JP2006304742A - Device and method for analyzing glycoprotein - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a proteinase capable of efficiently degrading a glycohemoglobin for quantifying the glycohemoglobin in high accuracy. <P>SOLUTION: A method for analyzing glycoprotein is provided, comprising the following procedure: A glycohemoglobin-containing specimen is treated with an Aspergillus proteinase, and then, the fructosylvaline concentration in the specimen is electrochemically sensed using a mechanism of sensing a fructosylamino acid oxidase acting on fructosyl L-valine and an electrochemically active substance varying in its amount by a reaction catalyzed by the fructosylamino acid oxidase. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to an analysis method and apparatus for measuring the amount of glycated hemoglobin at high speed and with high accuracy, and contributes to health management, clinical diagnosis, and the like.

  Glycated hemoglobin is widely used as an index for long-term blood glucose control in diabetic patients. Glycated hemoglobin is a kind of glycated protein in which sugar is non-enzymatically bound to hemoglobin. Among the glycated hemoglobins, particularly, a fraction called HbA1c formed a Schiff base at the valine residue at the N-terminal of the β-chain of hemoglobin A to form aldimine (unstable), and further undergoes Amadori transfer 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. Note that the N-terminal of the β chain after Amadori transition 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. In general, blood glucose levels tend to vary depending on the lifestyle before meals, meals, etc., but since HbA1c is an average value over a long period of time, it is suitable for use as a determinant for diagnosis and treatment of diabetes. It is said that there is.

  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 enzyme method, a glycated amino acid is cut out from a glycated protein by some technique, and then the amount of glycated amino acid generated is detected using an enzyme such as glycated amino acid oxidase. There is a possibility that more accurate analysis can be performed by utilizing the selectivity of the enzyme, but there are still many unsolved problems.

  First, as a first problem, a proteolytic enzyme that cuts out a glycated amino acid residue as quickly as possible is required. At the same time, proteolytic enzymes with high activity may degrade not only hemoglobin but also glycated amino acid oxidase, and a method for selectively degrading hemoglobin is being sought.

  The second problem is that the same problem remains in immunization, etc., but since analysis is performed on whole blood, glycated amino acids, especially fructosyl valine, should be selected from many coexisting components. It is necessary to measure selectively.

  Third, if fructosyl valine is quantitatively produced, there is no problem, but if quantification is performed in a partially decomposed system, it is necessary to devise a separate quantification method for estimating the total amount of hemoglobin.

  A method for measuring a glycated amino acid is described in Patent Document 2, but a method for cutting out a glycated amino acid from a glycated protein is not described.

  Proteolytic enzymes that cleave glycated amino acids from glycated proteins have been studied, and a method for measuring glycated amino acids in combination with fructosyl amino acid oxidase has been reported (Patent Document 3). However, as a result of various studies by the present inventors, an enzyme that produces a sufficient amount of fructosyl valine in a short time was not found from the proteolytic enzymes described.

  Furthermore, as a method of quantifying hydrogen peroxide generated when fructosyl amino acid oxidase is allowed to act on glycated amino acids, colorimetric combination with POD or the like, and applying a sample containing heme iron such as whole blood directly to the apparatus, The inventors have found that the system is very dirty and good reproducibility cannot be obtained.

  Conventionally, no proteolytic enzyme capable of efficiently degrading glycated hemoglobin has been found.

  It also eliminates various factors that cause problems when analyzing whole blood samples, such as concomitant interference components that occur when analyzing samples containing hemoglobin or analysis interference caused by heme iron, which has a high redox capacity. An effective method is not disclosed.

Furthermore, a method and apparatus for detecting the total amount of hemoglobin contained in a specimen and calculating the ratio of glycated hemoglobin simply and accurately has not been disclosed.
Japanese Patent Publication No. 5-59380 Japanese Patent Publication No. 5-33997 JP-A-11-196897

  An object of this invention is to provide the proteolytic enzyme which can decompose | disassemble glycated hemoglobin efficiently.

  It is another object of the present invention to provide a method and an apparatus for removing an analysis interfering factor accompanying analysis of a sample containing hemoglobin and detecting the amount of glycated hemoglobin with high accuracy.

  A further object of the present invention is to provide a method and apparatus for calculating the ratio of glycated hemoglobin by simply and accurately detecting the total amount of hemoglobin contained in a specimen.

