JP4622836B2 - Analysis equipment - Google Patents

Description

  The present invention relates to an analyzer for measuring a blood glucose level and a glycated hemoglobin amount 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 sugar level is a measurement item that rises due to ingestion of meals, etc., and fluctuates greatly. Therefore, 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, in particular, a fraction called HbA1c formed a Schiff base at the valine residue at the N-terminus of hemoglobin A to form an aldimine (unstable form), and further undergoes Amadori rearrangement to produce a ketoamine compound. Is. Those having an aldimine structure are called unstable glycated hemoglobin, and those having a ketoamine structure are called stable glycated hemoglobin. The N-terminal 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. When 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 to 2 months. Therefore, HbA1c is used as an index of the average value of blood glucose level over a long period. Since HbA1c is an average value over a long period of time, it is said that HbA1c is suitable for use as a decision material for definite diagnosis and treatment of diabetes.

  Therefore, various measurement methods have already been proposed for glycated hemoglobin. Typical examples include high performance liquid chromatography (HPLC), immunization, and enzymatic methods.

  The HPLC method is the most frequently used method at present. The hemoglobin is fractionated by a separation column, and the abundance ratio of HbA1c is calculated from the ratio of the peak eluted in the retention volume corresponding to HbA1c and the total peak area. In other words, since the relative area method is used, there is an advantage that the accuracy of the injection capacity can be ignored to some extent. However, there are problems such as a large and complicated apparatus and a heavy maintenance load. Moreover, since unstable type HbA1c and stable type HbA1c cannot be distinguished, it has the fault that a separation analysis must be performed after removing unstable type HbA1c beforehand (patent document 1). At the same time, when there is a congenital hemoglobin mutation, the separation pattern may change and show an abnormal value. In addition, there is a possibility that other biological components may include an error due to an overlap with the peak of HbA1c.

  The immunization method has a possibility that more accurate analysis can be achieved with a simpler mechanism by using an antibody corresponding to the structure near the N-terminus of the β chain of HbA1c. However, it is not intended for serum as in general immunoassay, but is intended for specimens in which whole blood has been hemolyzed, so it contaminates colorimetry used to detect non-specific reactions and reactions, It has been pointed out that satisfactory accuracy cannot always be obtained.

  On the other hand, in the enzymatic method, after glycated amino acid or glycated peptide is cut out from glycated protein by some technique, the amount of glycated amino acid or glycated peptide produced is detected using an enzyme such as fructosyl amino acid oxidase. By utilizing the selectivity of the enzyme, there is a possibility that more accurate analysis can be performed, and various studies have been made.

  A method for measuring a glycated amino acid with an enzyme is described in Patent Document 2, and a method for measuring a glycated peptide with an enzyme is described in Patent Document 3. Furthermore, as a method of cutting out a glycated amino acid or a glycated peptide from a glycated protein, there is a method using a proteolytic enzyme, and a proteolytic enzyme capable of acting on a glycated protein has been studied (Patent Documents 4, 5, 6, and 7). .

  Furthermore, since HbA1c, which is an index value used for clinical diagnosis of diabetes, is represented by a ratio between glycated hemoglobin and hemoglobin, it is necessary to measure the amount of glycated hemoglobin in the sample and quantify hemoglobin. There is an example in which hemoglobin is quantified by a known method and the ratio to glycated hemoglobin measured by an enzymatic method is calculated (Patent Document 8).

  As described above, three main methods, HPLC method, immunization method, and enzyme method, have been proposed for measuring glycated hemoglobin, and all of them measure only the glycated hemoglobin ratio. From the standpoint of clinical diagnosis, it is desirable that the blood glucose level and the glycated hemoglobin ratio are determined substantially simultaneously.

  Conventionally, when calculating these two index values at the same time, the blood glucose level is first measured with an appropriate blood glucose meter, and then the blood sample is transported to the HbA1c analyzer and HbA1c is separately measured with the HbA1c analyzer. Under such a configuration, two types of dedicated analyzers are required, and each device has a sample injection mechanism and a liquid feed mechanism, which requires a large installation space and is used in each analyzer. Since the solution compositions are completely different, an eluent and a buffer solution corresponding to each were required. In addition, it cannot be said that two index values are calculated simultaneously.

  Thus, an effective method for accurately calculating two index values simultaneously has not been proposed.

Further, when glycated hemoglobin is quantified by an enzymatic method, no effective method has been proposed for removing interference with enzymes such as glycated amino acid oxidase derived from blood components or products after proteolytic enzyme treatment.
Japanese Patent Publication No. 5-59380 Japanese Patent Publication No. 5-33997 JP 2001-95598 A JP-A-11-196897 JP 2003-235585 A JP 2004-344052 A JP2005-110657 WO2003 / 064683

  An object of this invention is to provide the apparatus which calculates the blood glucose level and glycated hemoglobin ratio contained in a sample simply and with high precision.

  The present invention immobilizes an immobilized body in which an enzyme that catalyzes the oxidation reaction of glucose is immobilized, an electrochemically active substance that increases or decreases by the glucose oxidation reaction, and a fructosyl amino acid oxidase that acts on fructosyl L-valine. A mechanism for detecting an electrochemically active substance that is increased or decreased by an oxidation reaction of a fructosyl amino acid or fructosyl peptide, and an immobilized form in which fructosyl peptide oxidase acting on fructosylvalylhistidine is immobilized; A first operation mechanism for obtaining HbA1c based on the measurement value of fructosyl L-valine and hemoglobin or fructosylvalylhistidine and the detection result of hemoglobin, and a whole blood / blood cell glucose and hemoglobin Whole blood based on detection results Discloses an analyzer of glucose and glycosylated hemoglobin, characterized in that it comprises a second operation mechanism for correcting a blood glucose plasma glucose.

