ. Microbiol., 61 (12), 4487-4489, 1995, Biosci. Biotech. Biochein., 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. Using these catalysts, hydrolysis products of glycated proteins such as glycated albumin and HbA1c are used. (WO 01/09735, WO 02/22698), Anal. Chim. Acta 435, 151-156, 2001).

However, the fructosylamine oxidation catalysts reported so far cannot oxidize unhydrolyzed sugarcane protein as a substrate. For this reason, glycated protein is first hydrolyzed, and the resulting low-molecular-weight glycated peptide or fructosylamine is oxidized using a fructosylamic acid catalyst to obtain a glycated protein. The protein was being measured. Therefore, there is a need in the art for a method for measuring a glycated protein without decomposing it.

An object of the present invention is to provide a novel method for measuring a saccharified protein and a saccharified protein in which the concentrations of glycated proteins and hemoglobin A 1c are measured without hydrolysis. More specifically, the present invention provides a method for measuring a glycated protein, particularly hemoglobin A1c (HbAlc) or glycated albumin, which is used in the field of clinical testing and the like, and a measuring reagent and a sensor constructed based on the method. The purpose is to: Disclosure of the invention

The present inventors have found that glycated proteins can competitively inhibit the fructosylamine oxidation reaction catalyzed by a fructosylamine oxidation catalyst, and have completed the present invention. That is, the present invention relates to a method for measuring a glycated protein in a sample, comprising contacting the sample with a fructosylamic acid catalyst in the presence of a fixed amount of fructosylamine. A method is provided wherein the amount of the glycated protein is measured by measuring the amount of fructosylamine oxidized by the catalyst. According to the present invention, glycated protein can be measured without hydrolyzing the glycated protein, using the competitive inhibition of the fructosylamine oxidation catalyst by the glycated protein as an index.

As used herein, the term "fructosylamine oxidation catalyst" refers to a catalyst that catalyzes the oxidation reaction of fructosylamine, which inhibits the reaction in a concentration-dependent manner in the presence of glycated protein. Represents the catalyst to be used. Examples of fructosylamine oxidation catalysts include fructosylamine oxidase and polymers that catalyze the oxidation of fructosylamine in the presence of an electron acceptor, for example, polymers containing imidazole groups.

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 is 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 performed by spectrophotometrically measuring the amount of an electron acceptor reduced as the fructosylamine is oxidized. Also preferably, in the method of the present invention, the step of measuring the amount of fructosylamine is performed by electrochemically measuring the amount of an electron acceptor reduced with the oxidation of fructosylamine using an electrode. Done. Further, the present invention provides a method for measuring a glycated protein, wherein the glycated protein described above is hemoglobin A1c.

The present invention also provides a glycated protein measurement kit based on the above principle. Preferably, the kit comprises a fructosylamine oxidation catalyst and fructosylamine. The present invention also provides a hemoglobin A1c measurement kit based on the above-described principle.

Further, the present invention provides a glycated protein measuring sensor based on the above principle. The present invention also provides a sensor for measuring hemoglobin A1c based on the principle described above. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the sensor system constructed in the second embodiment.

FIG. 2 shows a decrease in the steady-state current value when the HbAlc sample was added to the sensor system constructed in Example 2.

Fig. 3 shows that various proteins were added to the sensor shown in The graph shows the dependence of the decrease in the steady-state current value upon the addition of white matter and HbAlc on the sample concentration.

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 a glycated protein of the present invention is based on the discovery that glycated proteins can competitively inhibit the fructosylamine oxidation reaction catalyzed by a fructosylamine oxidation catalyst. That is, when a sample containing a glycated protein is brought into contact with a fructosylamine oxidation catalyst in the presence of a fixed amount of fructosylamine, the amount of fructosylamine oxidized by the fructosylamine oxidation catalyst becomes saccharified. It varies depending on the protein concentration. Therefore, by measuring the amount or oxidation rate of oxidized fructosylamine, the concentration of glycated protein in a sample can be determined.

