Method and device for evaluating protein glycation degree

Abstract

According to one embodiment of the present disclosure, provided is a method for measuring the glycation degree of protein, the method comprising: a feature of providing a solution possibly containing a target protein; a feature of causing the solution to contact a coloring indicator; a feature of causing the solution to contact a protease; a feature of using a first reaction between the coloring indicator and the target protein to determine the concentration of the target protein in the solution; a feature of using a second reaction between the protease and the target protein to determine the concentration of the protein that has been glycated; and a feature of determining the glycation degree of the target protein on the basis of the concentration of the target protein and the concentration of the target protein that has been glycated.

Description

Method and apparatus for evaluating the degree of glycation of proteins

[0001] The present disclosure relates to a method and apparatus for assessing the degree of glycosylation of a protein.

[0002] Conventionally, glycated proteins have been measured using high-performance liquid chromatography (HPLC), enzymatic methods, etc. While HPLC is a simple measurement method, it takes several tens of minutes to separate and quantify target components using multiple columns, and requires multiple high-pressure pumps, resulting in a large-scale instrument. Enzymatic methods are commonly used and simple, but require separate measurements of the total amount of protein (protein concentration) and the amount of glycated protein (glycated protein concentration), each using a different color reagent. Furthermore, when using colorimetric methods, the absorption peaks of non-glycated proteins and glycated proteins are close to each other. Therefore, in simultaneous measurements, one absorption peak is affected by the other peak, preventing accurate measurement of both. Therefore, simultaneous measurement of non-glycated proteins and glycated proteins using colorimetric methods requires a bleaching step after one measurement.

[0003] Alternatively, the concentrations of non-glycated and glycated proteins can be measured separately using conventional colorimetric methods. This requires combining two different measurement methods. However, the two measurements have different measurement errors. For example, different dilution rates result in different errors. That is, when calculating the degree of protein glycation, different errors may occur between the denominator (protein concentration) and the numerator (glycated protein concentration). Therefore, there is a risk of a large measurement error.

[0004] Some embodiments of the present disclosure provide a method for assessing the degree of glycosylation of a protein. The method comprises providing a solution potentially containing a protein of interest. In some embodiments, the method comprises contacting a color indicator with the solution and determining the concentration of the protein of interest in the solution using a first reaction between the color indicator and the protein of interest. In some embodiments, the method comprises contacting a protease with the solution and determining the concentration of the glycated protein of interest using a second reaction between the protease and the protein of interest. In some embodiments, the method further comprises determining the degree of glycosylation of the protein of interest based on the concentration of the protein of interest and the concentration of the glycated protein of interest.

[0005] According to some embodiments of the present disclosure, for example, albumin concentration may be measured using absorbance and glycated albumin may be measured using a hydrogen peroxide electrode. In other words, they can be measured using different detection methods. For example, while conventional colorimetric methods require separate measurements at absorbances of similar wavelengths (e.g., 546 nm and 600 nm), the method described above allows simultaneous measurements. Furthermore, for example, sample weighing and reagent dilution errors can be made uniform. For example, when calculating a ratio (GA value, GA%) from albumin concentration and glycated albumin concentration, errors in the numerator and denominator are canceled out, improving measurement accuracy. Alternatively, the degree of glycation of proteins can be easily and accurately evaluated.

[0006]

[0013] Further aspects and advantages of the present disclosure will become readily apparent to those skilled in the art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the present disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.

[0007] 1 is a flowchart of a method for evaluating the glycation degree of a protein according to one embodiment. FIG. 2 is a flowchart of a method for evaluating the glycation degree of a protein according to one embodiment. FIG. 3 is a graph showing the change in absorbance over time at multiple albumin concentrations according to one embodiment. FIG. 4 is a graph showing a calibration curve of albumin concentrations based on absorbance according to one embodiment. FIG. 5 is a graph showing the relationship between albumin concentrations in actual samples between a method according to one embodiment and a conventional method. FIG. 6 is a graph showing the effect of the presence or absence of BCP on GA concentration measurement according to one embodiment. FIG. 7 is a graph showing the change in absorbance over time at multiple albumin concentrations according to one embodiment. FIG. 8 is a graph showing the change in sensor output over time at multiple albumin concentrations according to one embodiment. FIG. 9 is a graph showing the relationship between GA measurement values ​​obtained by the method according to one embodiment and GA values ​​obtained by a conventional method. FIG. 10 is a graph showing the change in absorbance over time at multiple albumin concentrations according to one embodiment. FIG. 11 is a graph showing the relationship between GA values ​​obtained by the method according to one embodiment and GA values ​​obtained by a conventional method. FIG. 12 is a block diagram of an apparatus for evaluating the glycation degree of a protein according to one embodiment. FIG. 13 is a block diagram of an apparatus for evaluating the glycation degree of a protein according to one embodiment. 1 is a block diagram of an apparatus for evaluating the degree of glycation of a protein according to one embodiment. 2 is a graph of absorbance showing the effect of the presence or absence of SDS on the improved BCP method according to one example.

[0008] <Measurement Subject> In some embodiments, the subject (test subject) may include or may be a human. In some embodiments, the subject may include or may be a non-human animal. The subject may include or may be a mammal. The subject may be, for example, but not limited to, a working animal, a livestock animal, a pet, or a wild animal.

[0009] The sample to be measured may be a solution. The "solution" may be a body fluid, a solution derived from a body fluid, or a diluted solution of a body fluid. The solution may be a solution that is not a body fluid (non-body fluid-derived), or a mixture of a body fluid or a body fluid-derived solution and a non-body fluid-derived solution. The solution may be a solution used for sample measurement, or a solution used for calibration measurement. For example, the solution may be a standard solution or a calibration solution. For example, the solution may be a liquid that does not intentionally or deliberately contain the substance to be measured, so that it is used for calibration, etc. The sample to be measured may be a specimen. The solution may be a solution containing a chemical substance.

[0010] The "body fluid" may be lymph, tissue fluid such as interstitial fluid, intercellular fluid, or interstitial fluid, or may be body cavity fluid, serous cavity fluid, pleural fluid, ascites, pericardial fluid, cerebrospinal fluid (spinal fluid), synovial fluid, or aqueous humor (aqueous humor). The body fluid may be digestive fluid such as saliva, gastric juice, bile, pancreatic juice, or intestinal fluid, or may be sweat, tears, nasal mucus, urine, semen, vaginal fluid, amniotic fluid, or milk. The body fluid may be animal or human. The "body fluid" may also be a solution. The solution may contain a physiological buffer solution such as phosphate-buffered saline (PBS) or N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid buffer (TES) containing the substance to be measured. The solution is not particularly limited as long as it contains the substance to be measured.

[0011] The solution may contain a target substance. The solution may potentially contain a target substance. In some embodiments, the target substance may be a molecule, ion, polymer, biomolecule, etc. The target substance may comprise a biomolecule. The target substance may be a protein, a glycated protein, etc. For example, the solution may be tears, and the target substance may be albumin, glycated albumin, hemoglobin, and / or glycated hemoglobin contained in tears. Alternatively, the target substance may be albumin, glycated albumin, hemoglobin, or glycated hemoglobin in blood, serum, or plasma, or may be albumin, glycated albumin, hemoglobin, or glycated hemoglobin in interstitial fluid, urine, or saliva. Albumin may be oxidized albumin (HNA) or reduced albumin (HMA). In some embodiments, the substance to be measured may be AGEs (Advanced Glycation End Products). In some embodiments, the substance to be measured may be glycated lipids.

[0012] <Sensor> The sensor may be a chemical sensor, a biosensor, an ion sensor, etc. (hereinafter, these may be referred to as a "sensor," a "biochemical sensor," a "chemical sensor," or an "electrochemical sensor.") The sensor may include a plurality of sensors.

[0013] The sensor may include an electrode. The electrode may be an amperometric electrode. The electrode may include a hydrogen peroxide electrode. The electrode may include an oxygen electrode. The electrode may be a potentiometric electrode. The electrode may be an electrode for detecting ions (such as a pH electrode, a cyanide ion electrode, or an iodide ion electrode).

[0014] In some embodiments, the sensor may output an electrical signal. In some aspects, the sensor may output a current signal. The sensor may output a voltage signal or a charge. The sensor may be electrically connected to an ammeter, voltmeter, or the like.