  As a more preferable configuration, the fructosyl L- in which the influence of the glucose in the sample is eliminated based on the detection result of the glucose in the sample and fructosyl L-valine or the glucose in the sample and fructosylvalylhistidine is added to the analyzer having the above-described mechanism. Disclosed is an apparatus for analyzing glycated hemoglobin having a third calculation mechanism for obtaining a measurement value of valine or fructosyl valyl histidine.

  In the present invention, a mechanism for continuously feeding a buffer solution and a mechanism (4, 5, 7) for injecting a sample into a buffer solution downstream thereof, and a mechanism for injecting a sample (4, 5, 7). Downstream of the enzyme, an immobilized body in which an enzyme that catalyzes the oxidation reaction of glucose, a mechanism (17, 18) for detecting an electrochemically active substance that increases or decreases by the glucose oxidation reaction, and fructosyl that acts on fructosyl L-valine Electrochemical activity increased or decreased by the oxidation of fructosyl valine or fructosyl valyl histidine and immobilized form immobilized on amino acid oxidase or fructosyl peptide oxidase immobilized on fructosyl valyl histidine A mechanism (19, 20) for detecting a substance is disposed, and all hemogs are located upstream or downstream of the mechanism (4, 5, 7) for injecting the specimen. To the arrangement of the mechanism (16) for detecting a bin concentrations discloses a spectrometer of glucose and glycosylated hemoglobin characterized by.

Furthermore, the present invention discloses a method for analyzing glucose and glycated hemoglobin in a sample, comprising the following steps:
A step of electrochemically detecting a glucose concentration in a specimen 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 is increased or decreased by the glucose oxidation reaction;
Immobilization of immobilized fructosyl amino acid oxidase acting on fructosyl L-valine or immobilized fructosyl peptide oxidase acting on fructosylvalylhistidine, and oxidation reaction of fructosyl amino acid or fructosyl peptide Electrochemically detecting fructosyl L-valine or fructosylvalylhistidine in a specimen using a mechanism for detecting an electrochemically active substance that increases or decreases by
Detecting all hemoglobin in the sample using a mechanism for detecting all hemoglobin;
Obtaining HbA1c by the first calculation mechanism based on the detection result of fructosyl L-valine and hemoglobin or fructosylvalylhistidine and the detection result of hemoglobin; and by the second calculation mechanism based on the detection result of whole blood / blood glucose and hemoglobin. Correcting whole blood / blood cell glucose to plasma glucose.

  Further preferably, in the above analysis method, fructosyl L in which the influence of glucose in the sample is eliminated by the third calculation mechanism based on the detection results of glucose in the sample and fructosyl L-valine or glucose in the sample and fructosylvalylhistidine. -A step of obtaining a measurement value of valine or fructosyl valyl histidine is further included, and HbA1c is obtained by the first arithmetic mechanism based on the obtained measurement value and the detection result of hemoglobin.

  According to the present invention, the plasma glucose concentration and the glycated hemoglobin ratio (that is, HbA1c) in a blood sample can be quantified simply and with high accuracy.

  According to the apparatus of the present invention, glucose and HbA1c can be calculated simultaneously using the first and second calculation mechanisms. Further, the glucose concentration in the sample is converted into the plasma glucose concentration by the second calculation mechanism based on the hemoglobin concentration essential for the calculation of HbA1c. Since it is converted into HbA1c by the second calculation mechanism after being eliminated by the three calculation mechanisms, the plasma glucose and HbA1c can be measured efficiently and accurately as a whole.

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

  In the measurement of glucose in the present invention, an enzyme that catalyzes an oxidation reaction of glucose is immobilized and used. Applicable enzymes include glucose oxidase (EC 1.1.3.4), pyranose oxidase (EC 1.1.3.10), glucose dehydrogenase (EC 1.1.9.10.10) and the like. Among these, glucose oxidase is desirable because of its high durability and excellent substrate selectivity.

  Moreover, the glucose level in the field of clinical examination is usually evaluated by glucose in plasma. Since whole blood contains a component that cannot be a solvent such as hemoglobin, the whole blood glucose concentration value is different from the plasma glucose concentration value. In the measurement of the glycated hemoglobin ratio, a specimen containing hemoglobin is targeted, and the specimen to be used is whole blood or blood cells. The glucose value detected when measuring the glycated hemoglobin ratio and glucose simultaneously for whole blood is the whole blood glucose value.

  Note that since whole blood glucose and blood cell (erythrocyte) glucose are almost equivalent in fresh blood, in this specification, whole blood glucose and blood cell (erythrocyte) glucose may be expressed as “whole blood glucose”.

  As a method of calculating the plasma glucose value from the whole blood glucose value, there is a method of correcting the whole blood glucose value by measuring the hematocrit value by any method (hematocrit correction), but a separate device for measuring the hematocrit value is required. This is undesirable because the analyzer is large and complex. In the present invention, when measuring the glycated hemoglobin ratio, the hemoglobin concentration is always measured. Plasma glucose levels can be calculated without upsizing the device.

  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, does not substantially affect fructosyl lysine in which the ε-amino group is 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.

  However, the present inventors have discovered that fructosyl amino acid oxidase may act not only on glycated amino acids but also on glucose to a small extent. Since blood has approximately 100 times the glucose concentration of glycated hemoglobin, when fructosyl amino acid oxidase also acts on glucose, the detected fructosyl valine value includes a positive error due to glucose in the blood. Therefore, an accurate fructosyl valine concentration cannot be obtained. Therefore, in order to eliminate the interference of glucose in the sample with the detected value of fructosyl valine, a glucose oxidase immobilized body is arranged together with the fructosyl amino acid oxidase immobilized body, and fructosyl valine is measured simultaneously with glucose. The third arithmetic unit calculates a difference between the interference detected by the glucose in the specimen from the detected value of the silver valine.