  The present invention relates to an immobilized form in which fructosyl amino acid oxidase acting on fructosyl L-valine is immobilized, and a mechanism for detecting an electrochemically active substance that increases or decreases due to a reaction catalyzed by fructosyl amino acid oxidase. Before electrochemically detecting the concentration of silvaline and calculating the amount of glycated hemoglobin from the amount of fructosyl valine, before contacting the sample with the solidified form of fructosyl amino acid oxidase, the sample containing glycated hemoglobin should be treated with the Aspergillus proteolytic enzyme. The analysis method of glycated hemoglobin characterized by processing by this is disclosed.

  Further, it is desirable that the Aspergillus proteolytic enzyme is an alkaline proteolytic enzyme produced by Aspergillus oryzae.

  Further, when processing a sample containing glycated hemoglobin, the sample is mixed with an anionic surfactant-containing buffer, and the mixed sample is mixed with a carrier on which Aspergillus proteolytic enzyme is preferably chemically immobilized. It is a desirable form to obtain a sample that is contacted and decomposed with a buffer stream.

  It is possible to detect fructosyl amino acid and at least one free amino acid, calculate the ratio of the amount of fructosyl amino acid and the specific free amino acid concentration, and calculate the amount of glycated hemoglobin from this ratio easily and accurately. This is an effective method for calculating the ratio to total hemoglobin.

  To remove various interfering factors accompanying the analysis of samples containing heme iron, a sample containing glycated hemoglobin is treated with Aspergillus proteolytic enzyme, the treated solution is dialyzed, and the solution after dialysis is removed. It can be solved by contacting with the immobilized fructosyl amino acid oxidase.

  These analyzes include an immobilized form in which fructosyl amino acid oxidase acting on fructosyl L-valine is immobilized, and an electrochemical detection mechanism that detects electrochemically active substances that increase or decrease due to the reaction catalyzed by fructosyl amino acid oxidase. A first liquid feeding mechanism for feeding a buffer solution to the fructosyl amino acid oxidase immobilized body and the electrochemical detection mechanism; a second liquid feeding mechanism and a mechanism for injecting a specimen containing glycated hemoglobin; A column reactor in which a protease derived from Aspergillus is chemically bound is placed downstream of the injection mechanism, and after passing through the column reactor, the first and the first are passed through a semipermeable membrane that permeates a low molecular compound. The liquid fed by the liquid feeding mechanism 2 is allowed to run side by side for a certain time, and the substance that has permeated the semipermeable membrane is arranged downstream of the first liquid feeding mechanism. The glycated hemoglobin analyzer is brought into contact with the immobilized fructosyl amino acid oxidase.

  The fructosyl amino acid oxidase immobilized on the fructosyl amino acid oxidase acting on fructosyl L-valine and an electrochemical detection mechanism for detecting an electrochemically active substance that increases or decreases due to the reaction catalyzed by the fructosyl amino acid oxidase are provided. A first liquid-feeding mechanism for feeding a buffer solution to the tosylamino acid oxidase immobilized body and the electrochemical detection mechanism; a second liquid-feeding mechanism; a mechanism for injecting a specimen containing glycated hemoglobin; and the injection mechanism A column-shaped reactor in which a protease derived from Aspergillus is chemically bound is placed downstream of the first and second feeds through a semi-permeable membrane that passes through the column-shaped reactor and permeates low molecular weight compounds. The liquid fed by the liquid mechanism is allowed to run for a certain period of time, and the substance that has permeated the semipermeable membrane is disposed in the downstream of the first liquid feeding mechanism. It is desirable to contact the tosyl amino acid oxidase immobilized body and provide at least one free amino acid detection mechanism downstream of the first liquid feeding mechanism.

  According to the present invention, stable glycated hemoglobin in a blood sample can be quantified easily and accurately.

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

  The protease that can be used in the present invention is not particularly limited as long as it has a large effect of generating an N-terminal glycated amino acid from glycated hemoglobin or a fragment thereof. As a result of intensive studies by the present inventors, the action of Aspergillus proteolytic enzymes, particularly proteases derived from Aspergillus oryzae, was great. Further, since human hemoglobin precipitates at pH 5.0 or lower, a protease having an optimum pH range in which hemoglobin can be sufficiently dissolved is more preferable for the protease to act effectively. As the protease, neutral protease and alkaline protease can be preferably used, and most preferably alkaline protease derived from Aspergillus oryzae. An alkaline protease derived from Aspergillus oryzae is active at pH 7 or more and 9 or less.