[0015] <Measurement of Hydrogen Peroxide> In some embodiments, the produced glycated amino acid may be reacted with ketoamine oxidase to generate hydrogen peroxide. In some embodiments, the amount (concentration) of the generated hydrogen peroxide may be measured. In some embodiments, the amount (concentration) of hydrogen peroxide may be measured electrically.

[0016] "Ketoamine oxidase" generally refers to an oxidase that recognizes the ketoamine structure of a glycated amino acid or a peptide or peptide fragment containing a glycated amino acid residue and oxidizes the glycated amino acid to produce an amino acid, glucosone (α-ketoaldehyde), and hydrogen peroxide. Thus, ketoamine oxidase produces hydrogen peroxide at a concentration proportional to or related to the concentration of the glycated amino acid or peptide or peptide fragment containing the glycated amino acid residue that it recognizes.

[0017] The ketoamine oxidase may be a dehydrogenase, a kinase, or an oxidase, and may be fructosyl amino acid oxidase (FAOD), fructosyl peptide oxidase, fructosyl valylhistidine oxidase, fructosyl amine oxidase, amadoriase, fructosyl amine deglycase, or modified forms thereof.

[0018] In some embodiments, the ketoamine oxidase may be an oxidase (Group I ketoamine oxidase) that acts on an amino acid or peptide whose α-amino group is glycated. The amino acid or peptide may be valine, glycine, or valylhistidine. By using an oxidase that acts on an amino acid or peptide whose α-amino group is glycated, a glycated hemoglobin sensor or a glycated hemoglobin A1c (HbA1c) sensor can be constructed.

[0019] In some embodiments, the ketoamine oxidase may be an oxidase that acts on an amino acid or peptide whose ε-amino group is glycated (group II ketoamine oxidase). The amino acid may be lysine. The glycated amino acid may be fructosyl lysine. A glycoalbumin sensor can be constructed using an oxidase that selectively acts on an amino acid or peptide whose ε-amino group is glycated.

[0020] In some embodiments, the ketoamine oxidase may be an oxidase (Group III ketoamine oxidase) that acts on an amino acid or peptide whose α-amino group and / or ε-amino group is glycated. The amino acid or peptide may be lysine, valine, glycine, or valylhistidine. A glycated protein sensor can be constructed using an oxidase that acts on an amino acid or peptide whose α-amino group and / or ε-amino group is glycated.

[0021] In some embodiments, the sensor may include a detection unit. The detection unit may be a hydrogen peroxide detection unit. The "hydrogen peroxide detection unit" (hydrogen peroxide sensor) may be an electrochemical electrode or a hydrogen peroxide electrode. The hydrogen peroxide electrode may have a counter electrode, a reference electrode, and a working electrode. In certain embodiments, the detection unit may detect oxygen. For example, it may detect the amount or concentration of oxygen reduced by an enzyme reaction. Oxygen detection is considered to be relatively insensitive to molecules and ions that are noise sources and resistant to interference. Oxygen consumption may be measured by detecting oxygen. Since the detection unit is saturated with air, it may be used for enzyme sensing. The detection unit may be configured to perform multiple detection methods selectively or in combination.

[0022] Noise Reduction Using Ion Exchange Resin In some embodiments, the sensor may include an ion exchange resin on the detector. The sensor may include an ion exchange resin between the ketoamine oxidase layer and the hydrogen peroxide electrode.

[0023] For example, cation exchange resins such as Nafion (registered trademark) can be used to suppress or prevent ascorbic acid and uric acid, particularly negative ions, present in body fluids from permeating and reaching the detection unit, while anion exchange resins such as polypyrrole can be used to suppress or prevent dopamine, particularly positive ions, from permeating and reaching the detection unit.

[0024] The ion exchange resin may comprise one, multiple, or at least one type of ion exchange resin. The ion exchange resin may be configured with one, multiple, or at least one type of layer.

[0025] <Molecularly Imprinted Polymer Sensor> The sensor may include a molecularly imprinted polymer (MIP) membrane. The sensor may detect a change in charge or potential caused by molecular recognition of the MIP (potentiometry type). The sensor may also detect a change in current (e.g., mediator current) flowing through an electrode caused by molecular recognition of the MIP (amperometry type).

[0026] <Protease> "Protease" is generally a general term for peptide bond hydrolases that hydrolyze and catabolize proteins and polypeptides. Proteases may also be enzymes that break down proteins into peptide fragments. When a protein contains glycated amino acid residues, the peptide fragments produced by the action of the protease may include peptide fragments containing glycated amino acid residues and peptide fragments that are not glycated at all.

[0027] The "protease" may be an animal-derived protease, a plant-derived protease, or a microbial-derived protease. The protease may be an exopeptidase or an endopeptidase. The protease may be an aspartic protease, a metalloprotease, a serine protease, or a thiol protease.

[0028] The term "protease" may include multiple types or kinds of proteases, or may include a single type or kind of protease. For example, the protease may include either an endopeptidase or an exopeptidase, or may include both. Mixing multiple proteases may increase the decomposition efficiency.

[0029] The animal-derived protease may be trypsin, chymotrypsin, elastase, bovine pancreatic protease, cathepsin, calpain, protease type-I, protease type-XX, aminopeptidase N, carboxypeptidase, pancreatin (a mixture of multiple enzymes such as protease and amylase), or the like.

[0030] The plant-derived protease may be papain, bromelain, zingipain, kallikrein, ficin, chymopapain, or the like.

[0031] The protease derived from a microorganism may be a protease derived from Bacillus, a protease derived from Aspergillus, a protease derived from Penicillium, a protease derived from Streptomyces, a protease derived from Lysobacter, a protease derived from yeast, a protease derived from Tritirachium, a protease derived from Thermus, a protease derived from Pseudomonas, a protease derived from Achromobacter, or the like.

[0032] In some embodiments, the protease may be provided in liquid form or mixed with a solution. In some embodiments, the protease may be added to or mixed with a solution in solid form (e.g., a powder). In some embodiments, the protease may be supported on a support material and introduced into a solution. The protease may be supported on a flat plate, a curved surface, a sphere, beads, a biomolecule such as a protein, a polymer, etc.

[0033] Optical Measurements: As used herein, "optical measurement" generally refers to determining the optical properties of a substance using an optical element or device. In some embodiments, an optical measurement of a substance of interest may be measured. In some aspects, a property of a substance bound to or associated with the substance of interest (hereinafter, not the substance of interest itself, but a substance (e.g., a reagent) that is chemically, biologically, or physically bound to or associated with the substance of interest) may be measured. A property of a reagent may be measured. The reagent may be referred to as the "substance of interest."

[0034] In some embodiments, the optical measurement may include a spectroscopic measurement. For example, the absorbance of the substance of interest may be measured. In some embodiments, a color indicator corresponding to the substance of interest may be introduced. The color indicator may be detected or measured.

[0035] <Color Changing Indicator> As used herein, the term "color changing indicator" generally refers to a halochromic chemical compound. The color change may generally be reversible. Color changing indicators include, but are not limited to, acid-base indicators (pH indicators), redox indicators, adsorption indicators, TLC color indicators, and the like, which change the color of their solution depending on the pH. pH indicators may be, but are not limited to, bromocresol purple (BCP), bromocresol green (BCG), and the like.

[0036] In some embodiments, BCP or BCG can be used for albumin. For example, albumin binds to bromocresol green (BCG) to form an albumin-bromocresol green complex. At around pH 4, this complex exhibits a blue color. Therefore, the albumin concentration can be determined by measuring the absorbance (BCG method). A surfactant may be added to the measurement reagent. A surfactant may not be added to the measurement reagent. For example, a protein denaturant (such as SDS) may not be added.

[0037] For example, albumin binds to bromocresol purple (BCP) to form an albumin-bromocresol purple complex. This complex is blue in color. Therefore, the albumin concentration can be determined by measuring absorbance (BCP method). Generally, reduced albumin is converted to oxidized albumin using an oxidizing agent, and then the albumin concentration can be measured by a color reaction with BCP (improved BCP method). A surfactant may be added to the measurement reagent. For example, a protein denaturant (such as SDS) may not be added.

[0038] In some embodiments, the reaction in which albumin binds to bromocresol purple (BCP) or bromocresol green (BCG) to form a complex may be referred to as the "first reaction."