  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, which is 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.

  As with fructosyl amino acid oxidase, when fructosyl peptide oxidase also acts on glucose, it is used simultaneously with the glucose oxidase immobilized product to measure glycated peptide and glucose at the same time. Interference by glucose is calculated by a third calculation mechanism.

  Calculation method for removing glycated amino acid or glycated peptide detection system in the glycated amino acid or glycated peptide detection system using fructosyl amino acid oxidase or fructosyl peptide oxidase, if glucose in the sample interferes with the detected value of glycated amino acid or glycated peptide explain. Specifically, when a glycated amino acid or glycated peptide responds in the glucose detection system and glucose responds in the glycated amino acid or glycated peptide detection system, first, a calibration curve of glucose and glycated amino acid or glycated peptide is created in each detection system. . Thereafter, the difference between the response values for the sample obtained in each detection system is calculated using each calibration curve.

A glucose calibration curve obtained by a glucose detection system in which a glucose oxidase immobilized body and a first hydrogen peroxide electrode are combined is represented by y = a ₁₁ × x + b ₁₁
The calibration curve for fructosyl valine is y = a ₁₂ × x + b ₁₂
And a calibration curve of glucose obtained by a glycated amino acid or glycated peptide detection system in which a fructosyl amino acid oxidase or fructosyl peptide oxidase immobilized body and a hydrogen peroxide electrode are combined is represented by y = a ₂₁ × x + b ₂₁
A standard curve for fructosyl valine is represented by y = a ₂₂ × x + b ₂₂
Then, the response value by the glucose with x ₁ concentration and fructosyl valine with x ₂ concentration is as follows:
y ₁ = a ₁₁ × x ₁ + b ₁₁ + a ₁₂ × x ₂ + b ₁₂
In the second hydrogen peroxide electrode,
y ₂ = a ₂₁ × x ₁ + b ₂₁ + a ₂₂ × x ₂ + b ₂₂
It becomes.

When the concentrations of glucose and fructosyl valine are determined based on these values, the glucose concentration x ₁ is
_{x 1 = (a 12 × (} y 2 -b 21 -b 22) -a 22 × (y 1 -b 11 -b 12)) / (a 12 × a 21 -a 11 × a 22)
The concentration _{x 2} of fructosyl valine is _{x 2 = (a 11 × (} y 2 -b 21 -b 22) -a 21 × (y 1 -b 11 -b 12)) / (a 11 × a 22 -a ₁₂ xa ₂₁ )
It becomes.

  Even when fructosyl valine responds with fructosyl valine in the glucose detection system and with fructosyl amino acid oxidase or fructosyl peptide oxidase immobilized form, the concentrations of glucose and fructosyl valine can be calculated and fractional quantification is possible.

When fructosyl valine does not respond in the glucose detection system, a ₁₂ and b _{12 in the} above formula are 0, and the glucose concentration x ₁ is x ₁ = (y ₁ -b ₁₁ ) / a ₁₁
The concentration _{x 2} is _x 2 = the fructosyl valine _{(a 11 × (y 2 -b} 21 -b 22) -a 21 × (y 1 -b 11)) / (a 11 × a 22)
A calibration curve for glucose and fructosyl valine or fructosyl valyl histidine was prepared using the glucose detection system and the glycated amino acid or glycated peptide detection system. The glucose concentration and fructosyl valine concentration in the sample can be accurately calculated by the formula.

  Furthermore, the calculation of HbA1c, which is the ratio of glycated hemoglobin, is the ratio of the fructosyl valine concentration or fructosyl valyl histidine concentration obtained by subtracting the interference derived from glucose in the sample and the hemoglobin concentration in the sample. Is calculated by the first calculation mechanism.

  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.

  Furthermore, during immobilization, other types of enzymes, proteins such as gelatin and serum albumin, and synthetic polymers such as polyallylamine and polylysine are allowed to coexist to change the properties of the enzyme-immobilized product, that is, membrane strength, substrate permeability, etc. You can also. Known carriers such as clay minerals, diatomaceous earth, activated carbon, alumina ceramics, titanium oxide, cross-linked starch particles, cellulose-based polymers, chitin and chitosan derivatives can be used as the carrier when the enzyme is immobilized on an insoluble carrier. .

  Glucose peptide and glycated amino acid generated from glycated hemoglobin by the action of glucose and proteolytic enzyme in the sample are sequentially converted to hydrogen peroxide with glucose oxidase, fructosyl peptide oxidase and fructosyl amino acid oxidase (regardless of order), The concentration of each substance can be measured by electrochemically detecting hydrogen peroxide generated by each oxidase. 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 enzyme 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, but since hemoglobin itself has an absorption band in the vicinity of 400 to 600 nm, it is a peroxidation in these wavelength regions among the known methods. The hydrogen absorbance detection 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.

  The method of treating glycated hemoglobin with a proteolytic enzyme is as follows. First, a sample containing glycated hemoglobin is mixed with a surfactant-containing buffer, and then a proteolytic enzyme is added and reacted for a predetermined time, or a sample containing glycated hemoglobin is prepared. Examples include a method in which a sample mixed with a surfactant-containing buffer is brought into contact 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), etc.], 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%, preferably 0.05 to 1%, and the reaction time with the surfactant is about several seconds to 10 minutes.

  The buffer solution used when processing the specimen containing glycated hemoglobin is not particularly limited as long as it is a buffer solution in a pH range in which hemoglobin can be dissolved, and known buffers such as phosphate buffer and Tris buffer and salts thereof. Can be used. A salt such as sodium chloride or potassium chloride may be appropriately added to the buffer solution.

