Kit for quantitative analysis of glycated albumin, method for quantitative analysis of glycated albumin, and apparatus for implementing the same

Abstract

To provide a glycated albumin quantitative analysis kit, a glycated albumin quantitative analysis method, and / or a device for implementing the method, which allow a reaction for measuring the amount of total albumin and a reaction for measuring the amount of glycated albumin to take place in a single kit so as to enable miniaturization, reduce analysis time, and improve user convenience.SOLUTION: A kit for quantitative analysis of glycated albumin (GA) according to an embodiment of the present application comprises: a first composition containing a reaction buffer for inducing binding with albumin present in a blood sample; a second composition containing a decomposition reagent for a decomposition reaction to break down glycated albumin (GA) contained in the blood sample into glycated amino acids; a third composition containing a reaction reagent for an enzymatic reaction of the glycated amino acids present in the blood sample; and a fourth composition containing a chromogenic agent that is oxidized by hydrogen peroxide (H2O2) generated from the enzymatic reaction of the glycated amino acids.SELECTED DRAWING: Figure 7

Description

[Background technology]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of Korean Patent Application No. 10-2024-0029436, filed February 29, 2024, the entire disclosure of which is incorporated herein by reference.

[0002] 1. Field of the Invention The present application relates to the quantitative analysis of biological samples, and more particularly to a kit for the quantitative analysis of glycated albumin (GA), a GA quantitative analysis method, and / or a quantitative analysis device for carrying out the same. 2. Description of Related Technology In the diagnosis of diabetes, there is an increasing need to test for glycated albumin (hereinafter referred to as GA) alongside blood glucose measurement. Glycated albumin is a glycated substance that forms a ketoamine structure through a non-enzymatic oxidation reaction at the lysine site of albumin. The concentration of glycated albumin correlates with blood glucose levels and increases or decreases accordingly. Compared with HbA1c, which is widely used as an indicator for diagnosing diabetes, glycated albumin has the advantage of sensitively reflecting changes in blood glucose levels. Consequently, the development and research of techniques for quantitatively analyzing glycated albumin for diagnosing diabetes has attracted considerable attention.

[0003] Quantitative analysis of glycated albumin is quantified as the ratio of glycated albumin to total albumin (GA%) in blood. In conventional techniques for quantitative analysis of glycated albumin, the reaction for measuring the amount of total albumin and the reaction for measuring the amount of glycated albumin are separated and measured in separate spaces. This is necessary due to the different reaction environments of the reaction for measuring the amount of total albumin and the reaction for measuring the amount of glycated albumin. In conventional techniques, separating the two reactions limits miniaturization, and as a result, quantitative analysis of glycated albumin must mainly be performed on large biochemical instruments. Furthermore, in conventional techniques, separating the two reactions makes quantitative analysis of glycated albumin take a relatively long time, resulting in inconvenience for users.

[0004] Therefore, there is a need to develop and research a technology for quantitative analysis of glycated albumin that can be miniaturized and improves user convenience. Summary of the Invention

[0005] The problem to be solved by the present invention is to provide a glycated albumin quantitative analysis kit, a glycated albumin quantitative analysis method, and / or a device for carrying out the method, which allows miniaturization by implementing a reaction for measuring the amount of total albumin and a reaction for measuring the amount of glycated albumin in a single kit.

[0006] The problem to be solved by the present invention is to provide a glycated albumin quantitative analysis kit, a glycated albumin quantitative analysis method, and / or a device for performing the method, which implements a reaction for measuring the amount of total albumin and a reaction for measuring the amount of glycated albumin in a single kit, thereby reducing analysis time and increasing user convenience.

[0007] The objects of the present invention are not limited to the above objects, and other objects not described can be clearly understood by those skilled in the art from this specification and the accompanying drawings.

[0008] A kit for quantitative analysis of glycated albumin (GA) according to an embodiment of the present application may comprise: a first composition comprising a reaction buffer for inducing binding with albumin present in a blood sample; a second composition comprising a decomposition reagent for a decomposition reaction that decomposes glycated albumin (GA) contained in the blood sample into glycated amino acids; a third composition comprising a reaction reagent for an enzymatic reaction of glycated amino acids present in the blood sample; and a fourth composition comprising a color developer that is oxidized by hydrogen peroxide (H2O2) generated from the enzymatic reaction of glycated amino acids, wherein the GA quantitative analysis kit may comprise at least one reagent immobilization portion, and each of the second composition to the fourth composition may be independently immobilized on the reagent immobilization portion.

[0009] The solutions of the present invention are not limited to the solutions described above, and other solutions not described can be clearly understood by those skilled in the art from this specification and the accompanying drawings. [Brief explanation of the drawings]

[0010] These and other objects, features and advantages of the present invention will become more apparent to those skilled in the art from a more detailed description of illustrative embodiments thereof, taken in conjunction with the accompanying drawings.

[0011] [Figure 1] 1 is a schematic diagram of a glycated albumin quantitative analysis kit according to an embodiment of the present application.

[0012] [Figure 2] 1 is a schematic diagram of a sample collector of a glycated albumin quantitative analysis kit according to an embodiment of the present application. FIG.

[0013] [Figure 3] FIG. 1 is a schematic diagram of a main cartridge of a glycated albumin quantitative analysis kit according to an embodiment of the present application.

[0014] [Figure 4] 1 is a schematic diagram of a solution cell of a glycated albumin quantitative analysis kit according to an embodiment of the present application. FIG.

[0015] [Figure 5] 10A-10C illustrate how compositions stored in solution cells flow into a mixing region of a main cartridge when a sample collector is inserted into the main cartridge, according to an embodiment of the present application.

[0016] [Figure 6] 1 illustrates one aspect of an analytical method for quantifying GA, according to one embodiment of the present application.

[0017] [Figure 7] FIG. 1 is a diagram showing a specific structure of a glycated albumin quantitative analysis kit according to an embodiment of the present application. [Figure 8] FIG. 1 is a diagram showing a specific structure of a glycated albumin quantitative analysis kit according to an embodiment of the present application.

[0018] [Figure 9] FIG. 1 illustrates an analytical aspect for quantifying glycated albumin in a blood sample according to an embodiment of the present application.

[0019] [Figure 10] 1 is a table and graph showing results evaluating the performance of measuring albumin based on different reaction buffers, according to an embodiment of the present application.

[0020] [Figure 11] 1 is a table and graph showing results evaluating the measurement performance of glycated albumin based on different reaction buffers according to an embodiment of the present application.

[0021] [Figure 12] 1 is a table showing the results of evaluating the performance of measuring albumin and / or glycated albumin based on the surfactant added to the reaction buffer, according to an embodiment of the present application.

[0022] [Figure 13]1 is a table showing results evaluating the enhanced stability of a reaction reagent, an oxidase, and / or a degradation reagent based on stabilizers added thereto, according to an embodiment of the present application.

[0023] [Figure 14] 1 is a graph showing the results of evaluating the enhanced stability of a reaction reagent, an oxidase, and / or a degradation reagent based on a stabilizer added thereto, according to an embodiment of the present application.