  In the present specification, “Aspergillus proteolytic enzyme” or “protease derived from Aspergillus microorganism” may be a protease itself produced by an Aspergillus microorganism, and in the amino acid sequence of the protease, one or more These are variants obtained by substituting, adding, deleting, or inserting the amino acid, and variants that can increase the fructosyl valine concentration, such as Aspergillus proteolytic enzymes, are widely included.

  Furthermore, it was found that the protease derived from the microorganism of the genus Alpergillus can efficiently decompose hemoglobin in a short time by immobilizing it on an insoluble carrier at a high density. The amount of protease derived from Aspergillus microorganisms immobilized on an insoluble carrier is 1 to 20 mg / column, more preferably 5 to 10 mg / column.

When glycated hemoglobin is fragmented by the action of a protease-immobilized column, glycated amino acids and amino acids are generated. If this glycated amino acid and amino acid are sequentially converted to hydrogen peroxide with fructosyl amino acid oxidase and the amino acid oxidase (regardless of order), and the hydrogen peroxide produced by each oxidase is detected electrochemically, Glycated amino acids and amino acid concentrations can be measured.

The amount of glycated hemoglobin as a clinical diagnostic index is expressed as a ratio of a fraction in which a specific site is glycated in all hemoglobin. Therefore, once the hemoglobin concentration in the sample used for analysis is known, the ratio with the amount of fructosyl L-valine detected from the sample can be taken. Examples of the method for measuring the hemoglobin concentration include a method for determining from an absorbance near 500 nanometers, a method for measuring the amount of iron ions in a sample, and a method for measuring the amount of a specific amino acid or peptide produced by decomposition. Among them, the colorimetric method and the method of analyzing amino acids with amino acid oxidase can achieve the purpose with a simple mechanism.

Examples of the origin of the fructosyl amino acid oxidase include Corynebacterium, Aspergillus, Fusarium, Gibberella, Penicillium, Bacillus and the like. .

  For detecting fructosyl valine, a glycated amino acid at the N-terminal of glycated hemoglobin, it does not substantially act on glycated lysine in which the ε-amino group is glycated, but acts on a glycated amino acid in which the α-amino group is glycated Enzymes are preferred. The enzyme having such an action is preferably fructosyl amino acid oxidase derived from Corynebacterium. 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.

  In order to measure the amino acid produced together with the fructosyl amino acid, it may be converted into hydrogen peroxide by an amino acid oxidase using the amino acid as a substrate and detected electrochemically in the same manner as the fructosyl amino acid. As amino acids generated by the action of the protease of the present invention, valine, histidine, leucine, threonine, proline, glutamic acid, lysine, and serine are sequentially formed from the N-terminal of the β chain, and the amino acid oxidase may be used. For example, use of L-glutamate oxidase or L-lysine oxidase is desirable from the viewpoint of heat stability and sensitivity.

If these fructosyl amino acid oxidases and amino acid oxidases are also used in an immobilized manner, they can be used repeatedly, and are more preferable.
As enzyme immobilization methods such as protease derived from Aspergillus microorganisms used in the present invention, fructosyl amino acid oxidase, amino acid oxidase, etc., known as protein immobilization methods such as physical adsorption method, ionic bond method, inclusion method, and covalent bond method However, the covalent bond method is preferable 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 As the carrier for immobilizing the enzyme on an insoluble carrier, known carriers such as diatomaceous earth, activated carbon, alumina, titanium oxide, cross-linked starch particles, cellulose polymer, chitin and chitosan derivatives can be used.

Hydrogen peroxide can be measured directly or indirectly by a known method. However, since the saccharification rate of naturally occurring hemoglobin saccharified product is as low as about 5%, it is sufficiently expected that the amount of hydrogen peroxide derived from fructosyl valine generated by protease degradation is smaller than usual. Electrochemical methods such as amperometry are preferably used for highly sensitive measurement of hydrogen peroxide.
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, shown in FIG. 2, includes a mechanism (1,2) for forming a first buffer flow, a mechanism (3,4,6) for injecting a sample, and the sample injection. A protease-immobilized body (25) and a semipermeable membrane (26) are disposed downstream of the mechanism. A mechanism (21, 22) for forming a flow of the second buffer solution and an electrode (15, 17) capable of detecting the concentration of the electrochemically active substance downstream of the semipermeable membrane are arranged, and the fructosyl amino acid A detection electrode system (14, 15) and an amino acid detection electrode system (16, 17) are configured. Furthermore, the fructosyl amino acid detection electrode system has a fructosyl amino acid oxidase immobilized body (14), and an electrode (15) capable of detecting the concentration of the electrochemically active substance downstream thereof. Is an apparatus for measuring glycated hemoglobin, characterized in that an amino acid oxidase-immobilized body (16) suitable for the purpose of analysis and an electrode (17) capable of detecting the electrochemically active substance concentration are arranged.