  As the proteolytic enzyme that can be used, a proteolytic enzyme that has a large effect of generating a glycated amino acid or a glycated peptide from the N-terminus of glycated hemoglobin and is optimal in a pH range in which hemoglobin can be sufficiently dissolved can be preferably used. Specifically, it may be a proteolytic enzyme that has an optimum effect at pH 6.0 to 10.0, more preferably pH 6.0 to 8.0, and has a large action to generate a glycated amino acid or glycated peptide from the N-terminus of glycated hemoglobin. That's fine. As a proteolytic enzyme having such an action, a proteolytic enzyme derived from the genus Aspergillus having an optimum pH in a neutral range or an alkaline range, which has a large action to produce a glycated amino acid, and a neutral having a large action to produce a glycated peptide Examples thereof include proteolytic enzymes derived from the genus Bacillus having an optimum pH in a region or an acidic region.

  As used herein, “Aspergillus genus proteolytic enzyme” or “Protease derived from Aspergillus genus microorganism” or “Bacillus proteolytic enzyme” or “Protease derived from Bacillus genus microorganism” is produced by microorganisms belonging to the genus Aspergillus or Bacillus A protease obtained by substitution, addition, deletion, or insertion of one or more amino acids in the amino acid sequence of the protease, wherein the protease is Aspergillus proteolytic enzyme or Bacillus Variants that can increase fructosyl valine or fructosyl valyl histidine concentrations, such as proteolytic enzymes, are widely encompassed.

  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, and among them, the influence of various enzymes such as inhibition or inactivation The SLS-hemoglobin method is preferable because it has a low environmental impact when the reagent is discarded. In the SLS-hemoglobin method, a blood sample is treated with an alkyl sulfate solution (hereinafter referred to as a hemoglobin measuring reagent) that is an anionic surfactant, and then calculated from the change in absorbance of the sample at 540 nm. 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 or various salts. The concentration of the alkyl sulfate is 0.05 to 10%, preferably 0.05 to 1% with respect to the blood cell concentration of 0.25 to 20% 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 buffer used for the action of proteolytic enzymes is simultaneously coexisted in a hemoglobin measurement reagent containing alkyl sulfate, and hemolysis, hemoglobin denaturation, and SLS-hemoglobin formation reaction are simultaneously performed. Alternatively, a surfactant-containing buffer for proteolytic enzyme treatment may be added after the reaction of the specimen with the hemoglobin measurement reagent, or the reverse order may be used.

  The surfactant in the hemoglobin measuring 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 may be measured at any time within the time range in which the absorbance of the reaction solution is stable after completion of the reaction between the sample and the hemoglobin concentration measuring reagent. Either before or after the detection of glucose, 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.

  One preferred embodiment of the present invention is shown in FIG. A mechanism (1, 2) for forming a flow of buffer solution, a mechanism (4, 5, 7) for injecting a specimen, a proteolytic enzyme immobilized body (14) downstream of the sample injection mechanism, and a flow type for detecting hemoglobin concentration A colorimeter (16) and electrodes (18, 20) capable of detecting the electrochemically active substance concentration are arranged, a hemoglobin concentration detection system (16), a glucose detection electrode system (17, 18) and a glycated amino acid or glycated peptide It consists of a detection electrode system (19, 20).

  Specifically, the buffer A is sent from the buffer tank (1) by the pump (2). Insert the needle (7) into the sample (6), close the valve (10), open the valve (11), and pull the syringe pump (12) to pull the sample into the metering loop (5) of the metering valve (4) Pull in. Next, the metering valve (4) is switched, and the sample accumulated in the loop (5) is pushed out by the buffer A. Excess specimen once opens the valve (10) and closes the valve (11), draws the cleaning liquid (9) into the syringe pump (12), then closes the valve (10) and opens the valve (11) to push out the cleaning liquid. As a result, it is pushed out to the waste pot (8) and stored in the waste liquid bottle (22). The injected sample passes along the flow of the buffer A, passes through the proteolytic enzyme immobilized body (14) arranged in the thermostatic bath (13), and generates a glycated amino acid or a glycated peptide from the glycated protein in the sample. . The sample on which the proteolytic enzyme acts passes through the flow colorimeter (16) further downstream according to the flow of the buffer A, and the hemoglobin concentration in the sample is detected from the change in absorbance at a certain wavelength when the sample passes. . Next, the sample passes through the glucose oxidase immobilized body (17), and the hydrogen peroxide generated therein is detected by the hydrogen peroxide electrode (18). Subsequently, the hydrogen peroxide generated through the immobilized fructosyl amino acid oxidase or the immobilized fructosyl peptide oxidase (19) is detected by the downstream hydrogen peroxide electrode (20). The waste liquid passes through the back pressure coil (21) and accumulates in the waste liquid bottle (22).

  FIG. 2 is an example of a flow-type glucose and glycated amino acid or glycated peptide simultaneous measurement apparatus incorporating a dialysis module capable of continuously removing contamination of a measurement system by a blood sample. Buffer B is sent from the buffer tank (24) by the pump (25), and the sample (6) is injected into the flow of the buffer B by the sample injection mechanism (4, 5, 7). The injected sample passed through the flow colorimeter (16) installed in the thermostat (13) along the flow of the buffer B, and after the hemoglobin concentration was detected from the change in absorbance at a certain wavelength, Further, the glycated amino acid or the glycated peptide is generated from the glycated protein in the sample through the proteolytic enzyme immobilization column (14) arranged downstream. The sample on which the proteolytic enzyme acts is transported to a dialysis module (27) installed downstream, where only low molecular components in the sample are saccharified with glucose in a regenerated cellulose membrane having a film thickness of 20 μm and a molecular weight fraction of 12000 to 14000. Guided to amino acid or glycated peptide detection mechanism (17, 18, 19, 20). Since the buffer A is sent from the buffer tank (1) by the pump (2), the low-molecular components in the sample dialyzed by the dialysis module (27) are peroxidized with the glucose oxidase immobilized body (17). Hydrogen peroxide produced by the glucose oxidase immobilized body (17) passing through the hydrogen electrode (18) is detected. Subsequently, it passes through a fructosyl amino acid oxidase-immobilized column or a fructosyl peptide oxidase-immobilized column (19) and a hydrogen peroxide electrode (20), and the generated hydrogen peroxide is detected. The polymer component in the sample that has not passed through the dialysis membrane of the dialysis module (27) accumulates in the waste liquid bottle (22).