[0024] [Figure 15] 1 is a table showing measurement results and accuracy based on the type of disaccharide contained in the stabilizer added to the reaction reagent, oxidase, and / or decomposition reagent according to an embodiment of the present application.

[0025] [Figure 16] 10A and 10B are tables and graphs showing the results of evaluating linearity based on the type of disaccharide contained in a stabilizer added to a reaction reagent, an oxidase, and / or a degradation reagent according to an embodiment of the present application.

[0026] [Figure 17] FIG. 2 shows identification information included in a glycated albumin quantitative analysis kit according to an embodiment of the present application. DETAILED DESCRIPTION OF THE INVENTION

[0027] The above objects, features and advantages of the present invention will be clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which: While the invention may, however, be modified in various ways and take various alternative forms, specific embodiments thereof are shown by way of example in the drawings and will hereinafter be described in detail.

[0028] Generally, like reference numerals refer to like elements throughout the specification. Furthermore, elements with the same function within the same concept shown in the drawings of each embodiment will be described using the same reference numerals, and the description thereof will not be repeated.

[0029] When it is determined that a detailed description of related well-known functions or configurations may unnecessarily obscure the gist of the present invention, such detailed description will be omitted. Furthermore, ordinal numbers (e.g., first, second, etc.) used in the description of the specification are used only to distinguish one element from another.

[0030] Furthermore, the terms "module," "unit," "section," or "portion" of elements used in this specification are assigned or incorporated for the convenience of the description of the specification, and the terms themselves do not have any definite meaning or role.

[0031] As used herein, the singular forms "a" and "an" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0032] It will be further understood that the terms "comprises," "including," "comprises," and / or "comprises" as used herein specify the presence of stated features or elements, but do not exclude the presence or addition of one or more other features or elements.

[0033] The size of elements in the drawings may be exaggerated for the convenience of explanation. In other words, the size and thickness of elements in the drawings are arbitrarily illustrated for the convenience of explanation, and the following embodiments are not limited thereto.

[0034] When a particular embodiment may be implemented differently, the order of specific processes may be performed differently from the order described, for example, two processes described in succession may be performed substantially simultaneously or may be performed in the reverse order from that described.

[0035] In the following embodiments, when a "first element" is said to be "connected" to a second element, this does not only include the case where the two elements are "directly connected," but also includes the case where the two elements are "indirectly connected" with a third element sandwiched between them. For example, in this specification, when a first element is said to be "electrically connected" to a second element, this includes not only the case where the two elements are "electrically directly connected," but also the case where the two elements are "electrically indirectly connected" with a third element sandwiched between them.

[0036] A kit for quantitative analysis of glycated albumin (GA) according to an embodiment of the present application may comprise: a first composition comprising a reaction buffer for inducing binding with albumin present in a blood sample; a second composition comprising a decomposition reagent for a decomposition reaction that decomposes glycated albumin (GA) contained in the blood sample into glycated amino acids; a third composition comprising a reaction reagent for an enzymatic reaction of glycated amino acids present in the blood sample; and a fourth composition comprising a color developer that is oxidized by hydrogen peroxide (H2O2) generated from the enzymatic reaction of glycated amino acids, wherein the GA quantitative analysis kit may comprise at least one reagent immobilization portion, and each of the second composition to the fourth composition may be independently immobilized on the reagent immobilization portion.

[0037] According to an embodiment of the present application, the first composition may be selected from the group consisting of bromocresol purple (BCP) and bromocresol green (BCG).

[0038] According to an embodiment of the present application, the reaction buffer may be selected from the group consisting of HEPES, PB, Tris-HCl, Tris-Base and PBS.

[0039] According to an embodiment of the present application, the reaction buffer may be selected from the group consisting of Tris-HCl and Tris-Base, and the pH of the first composition may be in the range of 4.1 to 10.0.

[0040] According to an embodiment of the present application, the reaction buffer may be Tris-HCl, and the decomposition reaction of glycated albumin and the enzymatic reaction of glycated amino acids may be carried out under weakly alkaline pH conditions.

[0041] According to an embodiment of the present application, the first composition may further comprise a surfactant selected from the group consisting of CHAPSO, Triton® X-100, and Tween® 20.

[0042] According to embodiments of the present application, the first composition may further comprise Triton X-100 surfactant at a concentration greater than 0% and up to 0.1%.

[0043] According to an embodiment of the present application, the reaction buffer may contain Tris-HCl, the first composition may further contain Triton X-100 surfactant at a concentration greater than 0% and up to 0.1%, and the degradation reaction of glycated albumin and the enzymatic reaction of glycated amino acids may be carried out under weakly alkaline pH conditions.

[0044] According to an embodiment of the present application, the degradation reagent may be a proteinase.

[0045] According to an embodiment of the present application, the reaction reagent may include ketoamine oxidase (KAO) and peroxidase (POD).

[0046] According to an embodiment of the present application, the color former can be DA-67.

[0047] According to an embodiment of the present application, the GA quantitative analysis kit may further include a fifth composition comprising an oxidase to prevent interference from ascorbic acid present in the blood sample, and the oxidase may include ascorbic acid oxidase (ASOx).

[0048] According to an embodiment of the present application, the second composition may further comprise a stabilizer for immobilizing the degradation reagent in a solid form on at least one reagent immobilization portion, and the stabilizer may comprise a disaccharide, DEAE dextran, and NPS.

[0049] According to an embodiment of the present application, the third composition may further include a stabilizer for immobilizing the reaction reagent in a solid form on at least one reagent immobilization portion, and the stabilizer may include a disaccharide, DEAE dextran, and NPS.

[0050] According to an embodiment of the present application, the fourth composition may further comprise a stabilizer for immobilizing the color former in a solid form on the at least one reagent immobilizing portion, and the stabilizer may comprise a disaccharide.

[0051] According to an embodiment of the present application, the fifth composition may further comprise a stabilizer for immobilizing the ascorbic acid oxidase in a solid form on the at least one reagent immobilization portion, and the stabilizer may comprise a disaccharide, DEAE dextran, and NPS.

[0052] According to an embodiment of the present application, the third composition can be immobilized in a solid state on a first reagent immobilization portion arranged in a first region of the GA quantitative analysis kit, and the second composition can be immobilized in a solid state on a second reagent immobilization portion arranged in a second region of the GA quantitative analysis kit, separated from the first region.

[0053] According to an embodiment of the present application, the GA quantitative analysis kit may be configured so that the reaction buffer is mixed with the reaction reagent fixed to the first reagent fixing portion before being mixed with the decomposition reagent fixed to the second reagent fixing portion, and the reaction reagent may be characterized in that it performs an enzymatic reaction with the glycated amino acid from the point at which the glycated amino acid is produced by the decomposition reaction caused by the decomposition reagent.

[0054] According to an embodiment of the present application, the GA quantitative analysis kit may comprise a main body including an upper plate and a lower plate configured in an opposing arrangement, wherein the third composition may be fixed to a first reagent fixing portion arranged in a first region of the upper plate, and the fifth composition may be fixed to a third reagent fixing portion arranged in a first region of the lower plate, and the first reagent fixing portion and the third reagent fixing portion are configured to face each other.