[0039] In some embodiments, the color changing indicator may be provided in liquid form and mixed with the solution, hi some embodiments, the color changing indicator may be added to or mixed with the solution in solid form (e.g., powder).

[0040] <Measurement of Absorbance> In some embodiments, an absorptiometer (colorimeter) may be used. The absorptiometer has a light source and a light receiver. All or a portion of the light emitted from the light source enters the target solution. All or a portion of the light that has passed through the solution enters the light receiver. The light absorbed by the target substance can be analyzed.

[0041] The wavelength of the light source may be appropriately selected depending on the color indicator used. For example, the absorbance may be measured at a wavelength such as a dominant wavelength of 600 to 630 nm for BCP and at 565 to 660 nm for BCG.

[0042] In some embodiments, the change in absorbance of the color indicator over time may be measured. In some aspects, the absorbance may be measured at at least two (or more) times. For example, the initial protein concentration (before the start of protease degradation, t=0) may be determined based on measurements at two times. In some aspects, the time at which protease degradation begins may be measured, and the protein concentration may be determined based on measurements corresponding to the time from this start time. In some aspects, the protein concentration may be determined based on measurements at three or more times. From at least three or more measurements, it may be possible to determine the initial protein concentration even if the time from the start of protease degradation is not clear.

[0043] In some embodiments, the protein concentration may be determined by referring to a previously prepared calibration curve showing the relationship between absorbance and protein concentration. For example, a calibration curve showing the relationship between absorbance A(t) changing with time (t) and its change over time (ΔA(t) / Δt) is prepared in advance. In an actual measurement, the absorbance A(t1) and its slope (ΔA(t1) / Δt) at a certain time (t=t1) are determined. From this, the absorbance A(t=t0) at the time t=t0 when the protease starts decomposing can be determined.

[0044] In some embodiments, measurement of the glycated protein may be started after a predetermined or specified time has elapsed since the start of protease degradation. In some embodiments, measurement of the glycated protein may be started when the absorbance A becomes sufficiently small. In some embodiments, when the change in absorbance over time (ΔA / Δt) becomes substantially zero, it is determined that sufficient protease degradation has been completed, and measurement of the glycated protein may be started, or the measurement results of the glycated protein at that time may be adopted. In some embodiments, when the change in absorbance over time reaches a predetermined value, it is determined that the predetermined protease degradation has been achieved, and measurement of the glycated protein may be started, or the measurement results of the glycated protein at that time may be adopted.

[0045] In some embodiments, the absorbance measurement may be calibrated. The optical system including the optical measurement cell, the light-emitting element, the light-receiving element, etc. may be calibrated. For example, the absorbance may be measured before the solution is introduced into the optical system (e.g., when the optical measurement cell is empty, in its initial state, etc.) and after the solution is introduced into the optical system (e.g., when the measurement solution is introduced into the optical measurement cell). The difference between these absorbances may be calculated.

[0046] <Measurement Step> Some embodiments of the present disclosure provide a method for measuring proteins, the method comprising providing a solution that may contain a target protein, adding a color indicator to the solution, and subsequently or simultaneously adding a protease. The method can be used to measure glycated proteins. However, the application of the method is not limited thereto. The method can also be used for various other methods.

[0047] FIG. 1 shows a flowchart of a method for evaluating the glycation degree of albumin according to one embodiment. In step S101, a solution that may contain albumin as a protein to be measured is provided. In step S111, BCP is added as a color indicator. In step S121, the absorbance of BCP is measured. The albumin concentration is determined from the absorbance. In step S112, protease is added to the measurement solution to which BCP has already been added. The protease begins to decompose albumin into peptides or amino acids. In step S122, hydrogen peroxide is generated by the action of ketoamine oxidase, which specifically reacts with glycated amino acids among the peptides or amino acids produced by the protease digestion reaction. The hydrogen peroxide is measured using an electrode. The concentration of glycated albumin (GA) in the solution is thus determined. In step S131, the ratio of the determined glycated albumin (GA) to the albumin concentration is determined as the GA value.

[0048] 1, a color indicator is added (S111), absorbance is measured (S121), and then protease is added (S112). In some embodiments, the absorbance measurement may be completed before the protease is added. In some embodiments, the protease may be added before the absorbance measurement is completed or during the absorbance measurement.

[0049] FIG. 2 shows a flowchart of a method for evaluating the glycation degree of albumin according to one embodiment. In step S201, a solution that may contain albumin as a protein to be measured is provided. In step S211, BCP is added as a color indicator. In step S212, protease is added to the measurement solution to which BCP has already been added. The protease begins to decompose albumin into amino acids. In step S221, the absorbance of BCP is measured. The albumin concentration is determined from the absorbance. As the decomposition of albumin progresses, the absorbance decreases over time. For example, the concentration of albumin contained in the original measurement solution can be determined by measuring the absorbance at the time of protease introduction or the start of decomposition and a predetermined time after the start time. In step S222, hydrogen peroxide is generated by the action of ketoamine oxidase, which specifically reacts with glycated amino acids among the peptides or amino acids produced by the protease digestion reaction. The hydrogen peroxide is measured using an electrode. In this way, the concentration of glycated albumin (GA) in the solution is determined. In step S231, the ratio of the determined glycated albumin (GA) to the albumin concentration is determined as the GA value.

[0050] In Figure 2, the color indicator is added (S211), and then the protease is added (S212). However, the order of the steps is not limited to this. In some embodiments, the addition of the color indicator (S211) and the addition of the protease (S212) may be performed substantially simultaneously. For example, the protease and the color indicator may be mixed in advance, and the mixture may be added to the measurement solution.

[0051] In FIG. 2 , the albumin concentration is measured (S221), followed by the glycated albumin concentration (S222). However, the order of the steps is not limited to this. In some embodiments, the albumin concentration (S221) and the glycated albumin concentration (S222) may be measured substantially simultaneously. In some embodiments, the color indicator may be added (S211), the protease may be added (S212), and then the solution containing both may be split. The albumin concentration may be measured in one of the split solutions, and the glycated albumin concentration may be measured in the other. In some embodiments, the protease and the color indicator may not be added separately as in FIG. 2 . For example, the protease and the color indicator may be mixed in advance, and the resulting mixture may be added to the measurement solution.

[0052] A color indicator is contacted with a solution that may contain the target protein, and then a first reaction between the color indicator and the target protein is used to determine the concentration of the target protein in the solution.Similarly, a protease is contacted with a solution that may contain the target protein, and then a second reaction between the protease and the target protein is used to determine the concentration of the glycated target protein (also referred to as the concentration of the glycated target protein or the glycated protein concentration).

[0053] In some embodiments, the second reaction may comprise reacting the produced glycated amino acid with ketoamine oxidase to generate hydrogen peroxide.

[0054] Other than the above order, the order of the four items of introducing the color indicator, measuring the first reaction between the color indicator and the target protein, introducing the protease, and measuring the second reaction between the protease and the target protein is not limited.

[0055] In some embodiments, the order of steps may not be limited other than the order of mixing the measurement solution with an indicator, then measuring proteins for the indicator mixed solution, and mixing the measurement solution with a protease, then measuring glycated proteins for the protease mixed solution.

[0056] In some embodiments, after mixing the measurement solution with the indicator, a protease may be mixed with the indicator mixed solution. In some aspects, the measurement solution may be mixed with the indicator, proteins may be measured using the indicator mixed solution, a protease may be mixed with the indicator mixed solution, and glycated proteins may be measured using the indicator protease mixed solution (a solution in which the indicator and protease are mixed). In some aspects, the measurement solution may be mixed with the indicator, protease may be mixed with the indicator mixed solution, glycated proteins may be measured using the indicator protease mixed solution (a solution in which the indicator and protease are mixed), and proteins may be measured using the protease mixed solution. In some aspects, the following steps may be performed in that order: mixing the measurement solution with the indicator, mixing protease with the indicator mixed solution, measuring glycated proteins using the indicator protease mixed solution (a solution in which the indicator and protease are mixed), and measuring proteins using the indicator protease mixed solution.