  The membrane (27) used in the low-molecular component separation mechanism in the sample after protease degradation is difficult or does not permeate pentapeptides, hexapeptides or larger peptides that are considered to coexist in the process of degradation by proteolytic enzymes. Any fructosyl valine and fructosyl valyl histidine may be used. Examples of the membrane used include dialysis membranes made of regenerated cellulose, acetyl cellulose, polyvinylidene fluoride, and the like. The molecular weight fraction of the dialysis membrane is displayed as the minimum molecular weight that permeates when equilibrium dialysis is performed. On the other hand, when used for analytical purposes, separation is often based on the difference in the initial rate of dialysis, and the molecular weight desired for fractionation does not always match the performance display of the dialysis membrane. For the purpose of the present invention, those having a fractional molecular weight of 300 to 500,000 can be used. More preferably, it is 1,000 or more and 100,000 or less, and more preferably 10,000 or more and 20,000 or less.

  The blood sample (6) may be directly supplied into the analyzer after being treated with the hemoglobin measuring reagent, or may be supplied into the apparatus after being treated with the hemoglobin measuring reagent and further treated with a proteolytic enzyme. Good. When supplying a blood sample that has been treated with a hemoglobin measurement reagent and further processed with a proteolytic enzyme into the analyzer, for example, the sample treated with the proteolytic enzyme and the hemoglobin measurement reagent is supplied together, and the flow of the buffer solution is increased. It may be treated with a proteolytic enzyme in the tract. Glucose and glycated amino acid or glycated peptide by quantifying the hemoglobin concentration from the change in absorbance at a constant wavelength of the flow-type colorimeter (16) and detecting the change in the current value of the hydrogen peroxide electrodes (18) and (20) The 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 (14, 17, 19) is high (for example, pH 7-9). Further, it may contain antibacterial agents and surfactants of a kind and concentration range that do not negatively affect the immobilized enzyme (14, 17, 19) and the hydrogen peroxide electrode (18, 20). An activator of the immobilized enzyme (14, 17, 19) that does not interfere with the electrodes (18, 20) may be included.

Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is of course not limited thereto.
Example 1
(1) Production of fructosyl amino acid oxidase-immobilized column 150 mg of refractory brick (30-60 mesh) was dried well, immersed in an anhydrous toluene solution of 10% γ-aminopropyltriethoxysilane for 1 hour, and then thoroughly washed with toluene. And dry. The carrier thus treated with aminosilanization is immersed in 5% glutaraldehyde for 1 hour, then thoroughly washed with distilled water, and finally replaced with 100 mM sodium phosphate buffer at pH 7.0, and this buffer is removed as much as possible. 400 μl of a solution obtained by dissolving fructosyl amino acid oxidase (manufactured by Kikkoman Co., Ltd.) at a concentration of 18 units / ml in 100 mM sodium phosphate buffer was brought into contact with this formylated refractory brick at 0 to 4 ° C. Leave on the sun and fix. This enzyme-immobilized carrier is packed in a column having an inner diameter of 3.5 mm and a length of 30 mm to obtain a fructosyl amino acid oxidase-immobilized column.
(2) Production of hydrogen peroxide electrode The side surface of a platinum wire having a diameter of 2 mm is covered with heat-shrinkable Teflon (registered trademark), and one end of the wire is smoothed with a file and 1500-mm emery paper. Using this platinum wire as a working electrode, a 1 cm square platinum plate as a counter electrode, and a saturated calomel electrode as a reference electrode, electrolytic treatment is performed in 0.1 M sulfuric acid at +2.0 V for 10 minutes. Thereafter, the platinum wire is thoroughly washed with water, dried at 40 ° C. for 10 minutes, immersed in an anhydrous toluene solution of 10% γ-aminopropyltriethoxysilane for 1 hour, and then washed. 20 mg of bovine serum albumin (manufactured by Sigma, Fraction V) is dissolved in 1 ml of distilled water, and glutaraldehyde is added to 0.2% therein. This mixed solution is quickly put on 5 μl of the previously prepared platinum wire and dried and cured at 40 ° C. for 15 minutes. This is a hydrogen peroxide electrode.

An Ag / AgCl reference electrode was used as the reference electrode, and a conductive pipe was used as the counter electrode.
(3) Production of glucose electrode A hydrogen peroxide electrode is produced by the same procedure as in (2). Glucose oxidase (Sigma II, Type II) is dissolved in 100 mM phosphate buffer pH 6.0 so as to be 100 mg / ml. 100 mg / ml glucose oxidase solution, bovine serum albumin solution, glutaraldehyde solution, and 100 mM phosphate buffer pH 6 so that glucose oxidase is 20 mg / ml, bovine serum albumin is 5 mg / ml, and glutaraldehyde is 0.2%. 0 is mixed to obtain a glucose oxidase-immobilized enzyme solution. This glucose oxidase-immobilized enzyme solution is quickly put on a hydrogen peroxide electrode prepared earlier, 5 μl, and dried and cured at 40 ° C. for 15 minutes. This is a glucose oxidase electrode.