As a fructosylamine oxidation catalyst, for example, a synthetic polymer having an imidazole group or a fructosylamine oxidase can be used.A catalyst having an activity of oxidizing fructosylamine and being antagonized by glycated protein is used. If there is, it is not limited. For example, not limited to imidazole, it is possible to use a polymer obtained by polymerizing a functional group that functions as a general base catalyst, for example, a monomer containing bipyridyl containing pyridine.

The method of the present invention is particularly useful for measuring HbAlc. According to the method of the present invention, HbAlc has a fructosyl rubulin moiety in its molecule and acts antagonistically to a fructosylamine oxidation catalyst selective for fructosyl valine. This can be specifically quantified. Examples of such a catalyst include a catalytic polymer synthesized by allowing a synthetic polymer having an imidazole group to exist in the presence of fructosyl valine as a type III during polymerization, or a fructosyl valine selective for fructosyl valine. The enzyme can be used, but it is limited as long as it has the oxidizing activity of fructosyl valine and is subject to competitive inhibition by HbAlc. It is not something to be done. For example, not only fructosyl valine, but also a catalyst polymer having an imidazole group, which is synthesized using an analog, methyl valine, as 錡 -type can be used as the catalyst.

The measurement of the glycated protein in the present invention can be easily performed by measuring the amount or reduction rate of the electron acceptor reduced by the fructosylamine oxidation by the fructosylamine oxidation catalyst. For example, a sample is added to a solution containing a fixed amount of a fructosylamine oxidation catalyst, fructosylamine, and an electron acceptor, and the amount or reduction rate of the electron acceptor reduced by oxidation of fructosylamine is measured. By comparing the amount of the electron acceptor or the reduction rate in the absence of the sample, the glycated protein in the sample can be measured. Electron acceptors can be measured by spectroscopic methods based on the characteristic spectrum of the reduced state of the electron acceptor, or by reoxidizing the electron acceptor in the reduced state on the electrode. An electrochemical method by measuring the obtained current can be used.

When phenazine methosulfate (PMS) and dichlorophenol indphenol (DCIP) are used as electron acceptors and fructosyl valine is used as a substrate, fructosyl valine is oxidized with time and It is observed that DCIP is reduced and fades. The rate of this DCIP fade is proportional to the concentration of fructosylvaline present. Thus, for example, in the presence of HbAle, the oxidation of fructosyl valine is antagonistically inhibited, resulting in a decrease in the rate of bleaching of DCIP. The concentration of HbA1c can be measured using the rate of decrease in the fading speed 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 phenocyanide, phlegmene, an osmium derivative, or the like can be used.

On the other hand, when fructosylamine oxidase is used as a catalyst, oxygen can be used as an electron acceptor in addition to the above-mentioned electron acceptor. In this case, the amount of oxygen decrease and the rate of decrease or the amount of hydrogen peroxide produced The inhibition of the enzyme by glycated protein can be measured, and the concentration of glycated protein can be measured.

In another aspect, 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 oxidizing catalyst according to the present invention, fructosylamine, and an electron acceptor in an amount sufficient for at least one time of the assay. Typically, the kit contains a fructosylamic acid catalyst; a buffer adjusted to pH 6.0 to 10; a suitable mediator; and a glycated protein or a derivative thereof for preparing a calibration curve. Includes standard solutions, as well as guidelines for use. 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. Sensor

In another aspect, the present invention features a sensor for measuring a glycated protein such as glycated albumin, HbAle, or the like. The glycated protein sensor of the present invention electrochemically oxidizes a mediae, which is reduced as fructosylamine is oxidized by a fructosylamine oxidation catalyst, on an electrode, and determines a current value at this time. Measure. In the presence of glycated protein, the oxidation reaction is competitively inhibited depending on the concentration of glycated protein.Therefore, a current curve was measured in the presence of various concentrations of glycated protein, and a standard curve was prepared. Based on this, the glycated protein concentration in the sample can be determined. As the electrode, a carbon electrode, a gold electrode, a platinum electrode, or the like is used, and a fructosylamine oxidation catalyst is fixed on the electrode. Examples of the immobilization method 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.

