JP5742639B2 - Glycated protein analyzer and analysis method - Google Patents

Description

  The present invention relates to an analyzer that measures the amount of glycated hemoglobin at high speed and with high accuracy, and contributes to health management, clinical diagnosis, and the like.

  As diagnostic items for the purpose of diagnosis and treatment of diabetes, blood glucose level and glycated hemoglobin ratio are widely used. Due to the onset of diabetes, blood sugar levels rise. However, blood glucose level is a measurement item that rises due to ingestion of food and the like and is fluctuating. Accordingly, fasting blood glucose level is suitable for primary screening of diabetes and confirmation of daily treatment results, but it is difficult to grasp the exact pathology of diabetes only by blood glucose level. Therefore, many diagnostic indicators have been proposed. Among them, the glycation rate of hemoglobin is a powerful index. Glycated hemoglobin is a kind of glycated protein in which sugar is non-enzymatically bound to hemoglobin. Among the glycated hemoglobins, the fraction called HbA1c, in particular, formed a Schiff base on the valine residue at the N-terminal of the β-chain of hemoglobin A to form aldimine (unstable), and further undergoes Amadori rearrangement to produce a ketoamine compound. Is. Incidentally, those having an aldimine structure are called unstable glycated hemoglobin, and those having a ketoamine structure are called stable glycated hemoglobin. The N-terminus of the β chain after Amadori rearrangement is a fructosyl valine residue.

  Enzyme is not involved in this reaction process, and its amount increases according to the glucose concentration in the plasma. If the blood glucose level in the plasma shows a high value on average for a long period of time, HbA1c becomes high. Stable glycated hemoglobin does not disappear until the life of the red blood cells is exhausted. In general, the lifetime of hemoglobin molecules in vivo is about 2 months, and as a result, the value of HbA1c is considered to reflect the average blood glucose level over the past 1-2 months. Therefore, HbA1c is used as an index of the average value of long-term blood glucose level. Since HbA1c is an average value over a long period of time, it is said that HbA1c is suitable for use as a judgment material for definite diagnosis and treatment of diabetes.

  Therefore, various measurement methods have already been proposed for glycated hemoglobin (for example, Patent Documents 1 and 2).

  Patent Document 1 uses an immobilized body in which fructosyl peptide oxidase acting on fructosyl valylhistidine is immobilized, and a mechanism for detecting an electrochemically active substance that is increased or decreased by a reaction catalyzed by fructosyl peptide oxidase. In order to detect the fructosyl valyl histidine concentration electrochemically and calculate the glycated hemoglobin from the amount of fructosyl valyl histidine, the sample containing glycated hemoglobin should be analyzed before the solidified substance of fructosyl peptide oxidase contacts the sample. A method characterized by treatment with a proteolytic enzyme from the genus Bacillus is described. It has been reported that this method enables accurate quantification of the glycated product of hemoglobin β chain, which is the definition of HbA1c.

  Patent Document 2 discloses a mechanism for detecting an immobilized substance in which an enzyme that catalyzes an oxidation reaction of glucose is immobilized, an electrochemically active substance that is increased or decreased by the glucose oxidation reaction, and a fructosyl amino acid oxidase that acts on fructosyl L-valine. For detecting an electrochemically active substance that is increased or decreased by the oxidation reaction of fructosylamino acid or fructosyl peptide with the immobilized form immobilized with sucrose or the immobilized form with fructosyl peptide oxidase acting on fructosylvalylhistidine And an analyzer equipped with a mechanism for detecting total hemoglobin. It has been reported that the plasma glucose concentration and the glycated hemoglobin ratio (HbA1c) in a blood sample can be quantified easily and with high accuracy using this apparatus.

Japanese Patent No. 4622820 Japanese Patent No. 4622836

  An object of the present invention is to provide an apparatus and a method for calculating a glycated hemoglobin ratio with high accuracy.

  The present invention relates to an immobilized form in which fructosyl amino acid oxidase acting on fructosyl L-valine is immobilized or an immobilized form in which fructosyl peptide oxidase acting on fructosylvalylhistidine is immobilized and fructosyl amino acid or fructosyl peptide A mechanism for detecting electrochemically active substances that increase or decrease due to oxidation reactions of, a mechanism for detecting total hemoglobin, a mechanism for processing a sample containing glycated hemoglobin with a proteolytic enzyme, and before processing a sample with a proteolytic enzyme Disclosed is an apparatus for analyzing glycated hemoglobin, which is equipped with a calculation mechanism for obtaining HbA1c from which blanks have been eliminated based on the detection results of fructosyl L-valine or fructosyl valyl histidine and total hemoglobin.