[0057] In some embodiments, after mixing the measurement solution with a protease, an indicator may be mixed with the protease mixed solution. In some aspects, the following steps may be performed in that order: mixing the measurement solution with a protease, measuring glycated proteins using the protease mixed solution, mixing an indicator with the protease mixed solution, and measuring proteins using a protease-indicator mixed solution. In some aspects, the following steps may be performed in that order: mixing the measurement solution with a protease, mixing an indicator with the protease mixed solution, measuring glycated proteins using the indicator protease mixed solution, and measuring proteins using the indicator protease mixed solution. In some aspects, the following steps may be performed in that order: mixing the measurement solution with a protease, mixing an indicator with the protease mixed solution, measuring proteins using the indicator protease mixed solution, and measuring glycated proteins using the indicator protease mixed solution.

[0058] In some embodiments, the indicator and protease may be mixed into the measurement solution substantially simultaneously.

[0059] <Calculation of Glycation Degree> In some embodiments, the glycation degree of a target protein may be calculated based on the concentration of the target protein and the concentration of a glycated protein. The glycation degree of a protein may be calculated by dividing the concentration of the glycated protein by the concentration of the protein. The amount to be measured is not limited to concentration. The amount to be measured may be an absolute amount, a relative amount, a concentration, or any other amount related to mass or concentration.

[0060] <Others> In some embodiments, the protein concentration and the glycated protein concentration can be measured after the addition of a color indicator and a protease. In this case, the color indicator and / or protease are added in liquid form, and the volume changes from the initial solution. However, the variation caused by errors in the amount of color indicator and / or protease added occurs at the same rate for both the protein concentration and the glycated protein concentration. Therefore, it does not substantially affect the calculation of the degree of glycation. This allows for relatively accurate and reproducible measurements.

[0061] In some embodiments, after adding the color indicator, the protein concentration can be measured, a solid protease can be added, and the glycated protein concentration can be measured. In this case, as in the above, the variation caused by errors in the amount of color indicator and / or protease added occurs at the same rate for both the protein concentration and the glycated protein concentration. Therefore, the calculation of the glycation degree is not substantially affected. Therefore, relatively accurate and reproducible measurements are possible.

[0062] <Conditions for Color Indicator and Protease> In some embodiments, various conditions (eg, pH, temperature, etc.) in the reaction or measurement of the color indicator and the reaction or measurement of the protease may be adjusted or changed.

[0063] In some embodiments, the pH may be adjusted. The pH may be different between the addition or reaction of the color indicator (first reaction) and the decomposition by the protease (second reaction). For example, when BCP is added, the pH may be adjusted to a range that includes its general optimum range, such as pH 4.5 to 8.5 or pH 5.0 to 6.8. After the protease is introduced, the pH may be adjusted to a range that includes its general optimum range, such as pH 6.0 to 9.0 or pH 7.0 to 8.5.

[0064] In some embodiments, the first reaction and the second reaction may be carried out at substantially the same pH. For example, the pH of both the first reaction and the second reaction may be adjusted to a range of pH 5.0 to 9.0, pH 7.0 to 8.5, etc. In some embodiments, the pH value may differ from the generally optimal range. For example, the first reaction may be carried out at an alkaline pH (pH 7.0 to 9.0), and the second reaction may be carried out at an alkaline pH (pH 7.0 to 9.0). In some embodiments, the second reaction may be carried out after the first reaction.

[0065] In some embodiments, when the first and second reactions are carried out (at least partially) simultaneously, a single pH is applied to both reactions. The pH value may be adjusted to a range suitable for both reactions. For example, the pH may be adjusted to a range of pH 5.0-9.0, pH 7.0-8.5, etc. for both the first and second reactions. In some embodiments, the pH value may be different from the generally optimal range.

[0066] In some embodiments, the temperature may be adjusted. The temperature may be different between the addition or reaction of the color indicator (first reaction) and the decomposition by the protease (second reaction). For example, when BCP is added, the temperature may be adjusted to its general optimum temperature of 20°C to 70°C, and after the protease is introduced, the temperature may be adjusted to 25°C to 70°C, 50°C to 70°C, or 55°C to 65°C. After absorbance measurement and before the protease is introduced, the temperature may be raised once to the temperature for protease decomposition. After protease decomposition, the temperature may be adjusted to 25°C to 75°C, 30°C to 65°C, 35°C to 65°C, around 37°C, or around 55°C during the ketoamine oxidase reaction.

[0067] In some embodiments, when the first and second reactions are carried out (at least partially) simultaneously, a single temperature is applied to both reactions. This temperature may be adjusted to a suitable range for both reactions. For example, the temperature may be adjusted to a range of 25°C to 50°C, 25°C to 45°C, or 30°C to 45°C for both the first and second reactions. For example, the temperature may be adjusted to a range of 25°C to 70°C, 30°C to 70°C, 40°C to 70°C, 50°C to 70°C, or 55°C to 65°C for both the first and second reactions.

[0068] Example 1: ALB Concentration Measurement As an example, albumin concentration was measured using the absorbance method. A calibrator (GA-L Calibrator, Asahi Kasei Pharma Corporation) for a commercially available glycoalbumin measurement kit (Lucica® GA-L) was used as the albumin sample solution, and a dilution series with PBS (7.93 mg / mL, 3.97 mg / mL, 1.98 mg / mL) was prepared. A blank sample (a PBS-only solution) was also prepared. As a protease solution, 320 mg of Orientase 22BF (HITBI Corporation) was dissolved in 4 mL of HEPES buffer. A BCP solution (0.02 mM, pH 8.0) was used for the color reaction. Absorbance was measured over time at 12-second intervals using a DM-JACK Ex (Hitachi Chemical Diagnostic Systems Co., Ltd.) automated diabetes test analyzer. At time t = 0 minutes, 180 μL of BCP solution was added to 18 μL of sample solution. At t = approximately 4 minutes, 18 μL of protease solution was added. The absorbance was measured from t = 0 minutes to approximately t = 9 minutes.

[0069] <Generation of ALB calibration curve> Figure 3 shows the change over time in ΔA, which is the absorbance A obtained for a plurality of albumin concentrations minus the absorbance A0 of blank PBS. Since BCP is not present before t = 0 minutes, the absorbance ΔA = 0. A change in absorbance (ΔA) was observed after the addition of BCP, relative to the absorbance at t = 0 minutes (ΔA = 0). The amount of change in absorbance (ΔA) up to t = 4 minutes was substantially constant. This indicates that the absorbance of BCP can be measured stably.

[0070] The constant ΔA corresponds to the albumin concentration. In some embodiments, the albumin concentration may be determined based on the absorbance in a solution without added protease. In Figure 3, ΔA = 0.05, 0.1, and 0.18 for ALB = 1.98 mg / mL, 3.97 mg / mL, and 7.93 mg / mL, respectively, and the relationship between them was approximately linear.

[0071] When protease was added at t = 4 minutes, the absorbance ΔA decreased. This indicates that albumin was degraded by the protease, and the concentration of albumin reacting with BCP decreased. The decay curves were similar in all cases. At any given point, the proportional relationship between the three concentrations was constant.

[0072] In some embodiments, the albumin concentration may be determined based on the absorbance value (A or ΔA) at a predetermined time after the addition of the protease. For example, an end-point method may be used. In some embodiments, the albumin concentration may be determined based on the absorbance time conversion rate (dA / dt, or d(ΔA) / dt). For example, a rate assay may be used. In some embodiments, the albumin concentration may be determined based on a higher-order derivative of the absorbance (e.g., dA / dt, or d(ΔA) / dt).

[0073] In some embodiments, the albumin concentration may be determined based on the absorbance (ΔA) after a sufficient time has passed. In some embodiments, GA may be measured after ΔA stops changing. For example, after t=10 minutes (not shown), ΔA becomes constant, which is interpreted as the completion of the albumin degradation reaction. In some embodiments, GA may be measured at a predetermined time (for example, after t=10 minutes).

[0074] 4 shows a calibration curve obtained from the relationship between the albumin concentration of the sample solution and dA / dt. It was revealed that there was a very strong negative correlation.

[0075] <ALB Measurement in Actual Samples> Using the same method as above, albumin concentrations were measured in actual samples from 10 healthy individuals. The actual samples were prepared by diluting plasma collected from healthy individuals 10-fold with PBS. The albumin concentrations of these samples were measured using Lucica (registered trademark) GA-L, a commercially available glycoalbumin measurement kit, and the results were compared. Figure 5 shows the relationship between the two. It was revealed that a very strong positive correlation was observed.

[0076] Example 2 Measurement of Glycated Albumin Concentration As an example, the concentration of glycated albumin was measured using an enzyme electrode method.