According to the measurement principle of the present invention, a sensor for measuring HbAlc can be constructed as follows. Measured in an ambrometric system using a carbon electrode, gold electrode, platinum electrode, etc. In this case, use an electrode on which a fructosylamine oxidation catalyst is immobilized as a working electrode, and use a buffer solution containing a mediator together with a counter electrode (for example, a platinum electrode) and a reference electrode (for example, an AgZAgC1 electrode). And keep it at a constant temperature. Apply a constant voltage to the working electrode, add a fixed amount of fructosylamine and a sample, and measure the increase in current. As a medium, potassium ferricyanide, phlegmene, osmium derivatives, phenazine methosulfate and the like can be used.

Furthermore, as a method of measuring with an amometric method using a carbon electrode, a gold electrode, a platinum electrode, or the like, there is a system using an immobilized electron media. In other words, an electrode in which a fructosylamine oxidation catalyst and an electron mediator such as potassium ferricyanide, phenocene, osmium derivatives, and phenazine methosulfate are immobilized on a polymer matrix by adsorption or covalent bonding as a working electrode. Using a buffer, insert it into a buffer with a counter electrode (for example, a platinum electrode) and a reference electrode (for example, an Ag / AgCI electrode) and keep it at a constant temperature. Apply a constant potential to the working electrode, add a fixed amount of fructosylamine and sample, and measure the increase in current.

Regardless of which electrode is used, the concentration of glycated protein in the sample can be determined according to calibration curves prepared using glycated protein solutions of various concentrations.

The contents of all patents and references explicitly cited herein are hereby incorporated by reference. In addition, the entire contents described in the specification and drawings of Japanese Patent Application No. 2000-310900, which is the application on which the priority claim of the present application is based, are incorporated herein by reference. To quote. Example

Hereinafter, the present invention will be described in more detail by way of examples, but these examples do not limit the scope of the present invention. Example 1

1-Pinylimidazole 1.6 mmol, 4-vinylphenylporonic acid 0.8 mmol, ethylene glycol dimethacrylate (EGDMA) 2mmo 1 as a crosslinking reagent, 2,2,1-azobisisobutyronitrile 0.06mmo 1 as a polymerization initiator, and fructosyl valine 0 as a 铸 -type molecule. 2 mmo 1 was added, 522 n \ of methanol and 1741 water were added as solvents, and the atmosphere was replaced with argon. Then, a polymerization reaction was performed at 45 ° C for 12 hours. The polymer catalyst obtained here is called BI-I MIP. This polymer was ground in a mortar and sieved to prepare a polymer having a particle size of 40 m. BI-CP, a control polymer, was synthesized by performing exactly the same operation as above without adding fructosylvaline, a type I 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. Example 2

2 Omg of the polymer catalyst prepared in Example 1 was mixed with 5 Omg of carbon paste, and the mixture was filled in a carbon paste electrode. This electrode was immersed in a solution of ImM 1-methoxyphenazine methosulfate (mPMS) in 1 OmM phosphate buffered saline (pH 7.4), and the applied voltage was changed to lO OmV (vs. Ag / Ag C The response was observed when fructosyl valine was added to the substrate. Furthermore, a sensor system was constructed to observe the change in response value when HbAlc was added (Fig. 1). By adding fructosyl valine, fructosyl valine is oxidized by the polymer catalyst, and the 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. The redox reaction and the electrode reaction give a constant steady-state current value depending on the fructosyl valine concentration in the solution. When a certain amount of HbAlc is dropped here, the steady-state current value decreases (Fig. 2). This is based on the competitive inhibition of the polymer catalyst by HbA1c. If Hb Ale is dropped when the steady-state current value is reached, the steady-state current value further decreases. In other words, it was shown that depending on the HbAlc concentration, the oxidation reaction of fructosyl valine by the polymer catalyst was inhibited and the current value decreased. Example 3

In order to examine the selectivity of this sensor, we observed changes in sensor response when various proteins were added to this sensor system (Fig. 3). The proteins used for comparison were Hb AO, bovine serum albumin, and glycated albumin. There was some decrease in response when all proteins were added, but this was negligible compared to when HbAl c was added. Example 4