  The membrane (26) used in the low molecular component separation mechanism in the sample after protease degradation is difficult to permeate or does not permeate pentapeptides, hexapeptides and larger peptides that are thought to coexist in the process of protease degradation. Any material can be used as long as it can permeate silvaline and amino acids. 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.

  Further, from the viewpoint of removing interfering components, the sample can be treated with an ultrafiltration membrane after protease decomposition and then used for analysis.

  A surfactant has two functions when hemoglobin is decomposed into an amino acid and a glycated amino acid by a protease. Hemolysis and hemoglobin molecular structure change. Most of hemoglobin is present in erythrocytes, and hemoglobin comes 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 it is presumed that degradation by a protease is facilitated. 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 0.01 to 10%, preferably 0.05 to 5%.

  As a specific measuring device, FIG. 1 shows a flow type, one-channel measuring device. Buffer A is pumped from the buffer tank (1) by the pump (2). Insert the needle (6) into the sample (5), close the valve (9), open the valve (10), and pull the syringe pump (11) to pull the sample into the metering loop (4) of the metering valve (3). Pull in. Next, the metering valve (3) is switched, and the specimen accumulated in the loop (4) is pushed out by the buffer solution. Excess specimen once opens the valve (9) and closes the valve (10), draws the cleaning liquid (8) into the syringe pump (11), then closes the valve (9) and opens the valve (10) to push out the cleaning liquid. As a result, it is pushed out to the waste pot (7) and stored in the waste liquid bottle (20). The injected sample follows the flow of the buffer solution, passes through the mixing pipe (13) installed in the thermostat (12), is mixed with the temperature adjustment and the buffer solution, and the first immobilized enzyme. The hydrogen peroxide produced through the column (14) is detected by the hydrogen peroxide electrode (15). The sample then passes through the second immobilized enzyme column (16) and is detected by the downstream hydrogen peroxide electrode (17). The waste liquid accumulates in the waste liquid bottle (19) through the back pressure coil (18). The blood sample (5) may be directly supplied into the analyzer, but the sample (5) may be supplied into the device after being previously treated with protease. When the blood sample is directly supplied into the analyzer, it may be supplied together with, for example, protease and treated with protease in the flow path of the buffer solution.

The substance concentration is quantified by detecting changes in the current values of the hydrogen peroxide electrodes (15) and (17).
FIG. 2 shows a flow type fructosyl amino acid measuring apparatus incorporating a dialysis module. Buffer B is sent from the buffer tank (21) by the pump (22), the needle (6) is inserted into the sample (5), the valve (9) is closed, the valve (10) is opened, and the syringe pump By pulling (11), the specimen is drawn into the metering loop (4) of the metering valve (3). Next, the metering valve (3) is switched, and the specimen accumulated in the loop (4) is pushed out by the buffer solution. Excess specimen once opens the valve (9) and closes the valve (10), draws the cleaning liquid (8) into the syringe pump (11), then closes the valve (9) and opens the valve (10) to push out the cleaning liquid. As a result, it is pushed out to the waste pot (7) and stored in the waste liquid bottle (20). The injected sample follows the flow of the buffer B, passes through the protease immobilized enzyme column (25) arranged in the thermostat (12), and produces fructosyl amino acids and amino acids from the proteins in the sample. The sample on which the protease has acted is transported to a dialysis module (26) installed further downstream, where only low molecular components in the sample are fructosyl amino acids and amino acids in a regenerated cellulose membrane having a film thickness of 20 μm and a molecular weight fraction of 12000 to 14000. It is guided to the detection mechanism (14, 15, 16, 17). 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 (26) are peroxidized with the first immobilized enzyme column (14). Hydrogen peroxide produced by the first immobilized enzyme column (14) passing through the hydrogen electrode (15) is detected. The buffer solution containing the specimen that has passed through the first detection mechanism (14, 15) passes through the second immobilized enzyme column (16) and the hydrogen peroxide electrode (17), and passes through the second immobilized enzyme column ( The hydrogen peroxide produced in 16) is detected. The polymer component in the sample that has not passed through the dialysis membrane of the dialysis module (26) accumulates in the waste liquid bottle (19).