[0055] According to an embodiment of the present application, the second composition can be fixed to a second reagent fixing portion arranged in a second region of the upper plate, and the fourth composition can be fixed to a fourth reagent fixing portion arranged in a second region of the lower plate, and the second reagent fixing portion and the fourth reagent fixing portion are configured to face each other.

[0056] In the following, with reference to Figures 1 to 17, the structure of a kit for quantitative analysis of glycated albumin (GA), a method for quantitative analysis of GA, and / or an apparatus for quantitative analysis of GA according to embodiments of the present application will be described in more detail.

[0057] FIG. 1 is a schematic diagram of a glycated albumin quantitative analysis kit according to an embodiment of the present application.

[0058] A kit 10 for quantitative analysis of glycated albumin (hereinafter referred to as "GA") according to an embodiment of the present application may include a sample collector 100 for supplying a biological sample, and / or a main cartridge 200 into which the sample collector 100 can be inserted and received.

[0059] FIG. 2 is a schematic diagram of a sample collector 100 of a glycated albumin quantitative analysis kit according to an embodiment of the present application.

[0060] The sample collector 100 according to the embodiment of the present application may be configured to collect a predetermined amount of a biological sample (e.g., a blood sample) intended for analysis. Specifically, the sample collector 100 may include a capillary-type sample inlet 101, and may collect the biological sample (e.g., a blood sample) to be analyzed through a capillary provided in the sample inlet 101 and inject the collected biological sample into the main cartridge 200.

[0061] Furthermore, the sample collector 100 may include a protrusion 102 designed to contact and push inward a solution cell 301 inside the main cartridge 200 when the sample collector 100 is inserted into the receptacle 201 of the main cartridge 200, as will be described later. Specifically, the protrusion 102 may be positioned on the sample collector 100 so as to contact the solution cell 301 when the sample collector 100 is inserted into the receptacle 201 of the main cartridge 200. Consequently, when the sample collector 100 is inserted into the receptacle 201 of the main cartridge 200, the solution cell 301 may be moved inward toward the interior of the main cartridge 200, thereby removing or breaking the cover tape 302 of the solution cell 301. This will be described in more detail with reference to FIG. 5 .

[0062] In addition, the sample collector 100 may further include a handle 103 and / or a locking clip 104. Specifically, the handle 103 is a component designed to facilitate transportation or use of the sample collector 100 and is not limited to the structure shown in FIG. 2 . The locking clip 104 is provided on one side of the sample collector 100 and can secure the sample collector 100 to the main cartridge 200 when the sample collector 100 is inserted into the main cartridge 200. The locking clip 104 is designed to match the size and shape of a locking groove 211 provided in the receiving portion 201 of the main cartridge 200. The locking clip 104 of the sample collector 100 and the locking groove 211 of the main cartridge 200 can engage with each other. As a result, the inserted sample collector 100 can be fixed to the main cartridge 200, preventing the sample collector 100 from moving or falling off even if the GA quantitative analysis kit is rotated during analysis.

[0063] FIG. 3 is a schematic diagram of the main cartridge 200 of the glycated albumin quantitative analysis kit according to an embodiment of the present application.

[0064] The main cartridge 200 according to an embodiment of the present application may include a receptacle 201 into which the sample collector 100 may be inserted. Additionally, the receptacle 201 may include a locking groove 211 configured to engage with the locking clip 104 of the sample collector 100 to secure the sample collector 100, as described above.

[0065] The main cartridge 200 may also include a moving frame 203 configured to fix and move a solution cell 301 provided inside the main cartridge. Specifically, the moving frame 203 may be configured to fix the solution cell 301 before insertion of the sample collector 100. When the sample collector 100 is inserted, the solution cell 301 may be pushed by the protrusion 102 of the sample collector 100 and move along a moving path in the moving frame 203.

[0066] The main cartridge 200 may further include a cover tape breaking portion 202 configured to remove or break the cover tape 302 of the solution cell 301 as the solution cell 301 moves along the moving frame 203. This will be described in more detail with reference to FIG.

[0067] The main cartridge 200 may include a mixing section 204 (or mixing area) in which a biological sample (e.g., a blood sample) ejected from the sample collector 100 through the receiving section 201 mixes with a composition (hereinafter referred to as the first composition) ejected from the solution cell 301.

[0068] The main cartridge 200 may also include at least one reagent immobilization section 205 (referred to as a sample immobilization section) into which chemical reagents capable of reacting with the first composition and the biological sample mixed in the mixing section 204 are introduced to induce an enzymatic reaction and / or an antigen-antibody reaction.

[0069] The main cartridge 200 may further include a flow path 206 through which the mixed first composition and the biological sample in the mixing section 204 may move. Specifically, the analytical sample may move through the flow path 206 between the mixing section 204, the reagent fixing section 205, and / or a measurement unit 207 that optically measures the analytical sample. The structure of the flow path 206 is not limited as long as it is designed to allow the analytical sample to move by gravity when the main cartridge 200 is tilted.

[0070] The main cartridge 200 according to the embodiment of the present application may include a measurement unit 207 for measuring the result of a reaction carried out in the reagent fixing portion 205. The GA quantitative analysis device according to the embodiment of the present application may quantify an analytical sample (e.g., GA) through optical analysis such as UV / VIS via the measurement unit 207.

[0071] The main cartridge 200 may include a waste liquid processor 208 for collecting waste liquid after analysis is completed via the measurement unit 207. The waste liquid processor 208 enables collection and separate disposal of waste liquid, which is a type of medical waste. Collection of the waste liquid via the waste liquid processor 208 may be achieved by absorbing the waste liquid into highly absorbent cotton, an absorbent filter, or a polymer-based absorbent material disposed in the waste liquid processor 208.

[0072] The main cartridge 200 may further include a vent 209 to facilitate smooth transfer and absorption of waste liquid into the absorbent material, allowing for more efficient transfer of waste liquid to the waste treatment unit 208 and subsequent collection.

[0073] The main cartridge 200 may also include a handle 210 to facilitate its transportation and use. The structure of the handle 210 is not limited to the form shown in FIG.

[0074] 4 and 5, a more detailed description will be provided below of how the first composition in the solution cell 301 flows into the mixing section 204 of the main cartridge 200 when the sample collector 100 is inserted into the receiving section 201 of the main cartridge 200. FIG. 4 is a schematic diagram of the solution cell 301 of the GA quantitative analysis kit according to an embodiment of the present application. FIG. 5 is a diagram illustrating how the composition stored in the solution cell 301 flows into the mixing region of the main cartridge 200 when the sample collector 100 is inserted into the main cartridge 200 according to an embodiment of the present application.

[0075] The main cartridge 200 according to the embodiment of the present application may include a solution cell 301 that stores a first composition for reacting with a biological sample from the sample collector 100. The solution cell 301 includes an opening at one end that is sealed with a cover tape 302 to prevent leakage of the first composition stored therein. The opening of the solution cell 301 that is sealed with the cover tape 302 is positioned to face the cover tape break 202 of the main cartridge 200.