[0077] For the sample solutions, glucose was added to HSA (Fujifilm Wako Pure Chemical Industries, Ltd.) and incubated at 37°C to prepare GA solutions (40 mg / mL) with different glycation rates. The glycation rate of each sample was confirmed using a commercially available glycoalbumin measurement kit (Lucica (registered trademark) GA-L, Asahi Kasei Pharma Corporation). A protease solution was prepared by dissolving 2 mg of Amano K (Amano Enzyme Inc.) in 200 μL of TES buffer. 8 μL of BCP solution (1 mM) was added to 20 μL of the protease solution. This solution was heat-treated and stored at 4°C.

[0078] The sample was added to an electrochemical sensor with a hydrogen peroxide electrode immobilized with a ketoamine oxidase membrane, and the glycated albumin concentration was determined by measuring the output current.

[0079] 6 shows the relationship between the glycated albumin concentration and the enzyme sensor output in the presence or absence of BCP. The horizontal axis represents the glycated albumin concentration in the solution, and the vertical axis represents the measured current value. Furthermore, open circles represent measurements without BCP, and filled circles represent measurements with BCP.

[0080] All of these yielded similar GA values ​​and similar concentration-dependencies. This indicates that the presence of BCP does not substantially affect either the protease-mediated degradation of albumin or the measurement of glycated amino acids by ketoamine oxidase. Similar results were obtained with BCG (not shown). Therefore, it is believed that the addition of other color indicators does not substantially affect either the subsequent protease-mediated degradation of albumin or the measurement of glycated amino acids by ketoamine oxidase.

[0081] Example 3: GA Value Measurement-1 This example shows an example of GA value measurement. BCP was added to a sample containing glycated albumin, and the albumin concentration was measured by absorbance. Thereafter, protease was added to the solution, and the glycated albumin concentration was measured using an electrochemical sensor having a hydrogen peroxide electrode with an FAOD membrane immobilized thereon. This will be explained in more detail below.

[0082] <Generation of GA calibration curve> An example of measurement results used to generate a calibration curve of albumin concentration by absorbance measurement is shown in Figure 7. For the absorbance measurement, samples containing 1.3 mg / mL, 2.4 mg / mL, and 4.7 mg / mL of albumin were used. At 0 seconds in Figure 7, BCP solution (containing sodium dodecyl sulfate (SDS), Triton X-100, 5,5'-dithiobis (2-nitrobenzoic acid (DTNB), HEPES (pH 8.0), and NaCl) was introduced into the optical measurement cell. At approximately 30 seconds, the albumin sample was added to the BCP solution, which reduced the absorbance. The solution was then mixed by pipetting. The spikes and fluctuations visible up to approximately 40 seconds were caused by the movement of the liquid due to mixing and the obstruction of the light path by the pipette. After approximately 50 seconds, the absorbance of all albumin samples remained constant. As is clear from Figure 7, the absorbance showed albumin concentration dependence, so the relationship between albumin concentration and absorbance was determined and a calibration curve was generated (not shown).

[0083] FIG. 8 shows an example of measurement results used to generate a calibration curve for glycated albumin concentration using an electrochemical sensor. Samples containing 0.4 mg / mL, 0.7 mg / mL, and 1.5 mg / mL glycated albumin were used for the measurements. These solutions contained BCP. After absorbance measurement, protease was added to the solution, and a digestion reaction was carried out at 55°C for 30 minutes. Then, at 0 seconds in FIG. 8 , the solution after the digestion reaction was introduced into the electrochemical sensor. The output current of the electrochemical sensor was measured. It was found that, for all concentrations, a maximum value (inflection point) was reached approximately between 60 and 80 seconds. At this time, the relationship between the output current at 60 seconds and the glycated albumin concentration can be determined. Using this data, the relationship between glycated albumin concentration and the output current of the electrochemical sensor was determined, and a calibration curve was generated.

[0084] The GA value of an unknown sample can be determined using the calibration curve of albumin concentration and the calibration curve of glycated albumin concentration thus determined.

[0085] <GA Value Measurement of Unknown Samples> Next, the GA values ​​of unknown samples were measured using the same method as above. High-concentration GA value samples and low-concentration GA value samples were mixed in several ratios to prepare multiple measurement solutions. Human serum albumin reagent (Sigma-Aldrich) was used for the low-concentration GA value sample. High-concentration GA value samples were prepared by adding glucose to human serum albumin reagent (Sigma-Aldrich) and incubating. These were artificially prepared samples, but the actual GA values ​​were unknown.

[0086] The measurement method of this example was applied to these measurement samples to determine the GA values. The same measurement samples were also used to determine the GA values ​​(%) using a commercially available glycoalbumin measurement kit (Lucica (registered trademark) GA-L), an existing method. Figure 9 shows both measurements obtained for samples of four different unknown concentrations. The horizontal axis shows the GA values ​​measured using Lucica, and the vertical axis shows the GA values ​​(arbitrary units; GA values ​​(%) are not shown) obtained using the measurement method of this example. Thus, it has become clear that GA values ​​can be determined extremely well by using one of the methods disclosed herein.

[0087] Example 4: GA Value Measurement-2 This example shows an example of measuring a GA value (%). A mixed solution of BCP and protease was added to a sample containing glycated albumin, and then the albumin concentration was measured by optical measurement, while the glycated albumin concentration was measured using an electrochemical sensor having a hydrogen peroxide electrode with an FAOD membrane immobilized thereon. This will be explained in more detail below.

[0088] <Generation of ALB Standard Curve> Figure 10 shows an example of measurement results used to generate a standard curve of albumin concentration by absorbance measurement. Samples containing 1.2 mg / mL, 2.5 mg / mL, and 5.0 mg / mL albumin were used for the measurement. Before -10 seconds in Figure 10, a BCP and protease mixture (containing SDS, Triton X-100, DTNB, HEPES (pH 8.0), NaCl, and protease) was introduced into the optical measurement cell. At 0 seconds, the albumin sample was added to the BCP and protease mixture, which resulted in a decrease in absorbance. The solution was then mixed by pipetting. The spikes and fluctuations visible up to approximately 15 seconds were due to the movement of the liquid during mixing and the obstruction of the light path by the pipette. After approximately 20 seconds, the absorbance showed a steady decrease.

[0089] In this way, values ​​related to absorbance, such as the absorbance at 20 seconds, the difference between the absorbance at 20 seconds and a later time point (for example, 90 seconds), and the change in absorbance over time (time derivative), are determined by the initial albumin concentration. Using such data, the relationship between albumin concentration and absorbance was determined, and a calibration curve was generated.

[0090] Using the same method as in Figure 8, the relationship between the glycated albumin concentration and the output current of the electrochemical sensor was determined, and a calibration curve was generated (not shown).

[0091] <Measurement of GA Value (%) of Unknown Sample> A measurement sample was prepared in the same manner as in Example 3.

[0092] The measurement method of this example was applied to these measurement samples, and the GA values ​​(%) were determined. The same measurement samples were also subjected to an existing method, using a commercially available glycoalbumin measurement kit (Lucica (registered trademark) GA-L), to determine the GA values ​​(%). Figure 11 shows the relationship between the two measurement values ​​obtained for five samples with different unknown concentrations. The horizontal axis shows the GA values ​​(%) obtained by the existing measurement method, and the vertical axis shows the GA values ​​(%) obtained by the measurement method of this example. Thus, it was revealed that the GA values ​​(%) can be determined extremely well by using one of the methods disclosed herein.

[0093] Example 5: An example of a weakly alkaline modified BCP method In the above example, SDS is included, but in other embodiments, SDS may not be included. This example shows an example in which both the first and second reactions are carried out in a weakly alkaline environment (in this example, pH = 8.0). The first reaction will be described below.

[0094] Albumin measurement using the improved BCP method is generally performed under acidic conditions using an oxidizing agent and SDS. Protein denaturants such as SDS are thought to make albumin more susceptible to oxidation when an oxidizing agent such as DTNB is introduced. BCP is then introduced under these conditions.

[0095] On the other hand, the protease reaction as the second reaction generally requires a weak alkaline environment. In order to carry out the first and second reactions simultaneously, consecutively, or serially, it is preferable to carry out the first reaction in a similar alkaline environment. This eliminates the need to change the pH.