  The buffer solution to be passed through this apparatus is not particularly limited, but may be selected so that the pH of the immobilized enzyme (14, 16, 25) is high (for example, pH 7 to 9). Specific buffer B contains, for example, 50 mM phosphoric acid and 0.1% sodium dodecyl sulfate, has a pH of 8.0, and a flow rate of 0.8 ml / min. Alternatively, Buffer A contains 100 mM phosphate, 50 mM potassium chloride, 1 mM sodium azide, has a pH of 8.0 and a flow rate of 0.8 ml / min.

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 (80-100 mesh) was thoroughly dried, 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 prepared by dissolving fructosyl amino acid oxidase (manufactured by Kikkoman) at a concentration of 18 units / ml in a pH 7.0, 100 mM sodium phosphate buffer solution was brought into contact with this formylated refractory brick and left at 0 to 4 ° C. for 1 day. 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) Measuring device 1
FIG. 1 shows a flow type single-channel measuring device. Buffer A is sent from the buffer tank (1) by the pump (2), and 5 μl of sample is injected using the metering valve (3). A fructosyl amino acid oxidase-immobilized column is disposed in the first immobilized enzyme column (14), and a hydrogen peroxide electrode (15) is disposed downstream thereof, and the second immobilized enzyme column (16) and the second peroxidation are disposed. The hydrogen electrode (17) is not arranged. The injected sample follows the flow of the buffer solution, passes through the mixing pipe (13) installed in the thermostat (12), is mixed with the temperature adjustment and the buffer solution, and immobilizes the fructosyl amino acid oxidase. Through the column (14) and the hydrogen peroxide electrode (15), hydrogen peroxide is generated from fructosyl valine in the sample, and a change in 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 8.0.

  The flow rate of the buffer solution was 1.0 ml / min, and the temperature of the thermostatic bath was 37 ° C.

(4) Measuring device 2
FIG. 2 shows a flow type fructosyl amino acid measuring apparatus incorporating a dialysis module. Buffer B is sent from the buffer tank (21) by the pump (22), and 100 μl of sample is injected using the metering valve (3). The protease immobilized enzyme column (25), the second immobilized enzyme column (16), and the hydrogen peroxide electrode (17) are not arranged, and the first immobilized enzyme column (17) is immobilized with fructosyl amino acid oxidase. Arrange the column. The injected sample was carried along the flow of buffer B to the dialysis module (26) installed in the thermostatic bath (12), and the sample was formed on a regenerated cellulose membrane having a film thickness of 20 μm and a molecular weight fraction of 12000 to 14000. Only the low molecular components are guided to the fructosyl amino acid detection mechanism (14, 15). 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 (26) are the fructosyl amino acid oxidase immobilized column (14) and Hydrogen peroxide produced from the fructosyl amino acid in the sample is detected after passing through the hydrogen peroxide electrode (15).

  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 0.8 ml / min.

The temperature of the thermostatic bath was 37 ° C.
(5) Characteristics of Fructosyl Amino Acid Oxidase Immobilized Column 5 μl of fructosyl glycine was injected using the measuring device 1 of (3), and the results of examining the characteristics of the fructosyl amino acid oxidase column are shown below.
pH Characteristics FIG. 3 shows the measurement results of fructosylglycine oxidation activity when the pH of the buffer is 7.0, 7.5, 8.0, 8.5, and 9.0. High activity was exhibited at pH 8.0 to 9.0.
Substrate specificity Table 1 shows the substrate specificity of the fructosyl amino acid oxidase-immobilized column.

Stability The fructosyl amino acid oxidase-immobilized column maintains the initial activity for more than 1 month under the conditions of pH 8.0 of the buffer, 1.0 ml / min of the buffer flow rate, and 37 ° C. And stable.

  The fructosyl amino acid-immobilized column used does not substantially act on ε-fructosyl lysine but selectively acts on fructosylglycine and fructosyl valine in which the α-position is glycated. In addition, since the optimum pH is in the alkaline region, it is desirable that the proteolytic enzyme used for measuring the glycated amino acid of the protein has a high activity in the alkaline region.

(6) Separation of low molecular components by semipermeable membrane Amino acids and glycated amino acids generated by degrading human hemoglobin with proteolytic enzymes are measured using amino acid electrodes or fructosyl amino acid oxidase immobilized enzyme columns and hydrogen peroxide electrodes. On the other hand, if only low molecular components are separated by a semipermeable membrane after degradation with a proteolytic enzyme, amino acids and glycated amino acids in human hemoglobin can be measured with good reproducibility.