  As a more preferable configuration, the analyzer having the above-described mechanism further includes an immobilized body on which an enzyme that catalyzes the oxidation reaction of glucose is immobilized, and a mechanism that detects an electrochemically active substance that increases or decreases due to the glucose oxidation reaction. An apparatus for analyzing glycated hemoglobin is disclosed.

  A preferred example of a mechanism for treating a sample containing glycated hemoglobin with a proteolytic enzyme is a mechanism in which a sample containing glycated hemoglobin and a proteolytic enzyme are brought into contact with each other for an arbitrary time and a part thereof is injected.

  More specifically, the calculation mechanism includes the ratio of the electrochemically active substance after the sample is treated with the proteolytic enzyme and the detection result of the total hemoglobin to the electrochemically active substance before the proteolytic enzyme is treated. This is a calculation mechanism that corrects the ratio of the detection results of all hemoglobin as a blank.

The present invention also discloses a method for analyzing glycated hemoglobin in a specimen, which comprises the following steps:
Immobilization of fructosyl amino acid oxidase acting on fructosyl L-valine, or immobilization of fructosyl peptide oxidase acting on fructosylvalylhistidine, and fructosyl amino acid or fructosyl peptide oxidation reaction Detecting the electrochemically active substance in the specimen before the proteolytic enzyme treatment using a mechanism for detecting the electrochemically active substance that increases or decreases depending on the condition;
Detecting the total hemoglobin in the sample before the proteolytic enzyme treatment using a mechanism for detecting the total hemoglobin;
Treating a sample containing glycated hemoglobin with a proteolytic enzyme;
Immobilization of fructosyl amino acid oxidase acting on fructosyl L-valine, or immobilization of fructosyl peptide oxidase acting on fructosylvalylhistidine, and fructosyl amino acid or fructosyl peptide oxidation reaction Detecting the electrochemically active substance in the specimen after the proteolytic enzyme treatment using a mechanism for detecting the electrochemically active substance that increases or decreases depending on the condition;
Detecting the total hemoglobin in the specimen after the proteolytic enzyme treatment using a mechanism for detecting total hemoglobin;
A process of obtaining HbA1c from which blanks are excluded by a calculation mechanism based on the detection results of electrochemically active substances and total hemoglobin before and after the sample is treated with a proteolytic enzyme.

  As a preferred configuration, in the analysis method described above, detection of the electrochemically active substance and the total hemoglobin in the specimen before the proteolytic enzyme treatment is performed after at least 1 minute has elapsed since the dilution of the specimen with the surfactant solution, And a method for analyzing glycated hemoglobin in a specimen, wherein the detection of the electrochemically active substance and total hemoglobin in the specimen after the proteolytic enzyme treatment is performed after at least 2 minutes have elapsed since the start of the proteolytic enzyme treatment. Disclose.

  As a more preferred configuration, in the analysis method described above, a proteolytic enzyme using an immobilized body in which an enzyme that catalyzes an oxidation reaction of glucose is immobilized, and a mechanism that detects an electrochemically active substance that increases or decreases due to the glucose oxidation reaction. Disclosed is a method for analyzing glycated hemoglobin in a specimen, further comprising the step of electrochemically detecting the glucose concentration in the specimen before treatment.

  According to the apparatus and method of the present invention, HbA1c from which blanks have been excluded can be calculated using an arithmetic mechanism, so that HbA1c can be measured efficiently and accurately.

It is a figure which shows the technical outline of a prior art and a novel technique (this invention). It is the schematic of a measuring apparatus. It is a graph which shows the detection electric current value (pA) of the hydrogen peroxide electrode with respect to the elapsed time (minutes) after hemolysis. 6 is a graph showing the detected current value (pA) of a hydrogen peroxide electrode with respect to the elapsed time (minutes) after hemolysis for four types of specimens. It is a graph which shows the action time (minute) of a proteolytic enzyme, and the detected current value (pA) of a hydrogen peroxide electrode. It is a graph which shows the relationship between the measured value (HbA1c /%) and a reference example (HbA1c /%) at the time of calculating only after making a proteolytic enzyme act without performing a blank measurement. It is a graph which shows the relationship between the measured value (HbA1c /%) at the time of performing a blank calculation, and a reference example (HbA1c /%).

  Hereinafter, the configuration and preferred embodiments of the present invention will be described in more detail.