An Ag / AgCl reference electrode was used as the reference electrode, and a conductive pipe was used as the counter electrode.
(4) Measuring device In the measuring device of FIG. 2, the flow-type colorimeter (16), the proteolytic enzyme-immobilized column (14) and the first immobilized enzyme column (17) are not arranged, but a hydrogen peroxide electrode A glucose electrode is disposed in (18), a fructosyl amino acid oxidase immobilized column is disposed in the second immobilized enzyme column (19), and a hydrogen peroxide electrode is disposed in the hydrogen peroxide electrode (20). Buffer B is sent from the buffer tank (24) by the pump (25), and 100 μl of sample is injected using the metering valve (4). The injected sample is carried along the flow of buffer B to the dialysis module (27) installed in the thermostatic chamber (13), and the sample is formed with a regenerated cellulose membrane having a film thickness of 20 μm and a molecular weight fraction of 12000 to 14000. Only the low molecular weight component is led to the glucose detection system (18) and the glycated amino acid detection system (19, 20). Since the buffer A is sent from the buffer tank (1) by the pump (2), the low molecular components in the sample dialyzed by the dialysis module (27) are fixed to the glucose electrode (18) and the fructosyl amino acid oxidase. Hydrogen peroxide generated from glucose and glycated amino acid in the sample is detected after passing through the glycation column (19) and the hydrogen peroxide electrode (20).

  The composition of the buffer solution to be passed through this apparatus is that buffer A contains 100 mM phosphoric acid, 50 mM potassium chloride, 1 mM sodium azide, pH is 8.0, and the flow rate is 0.8 ml / min. Buffer B contains 50 mM phosphate, 0.1% sodium dodecyl sulfate, has a pH of 8.0 and a flow rate of 1.0 ml / min.

The temperature of the thermostatic bath was 37 ° C.
(5) Substrate specificity of the fructosyl amino acid oxidase-immobilized column Using the measuring device of (4), fructosyl amino acids for various glycated amino acids, glycated peptides, amino acids, and sugars when the pH of the buffer is 8.0 Table 1 shows the results of examining the substrate specificity of the oxidase-immobilized column. The values in the table are relative values when the response to fructosylglycine is 100.

  Great response to fructosylglycine and fructosyl valine. In addition, it hardly responded to fructosyl lysine and glycated peptide fructosyl valyl histidine. Other amino acids and sugars responded very little to glutamine, methionine, and glucose.

  This fructosyl amino acid oxidase-immobilized column does not substantially act on a glycated peptide and fructosyl lysine glycated at the ε position.

  However, since it responds to glucose, a positive error may occur if the sample contains a large amount of glucose. In normal human blood, glucose is about 10 mM, glycated amino acid is about 0.1 mM, and there is a concentration difference of about 100 times in blood. Therefore, blood glucose interferes with the same magnitude as fructosyl valine. May give. Such interference can be eliminated by measuring glucose simultaneously with the glycated amino acid and calculating the difference.

(6) Simultaneous measurement of glucose and fructosyl valine Using the measuring device of (4), 100 μl each of 2, 5, 10 mM glucose and 20, 50, 100 μM fructosyl valine were injected, and the detected value was measured. Obtained.

  The detected values of the glucose electrode and the glycated amino acid electrode for glucose are as shown in FIG. 3, and the detected values of the glucose electrode and the glycated amino acid electrode for fructosyl valine are as shown in FIG. 4, and the following calibration curve was obtained. Where Y is the detected value, X is the concentration in the sample, and r is the correlation coefficient.

Glucose electrode Glucose calibration curve Y (nA) = 2.44X (mM) -0.25 r = 0.9998
Fructosylvaline calibration curve Y (nA) = 0.00X (μM) −0.00
Glycated amino acid electrode Glucose calibration curve Y (nA) = 0.030X (mM) +0.029 r = 0.9778
Fructosylvaline calibration curve Y (nA) = 0.014X (μM) +0.007 r = 0.9999
When a mixed standard solution of glucose 5 mM and fructosyl valine 80 μM was measured using this measuring apparatus, a response value of 11.97 nA was obtained at the glucose electrode and a response value of 1.29 nA was obtained at the glycated amino acid electrode.

The concentration x ₁ of glucose is x ₁ = 5.00 mM from the formula x ₁ = (y ₁ −b ₁₁ ) / a ₁₁ , and the concentration x ₂ of fructosyl valine is expressed by the formula x ₂ = (a ₁₁ × (y ₂₋ b ₂₁ −b ₂₂ ) −a ₂₁ × (y ₁ −b ₁₁ )) / (a ₁₁ × a ₂₂ ), x ₂ = 80.0 μM was obtained.
Comparative Example 1
In the same manner as in Example 1 (6), a sample containing glucose and fructosyl valine was measured using only a glycated amino acid electrode.
(1) Production of fructosyl amino acid oxidase-immobilized column A fructosyl amino acid oxidase-immobilized column was produced in the same manner as in Example 1 (1).
(2) Production of hydrogen peroxide electrode A hydrogen peroxide electrode was produced in the same manner as in Example 1 (2).
(3) Measuring apparatus Measurement was performed using the measuring apparatus of FIG. 2 in the same manner as in Example 1 (4). The flow-type colorimeter (16), the proteolytic enzyme-immobilized column (14), the first immobilized enzyme column (17), and the hydrogen peroxide electrode (18) are not arranged, and the second immobilized enzyme column ( In 19), a hydrogen peroxide electrode was placed on a fructosyl amino acid oxidase-immobilized column and a hydrogen peroxide electrode (20). 100 μl of sample is injected through a metering valve.