(7) Proteolytic degradation of human hemoglobin Since fructosyl amino acid oxidase-immobilized column, which is optimal in the alkaline region, is used for the measurement of fructosyl amino acids generated by degrading human hemoglobin with proteolytic enzymes, it is active in the alkaline region. Alkaline proteolytic enzyme Ummamizyme G (manufactured by Amano Enzyme) produced by Aspergillus oryzae having a high A

  The protease-treated solution is prepared by weighing human hemoglobin (manufactured by Sigma), dissolving it in 20 mM phosphate buffer pH 8.0 containing 2% sodium dodecyl sulfate, and adding 1 mg / ml equinezyme G. The concentration of human hemoglobin in the protease treatment solution is 30 mg / ml.

  The protease treatment solution thus prepared was reacted at 37 ° C. for 8, 17, 25, 33, 42, and 51 minutes, and then 100 μl of the protease treatment solution was injected into the measuring device 2 of (4). Fructosylvaline is measured.

The fructosyl valine concentration at each reaction time changes as shown in FIG. When human hemoglobin was not treated with protease, fructosyl valine was not detected at all. However, it was found that fructosyl valine was released from hemoglobin by treatment with protease for a predetermined time. This protease effectively acts on human hemoglobin.
Comparative Example 1
We studied the degradation of human hemoglobin using a protease whose origin and optimum pH are different from those of horse zyme.

  The proteolytic enzymes used were horse zyme G (Aspergillus oryzae), protease A “Amano” G (Aspergillus oryzae), protease N “Amano” G (Bacillus subtilis), bromelain F (Ananas comosus M.), peptidase R (Rhizopus oryzae). ), Protease P “Amano” 3G (Aspergillus melleus) (all manufactured by Amano Enzyme), proteinase K (Tritirachium album, manufactured by Sigma), protease XIV (Streptomyces griseus, manufactured by Sigma), Sumiteam MP (Aspergillus sp., New Nippon Chemical Industry).

  In the same manner as in Example 1 (6), human hemoglobin was prepared as a protease treatment solution of the above-mentioned various proteolytic enzymes at 1 mg / ml, reacted at 37 ° C. for 30 minutes, and then placed in the measuring device 2 of Example 1 (4). 100 μl of protease treatment solution was injected. The results are shown in Table 2. In the table, ◯ indicates that a detected value of fructosyl amino acid of 40 pA or more is obtained, Δ indicates that a fructosyl amino acid of about 25 pA is detected, and × indicates that the detected value is less than 20 pA.

Large detection values were obtained with the protease derived from Aspergillus oryzae, but fructosyl amino acids were not detected with other enzymes. This indicates that a proteolytic enzyme derived from Aspergillus oryzae is effective in degrading human hemoglobin. Furthermore, among the Aspergillus oryzae-derived proteolytic enzymes, the alkaline pH gives a larger detection value than the neutral pH, so the alkaline Alpergillus oryzae-derived proteolytic enzyme can be said to be optimal for the degradation of human hemoglobin. .

Example 2
As shown in Example 1, human hemoglobin can be decomposed by using a proteolytic enzyme derived from Aspergillus oryzae, and amino acids and glycated amino acids generated from human hemoglobin are detected by a measuring device through a semipermeable membrane. be able to. Furthermore, in order to simplify the processing and shorten the analysis time, analysis was performed using a column in which the present proteolytic enzyme was immobilized on a carrier at a high density. Details are shown below.

(1) Production of fructosyl amino acid oxidase-immobilized column A fructosyl amino acid oxidase (manufactured by Kikkoman) immobilized column was prepared in the same manner as in Example 1.

(2) Production of lysine oxidase-immobilized column Example 1 (1) 150 mg of refractory brick (30-60 mesh) is formylized in the same manner as in the production method of a fructosyl amino acid oxidase-immobilized column. Then, 50 μL of a solution prepared by dissolving lysine oxidase at a concentration of 50 units / ml is brought into contact with this refractory brick at pH 7.0 and 100 mM sodium phosphate buffer, and allowed to stand at 0 to 4 ° C. for 1 day for immobilization. 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 lysine oxidase-immobilized column.