  The measurement of glycated amino acid is performed by immobilizing fructosyl amino acid oxidase that oxidizes glycated amino acid to produce hydrogen peroxide. The characteristics required of fructosyl amino acid oxidase when detecting fructosyl valine, a glycated amino acid at the N-terminal of glycated hemoglobin, is not substantially affected by fructosyl lysine with ε-amino group glycated. It is preferable to act specifically on kutosylvaline. This is because lysine in which ε-amino group is glycated is released from glycated albumin contained in serum, or lysine glycated product in hemoglobin may cause a positive error in measurement results. Because there is. Examples of the fructosyl amino acid oxidase having such properties include fructosyl amino acid oxidase derived from Corynebacterium.

  The measurement of the glycated peptide is performed by immobilizing fructosyl peptide oxidase that oxidizes the glycated peptide to generate hydrogen peroxide. When detecting fructosyl valyl histidine, a glycated dipeptide at the N-terminal of glycated hemoglobin, the characteristics required for fructosyl peptide oxidase are fructosyl valyl leucine and ε- An enzyme that does not substantially act on ε-fructosyl lysine in which the amino group is glycated and acts specifically on fructosyl valyl histidine, which is a glycated dipeptide at the N-terminal of glycated hemoglobin β chain, is preferred.

  In the measurement of glucose in the present invention, an enzyme that catalyzes an oxidation reaction of glucose is immobilized and used. Such enzymes include glucose oxidase (EC 1.1.3.4), pyranose oxidase (EC 1.1.3.10), glucose dehydrogenase (EC 1.1.99.10) and the like. Among these, glucose oxidase is desirable because of its high durability and excellent substrate selectivity.

  As enzyme immobilization methods such as glucose oxidase, fructosyl amino acid oxidase, fructosyl peptide oxidase, etc., methods known as protein immobilization methods such as physical adsorption method, ionic bond method, inclusion method, and covalent bond method can be used. Of these, the covalent bond method is desirable because of its long-term stability. As a method for covalently binding a protein, various methods such as a method using a compound having an aldehyde group such as formaldehyde, glyoxal, and glutaraldehyde, a method using a polyfunctional acylating agent, a method of crosslinking a sulfhydryl group, and the like are used. it can. As the shape of the enzyme-immobilized body, it can be immobilized in a film shape and placed on an electrode made of platinum, gold, carbon or the like, or it can be immobilized on an insoluble carrier and packed in a column reactor for use.

  In addition, other types of enzymes or proteins such as gelatin and serum albumin and synthetic polymers such as polyallylamine and polylysine coexist at the time of immobilization to change the properties of the enzyme-immobilized product, that is, membrane strength, substrate permeability, etc. You can also. As the carrier for immobilizing the enzyme on an insoluble carrier, known carriers such as clay minerals, diatomaceous earth, activated carbon, alumina ceramics, titanium oxide, silica gel, cross-linked starch particles, cellulose-based polymers, chitin and chitosan derivatives are used. Available.

  The glycated peptide and glycated amino acid produced from glycated hemoglobin by the action of glucose and proteolytic enzyme in the sample are converted into hydrogen peroxide by glucose oxidase, fructosyl peptide oxidase and fructosyl amino acid oxidase, and the oxidase produced by each oxidase. The concentration of each substance can be measured by detecting hydrogen oxide. However, the saccharification rate of naturally occurring hemoglobin saccharified products is as low as about 5%, and the amount of hydrogen peroxide derived from fructosyl valine or fructosyl valyl histidine generated by the treatment with proteolytic enzymes is less than usual. Since it is sufficiently expected, hydrogen peroxide is detected with high sensitivity when measuring glycated peptides and glycated amino acids. Hydrogen peroxide can be measured directly or indirectly by a known method. However, since hemoglobin itself has an absorption band in the vicinity of 400 to 600 nm, the excess in these wavelength regions is known among known methods. The absorbance detection of hydrogen oxide is largely disturbed by the absorption of hemoglobin, and is likely not applicable to actual glycated hemoglobin measurement. For these reasons, it is more preferable to use an electrochemical method such as amperometry for highly sensitive measurement of hydrogen peroxide. In addition, a known method combining various mediators can be used for electrochemical detection of hydrogen peroxide. Furthermore, it can replace with the detection of hydrogen peroxide by detecting the oxidation-reduction reaction of an enzyme directly or indirectly electrochemically.