[0076] Before the sample collector 100 is inserted into the receptacle 201 of the main cartridge 200, the opening of the solution cell 301 sealed with the cover tape 302 may be located away from the cover tape break 202. Before the sample collector 100 is inserted into the receptacle 201 of the main cartridge 200, the opening of the solution cell 301 sealed with the cover tape 302 may be located away from the cover tape break 202. When the sample collector 100 is inserted into the receptacle 201 of the main cartridge 200, the protrusion 102 of the sample collector 100 applies pressure to the opposite end of the solution cell 301, away from the opening sealed with the cover tape 302. This applied pressure causes the solution cell 301 to move along the movement path of the moving frame 203.

[0077] As a result, when the cover tape 302 of the solution cell 301 comes into contact with the cover tape break 202, the cover tape 302 of the solution cell 301 is removed or broken. The first composition stored in the solution cell 301 is then transferred into the mixing section 204 of the main cartridge 200 through the channel or hollow structure formed in the cover tape break 202. In the mixing section 204, the reaction buffer mixes with the biological sample (e.g., a blood sample) supplied from the sample collector 100.

[0078] Specifically, the first composition flowing into the mixing section 204 can be designed to contact the sample inlet 101 of the sample collector 100. This contact allows the biological sample contained in the capillary-type sample inlet 101 to move through the sample inlet 101 into the mixing section 204 of the main cartridge 200. As a result, the first composition stored in the solution cell 301 and the biological sample collected by the sample collector 100 can be mixed in the mixing section 204 of the main cartridge 200.

[0079] 4 and 5, the shape of solution cell 301 is illustrated as a specific example. However, this is merely an example, and the shape of solution cell 301 is not limited as long as it provides a suitable structure for storing the first composition. Similarly, the material of cover tape 302 is not particularly limited as long as it prevents leakage of the first composition while allowing for easy removal or tearing.

[0080] 5 illustrates the cover tape break 202 in a particular shape for purposes of explanation. However, this is merely an example, and the shape of the cover tape break 202 is not limited as long as it facilitates removal or breaking of the cover tape. Suitable structures include, but are not limited to, needle-shaped or edge-shaped configurations.

[0081] The GA quantitative analysis kit 10 according to an embodiment of the present application can be used to quantitatively analyze GAs present in blood (e.g., plasma, serum). According to one embodiment, the GA quantitative analysis kit 10 can be configured to quantitatively analyze GAs using a BCP method and / or an enzymatic method. Below, with reference to Figures 7 to 9, a more detailed description of the quantitative analysis of GAs using the GA quantitative analysis kit 10 according to an embodiment of the present application is provided.

[0082] The analytical method for quantifying GA can be broadly divided into four steps, as shown in Figure 6 below. FIG. 6 illustrates one aspect of an analytical method for quantifying GA according to one embodiment of the present application.

[0083] The first step involves quantifying the total amount (or concentration) of albumin present in blood. According to one embodiment, the total amount of albumin in blood (plasma or serum) can be quantified using the bromocresol purple (BCP) method. Specifically, BCP binds to albumin to form a BCP complex. The total amount of albumin can be quantified by measuring the absorbance of a sample containing the BCP complex.

[0084] The second step involves degrading albumin (including glycated albumin) having a tertiary structure. Specifically, the tertiary structure of albumin can be degraded into at least one glycated amino acid (e.g., fructosyl-lysine) using a degradative enzyme such as a protease or proteinase.

[0085] The third step involves generating hydrogen peroxide through an enzymatic reaction of glycated amino acids. According to one embodiment, glycated amino acids can be degraded by a reaction enzyme (e.g., ketoamine oxidase (KAO)) into amino acids and glucose, during which hydrogen peroxide is generated.

[0086] The fourth step involves using the generated hydrogen peroxide to oxidize a color-forming agent (e.g., DA-67) in the presence of a reactive enzyme (e.g., peroxidase (POD)). The oxidation of the color-forming agent results in a color change. The amount of glycated albumin can be quantified by measuring the absorbance at a specific wavelength corresponding to this color change.

[0087] However, the above analytical mechanism for quantifying GA is merely an example, and any suitable GA quantitative analysis method can be used. Depending on the GA quantitative analysis method, the GA quantitative analysis kit according to the embodiment of the present application can be appropriately modified.

[0088] 7 and 8 are diagrams showing a specific structure of a GA quantitative analysis kit 10 according to an embodiment of the present application.

[0089] The solution cell 301 of the GA quantitative analysis kit 10 according to an embodiment of the present application can store a composition (hereinafter referred to as the "first composition") containing a reagent for inducing binding with albumin present in a blood sample. According to one embodiment, the first composition can include bromocresol purple (BCP) for forming a complex by binding with albumin present in the blood sample, and a reaction buffer (B) for establishing a reaction environment. Although bromocresol purple (BCP) is exemplified as a substance for forming a complex by binding with albumin, any suitable substance having similar characteristics to BCP, including bromocresol green, can be substituted.

[0090] In one embodiment, reaction buffer B may be selected from the group consisting of HEPES, PB, Tris-HCl, Tris-Base, and PBS. In this case, the pH of the first composition containing reaction buffer B may be within a pH range of 4.1 to 10.0. More preferably, the pH of the first composition may be within a pH range of 5.8 to 8.0.

[0091] In one embodiment, reaction buffer B may be selected from the group consisting of Tris-HCl and Tris-Base. In this case, the pH of the first composition containing reaction buffer B may be within a pH range of 4.1 to 10.0. More preferably, the pH of the first composition may be within a pH range of 5.8 to 8.0.

[0092] In one embodiment, reaction buffer B may be Tris-HCl. In this case, the pH of the first composition containing reaction buffer B may be within a pH range of 4.1 to 10.0. More preferably, the pH of the first composition may be within a pH range of 4.1 to 8.0. Preferably, the pH of the first composition may be within a pH range of 5.8 to 8.0.

[0093] More preferably, the analytical environment established by reaction buffer B (e.g., the analytical environment for the decomposition reaction of glycated albumin and the enzymatic reaction of glycated amino acids, which will be described later) can be maintained under weakly alkaline conditions (e.g., pH 8). Specifically, the decomposition reaction of glycated albumin and the enzymatic reaction of glycated amino acids, which will be described later, can be carried out under weakly alkaline conditions (e.g., pH 8).

[0094] According to the embodiments of the present application, the glycated albumin quantitative analysis kit, the glycated albumin quantitative analysis method, and / or the device for carrying out the method, maintaining the pH of the analysis environment at a weakly alkaline level, which is the optimal reaction environment for the enzyme ketoamine oxidase (KAO), can provide the effect of improving the accuracy of glycated albumin quantified by the enzymatic method.

[0095] The first composition according to one embodiment may further comprise a surfactant to create an optimal analytical environment, more specifically, the first composition may further comprise a surfactant to prevent protein precipitation.