(3) Manufacture of equinezyme G-immobilized column 300 mg of toyonite 200 (average particle size 170 μm, manufactured by Toyo Denka Kogyo Co., Ltd.) was immersed in a 20% ethanol solution of 10% γ-aminopropyltriethoxysilane for 1 hour. Wash 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. This formylated toyonite 200 was contacted with 800 μl of a solution prepared by dissolving Umamizyme G (manufactured by Amano Enzyme) at a concentration of 100 mg / ml in a pH 7.0, 100 mM sodium phosphate buffer, and allowed to stand at 0 to 4 ° C. for 1 day. Turn into. 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 an equinezyme G-immobilized column.

(4) Method for Producing Hydrogen Peroxide Electrode A hydrogen peroxide electrode was produced in the same manner as in Example 1.

(5) Measuring device The immobilized enzyme column (25) of FIG. 2 is equipped with the equine enzyme G immobilized column, the first immobilized enzyme column (14) with the fructosyl amino acid oxidase immobilized column, and the second immobilized enzyme. An lysine oxidase-immobilized column is incorporated into the column (16), so that a flow type fructosyl amino acid and L-lysine can be measured simultaneously. Buffer B is sent from the buffer tank (21) by the pump (22), and 100 μl of sample is injected using the metering valve (3). The injected sample is carried along the flow of the buffer B to the equinezyme G immobilization column arranged in the thermostat (12), and glycated amino acid and amino acid are produced by the action of equinezyme G. The sample on which the Umamizyme G acted is transported to the dialysis module (26) installed further downstream by the flow of the buffer B, and the low molecular component in 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 lead to fructosyl amino acids and amino acid detection mechanisms. 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 (26) are the fructosyl amino acid oxidase immobilized column (14) and Hydrogen peroxide generated from fructosyl amino acids in the sample is detected after passing through the hydrogen peroxide electrode (15). Next, the buffer solution containing the sample that passed through the fructosyl amino acid detection mechanism (14, 15) passed through the lysine oxidase-immobilized column (16) and the hydrogen peroxide electrode (17), and the excess solution generated from the lysine in the sample. Detect hydrogen oxide.

  The buffer solution to be flowed to the apparatus has a buffer A containing 100 mM phosphoric acid, 50 mM potassium chloride, 1 mM sodium azide, pH 8.0, and a flow rate of 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 0.8 ml / min.

  The temperature of the thermostatic bath was 37 ° C.

(6) Measurement of fructosyl amino acid and amino acid in human hemoglobin 100 μl of human hemoglobin solution is injected with the measuring device of (5), and fructosyl valine concentration and lysine concentration in the sample are measured simultaneously.

  A human hemoglobin solution is prepared by weighing a predetermined amount of human hemoglobin (manufactured by Sigma) so that the hemoglobin concentration is 30 mg / ml, and sufficiently dissolving it in 20 mM phosphate buffer pH 8.0 containing 2% sodium dodecyl sulfate. To do.

  When the fructosyl valine concentration and the lysine concentration in the 30 mg / ml human hemoglobin solution were measured with the measuring device (5), fructosyl valine was 15.1 μM and lysine was 12.6 μM.

In Example 1, which was reacted with a solution enzyme, a reaction time of about 20 minutes was required to produce about 15 μM fructosyl valine. However, in this measurement apparatus, the contact time between the equine zymme G immobilized column and the sample ( Fructosylvaline of about 15 μM is produced in about 10 seconds). Human hemoglobin could be efficiently degraded by immobilizing equinezyme G at high density.

  According to the present invention, stable glycated hemoglobin in a blood sample can be quantified easily and accurately, contamination of the measuring device can be avoided, and diabetes can be easily tested.

Schematic diagram of measuring device Schematic diagram of a measuring device for dialysis PH characteristics of immobilized enzyme Time course of solution proteolysis

Explanation of symbols

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

Claims (11)