  The method of treating glycated hemoglobin with a proteolytic enzyme is as follows. First, a sample containing glycated hemoglobin is mixed with a surfactant solution, and then a proteolytic enzyme is added to react for a predetermined time, or a sample containing glycated hemoglobin is interfaced. Examples of the method include contacting the sample mixed with the activator solution with a carrier on which a proteolytic enzyme is immobilized for a predetermined time, and any method may be used.

  A surfactant used for processing a specimen containing glycated hemoglobin needs two actions to change the molecular structure of hemolysis and hemoglobin. Most of hemoglobin is present in erythrocytes, and hemoglobin is released out of erythrocytes in the presence of an appropriate concentration of surfactant. In addition, hemoglobin is usually present in a folded state, but is present in a relaxed state in an appropriate concentration of surfactant, and this action is presumed to facilitate degradation by proteolytic enzymes. Surfactants include nonionic polyoxyethylene alkyl ethers [for example, polyoxyethylene (10) octylphenyl ether (Triton X-100), polyoxyethylene (23) lauryl ether, etc.] and polyoxyethylene sorbitan fatty acids Esters [eg, polyoxyethylene sorbitan monolaurate (Tween 20), polyoxyethylene sorbitan monopalmitate (Tween 40)], anionic polyoxyethylene alkyl ethers and alkyl sulfates [sodium dodecyl sulfate (SDS ) Etc.]], cationic and zwitterionic, but anionic surfactants are desirable because they have the two effects described above. The concentration is 0.05 to 10% by weight, preferably 0.1 to 5% by weight, and the reaction time with the surfactant is about several seconds to 10 minutes.

  Examples of the surfactant solution include an aqueous solution and a buffer solution containing a surfactant. The buffer solution is not particularly limited as long as it is a buffer solution in a pH range in which hemoglobin can be dissolved, and a known buffer solution such as a phosphate buffer solution or a Tris buffer solution and a salt thereof may be used. A salt such as sodium chloride or potassium chloride may be appropriately added to the buffer solution.

  The proteolytic enzyme that can be used is not particularly limited as long as it can act on the β-chain N-terminus of glycated hemoglobin and release fructosyl amino acid or fructosyl peptide, but it reaches a pH range in which hemoglobin can be sufficiently dissolved. Suitable proteolytic enzymes can be preferably used. The proteolytic enzyme may be derived from any of microorganisms, animals, and plants. Specifically, commercial products for research such as carboxypeptidase, papain, pepsin, aminopeptidase, proteinase K, trypsin, morsine, AO protease, acid protease, alkaline protease, peptidase (above, manufactured by Kikkoman), Sumiteam LP50D, Sumiteam CP, Sumiteam TP (above, Shin Nippon Chemical Industry Co., Ltd.), Pronase E (above, Roche), Protease S “Amano” G, Protease A “Amano” G, Protease P “Amano” 3G, Umamizyme (above) , Manufactured by Amano Enzyme), Protin PC, Protin PC10F, Protin PS10, Protin NY10, Protin NL10, Protin NC25, Samoaase PC10F (above, manufactured by Daiwa Kasei Co., Ltd.), Actinase AS (above, manufactured by Kaken Pharmaceutical Co., Ltd.), Toyoteam NEP, Industrial commercial products such as neutral proteinase (Toyobo Co., Ltd.) Is mentioned. Among them, preferred are those derived from microorganisms belonging to the genus Bacillus, Aspergillus or Streptomyces, or belonging to neutral protease, acidic protease, basic protease or metalloproteinase. Examples of proteases derived from the genus Bacillus include Toyoteam NEP (above, manufactured by Toyobo Co., Ltd.), Protin PC10F, Protin NC25 (above, manufactured by Daiwa Kasei Co., Ltd.), etc., Aspergillus derived proteases, Morsin (above, manufactured by Kikkoman) Examples of proteases derived from the genus Streptomyces include actinase AF, actinase E, actinase AS (above, manufactured by Kaken Pharmaceutical Co., Ltd.), protease Type-XIV (above, made by Sigma) and the like.

  In the present specification, the protease derived from microorganisms, animals and plants may be a protease itself produced by the microorganisms, animals and plants, and further, one or more amino acids are substituted in the amino acid sequence of the protease, Variants obtained by addition, deletion, or insertion, which are capable of releasing fructosyl valine or fructosyl valyl histidine, are widely included.