[0096] At high pH (weakly alkaline, e.g., pH 8.0), the oxidation rate is sufficiently high for general measurement efficiency, so albumin can be oxidized without SDS. Furthermore, the absence of SDS is believed to prevent deformation of the protein's higher-order structure, e.g., a decrease in protease activity. In some embodiments, in the improved BCP method performed at weakly alkaline conditions, the solution preferably does not contain SDS.

[0097] Therefore, in this example, it was confirmed whether the improved BCP method would function at pH = 8.0. First, GA-L calibrator (Asahi Kasei Pharma Corporation) was diluted to prepare samples with albumin concentrations of 0 g / dL, 0.23 g / dL, and 0.45 g / dL. A succinic acid solution (pH 5.4) was used as the weakly acidic buffer solution, and a HEPES solution (pH 8.0) and a Tricine solution (pH 8.0) were used as the weakly alkaline buffer solutions. HEPES is frequently used for similar measurements, but it has a sulfonic acid group (SO ３ －), which may react with DTNB. Therefore, Tricine was also used. 0.1 mM DTNB was used as the oxidation reagent. The reaction solution containing SDS was adjusted to a concentration of 0.02% SDS. In this way, a total of six reaction reagents were prepared, with three types of buffer solutions and the presence or absence of SDS.

[0098] After mixing the sample and reaction reagent, the mixture was heated to 37°C, and the absorbance at 412 nm was measured after 5 minutes. The compound generated after DTNB reacts with the reduced cysteine ​​residues of albumin has a maximum absorption wavelength of 412 nm. Figure 16 shows the absorbance for three types of buffer solution: succinic acid solution, HEPES solution (HEPES, circles), and Tricine solution (Tricine, triangles), with and without SDS (SDS+, solid) and without SDS (SDS-, open).

[0099] For the succinic acid solution (Suc, squares) at pH 5.4, a difference in absorbance was observed between the presence (SDS+, solid) and absence (SDS-, open) of SDS. This indicates that the oxidation reaction does not proceed sufficiently in a weakly acidic solution without SDS.

[0100] On the other hand, in the HEPES solution (HEPES, circles) and Tricine solution (Tricine, triangles) at pH 8.0, no substantial difference in absorbance was observed either with (SDS+, solid) or without (SDS-, open) SDS. In other words, it was confirmed that oxidation proceeded sufficiently in weakly alkaline conditions, regardless of the presence or absence of SDS. The absence of SDS can avoid deformation of the higher-order structure of proteins, and can avoid, for example, a decrease in protease activity.

[0101] <Apparatus Configuration Example 1> Fig. 12 schematically shows the configuration of an apparatus 100 according to one embodiment. The apparatus 100 includes an optical measurement unit 110, a protease reaction unit 120, and a glycated protein measurement unit 130. A sample solution is introduced into the optical measurement unit 110 from an introduction unit 140 and sequentially flows through the protease reaction unit 120 and the glycated protein measurement unit 130. A color indicator tank 150 is connected to the optical measurement unit 110 and configured to provide a color indicator to the optical measurement unit 110. A protease solution tank 160 is connected to the protease reaction unit 120 and configured to provide a protease solution to the protease reaction unit 120.

[0102] The introduction and delivery of the solutions are controlled by a solution delivery mechanism 170. The solution delivery mechanism 170 can control the timing and amount of the sample solution introduced into the optical measurement unit 110. Similarly, the solution delivery mechanism 170 can control the timing and amount of the color indicator solution introduced from the color indicator solution tank 150 to the optical measurement unit 110, and the timing and amount of the protease solution introduced from the protease solution tank 160 to the protease reaction unit 120.

[0103] The liquid delivery mechanism 170 may include, for example, a pump. When the optical measurement unit 110, the protease reaction unit 120, and the glycated protein measurement unit 130 are connected via a closed flow path, the movement of the solution can be controlled by applying positive pressure to the upstream side. The liquid delivery mechanism 170 may be a pressing mechanism attached to each solution tank. When each solution tank is a deformable container, a predetermined amount of solution can be delivered to the outside by deforming the container.

[0104] Temperature control units 118, 128, and 138 having temperature sensors and heaters (not shown) are respectively disposed in the optical measurement section 110, the protease reaction section 120, and the glycated protein measurement section 130. The temperature control units 118, 128, and 138 are connected to a temperature control mechanism (driver) 180, which receives measured temperature information from the temperature sensors and can send heating on / off instructions and power to the heaters.

[0105] An optical measurement unit 119 equipped with a light-emitting element and a light-receiving element (not shown) is disposed in the optical measurement section 110. Light emitted from the light-emitting element passes through the solution in the optical measurement section 110, and the light that emerges is detected by the light-receiving element. The optical measurement unit 119 can output absorbance information at that time.

[0106] A sensor 139 is disposed in the glycated protein measuring unit 130. The sensor 139 is disposed inside the glycated protein measuring unit 130 and can directly contact the solution therein. The sensor 139 is configured to measure the concentration of glycated proteins.

[0107] The apparatus 100 further includes a computer system 190. The computer system 190 includes at least a central processing unit (CPU) 195, which performs necessary calculations. The computer system 190 includes communication interfaces 197 and 198 with the liquid delivery mechanism 170 and the temperature control mechanism 180, respectively. The computer system 190 also includes communication interfaces 191 and 193 with the optical measurement unit 119 and the sensor 139, respectively.

[0108] The CPU 195 sends necessary information, such as the required operation items (the solution to be introduced and the component to which it is introduced), the timing, and the amount, to the liquid delivery mechanism 170 via the liquid delivery mechanism communication interface 197. The liquid delivery mechanism 170 executes the required operation based on the received information. The liquid delivery mechanism 170 can send information to the communication interface 197 that each operation has been executed, information on execution failure, and the like.

[0109] The CPU 195 sends necessary information, such as the required operation items (temperature measurement, the temperature required for each reaction, etc.), the timing, and the temperature and time, to the temperature control mechanism 180 via the temperature control mechanism communication interface 198. The temperature control mechanism 180 executes the required operations based on the received information. The temperature control mechanism 180 can send information to the communication interface 198, such as information that each operation has been executed, or information on execution failure.

[0110] The optical measurement unit communication interface 191 receives information about absorbance from the optical measurement unit 119 arranged in the optical measurement section 110. The glycated protein sensor communication interface 193 receives information about the concentration of glycated protein from the sensor 139 arranged in the glycated protein measurement section 130.

[0111] The optical measurement unit communication interface 191 issues instructions such as start and end of absorbance measurement to the optical measurement unit 119 arranged in the optical measurement section 110, and then receives information on the protein concentration obtained by the measurement. The glycated protein sensor communication interface 193 issues instructions such as start and end of current measurement to the sensor 139 arranged in the glycated protein measurement section 130, and then receives information on the glycated protein concentration obtained by the measurement.

[0112] The CPU 195 receives information relating to absorbance and information relating to glycated proteins via the communication interfaces 191 and 193, performs a predetermined conversion, and calculates the degree of glycation.

[0113] <Apparatus Configuration Example 2> Fig. 13 shows a schematic diagram of an apparatus 200 according to one embodiment. The apparatus 200 includes an optical measurement unit 210 and a glycated protein measurement unit 230. The sample solution is introduced into the optical measurement unit 210 from an introduction unit 240 thereof and then flows into the glycated protein measurement unit 230. A measurement solution (color indicator protease solution) tank 250 is connected to the optical measurement unit 210 and configured to provide a mixed solution of a color indicator and protease to the optical measurement unit 210.

[0114] The introduction and delivery of the solution is controlled by a solution delivery mechanism 270 .

[0115] Temperature control units 218 and 238 having temperature sensors and heaters (not shown) are disposed in the optical measurement section 210 and the glycated protein measurement section 230, respectively.

[0116] An optical measurement unit 219 is disposed in the optical measurement section 210 and is capable of measuring absorbance and outputting information about the measured absorbance.

[0117] A sensor 239 is disposed in the glycated protein measuring section 230 and is configured to measure the concentration of glycated proteins and output information relating to the measured glycated proteins.

[0118] The apparatus 200 further includes a computer system 290. The computer system 290 includes at least a central processing unit (CPU) 295, which performs necessary calculations. The computer system 290 can communicate with the liquid delivery mechanism 270 and the temperature control mechanism 280 via communication interfaces 297 and 298. The computer system 290 can communicate with the optical measurement unit 219 and the sensor 239 via communication interfaces 291 and 293.