[0096] In one embodiment, the surfactant may be selected from the group consisting of CHAPSO, Triton X-100, and Tween 20. Preferably, the surfactant may be Triton X-100, and the concentration of Triton X-100 may be included in the first composition at a concentration greater than 0% and up to 0.1%.

[0097] According to the glycated albumin quantitative analysis kit, glycated albumin quantitative analysis method, and / or device for carrying out the method according to the embodiments of the present application, the optimal combination of reaction buffer and surfactant can provide the effect of implementing the BCP reaction for quantifying the total amount of albumin and the enzymatic reaction for quantifying the amount of glycated albumin in a single kit. This further enables the miniaturization of the glycated albumin quantitative analysis kit. In addition, this can provide the effect of reducing the analysis time for glycated albumin and enhancing user convenience.

[0098] According to an embodiment of the present application, the GA quantitative analysis kit 10 may include a composition (hereinafter referred to as the second composition) containing a degradation reagent (R2) for a degradation reaction that degrades glycated albumin (GA) contained in a blood sample into glycated amino acids. The degradation reagent (R2) may be a proteinase. The degradation reagent (R2) performs the function of degrading the tertiary protein structure of glycated albumin and converting it into glycated amino acids, which are in a form that can participate in the enzymatic reaction described below.

[0099] According to an embodiment of the present application, the GA quantitative analysis kit 10 may include a composition (hereinafter referred to as the third composition) containing a reaction reagent (R1) for an enzymatic reaction of glycated amino acids contained in a blood sample. The reaction reagent (R1) may include ketoamine oxidase (KAO) and / or peroxidase (POD). The reaction reagent (R1) performs an enzymatic reaction with the glycated amino acids generated by the degradation reagent (R2) and oxidizes the result of the enzymatic reaction. Specifically, the ketoamine oxidase (KAO) in the reaction reagent (R1) functions to generate hydrogen peroxide through an enzymatic reaction with the glycated amino acids, while the peroxidase (POD) reacts with the hydrogen peroxide to oxidize a color developer, as described below.

[0100] According to an embodiment of the present application, the GA quantitative analysis kit 10 may include a composition (hereinafter referred to as a fourth composition) containing a color former that is oxidized by hydrogen peroxide generated from the enzymatic reaction of glycated amino acids. The color former may be DA-67.

[0101] According to an embodiment of the present application, the GA quantitative analysis kit 10 may include a composition (hereinafter referred to as a fifth composition) containing an oxidase (E) to prevent interference from ascorbic acid present in a blood sample. The oxidase (E) may include ascorbic acid oxidase (ASOx).

[0102] According to an embodiment of the present application, the GA quantitative analysis kit 10 may further include a stabilizer for immobilizing the second to fifth compositions in a solid state on at least one of the reagent immobilizing portions 205 .

[0103] According to one embodiment, the second composition may further comprise a stabilizer for immobilizing the degradation reagent (R2) in the solid state, wherein the stabilizer may be selected from the group consisting of disaccharides (e.g., trehalose, sucrose), DEAE dextran, and NPS. More preferably, the stabilizer for the second composition may comprise trehalose, DEAE dextran, and NPS.

[0104] According to one embodiment, the third composition may further comprise a stabilizer for immobilizing the reaction reagent (R1) in a solid state, wherein the stabilizer may be selected from the group consisting of disaccharides (e.g., trehalose, sucrose), DEAE dextran, and NPS. More preferably, the stabilizer for the third composition may comprise trehalose, DEAE dextran, and NPS.

[0105] According to one embodiment, the fourth composition may further comprise a stabilizer for immobilizing the color former (D) in a solid state, wherein the stabilizer may be a disaccharide (e.g., trehalose, sucrose). More preferably, the stabilizer for the fourth composition may be trehalose.

[0106] According to one embodiment, the fifth composition may further comprise a stabilizer for immobilizing the oxidase (E) in a solid state, wherein the stabilizer may be selected from the group consisting of disaccharides (e.g., trehalose, sucrose), DEAE dextran, and NPS. More preferably, the stabilizer for the fifth composition may comprise trehalose, DEAE dextran, and NPS.

[0107] As described above, the GA quantitative analysis kit 10 may include at least one reagent immobilizing portion 205. According to one embodiment, the second composition to the fifth composition may each be independently immobilized on at least one reagent immobilizing portion 205.

[0108] According to one embodiment, the third composition including the reaction reagent (R1) can be immobilized on a first reagent immobilizing section 205-1 disposed in a first region of the GA quantitative analysis kit 10. The first region can be an area located above the mixing section 204. In contrast, the second composition including the degradation reagent (R2) can be immobilized on a second reagent immobilizing section 205-2 disposed in a second region of the GA quantitative analysis kit 10. The second region is a separate area separated from the first region. The first and second regions can be connected via a flow path 206, and the GA quantitative analysis kit 10 can be configured such that the measurement unit 207 is located midway along the path along which the blood sample moves from the first region to the second region.

[0109] Referring to FIG. 8, the GA quantitative analysis kit 10 may include a body including an upper plate and a lower plate configured in an opposing arrangement.

[0110] According to one embodiment, a third composition containing a reaction reagent (R1) may be dispensed and dried on a first reagent immobilizing portion 205-1 provided in a first region of an upper plate of the main body. In contrast, a fifth composition containing an oxidase (E) may be dispensed and dried on a third reagent immobilizing portion 205-3 provided in a first region of a lower plate of the main body. Furthermore, the first reagent immobilizing portion 205-1 provided on the upper plate of the main body and the third reagent immobilizing portion 205-3 provided on the lower plate may be configured in an opposing arrangement. In FIG. 8 , the third composition containing a reaction reagent (R1) is described as being provided on the upper plate of the main body, while the fifth composition containing an oxidase (E) is described as being provided on the lower plate. However, this is merely an example, and compositions may be provided on multiple reagent immobilizing portions 205 in any suitable configuration. For example, the third composition containing a reaction reagent (R1) may be provided on the lower plate, and the fifth composition containing an oxidase (E) may be provided on the upper plate.

[0111] According to one embodiment, a second composition containing a decomposition reagent (R2) may be dispensed and dried on a second reagent immobilizing portion 205-2 provided in a second region of the upper plate of the main body. In contrast, a fourth composition containing a color former (D) may be dispensed and dried on a fourth reagent immobilizing portion 205-4 provided in a second region of the lower plate of the main body. Furthermore, the second reagent immobilizing portion 205-2 provided on the upper plate of the main body and the fourth reagent immobilizing portion 205-4 provided on the lower plate may be configured in an opposing arrangement. In FIG. 8 , the second composition containing a decomposition reagent (R2) is provided on the upper plate of the main body, while the fourth composition containing a color former (D) is provided on the lower plate. However, this is merely an example, and compositions may be provided on multiple reagent immobilizing portions 205 in any suitable configuration. For example, the second composition containing a decomposition reagent (R2) may be provided on the lower plate of the main body, and the fourth composition containing a color former (D) may be provided on the upper plate of the main body.