Conditions such as the composition of the buffer solution flowing through the apparatus, the flow rate of the buffer solution, and the temperature of the thermostatic bath were the same as in Example 1 (4).
(4) Measurement of glucose and fructosyl valine mixed standard solution Using the measuring device of (3), 100 μl of 20, 50, 100 μM fructosyl valine was injected to obtain a detection value. The calibration curve for fructosyl valine is
Y (nA) = 0.014X (μM) +0.007 r = 0.9999
It became. Where Y is the detected value, X is the concentration in the sample, and r is the correlation coefficient.

When a mixed standard solution of glucose 5 mM and fructosyl valine 80 μM was measured using this measuring apparatus, a response value of 1.28 nA was obtained, and the concentration x ₂ of fructosyl valine in the sample was expressed by the formula x ₂ = From (y ₂ −b ₂₂ ) / a ₂₂ , x ₂ = 92.3 μM was obtained, but a positive error due to glucose was included.
Example 2
(1) Production of fructosyl peptide oxidase-immobilized column As in Example 1 (1), 150 mg of refractory brick (30 to 60 mesh) is formylated. This formylated refractory brick was brought into contact with 200 μl of a solution prepared by dissolving fructosyl peptide oxidase (manufactured by Kikkoman Co., Ltd.) at a concentration of 140 units / ml in a pH 7.0, 100 mM sodium phosphate buffer solution. Leave on the sun and fix. The enzyme-immobilized carrier is packed in a column having an inner diameter of 3.5 mm and a length of 30 mm to obtain a fructosyl peptide oxidase-immobilized column.
(2) Production of hydrogen peroxide electrode A hydrogen peroxide electrode was produced in the same manner as in Example 1 (2).
(3) Production of glucose electrode A glucose electrode was produced in the same manner as in Example 1 (3).
(4) Measuring device In the measuring device of FIG. 1, the proteolytic enzyme-immobilized column (14), the flow-type colorimeter (16), and the first immobilized enzyme column (17) are not arranged, but a hydrogen peroxide electrode A glucose electrode is disposed in (18), a fructosyl peptide oxidase immobilized column is disposed in the second immobilized enzyme column (19), and a hydrogen peroxide electrode is disposed in the second hydrogen peroxide electrode (20). Buffer A is sent from the buffer tank (1) by the pump (2), and 5 μl of sample is injected using the metering valve (4). The injected sample flows along the flow of the buffer A, passes through the mixing pipe (15) installed in the thermostatic chamber (13), is mixed with the temperature adjustment and the buffer, and the glucose electrode downstream thereof. (18), through a fructosyl peptide oxidase immobilization column (19) and a hydrogen peroxide electrode (20), hydrogen peroxide is generated from glucose and fructosylvalylhistidine in the sample, and a change in the current value is detected. .

  The composition of buffer A used in this measuring apparatus contains 100 mM phosphoric acid, 50 mM potassium chloride, and 1 mM sodium azide, and has a pH of 7.0.

The flow rate of the buffer solution was 1.0 ml / min, and the temperature of the thermostatic bath was 30 ° C.
(5) Substrate specificity of fructosyl peptide oxidase-immobilized column Fructosyl for various glycated amino acids, glycated amino acids, glycated peptides, amino acids, and sugars when pH of buffer A is 7.0 using the measuring device of (4) The results of examining the substrate specificity of the amino acid oxidase-immobilized column are shown in Table 2. The values in the table are relative values when the response to fructosyl valine is 100.

  It was specific for fructosyl valylhistidine and fructosyl valine, hardly responded to α, ε-fructosyl lysine, and did not respond to various amino acids and saccharides at all. Therefore, in the measurement of glycated hemoglobin, there is no possibility that a positive error derived from amino acids or blood glucose will occur if the degradation of glycated hemoglobin by proteases has progressed considerably. Absent.

(6) Simultaneous measurement of glucose and fructosyl valine Using the measuring device of (4), 5 μl each of 2, 5, 10 mM glucose and 10, 20, 50, 100 μM fructosyl valine were injected and detected. Got the value.

  Each calibration curve is shown below. Where Y is the detected value, X is the concentration in the sample, and r is the correlation coefficient.

Glucose electrode Glucose calibration curve Y (nA) = 6.74X (mM) -0.28 r = 1.0000
Fructosylvaline calibration curve Y (nA) = 0.00X (μM) +0.00
Glycated peptide electrode Glucose calibration curve Y (nA) = 0.00X (mM) +0.00
Fructosylvaline calibration curve Y (nA) = 0.035X (μM) +0.087 r = 0.9992
When a mixed solution of glucose 5 mM and fructosyl valine 80 μM was measured using this measuring apparatus, a response value of 34.1 nA was obtained at the glucose electrode and a response value of 2.87 nA was obtained at the fructosyl peptide electrode.

The concentration x ₁ of glucose is x ₁ = 5.01 mM from the formula x ₁ = (y ₁ −b ₁₁ ) / a ₁₁ , and the concentration x ₂ of fructosyl valine is expressed by the formula x ₂ = (y ₂ −b ₂₂ ) / A ₂₂ to obtain x ₂ = 80.0 μM.

  When the immobilized fructosyl peptide oxidase is used, since it does not respond to glucose, fructosyl amino acid or fructosyl peptide in the sample can be accurately measured without performing a difference calculation.

  According to the present invention, stable glycated hemoglobin and glucose in a blood sample can be quantified easily and accurately at the same time, and a test for diabetes can be easily performed.