[0119] The CPU 295 receives information relating to absorbance and information relating to glycated proteins via the communication interfaces 291 and 293, performs a predetermined conversion, and calculates the degree of glycation.

[0120] 14 is a schematic diagram showing the configuration of an apparatus 300 according to one embodiment. The apparatus 300 includes a color indicator reaction section 305, an optical measurement section 310, a protease reaction section 320, and a glycated protein measurement section 330. The sample solution is introduced into the color indicator reaction section 305 from an introduction unit 340, and then flows sequentially through the optical measurement section 310, the protease reaction section 320, and the glycated protein measurement section 330.

[0121] The color indicator reaction section 305 contains a color indicator in advance. The solution introduced into the color indicator reaction section 305 is mixed with the color indicator and reacts with it. The protease reaction section 320 contains a protease in advance. The solution introduced into the protease reaction section 320 comes into contact with the protease and reacts with it.

[0122] The introduction and delivery of the solution is controlled by a solution delivery mechanism 370 .

[0123] Temperature control units 318, 328, and 338 having temperature sensors and heaters (not shown) are arranged in the optical measurement section 310, the protease reaction section 320, and the glycated protein measurement section 330, respectively.

[0124] An optical measurement unit 319 is disposed in the optical measurement section 310 and is capable of measuring absorbance and outputting information relating to the measured absorbance.

[0125] A sensor 339 is disposed in the glycated protein measuring section 330 and configured to measure the concentration of glycated proteins and output information relating to the measured glycated proteins.

[0126] The apparatus 300 further includes a computer system 390. The computer system 390 includes at least a central processing unit (CPU) 395, which performs necessary calculations. The computer system 390 can communicate with the liquid delivery mechanism 370 and the temperature control mechanism 380 via communication interfaces 397 and 398. The computer system 390 can communicate with the optical measurement unit 319 and the sensor 339 via communication interfaces 391 and 393.

[0127] The CPU 395 receives information relating to absorbance and information relating to glycated proteins via the communication interfaces 391 and 393, performs a predetermined conversion, and calculates the degree of glycation.

[0128] 15 is a schematic diagram showing the configuration of an apparatus 400 according to one embodiment. The apparatus 400 includes a color indicator protease solution reaction section 405, an optical measurement section 410, and a glycated protein measurement section 430. The sample solution is introduced into the color indicator reaction section 405 from an introduction unit 440, and then flows sequentially through the optical measurement section 410 and the glycated protein measurement section 430.

[0129] A color indicator and a protease are previously stored in the color indicator / protease solution reaction section 405. The solution introduced into the color indicator / protease solution reaction section 405 is mixed with and reacts with the color indicator, and also comes into contact with and reacts with the protease.

[0130] The introduction and delivery of the solution is controlled by a solution delivery mechanism 470 .

[0131] Temperature control units 418 and 438 having temperature sensors and heaters (not shown) are disposed in the optical measurement section 410 and the glycated protein measurement section 430 .

[0132] An optical measurement unit 419 is disposed in the optical measurement section 410 and is capable of measuring absorbance and outputting information about the measured absorbance.

[0133] A sensor 439 is disposed in the glycated protein measuring section 430 and is configured to measure the concentration of glycated proteins and output information relating to the measured glycated proteins.

[0134] The apparatus 400 further includes a computer system 490. The computer system 490 includes at least a central processing unit (CPU) 495, which performs necessary calculations. The computer system 490 can communicate with the liquid delivery mechanism 470 and the temperature control mechanism 480 via communication interfaces 497 and 498. The computer system 490 can communicate with the optical measurement unit 419 and the sensor 439 via communication interfaces 491 and 493.

[0135] The CPU 495 receives information relating to absorbance and information relating to glycated proteins via the communication interfaces 491 and 493, performs a predetermined conversion, and calculates the degree of glycation.

[0136] The device configuration is not limited to the above examples. In some embodiments, the device or system may be an automatic pipetting device or system (sometimes simply referred to as an automatic pipetting device). The automatic pipetting device may have wells that contain reagents or mixtures of at least two of them. In some embodiments, a cartridge (microplate) having multiple wells may be provided. The device or cartridge may be equipped with an electrochemical sensor or may be configured to have an electrochemical sensor attached. Samples and solutions may be metered, transferred from one well to another, or mixed by pipetting. The automatic pipetting device may be equipped with a heater, and the heater may control the temperature of the wells or the solutions in the wells. The automatic pipetting device may be equipped with an optical measurement system.

[0137] In some embodiments, the device may include a waste tank. The solution that has passed through the optical measurement unit, the glycated protein measurement unit, etc. may be collected in the waste tank. This reduces the risk of the solution containing the body fluid leaking to the outside.