[0112] According to an embodiment of the present application, a third composition containing a reaction reagent (R1) is immobilized in a first reagent immobilizing portion 205-1 provided on the mixing portion 204, and a second composition containing a degradation reagent (R2) is immobilized in a second reagent immobilizing portion 205-2 separated from the mixing portion 204. This configuration ensures that the reaction buffer solution released from the solution cell 301 (and / or the blood sample released from the sample collector 100) is mixed with the reaction reagent (R1) immobilized in the first reagent immobilizing portion 205-1 before contacting the degradation reagent (R2) immobilized in the second reagent immobilizing portion 205-2. However, the reaction reagent (R1) does not perform an enzymatic reaction from the point at which the reaction buffer solution and the blood sample are mixed. Rather, the enzymatic reaction with glycated amino acids is carried out from the point at which glycated amino acids are produced through a degradation reaction by the degradation reagent (R2), i.e., from the point at which glycated albumin contained in the analysis sample is decomposed by the degradation reagent (R2) in the second reagent immobilizing portion (205-2) to produce glycated amino acids. According to the glycated albumin quantitative analysis kit, the glycated albumin quantitative analysis method, and / or the device for performing the method described in the embodiments of the present application, immobilizing the second composition containing the degradation reagent (R2) to the second reagent immobilizing portion 205-2 rather than the first reagent immobilizing portion 205-1 prevents the loss of BCP binding sites present on albumin, thereby improving the accuracy of total albumin quantification using the BCP analysis method.

[0113] FIG. 9 illustrates an analytical embodiment for quantifying glycated albumin in a blood sample according to an embodiment of the present application.

[0114] Referring to FIG. 9, the method for quantitative analysis of glycated albumin according to an embodiment of the present application can be implemented as follows. 1) Prepare the main cartridge 200. 2) Position the sample collector 100 on the receiving portion 201 of the main cartridge 200. 3) Apply pressure to the sample collector 100 in a direction toward the receiving portion 201 of the main cartridge 200. 4) When the protrusion 102 of the sample collector 100 applies pressure to the solution cell 301, the solution cell 301 moves through the moving frame 203 in the direction of the cover tape breaking portion 202, as described above. The cover tape breaking portion 202 comes into contact with the cover tape 302 of the solution cell 301, and as a result, the solution (first composition) stored inside the solution cell 301 flows into the mixing portion 204 of the main cartridge 200. 5) As the first composition flows into the mixing section 204 , it comes into contact with the blood sample from the sample collector 100 , and as a result, the blood sample (eg, plasma, serum) also flows into the mixing section 204 of the main cartridge 200 . 6) In this step, a first composition containing a reaction buffer (B) and bromocresol purple (BCP) is mixed with the blood sample on the mixing section 204. In this process, albumin (meaning both non-glycated albumin and glycated albumin) in the blood sample binds to BCP to form a BCP complex.

[0115] Meanwhile, on the mixing unit 204, the reaction reagent (R1) and oxidase (E) immobilized on reagent immobilizing units 205-1 and 205-2 provided on the mixing unit 204 are also mixed with the blood sample. However, the reaction reagent (R1) does not react with albumin or glycated albumin, which have a tertiary structure. The enzymatic reaction proceeds only when the blood sample reaches the reagent immobilizing units 205-3 and 205-4, on which the decomposition reagent (R2) is immobilized, and the tertiary structure of albumin and / or glycated albumin is decomposed by the decomposition reagent (R2) to produce glycated amino acids.

[0116] On the other hand, according to the embodiment of the present application, the GA quantitative analysis kit 10 rotates by a predetermined angle due to an external force (e.g., a rotational force applied by a quantitative analysis device, as described later). As a result, the analysis sample, including the blood sample, inside the main cartridge 200 moves through the flow path 206 of the main cartridge 200 under the influence of gravity. 7) An external force applied to the GA quantitative analysis kit 10 moves the blood sample to the measurement unit 207. While the sample is positioned on the measurement unit 207, an absorbance (hereinafter referred to as a first absorbance) corresponding to the total amount (or concentration) of albumin (total albumin) in the blood sample can be measured.

[0117] Meanwhile, as described above, the GA quantitative analysis kit 10 may include a measuring unit 207 for quantifying GA contained in a blood sample. According to one embodiment, the GA quantitative analysis kit 10 may be configured so that the blood sample passes through the measuring unit 207 while moving from the mixing section 204 through the flow path 206 to the reagent immobilizing sections 205-3 and 205-4 where the enzyme reactions are carried out. In this embodiment, both the absorbance before the enzyme reaction, which corresponds to the concentration of total albumin, and the absorbance after the enzyme reaction, which corresponds to the concentration of glycated albumin, can be easily measured. This may provide the advantage of enabling the glycated albumin ratio to be quantified more conveniently. 8) Due to an external force applied to the GA quantitative analysis kit 10, the analysis sample containing the blood sample moves from the measurement unit 207 to the reagent immobilization sections 205-3 and / or 205-4 where a decomposition reagent (R2, e.g., protease) is immobilized. On the reagent immobilization sections 205-3 and / or 205-4, glycated albumin contained in the blood sample is decomposed into glycated amino acids by the decomposition reagent (R2).

[0118] 9 occurs between the decomposed glycated amino acids and the reaction reagent (R1, e.g., KAO) previously mixed in the reagent immobilizing section 205-1 and / or 205-2. Hydrogen peroxide is generated through this enzyme reaction, and when the generated hydrogen peroxide is oxidized by the reaction reagent (R1, e.g., POD), electrons are released, causing a change in the color of the color developer (D), resulting in a change in absorbance. 9) An external force applied to the GA quantitative analysis kit 10 causes the post-enzyme reaction sample to move from the reagent fixing section 205-3 and / or 205-4 to the measurement unit 207. While the sample is located on the measurement unit 207, a measurement value for the absorbance of the post-enzyme reaction sample (second absorbance) can be measured. The second absorbance can correspond to the amount (or concentration) of glycated albumin present in the blood sample.

[0119] Furthermore, the ratio of glycated albumin to total albumin in the blood sample can be quantified based on the first absorbance related to the total albumin concentration measured in the measurement unit 207 before the enzymatic reaction and the second absorbance related to the glycated albumin concentration measured in the measurement unit 207 after the enzymatic reaction. 10) Waste from the completed quantitative analysis moves from the measurement unit 207 to the waste liquid treatment unit 208 due to an external force applied to the GA quantitative analysis kit 10, where it is collected.

[0120] The present invention will now be described in detail through experimental examples with reference to Figures 10 to 16. However, the following experimental examples are merely illustrative and should not be construed as limiting.

[0121] <Experimental Example 1: Evaluation of albumin measurement performance based on reaction buffer> 1. Experimental Method To determine the optimal reaction buffer and reaction conditions (pH) for albumin analysis, five candidate buffers (HEPES, phosphate buffer, Tris-HCl, Tris-Base, and phosphate-buffered saline) were selected. For each candidate buffer, five pH conditions (pH 4.1, 5.8, 6.5, 8.0, and 10.0) were tested, resulting in a total of 25 buffer and pH combinations. The measurement performance of the albumin-BCP assay was evaluated for all combinations. Specifically, the candidate buffers were supplemented with appropriate concentrations of bromocresol purple (BCP) reagent and the nonionic surfactant Triton X-100.