  As a method for measuring the hemoglobin concentration, a known method such as cyan methemoglobin method, methemoglobin method, azide hemoglobin method, SLS-hemoglobin method may be used. The SLS-hemoglobin method is preferable because it has a small amount and has a low environmental impact when the reagent is discarded. The SLS-hemoglobin method is calculated from the change in absorbance of a sample at 540 nm after treating a blood sample with an alkyl sulfate solution that is an anionic surfactant (hereinafter referred to as a hemoglobin measuring reagent). . As the alkyl sulfate, sodium lauryl sulfate (SLS), polyoxyethylene sodium lauryl sulfate or the like is appropriately selected and used, and the hemoglobin measuring reagent may contain a buffer solution and various salts. The concentration of the alkyl sulfate is 0.05 to 10% by weight for a blood cell concentration of 0.25 to 20% by volume in the reaction solution, preferably 0.1 to 5% by weight for a blood cell concentration of 0.5 to 10% by volume in the reaction solution. . The reaction between the blood sample and the hemoglobin measuring reagent is completed in about several seconds to several minutes.

  In addition, a surfactant-containing solution used when a proteolytic enzyme is allowed to coexist in a hemoglobin measurement reagent containing an alkyl sulfate simultaneously to perform hemolysis, hemoglobin denaturation, and SLS-hemoglobin formation reaction simultaneously. Alternatively, a surfactant-containing solution for proteolytic enzyme treatment may be added after the reaction of the specimen with the hemoglobin measuring reagent, or the reverse order may be used.

  The surfactant in the hemoglobin measurement reagent and the surfactant for proteolytic enzyme treatment may be different or the same as long as they satisfy the above conditions.

  The hemoglobin concentration before proteolytic enzyme treatment may be measured at any time as long as the absorbance of the reaction solution is stable after completion of the reaction between the specimen and the hemoglobin concentration measuring reagent, preferably at the interface. After at least one minute has passed since the sample was diluted with the activator solution. The measurement may be performed before or after the detection of glucose, fructosyl valine and fructosyl valyl histidine.

  The hemoglobin concentration after the proteolytic enzyme treatment may be measured at any time as long as the absorbance of the reaction solution is stable, preferably at least 2 minutes from the start of the proteolytic enzyme treatment. Is after 3 minutes. The measurement may be performed before or after detection of fructosyl valine and fructosyl valyl histidine.

  As a method for measuring the absorbance of a specimen treated with a hemoglobin measuring reagent, a batch method in which the sample is dispensed into a cuvette and the absorbance at a specific wavelength is measured, or a sample is used as a Teflon (registered trademark) tube as a cell. A flow method for measuring a change in absorbance at a specific wavelength when passing through a (registered trademark) tube may be used, and the flow method is more preferable because the operation is simple. In order to advance the reaction by contacting the sample with the immobilized enzyme for a certain period of time, a batch method in which the reaction is caused while stirring the sample liquid for a certain period of time is possible, but in order to carry out a more accurate measurement. It is desirable to use flow based measurements. In the present invention, a flow type apparatus capable of performing measurement with higher accuracy is disclosed.

  The present inventors have discovered that the blank value varies from sample to sample, and have found that HbA1c can be obtained more stably and accurately with high accuracy by performing blank measurement before proteolytic enzyme treatment. The technique of the present invention and the prior art are shown in FIG. In the prior art, the amount of hemoglobin, fructosyl valyl histidine and glucose were detected after the proteolytic enzyme treatment (upper part of FIG. 1). However, in the present invention, the amount of hemoglobin and fructosyl valyl histidine are detected before proteolytic enzyme treatment, and the difference between the amount of hemoglobin and fructosyl valyl histidine detected after proteolytic enzyme treatment is calculated. HbA1c from which blanks are excluded is obtained (FIG. 1 bottom). That is, in the present invention, before and after the proteolytic enzyme treatment, total hemoglobin and fructosyl L-valine or fructosyl valyl histidine are detected, and a blank is difference-calculated by an arithmetic mechanism.

  A calculation method for eliminating the blank will be described. Specifically, using the detection results of total hemoglobin and electrochemically active substance before and after the proteolytic enzyme treatment, a measurement calculation value is obtained from the following equation.

Measurement calculation value = [Current value of hydrogen peroxide electrode after decomposition] / [Measurement value of colorimeter after decomposition]
− A × [Current value of hydrogen peroxide electrode before decomposition] / [Measurement value of colorimeter before decomposition]
Here, A is a coefficient determined by the type of sample, the type of proteolytic enzyme, reaction conditions, and the like, and is a value set as appropriate according to the experimental conditions to be performed. The value of A is preferably 0.1 to 2.0.