Fructosyl amino acid oxidase that acts on fructosyl L-valine and a mechanism that detects electrochemically active substances that increase or decrease due to the reaction catalyzed by fructosyl amino acid oxidase are used to detect fructosyl amino acids in the specimen. A method for analyzing glycated hemoglobin, wherein a specimen containing glycated hemoglobin is treated with an Aspergillus proteolytic enzyme before the sample is brought into contact with the solidified form of fructosyl amino acid oxidase for electrochemical detection of phosphorus concentration . The method for analyzing glycated hemoglobin according to claim 1, wherein the Aspergillus proteolytic enzyme is an alkaline proteolytic enzyme produced by Aspergillus oryzae or a variant thereof. The glycated hemoglobin according to claim 1, wherein the glycated hemoglobin is treated by mixing a sample containing glycated hemoglobin with an anionic surfactant-containing buffer and then contacting the sample with a carrier on which an Aspergillus proteolytic enzyme is immobilized. Analysis method. The fructosyl amino acid and at least one kind of free amino acid are detected, the ratio of the amount of fructosyl amino acid and the specific free amino acid concentration is calculated, and the amount of glycated hemoglobin is calculated from the ratio. Analysis method of glycated hemoglobin. The sample containing glycated hemoglobin is treated with an Aspergillus proteolytic enzyme, the treated solution is dialyzed, and the dialyzed solution is brought into contact with the immobilized fructosyl amino acid oxidase. Analysis method. An immobilized form (14) on which fructosyl amino acid oxidase acting on fructosyl L-valine is immobilized; and an electrochemical detection mechanism (15) for detecting an electrochemically active substance that increases or decreases by a reaction catalyzed by fructosyl amino acid oxidase; , A glycated hemoglobin analyzer equipped with a mechanism (3,4,6) for injecting a specimen containing glycated hemoglobin, and having a column shape in which a protease derived from the genus Aspergillus is bound downstream of the mechanism for injecting the specimen An apparatus for analyzing glycated hemoglobin, comprising a reactor (25). An immobilized form (14) on which fructosyl amino acid oxidase acting on fructosyl L-valine is immobilized; and an electrochemical detection mechanism (15) for detecting an electrochemically active substance that increases or decreases by a reaction catalyzed by fructosyl amino acid oxidase; , A glycated hemoglobin analyzer equipped with a mechanism (3, 4, 6) for injecting a specimen containing glycated hemoglobin, wherein at least one free amino acid is provided downstream of the first liquid feeding mechanism (1, 2) 7. The glycated hemoglobin analyzer according to claim 6, further comprising a detection mechanism (16, 17). An immobilized body (14) in which fructosyl amino acid oxidase acting on fructosyl L-valine is immobilized, and an electrochemical detection mechanism (15) for detecting an electrochemically active substance that is increased or decreased by a reaction catalyzed by fructosyl amino acid oxidase. A fructosyl amino acid oxidase immobilized body (14) and an electrochemical detection mechanism (15), a first liquid feeding mechanism (1, 2) for feeding a buffer solution, and a second liquid feeding mechanism ( 21 and 22) and a column reactor (25) to which a protease derived from the genus Aspergillus is bound downstream of the liquid feeding mechanism, and after passing through the column reactor (25), a semi-permeating compound of low molecular weight is placed. Through the permeable membrane (26), the liquids sent by the first and second liquid feeding mechanisms are allowed to run for a certain period of time, and the substance that has permeated the semipermeable membrane is allowed to flow through the first liquid feeding mechanism (1,2 The apparatus for analyzing glycated hemoglobin, wherein the fructosyl amino acid oxidase-immobilized body (14) disposed downstream of the glycated hemoglobin is contacted. An immobilized body (14) in which fructosyl amino acid oxidase acting on fructosyl L-valine is immobilized, and an electrochemical detection mechanism (15) for detecting an electrochemically active substance that is increased or decreased by a reaction catalyzed by fructosyl amino acid oxidase. A fructosyl amino acid oxidase immobilized body (14) and a first liquid feeding mechanism (1, 2) for feeding a buffer solution to the electrochemical detection mechanism, and a second liquid feeding mechanism (21, 22). ) And a glycated hemoglobin-containing specimen (3,4,6), and a column reactor (25) chemically coupled with a protease derived from the genus Aspergillus downstream of the injection mechanism, After passing through the column-shaped reactor (25), the liquid fed by the first and second liquid feeding mechanisms is fed through the semipermeable membrane (26) that permeates the low molecular weight compound. The substance that was allowed to run for a fixed time and permeated through the semipermeable membrane (26) was brought into contact with the immobilized fructosyl amino acid oxidase (14) disposed downstream of the first liquid feeding mechanism (1, 2), and The glycated hemoglobin analyzer according to claim 7, wherein at least one type of free amino acid detection mechanism (16, 17) is provided downstream of one liquid feeding mechanism (1, 2). Use of an Aspergillus proteolytic enzyme to process a sample containing glycated hemoglobin to measure glycated hemoglobin. 2. The method for analyzing glycated hemoglobin according to claim 1, wherein the ratio of fructosyl amino acid and the amount of hemoglobin obtained from light absorption of the sample is taken, and the amount of glycated hemoglobin is calculated from the ratio.

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