[0122] Test samples were prepared using albumin powder (human serum-derived albumin, lyophilized powder, Sigma-Aldrich; A3782). Specifically, a low-concentration sample (16.6 g / L) was prepared by dissolving 83 mg of albumin powder in 5 mL of deionized water (DIW), and a high-concentration sample (64.8 g / L) was prepared by dissolving 130 mg of albumin powder in 5 mL of DIW. A medium-concentration sample (40.4 g / L) was prepared by mixing the low-concentration and high-concentration samples in a 1:1 ratio.

[0123] Furthermore, a medium-low concentration sample (28.4 g / L) was prepared by mixing the low-concentration sample and the medium-concentration sample in a 1:1 ratio, and a medium-high concentration sample (52.6 g / L) was prepared by mixing the medium-concentration sample and the high-concentration sample in a 1:1 ratio, resulting in a total of five sample concentrations. The albumin content (g / L) of the prepared samples was verified using a reference reagent (Lucica GA-L, Asahi Kasei Pharma) and an automated biochemistry analyzer (RX Imola, RANDOX). Each candidate buffer was applied to an albumin sample (Sigma-Aldrich) under five different pH conditions. Furthermore, 25 candidate buffers and five test samples were mixed in a 20:1 ratio in a 96-microwell plate (specifically, 195 μL of candidate buffer and 5 μL of test sample). After 1 min of reaction, absorbance-related measurements were analyzed using a spectrophotometer (Thermo Scientific; Multiskan GO Microplate Spectrophotometer) at a wavelength of 610 nm, where the maximum peak of the albumin-BCP complex was observed.

[0124] Linearity (RQS) and slope were calculated based on absorbance-related measurements for each candidate buffer under five pH conditions.

[0125] The five candidate buffer solutions and five pH conditions are shown in Table 1. Table 1 shows the five candidate reaction buffer solutions and pH conditions for evaluating albumin measurement performance. For each sample, if the linearity (RQS) was measured to be greater than 0.98 and the slope was 0.003 or greater, the corresponding candidate buffer solution and pH condition were evaluated as providing sufficient measurement performance for albumin-BCP analysis.

[0126] Table 1: Candidate reaction buffers and pH conditions for evaluating albumin measurement performance. [Table 1]

[0127] 2. Experimental Results FIG. 10 is a table and graph showing the results of evaluating the performance of measuring albumin based on different reaction buffers according to an embodiment of the present application.

[0128] For HEPES buffer: 1) Under conditions of pH 5.8, pH 6.5, and pH 8.0, the linearity (RQS) was measured to be greater than 0.98, and the slope was measured to be 0.003 or greater. 2) Under conditions of pH 4.1 and pH 10.0, the slope was measured to be less than 0.003.

[0129] For PB buffer: 1) Under conditions of pH 5.8, pH 6.5, and pH 8.0, the linearity (RQS) was measured to be greater than 0.98, and the slope was measured to be 0.003 or greater. 2) Under conditions of pH 4.1 and pH 10.0, the slope was measured to be less than 0.003.

[0130] For Tris-HCl buffer: 1) Under conditions of pH 5.8, pH 6.5, and pH 8.0, the linearity (RQS) was measured to be greater than 0.98, and the slope was measured to be 0.003 or greater. 2) Under pH 4.1 conditions, the linearity (RQS) was measured to be less than 0.98, and under pH 10.0 conditions, the slope was measured to be less than 0.003.

[0131] For Tris-Base buffer: 1) Under conditions of pH 4.1, pH 5.8, pH 6.5 and pH 8.0, the linearity (RQS) was measured to be greater than 0.98 and the slope was measured to be 0.003 or greater. 2) Under conditions of pH 10.0, the slope was measured to be less than 0.003.

[0132] For PBS buffer: 1) Under conditions of pH 5.8 and pH 6.5, the linearity (RQS) was measured to be greater than 0.98, and the slope was measured to be 0.003 or greater. 2) Under conditions of pH 4.1, pH 8.0 and pH 10.0, the slope was measured to be less than 0.003.

[0133] Through Experimental Example 1, it was confirmed that the reaction buffer (B) according to an embodiment of the present application may be selected from the group consisting of HEPES, PB, Tris-HCl, Tris-Base, and PBS, and the pH of the first composition containing the reaction buffer (B) may be in the range of 4.1 to 10.0. Preferably, the pH of the first composition containing the reaction buffer (B) may be in the range of 5.8 to 8.0.

[0134] <Experimental Example 2: Evaluation of glycated albumin measurement performance based on reaction buffer> 1. Experimental Method To select the optimal reaction buffer and reaction conditions (pH) for both the BCP analytical method and the enzymatic method for glycated albumin analysis, five candidate buffers (HEPES, phosphate buffer, Tris-HCl, Tris-Base, and phosphate-buffered saline) were selected. Using these five candidate buffers, the measurement performance of the albumin-BCP assay was evaluated under five different pH conditions (pH 4.1, 5.8, 6.5, 8.0, and 10.0), resulting in a total of 25 buffer and pH combinations. Specifically, appropriate concentrations of bromocresol purple (BCP) reagent and the nonionic surfactant Triton X-100 were added to each candidate buffer.

[0135] Test samples were prepared by mixing low-concentration (4.51 g / L) and high-concentration (17.59 g / L) standard solutions of Lucica GA-L Control (Asahi Kasei Pharma), a standard solution used as a reference reagent by i-SENS. Specifically, the medium-concentration sample (11.37 g / L) was prepared by mixing the low-concentration and high-concentration samples of the standard solution in a 1:1 ratio, the medium-low-concentration sample (8.01 g / L) was prepared by mixing the low-concentration and medium-concentration samples in a 1:1 ratio, and the medium-high-concentration sample (14.66 g / L) was prepared by mixing the medium-concentration and high-concentration samples in a 1:1 ratio.

[0136] The glycated albumin (g / L) content of the prepared samples was verified before use using a reference reagent (Lucica GA-L, Asahi Kasei Pharma) and a reference instrument, an automatic biochemistry analyzer (RX Imola, RANDOX).

[0137] Furthermore, the absorbance measurement results for the color development of the color former (DA-67) by the glycosylated albumin enzymatic assay were analyzed using the i-SENS A1Care analyzer (in vitro diagnostic medical device approval number 21-1020) at a wavelength of 660 nm, where the maximum peak of the color former (DA-67) was observed.

[0138] Additionally, linearity (RQS) and slope were calculated based on absorbance-related measurements for each candidate buffer under different pH conditions.

[0139] The five candidate buffer solutions and five pH conditions are as shown in Table 2. Table 2 shows the five candidate reaction buffer solutions and pH conditions for evaluating the glycated albumin measurement performance.