Schematic diagram of measuring device Schematic diagram of a measuring device for dialysis Glucose calibration curve of glucose electrode and fructosyl amino acid oxidase immobilized body Calibration curve for fructosyl valine, a glucose electrode and fructosyl amino acid oxidase immobilized form

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Buffer solution tank 2 Buffer solution feed pump 3 Damper 4 Metering valve 5 Metering loop 6 Sample tube 7 Sample suction needle 8 Waste pot 9 Washing solution tank
10 Valve 11 Valve 12 Syringe pump 13 Thermostatic chamber 14 Proteolytic enzyme immobilization column 15 Mixing pipe 16 Flow colorimeter 17 First immobilization enzyme column 18 Hydrogen peroxide electrode 19 Second immobilization enzyme column 20 Hydrogen oxide electrode 21 Back pressure coil 22 Waste liquid bottle 23 Waste liquid bottle 24 Buffer liquid tank 25 Buffer liquid feed pump 26 Constant temperature piping 27 Dialysis module

Claims (5)

A mechanism for detecting an electrochemically active material which increases or decreases the oxidation fixing embodying the glucose oxidation reaction with immobilized enzyme that catalyzes the glucose, fixed immobilized fructosyl peptide oxidase which acts on the full torque tosyl valyl histidine a mechanism for detecting an electrochemically active material which increases or decreases the oxidation of embodying the full torque tosyl peptide, a mechanism for detecting the total hemoglobin, a mechanism for processing a sample containing glycosylated hemoglobin with proteolytic enzymes, Furukutoshiruba A first calculation mechanism for obtaining HbA1c based on the detection result of rilhistidine and the detection result of hemoglobin, and a second calculation mechanism for correcting whole blood / blood cell glucose to plasma glucose based on the detection result of whole blood / blood cell glucose and hemoglobin Glucose and glycated hemoglobin content characterized by comprising Apparatus. Immobilized form in which an enzyme that catalyzes the oxidation reaction of glucose is immobilized, a mechanism for detecting an electrochemically active substance that is increased or decreased by the glucose oxidation reaction, and immobilization in which fructosyl amino acid oxidase acting on fructosyl L-valine is immobilized Incorporates an immobilized form of fructosyl peptide oxidase that acts on the body or fructosyl valyl histidine, a mechanism for detecting electrochemically active substances that increase or decrease due to the oxidation reaction of fructosyl amino acids or fructosyl peptides, and glycated hemoglobin Excludes the effects of glucose in the sample based on the mechanism for treating the sample with proteolytic enzyme, the mechanism for detecting total hemoglobin, and the detection result of glucose and fructosyl L-valine in the sample or glucose and fructosylvalylhistidine in the sample Fructosyl L- A third calculation device for obtaining a measure of phosphorus or fructosyl valyl histidine, HbA1c based on the measured value and the hemoglobin detection result of the measurement of fructosyl L- valine and hemoglobin detection result or fructosyl valyl histidine And a glycated hemoglobin characterized by comprising: a first computing mechanism for obtaining a second computing mechanism for correcting whole blood / blood cell glucose to plasma glucose based on detection results of whole blood / blood cell glucose and hemoglobin Analysis equipment. A mechanism for continuously feeding the buffer solution and a mechanism (4, 5, 7) for injecting the sample into the buffer solution downstream thereof, and a mechanism for injecting glucose downstream of the mechanism (4, 5, 7) for injecting the sample. Immobilized enzyme in which an enzyme that catalyzes the oxidation reaction is immobilized, a mechanism (17, 18) for detecting an electrochemically active substance that increases or decreases due to glucose oxidation reaction, and 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 fructosyl valine or fructosyl valyl histidine with the immobilized form or immobilized form of fructosyl peptide oxidase acting on fructosyl valyl histidine with arranging the (19, 20) comprises a mechanism (4,5,7) for injecting the sample, a mechanism for injecting a sample (4,5,7) To the arrangement of the mechanism for detecting the total hemoglobin concentration (16) glucose and analyzer of glycated hemoglobin according to claim 1 or 2, wherein the flow. A method for analyzing glucose and glycated hemoglobin in a sample, comprising the following steps:
Treating a sample containing glycated hemoglobin with a proteolytic enzyme;
A step of electrochemically detecting a glucose concentration in a specimen 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 is increased or decreased by the glucose oxidation reaction ;
Full torque tosyl immobilized and body immobilized fructosyl peptide oxidase which valyl acting on histidine, full in a sample using a mechanism for detecting an electrochemically active material which increases or decreases the oxidation of full torque tosyl peptide Rukutoshirubariru Electrochemically detecting histidine;
Detecting all hemoglobin in the sample using a mechanism for detecting all hemoglobin ;
Step of correcting and whole blood / blood glucose and hemoglobin detection result to-out based whole blood / blood glucose plasma glucose; full torque tosyl the valyl histidine detection result and hemoglobin detection result obtained based on the Evaluation Technical H BA1c step . A method for analyzing glucose and glycated hemoglobin in a sample, comprising the following steps:
Treating a sample containing glycated hemoglobin with a proteolytic enzyme;
A step of electrochemically detecting a glucose concentration in a specimen 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 is increased or decreased by the glucose oxidation reaction;
Immobilization of immobilized fructosyl amino acid oxidase acting on fructosyl L-valine or immobilized fructosyl peptide oxidase acting on fructosylvalylhistidine, and oxidation reaction of fructosyl amino acid or fructosyl peptide Electrochemically detecting fructosyl L-valine or fructosylvalylhistidine in a specimen using a mechanism for detecting an electrochemically active substance that increases or decreases by
Detecting all hemoglobin in the sample using a mechanism for detecting all hemoglobin;
Obtaining a measurement value of fructosyl L-valine or fructosyl valyl histidine excluding the influence of glucose in the sample based on the detection result of glucose in the sample and fructosyl L-valine or glucose in the sample and fructosyl valyl histidine;
Obtaining HbA1c based on the measurement value of fructosyl L-valine and the detection result of hemoglobin or the measurement value of fructosyl valylhistidine and the detection result of hemoglobin; and
A step of correcting whole blood / blood cell glucose to plasma glucose based on detection results of whole blood / blood cell glucose and hemoglobin.

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