Based on Example 3, HbAlc was measured using the inhibition of fructosyl valine response observed in the present sensor system as an index. That is, in this sensor system, the response current value to fructosyl valine 0. ImM in the absence of HbAlc decreases as the added HbAlc concentration increases. From the response current value for fructosyl valine 0.1 mM, the decrease in the non-specific response value observed in the presence of various proteins was subtracted to obtain the response current value for fructosyl palin 0.1 ImM. Fructosylvaline, which is observed in the presence of various concentrations of HbA1c, is subtracted from this value. The response current to ImM is subtracted, and the difference in fructosylvaline in the absence of HbA1c is calculated. The ratio of the response current to sylvaline 0.1 mM was defined as the inhibition rate. The inhibition rate is plotted on the vertical axis, and the results of measurement of HbAlc are shown in FIG. Thus, HbAlc could be detected at a sensitivity of 10 nM without denaturing or hydrolyzing using this sensor. Example 5

Using a sensor system that uses a platinum electrode as the working electrode, a platinum wire as the counter electrode, and an AgAgC1 electrode as the reference electrode, the ratio of hemoglobin Alc (HbAlc) to hemoglobin Ao (HbAo) in the sample is determined. It was measured. The principle of this measurement is based on the fact that HbAlc inhibits the oxidation of fructosyl valin by fructosylamine oxidase (FAOD) in a concentration-dependent manner.

FAOD 20mU, Hb A 1 cZHb Ao mixture (Total 0. Add 47 o 1) to 600 1 of 5 OmM potassium phosphate buffer, incubate at 8 ° C for 30 minutes, and apply a potential of 600 mV Vs.Ag/AgC1 to the working electrode. Incubated at 25 ° C for 20 minutes. Next, 0.1 lmmol of fructosyl valine was added (total amount of 650 H 1), and the response current value (IC) was measured. FIG. 5 shows the result of plotting the increased current value against the Hb A 1c concentration. This measurement was carried out by varying the mixing ratio of 1181 cZHbAo in the HbA1.711180 mixture, which is the sample to be added. As is clear from the figure, the response current value to fructosyl valine decreases as the HbAlc concentration in this sample increases. Using this decrease in response current as an index, HbA1c of 0 to 800 η η could be detected using FAOD.

Claims

The scope of the claims

1. A method for measuring glycated protein in a sample, comprising contacting the sample with a fructosylamine oxidation catalyst in the presence of a fixed amount of fructosylamine, and measuring the amount of fructosylamine oxidized by the catalyst. A method comprising measuring the amount of the glycated protein by measuring the amount.

2. The method for measuring a glycated protein according to claim 1, wherein the fructosylamine oxidation catalyst is a molecule having an imidazole group.

3. The method for measuring a glycated protein according to claim 1, wherein the fructosylamine oxidation catalyst is a 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 any one of claims 1 to 4, wherein the step of measuring the amount of fructosylamine is performed by spectroscopically measuring the amount of an electron acceptor reduced as the fructosylamine is oxidized. A method for measuring a glycated protein that is a feature.

6. The method according to any one of claims 1 to 4, wherein the step of measuring the amount of fructosylamine comprises electrochemically measuring, using an electrode, the amount of the electron acceptor reduced along with the oxidation of fructosylamine. A method for measuring a glycated protein, characterized in that the method is performed by the following method.

7. The method for measuring a glycated protein according to claims 1 to 6, wherein the glycated protein is hemoglobin A1c.

8. A glycated protein measurement kit for performing the method according to any one of claims 1 to 7. '

9. A kit for measuring a sugarcane protein for carrying out the method according to any one of claims 1 to 7, comprising a fructosylamine oxidation catalyst and fructosylamine.

10. The measurement kit according to claim 8, wherein the glycated protein is hemoglobin A1c.

11. A sensor for measuring a glycated protein for performing the method according to claims 1 to 4 and 6.

12. A sensor for measuring hemoglobin Alc for performing the method according to claims 1 to 4 and 6.