  HbA1c is calculated from a calibration curve obtained in advance using the measurement calculation value obtained from the above equation. Thus, the calculation mechanism performs blank difference calculation and HbA1c calculation.

  One preferred embodiment of the present invention is shown in FIG. A mechanism (12, 13) for forming a buffer flow, a mechanism (5, 7) for injecting a specimen, a flow colorimeter (6) for detecting hemoglobin concentration, and an electrode (18) for detecting the concentration of electrochemically active substance 19), and is composed of a hemoglobin concentration detection system (6), a glycated amino acid or glycated peptide detection electrode system (17, 18) and a glucose detection electrode system (19).

  Specifically, the buffer solution is sent from the buffer solution tank (12) by the pump (13), and the air in the buffer solution is trapped by the air trap damper (14). A nozzle (7) is inserted into the sample (8), the valve (2) is closed, the valve (4) is opened, a syringe pump (3) is pulled to collect a certain amount of sample, and a reaction thermostat (9) Pour into the solution and dilute. The diluted specimen in the reaction thermostat is pulled into the metering valve (5) by the nozzle (7) after a certain time has elapsed or after protease has been added and reacted for a certain time. Next, the metering valve (5) is switched, and the sample in the metering valve is pushed out by the buffer solution and injected into the buffer solution line. The injected sample passes through the fructosyl amino acid oxidase-immobilized substance or fructosyl peptide oxidase-immobilized substance (17) according to the flow of the buffer solution, and the generated hydrogen peroxide is transferred to the downstream hydrogen peroxide electrode (18). Detected. Subsequently, hydrogen peroxide generated at the glucose electrode (19) is detected. The waste liquid passes through the back pressure coil (20) and accumulates in the waste liquid bottle (21).

  Saccharification that eliminates glucose and blanks by quantifying the hemoglobin concentration from the change in absorbance at a constant wavelength of the flow-type colorimeter (6) and detecting the change in the current value of the hydrogen peroxide electrodes (18) and (19) The amino acid or glycated peptide concentration can be quantified.

  The buffer solution to be passed through these devices is not particularly limited, but may be selected so that the pH of the immobilized enzyme (17, 19) is high (for example, pH 7-9). It may also contain antibacterial agents and surfactants of a kind and concentration range that do not negatively affect the immobilized enzyme (17, 19) and the hydrogen peroxide electrode (18, 19). An activator of the immobilized enzyme (17, 19) that does not interfere with 18, 19) may be included.

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated further in detail, of course, this invention is not limited to these.

-About the electrode used The immobilized enzyme reactor (17) is a column-shaped reactor having an inner diameter of 3 mm and a length of 15 mm of a carrier packed part, and a porous silica gel having an average particle diameter of 250 μm as a carrier, and fructosyl peptide oxidase (Kikkoman). Used) was used. For immobilization, the silica gel surface is modified with a silane coupling agent γ-aminopropyltrimethoxysilane (manufactured by Shin-Etsu Chemical Co., Ltd.) to introduce an amino group, and glutaraldehyde (manufactured by Kishida Chemical Co., Ltd.) is used as a crosslinking agent. Fructosyl peptide oxidase was chemically conjugated so that 0.25 U could be immobilized per 1 mg of silica gel.

  The hydrogen peroxide electrode (18) used a three-electrode system in which platinum as the working electrode, platinum as the counter electrode, and silver / silver chloride as the reference electrode were arranged on the same substrate plane. Cover the electrode surface with an acetylcellulose membrane to suppress permeation of oxidizable substances other than hydrogen peroxide, place it in the flow cell, and apply a potential of +0.65 V to the working electrode. Thus, hydrogen peroxide can be selectively oxidized to obtain the oxidation current value.

  The glucose electrode (19) is obtained by coating a glucose oxidase-immobilized film on the acetylcellulose film of the hydrogen peroxide electrode, and detecting hydrogen peroxide generated when glucose oxidase decomposes glucose as an oxidation current value. To do. Glucose oxidase-immobilized membrane was coated with an aqueous solution containing glucose oxidase (Sigma, Type II) 50 mg / ml, bovine serum albumin 10 mg / ml, glutaraldehyde 1 (w / v)% in the form of an acetylcellulose membrane, It was prepared by drying and curing at 40 ° C. for 15 minutes. This electrode was placed in the flow cell to form a glucose oxidase electrode.