[0138] The present disclosure also provides the following embodiments. A001: A method for evaluating the glycation degree of a protein, comprising: providing a solution possibly containing a target protein; contacting the solution with a color indicator; contacting the solution with a protease; determining the concentration of the target protein in the solution using a first reaction between the color indicator and the target protein; determining the concentration of the glycated target protein using a second reaction between the protease and the target protein; and determining the glycation degree of the target protein based on the concentration of the target protein and the concentration of the glycated target protein. A011: The method of embodiment A001, wherein contacting the solution with the protease comprises adding the protease to a color indicator mixture produced by contacting the solution with the color indicator. A012: The method of embodiment A001, wherein contacting the solution with the color indicator and contacting the solution with the protease are performed substantially simultaneously. A012b The method of embodiment A001, comprising mixing the color indicator and the protease with the solution substantially simultaneously. A013 The method of embodiment A001, wherein contacting the solution with the color indicator comprises adding the color indicator to a protease mixture produced by contacting the solution with the protease. A021 The method of any one of embodiments A001 to A013, wherein measuring the first reaction between the color indicator and the target protein comprises measuring the absorbance of the color indicator. A022 The method of embodiment A021, wherein measuring the absorbance of the color indicator comprises measuring the change in absorbance over time. A023 The method of embodiment A022, wherein measuring the change in absorbance over time comprises measuring the absorbance at at least two (or more) times.A024 The method of embodiment A022 or A023, wherein the concentration of the glycated protein of interest is determined based on the change in absorbance over time. A031 The method of any one of embodiments A001 to A024, wherein the protein of interest is albumin. A032 The method of embodiment A031, wherein determining the concentration of glycated albumin using the second reaction of the protease and the albumin comprises: degrading the albumin with the protease to generate peptides; reacting the generated peptides with ketoamine oxidase to generate hydrogen peroxide; measuring the concentration of the generated hydrogen peroxide; and determining the concentration of the glycated albumin based on the concentration of the hydrogen peroxide. A033 The method of embodiment A032, wherein measuring the concentration of hydrogen peroxide includes using a hydrogen peroxide electrode. A034 The method of embodiment A033, wherein measuring the concentration of hydrogen peroxide comprises contacting the hydrogen peroxide that has passed through an ion exchange resin with the hydrogen peroxide electrode. A041 The method of any one of embodiments A001 to A034, wherein the color changing indicator is a pH indicator. A042 The method of any one of embodiments A031 to A034, wherein the color changing indicator is bromocresol purple (BCP) or bromocresol green (BCG). A051 The method of embodiment A042, wherein the color changing indicator is bromocresol purple (BCP) or bromocresol green (BCG), further comprising adjusting the pH of the solution to a range of 4.5 to 8.5 during the first reaction and adjusting the pH of the solution to a range of 6.0 to 9.0 during the second reaction.A052 The method of any one of embodiments A001 to A042, wherein when a first reaction between the color indicator and the target protein and a second reaction between the color indicator and the target protein are carried out (at least partially) simultaneously, the pH of the solution is adjusted to a range suitable for both the first reaction and the second reaction. A053 The method of embodiment A052, wherein the color indicator is bromocresol purple (BCP) or bromocresol green (BCG), and the pH is adjusted to a range of 7.0 to 8.5. A054 The method of embodiment A051 or A053, wherein the pH is adjusted to a range of 7.0 to 8.5, and during the first reaction, the mixture of solutions does not contain (is free of) a protein denaturant (SDS). A055 The method of any one of embodiments A032 to A054, wherein the color changing indicator is bromocresol purple (BCP) or bromocresol green (BCG), and further comprising adjusting the temperature of the solution to a range of 20°C to 70°C during the first reaction, and adjusting the temperature of the solution to a range of 25°C to 70°C during the second reaction. A056 The method of any one of embodiments A001 to A054, wherein the first reaction between the color changing indicator and the target protein and the second reaction between the color changing indicator and the target protein are carried out (at least partially) simultaneously, and the temperature of the solution is adjusted to a range suitable for both the first reaction and the second reaction. A057 The method of embodiment A056, wherein the temperature is adjusted to a range of 25°C to 70°C. A058 The method of any one of embodiments A032 to A034, further comprising adjusting the temperature of the ketoamine oxidase to the range of 25°C to 75°C.A061 The method of any one of embodiments A032 to A058, wherein the ketoamine oxidase is an oxidase acting on an amino acid or peptide having a glycated α-amino group (group I ketoamine oxidase), and measuring the concentration of the generated hydrogen peroxide comprises calibrating the measurement using a standard solution containing at least one of fructosyl valine, fructosyl glycine, and fructosyl valyl histidine. A062 The method of any one of embodiments A032 to A058, wherein the ketoamine oxidase is an oxidase acting on an amino acid or peptide having a glycated ε-amino group (group II ketoamine oxidase), and measuring the concentration of the generated hydrogen peroxide comprises calibrating the measurement using a standard solution containing fructosyl lysine. A063 The method according to any one of embodiments A032 to A058, wherein the ketoamine oxidase is an oxidase acting on an amino acid or peptide whose α-amino group and / or ε-amino group is glycated (Group III ketoamine oxidase), and measuring the concentration of the generated hydrogen peroxide comprises calibrating the measurement using a standard solution containing at least one of fructosyl lysine, fructosyl valine, fructosyl glycine, and fructosyl valyl histidine. B01 A program for causing a computer system to execute the method according to any one of embodiments A001 to A063. B11 A storage medium for storing the program according to embodiment B01. C01 An apparatus for evaluating the degree of glycation of a protein, comprising: an introduction unit (or introduction port) for a body fluid; a mechanism for mixing the body fluid with a color indicator solution; a protein concentration measurement unit having an optical measurement unit that measures the concentration of protein in a mixture of the body fluid and the color indicator solution; a mechanism for mixing the body fluid with a protease solution; a glycated protein measurement unit having an electrode unit (sensor) that measures the concentration of glycated protein in a mixture of the body fluid and the protease; and an arithmetic processing unit that communicates with the albumin concentration measurement unit and the glycated protein measurement unit.C02 The device according to embodiment C01, wherein mixing the body fluid with the protease solution comprises mixing the protease solution with a mixture of the body fluid and the color indicator solution. C11 The device according to embodiment C01 or C02, wherein the protein concentration measurement unit and the glycated protein measurement unit are fluidly connected in series. C12 The device according to embodiment C11, wherein the body fluid introduction unit, the protein concentration measurement unit, and the glycated protein measurement unit are fluidly connected in series in that order. C13 The device according to embodiment C01 or C02, wherein the protein concentration measurement unit and the glycated protein measurement unit are connected in parallel to each other and to the introduction unit. C21 The device according to any one of embodiments C01 to C13, further comprising a waste liquid unit (tank) fluidly connected downstream of the protein concentration measurement unit and the glycated protein measurement unit. C31 The device of any one of embodiments C01 to C21, wherein the optical measurement unit comprises an absorbance meter. C41 The device of any one of embodiments C01 to C31, wherein the electrode unit (sensor) for measuring the concentration of glycated proteins comprises a ketoamine oxidase and a hydrogen peroxide electrode. C42 The device of embodiment C41, wherein the sensor further comprises an ion exchange membrane covering the hydrogen peroxide electrode. C43 The device of embodiment C41 or C42, further comprising an amperometer for measuring the current generated at the hydrogen peroxide electrode. C51 The device of any one of embodiments C01 to C043, further comprising a color indicator solution tank for containing the BCP solution and a protease solution tank for containing the protease solution. C52 The device of any one of embodiments C01 to C043, further comprising a processing solution tank containing a mixed solution of the color indicator solution and the protease solution. C61 The device of any one of embodiments C01 to C52, wherein the protein is albumin.C62 The apparatus of embodiment C61, wherein the color changing indicator is BCP or BCG. C71 The apparatus of any one of C01 to C62, wherein the apparatus is or comprises an automated pipetting apparatus or device.

[0139] Several embodiments and examples of the present disclosure have been described above, but these embodiments and examples exemplify the present disclosure. For example, the above embodiments have been described in detail to clearly explain the present disclosure, and additional changes in dimensions, configurations, materials, and circuits may be made as necessary. Note that embodiments that combine any one or more features of the present disclosure described above are also included within the scope of the present disclosure. The claims encompass numerous modifications to the embodiments without departing from the technical spirit of the present disclosure. Therefore, the embodiments and examples disclosed herein are provided for illustrative purposes and should not be considered to limit the scope of the present disclosure.

Claims

1. A method for evaluating the degree of glycation of albumin, comprising: (a) providing a solution that may contain albumin; (b) contacting the solution with a color changing indicator; (c) subsequently or simultaneously contacting the solution with a protease; (d) determining the concentration of albumin in the solution using a first reaction between the color indicator and the albumin; (e) determining a concentration of glycated albumin using a second reaction of the protease with the albumin, wherein step (e) comprises: allowing the protease to degrade the albumin to produce peptides; reacting the generated peptide with ketoamine oxidase to generate hydrogen peroxide; measuring the concentration of the generated hydrogen peroxide using a hydrogen peroxide electrode; and determining a concentration of the glycated albumin based on the concentration of the hydrogen peroxide; including; and (f) determining a degree of glycation of the albumin based on the concentration of the albumin and the concentration of the glycated albumin; A method for providing the above.

2. 2. The method of claim 1 , Measuring the first reaction between the color indicator and the albumin comprises measuring the absorbance of the color indicator. method.

3. 3. The method of claim 2, Measuring the absorbance of the color indicator comprises measuring a change in the absorbance over time. method.

4. 2. The method of claim 1 , Measuring the concentration of hydrogen peroxide includes contacting the hydrogen peroxide that has passed through an ion exchange resin with the hydrogen peroxide electrode. method.

5. 2. The method of claim 1 , The color indicator is a pH indicator. method.

6. 2. The method of claim 1 , The color indicator is bromocresol purple (BCP) or bromocresol green (BCG); method.

7. 2. The method of claim 1 , the color indicator is bromocresol purple (BCP) or bromocresol green (BCG); The method further comprising adjusting the pH of the solution during the first reaction to a range of 4.5 to 8.5, and adjusting the pH of the solution during the second reaction to a range of 6.0 to 9.

0.

8. 2. The method of claim 1 , When the first reaction between the color indicator and the albumin and the second reaction between the color indicator and the albumin are carried out simultaneously, the pH of the solution is adjusted to a range suitable for both the first reaction and the second reaction. method.

9. 9. The method of claim 8, the color indicator is bromocresol purple (BCP) or bromocresol green (BCG); The pH is adjusted to a range of 7.0 to 8.

5. method.

10. 10. The method of claim 7 or 9, The pH is adjusted to a range of 7.0 to 8.5; During the first reaction, the mixture of solutions does not contain a protein denaturant; method.

11. 2. The method of claim 1 , the color indicator is bromocresol purple (BCP) or bromocresol green (BCG); The method further comprising adjusting a temperature of the solution during the first reaction to a range of 20° C. to 70° C., and adjusting a temperature of the solution during the second reaction to a range of 25° C. to 70° C.

12. 2. The method of claim 1 , When the first reaction between the color indicator and the albumin and the second reaction between the color indicator and the albumin are carried out at least partially simultaneously, the temperature of the solution is adjusted to a range suitable for both the first reaction and the second reaction. method.

13. 13. The method of claim 12, The temperature is adjusted to a range of 25° C. to 70° C. method.

14. 2. The method of claim 1 , The method further comprising adjusting the temperature of the ketoamine oxidase to a range of 25°C to 75°C.

15. 2. The method of claim 1 , the ketoamine oxidase is an oxidase (group II ketoamine oxidase) acting on an amino acid or peptide whose ε-amino group is glycated, and measuring the concentration of the generated hydrogen peroxide comprises calibrating the measurement using a reference solution containing fructosyl lysine. method.