-About the buffer line (sensor system) The buffer used for the measurement contains 100 mM phosphoric acid and 50 mM KCl, and pH is 7.0. The buffer solution in the buffer tank (12) is sent at a flow rate of 1 mL / min by the buffer solution pump (13), and the air in the buffer solution is trapped by the air trap damper (14). Is absorbed and sent to the metering valve (5). When a fixed amount (4 μL) of sample is injected with the metering valve (5), it diffuses into the buffer solution through the mixing pipe in the thermostat (15) and is immobilized with the sample in the immobilized enzyme column (17). The enzyme contacts and reacts to generate hydrogen peroxide, and the hydrogen peroxide electrode (18) detects the hydrogen peroxide as an oxidation current value. Further, the sample is sent to the glucose electrode (19), where glucose in the sample is detected as an oxidation current value. The sample and buffer are passed through the back pressure coil (20) and finally sent to the waste bottle.

-An aqueous solution containing 1% of an anionic surfactant was used for the dilution of the sample and the reaction that causes the protease to act on the treatment liquid line . The treatment liquid in the treatment liquid tank (1) is sucked and discharged by combining the syringe pump (3) and the valves (2, 4). The sample (40 μL) in the sample tube (8) is discharged into the reaction thermostat (9) together with a fixed amount of processing liquid (745 μL) by the sample suction nozzle (7) and diluted and mixed. Part of the diluted sample in the thermostat (9) is sucked with the sample suction nozzle (7), the absorbance of the sample is measured with the flow-type colorimeter (6), and further to the measuring valve (5) Aspirate and inject into buffer line. Proteolytic enzyme (15 μL) is added to the sample diluent remaining in the reaction thermostat (9) and allowed to act at a constant temperature (40 ° C.) for a fixed time (3 minutes). The sample decomposed by the proteolytic enzyme is sucked by the sample suction nozzle (7), the absorbance of the sample is measured by the flow type colorimeter (6), and further sucked to the measuring valve (5) to the buffer line. inject.

-About reaction sequence By diluting 40 μL of human whole blood as a sample with 745 μL of treatment solution, red blood cells are hemolyzed and hydrogen peroxide is generated. The hydrogen peroxide disappears for 3 minutes, and 200 μL of the diluted solution is sampled and injected into the absorbance measurement and buffer solution line, and the blank before the proteolytic enzyme is allowed to act on the hydrogen peroxide electrode (18 ) And the glucose concentration in the sample is measured with the glucose electrode (19).

  Thereafter, 15 μL of a solution containing a proteolytic enzyme is added to make a total volume of 600 μL. By degrading hemoglobin in erythrocytes at 40 ° C. for 5 minutes, fructosyl valyl histidine is produced from glycated hemoglobin acting on the glycated N-terminus. When the sample diluted solution after the reaction is collected, the absorbance is measured, and further injected into the buffer solution line, the fructosylvalylhistidine derived from glycated hemoglobin is immobilized enzyme reactor (17) in which fructosyl peptide oxidase is immobilized. When passing through, hydrogen peroxide corresponding to the concentration is generated and can be detected as an oxidation current value by the hydrogen peroxide electrode (18).

  The reason for waiting for 1 minute when the sample is diluted and hemolyzed is to perform an accurate blank measurement, because it takes at least 1 minute for hydrogen peroxide generated from erythrocytes to disappear during hemolysis. FIG. 3 shows a state in which the amount of hydrogen peroxide from the time after diluting human whole blood to hemolysis until the disappearance was detected by a hydrogen peroxide electrode. It can be seen that under this condition, it is necessary to wait for at least one minute before hydrogen peroxide generated immediately after hemolysis disappears. FIG. 4 shows the same data as in FIG. 3 and shows the results of studying other samples. As in FIG. 3, it is understood that it is necessary to wait for at least one minute, but the value that settles as the blank value differs for each sample, and it is understood that it is important to perform blank measurement for each sample.

  Using a proteolytic enzyme, the time when hemoglobin was decomposed, and the current value detected at the hydrogen peroxide electrode for hydrogen peroxide produced when the degradation product fructosylvalylhistidine was oxidized with fructosyl peptide oxidase Is shown in FIG.

  Under these conditions, it can be seen that the reaction is completed when the reaction exceeds 3 minutes, and it is appropriate to set the reaction time to at least 3 minutes.

・ With or without blank measurement When measuring and calculating only after applying proteolytic enzyme without performing blank measurement using actual human whole blood, and when performing difference calculation with blank measurement The results are shown in FIGS. 6 and 7, respectively. Table 1 shows the correlation coefficient when the blank measurement is not performed and when the blank measurement is performed. These were all calibrated by measuring and calibrating certified practical reference materials (manufactured by National Institute of Standards and Technology) as real samples, and calculating glycated hemoglobin values of human whole blood. Reference data as a reference was measured using HA-8160 (Arkray). In addition, the difference calculation at the time of performing a blank measurement was performed using the following formula | equation.

Measurement calculation value = [Current value of hydrogen peroxide electrode after decomposition] / [Measurement value of colorimeter after decomposition]
−0.3 × [Current value of hydrogen peroxide electrode before decomposition] / [Measurement value of colorimeter before decomposition]

  As is clear from these results, it was confirmed that the correlation with the reference example was better in the result with the calculation for performing the blank measurement compared with the result without the calculation for which the blank measurement was not performed.

  According to the present invention, stable sugar hemoglobin in a blood sample can be quantified easily and accurately, and a test for diabetes can be easily performed.

DESCRIPTION OF SYMBOLS 1 Process liquid tank 2 Valve 3 Syringe pump 4 Valve 5 Metering valve 6 Flow type colorimeter 7 Sample suction nozzle 8 Sample tube 9 Reaction thermostat 10 Waste / washing port 11 Waste liquid bottle 12 Buffer liquid tank 13 Buffer liquid feed pump 14 Air trap damper 15 Thermostatic bath 16 Mixing pipe 17 Immobilized enzyme reactor 18 Hydrogen peroxide electrode 19 Glucose electrode 20 Back pressure coil 21 Waste liquid bottle

Claims (5)

Immobilization of fructosyl amino acid oxidase acting on fructosyl L-valine or immobilization of fructosyl peptide oxidase acting on fructosylvalylhistidine and fructosyl amino acid or fructosyl peptide oxidation reaction a mechanism for detecting an electrochemically active material for good Ri decrease, a mechanism for detecting the total hemoglobin, a mechanism for processing a sample containing glycosylated hemoglobin with proteolytic enzymes, and prior to processing the sample with proteolytic enzymes later An apparatus for analyzing glycated hemoglobin, comprising an electrochemically active substance and a calculation mechanism for obtaining HbA1c from which blanks have been eliminated based on the detection results of total hemoglobin.   The analyzer according to claim 1, further comprising: an immobilized body on which an enzyme that catalyzes an oxidation reaction of glucose is immobilized; and a mechanism for detecting an electrochemically active substance that is increased or decreased by the glucose oxidation reaction. A method for analyzing glycated hemoglobin in a specimen, comprising the following steps:
Immobilization of fructosyl amino acid oxidase acting on fructosyl L-valine, or immobilization of fructosyl peptide oxidase acting on fructosylvalylhistidine, and fructosyl amino acid or fructosyl peptide oxidation reaction Detecting the electrochemically active substance in the specimen before the proteolytic enzyme treatment using a mechanism for detecting the electrochemically active substance that increases or decreases depending on the condition;
Detecting the total hemoglobin in the sample before the proteolytic enzyme treatment using a mechanism for detecting the total hemoglobin;
Treating a sample containing glycated hemoglobin with a proteolytic enzyme;
Immobilization of fructosyl amino acid oxidase acting on fructosyl L-valine, or immobilization of fructosyl peptide oxidase acting on fructosylvalylhistidine, and fructosyl amino acid or fructosyl peptide oxidation reaction Detecting the electrochemically active substance in the specimen after the proteolytic enzyme treatment using a mechanism for detecting the electrochemically active substance that increases or decreases depending on the condition;
Detecting the total hemoglobin in the specimen after the proteolytic enzyme treatment using a mechanism for detecting total hemoglobin;
A process of obtaining HbA1c from which blanks are excluded by a calculation mechanism based on the detection results of electrochemically active substances and total hemoglobin before and after the sample is treated with a proteolytic enzyme.   Detection of the electrochemically active substance and total hemoglobin in the sample before proteolytic enzyme treatment is performed after at least 1 minute has passed since the sample was diluted with the surfactant solution. The method according to claim 3, wherein the detection of the chemically active substance and the total hemoglobin is performed after at least 2 minutes have elapsed from the start of the proteolytic enzyme treatment.   A process for detecting glucose concentration in a sample before proteolytic enzyme treatment using an immobilized body in which an enzyme that catalyzes an oxidation reaction of glucose is immobilized, and a mechanism for detecting an electrochemically active substance that increases or decreases due to the glucose oxidation reaction The method according to claim 3 or 4, further comprising:

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