[0140] For each sample, if the linearity (RQS) was measured to be greater than 0.98 and the slope was 0.0003 or greater, the corresponding candidate buffer and pH conditions were assessed as providing sufficient measurement performance for the glycated albumin enzymatic analysis.

[0141] Table 2: Candidate reaction buffers and pH conditions for evaluating glycated albumin measurement performance. [Table 2]

[0142] 2. Experimental Results FIG. 11 is a table and graph showing the results of evaluating the measurement performance of glycated albumin based on different reaction buffers according to an embodiment of the present application.

[0143] For HEPES buffer: 1) Under conditions of pH 6.5 and pH 8.0, the linearity (RQS) was measured to be greater than 0.98, and the slope was measured to be 0.0003 or greater. 2) Under conditions of pH 4.1, pH 5.8 and pH 10.0, the slope was measured to be less than 0.0003.

[0144] For PB buffer, the linearity (RQS) was measured to be less than 0.98 and the slope was measured to be less than 0.0003 under conditions of pH 4.1, pH 5.8, pH 6.5, pH 8.0, and pH 10.0. Note that for PB buffer, the slope was measured to be essentially zero under the condition of pH 5.8, and the slope was measured to be essentially negative under the conditions of pH 6.5 and pH 8.0. This confirmed that, for PB buffer, the enzymatic reaction did not proceed, or the signal intensity decreased as the concentration of glycated albumin (or glycated amino acid) increased, indicating that PB buffer is not suitable for use in enzymatic analysis of glycated albumin.

[0145] For Tris-HCl buffer: 1) Under conditions of pH 4.1, pH 5.8, pH 6.5 and pH 8.0, the linearity (RQS) was measured to be greater than 0.98 and the slope was measured to be 0.0003 or greater. 2) Under conditions of pH 10.0, the slope was measured to be less than 0.0003.

[0146] For Tris-Base buffer: 1) Under conditions of pH 4.1, pH 5.8, pH 6.5 and pH 8.0, the linearity (RQS) was measured to be greater than 0.98 and the slope was measured to be 0.0003 or greater. 2) Under conditions of pH 10.0, the linearity (RQS) was measured to be less than 0.98 and the slope was measured to be less than 0.0003.

[0147] For PBS buffer: Under conditions of pH 4.1, pH 5.8, pH 6.5, pH 8.0 and pH 10.0, the slope was measured to be less than 0.0003.

[0148] Through Experimental Example 2, it was confirmed that the reaction buffer (B) according to an embodiment of the present application may be selected from the group consisting of Tris-HCl and Tris-Base, and the pH of the first composition containing the reaction buffer (B) may be within the range of 4.1 to 10.0. Preferably, the pH of the first composition containing the reaction buffer (B) may be implemented within the range of 4.1 to 8.0. More preferably, the pH of the first composition containing the reaction buffer (B) may be implemented within the range of 5.8 to 8.0.

[0149] On the other hand, under conditions of pH 6.5 and pH 8.0, the slope of the Tris-HCl buffer was observed to be relatively greater than that of the Tris-Base buffer. Therefore, in a preferred embodiment of the present application, the reaction buffer (B) may be Tris-HCl. Furthermore, the pH of the first composition containing Tris-HCl as reaction buffer (B) may be implemented within the range of 4.1 to 10.0. More preferably, the pH of the first composition containing Tris-HCl as reaction buffer (B) may be implemented within the range of 4.1 to 8.0, and even more preferably within the range of 5.8 to 8.0.

[0150] On the other hand, the pH of the first composition containing Tris-HCl as the reaction buffer solution (B) can be preferably provided in the weak alkaline pH range, taking into consideration the optimal reaction environment for the ketoamine oxidase (KAO) of the reaction reagent (R1).

[0151] <Experimental Example 3: Evaluation of measurement performance using surfactants> 1. Experimental Method To select surfactants that provide optimal analytical conditions for both the BCP analytical method and the enzymatic method, the following evaluations were performed: 1) Measurement performance of the albumin-BCP assay for different concentrations of each candidate surfactant. 2) Measurement performance of the glycated albumin enzymatic assay for different concentrations of each candidate surfactant.

[0152] Specifically, 1) for the measurement performance of the albumin-BCP assay, each candidate surfactant was applied to an albumin sample (Sigma Aldrich) at concentrations of 0.1%, 0.01%, and 0.001%.

[0153] Measurement samples were prepared using albumin powder (human serum-derived albumin, freeze-dried powder, Sigma Aldrich; A3782).

[0154] Specifically, a low-concentration sample (16.6 g / L) was prepared by dissolving 83 mg of albumin powder in 5 mL of deionized water (DIW), a high-concentration sample (64.8 g / L) was prepared by dissolving 130 mg of albumin powder in 5 mL of deionized water (DIW), a medium-concentration sample (40.4 g / L) was prepared by mixing the low-concentration sample and the high-concentration sample in a 1:1 ratio, a medium-low-concentration sample (28.4 g / L) was prepared by mixing the low-concentration sample and the medium-concentration sample in a 1:1 ratio, and a medium-high-concentration sample (52.6 g / L) was prepared by mixing the medium-concentration sample and the high-concentration sample in a 1:1 ratio, for a total of five sample concentrations.

[0155] The albumin (g / L) values ​​of the prepared samples were verified before use using a reference reagent (Lucica GA-L, Asahi Kasei Pharma) and a reference device, an automatic biochemistry analyzer (RX Imola, RANDOX).

[0156] Furthermore, the absorbance-related measurements of each sample were analyzed using the i-SENS A1Care analyzer at 610 nm, where the maximum peak of the BCP complex was observed. The linearity (RQS) and slope were calculated based on the absorbance-related measurements for each candidate surfactant at various concentrations. For each sample, if the linearity (RQS) was measured to be greater than 0.98 and the slope was 0.003 or greater, the corresponding surfactant and its concentration were deemed to provide sufficient measurement performance for albumin-BCP analysis.

[0157] Furthermore, for 2) the measurement performance of the glycated albumin enzymatic assay, each candidate surfactant was applied to a glycated albumin sample (Sigma-Aldrich) at concentrations of 0.1%, 0.01%, and 0.001%. Test samples were prepared by mixing low-concentration (4.51 g / L) and high-concentration (17.59 g / L) standard solutions of Lucica GA-L Control (Asahi Kasei Pharma), a standard solution used as a reference reagent by i-SENS. Specifically, the medium-concentration sample (11.37 g / L) was prepared by mixing the low-concentration and high-concentration standard solutions in a 1:1 ratio, the medium-low-concentration sample (8.01 g / L) was prepared by mixing the low-concentration sample and the medium-concentration sample in a 1:1 ratio, and the medium-high-concentration sample (14.66 g / L) was prepared by mixing the medium-concentration sample and the high-concentration sample in a 1:1 ratio.

[0158] The glycated albumin (g / L) values ​​of the prepared samples were verified before use using a reference reagent (Lucica GA-L, Asahi Kasei Pharma) and a reference device, an automatic biochemistry analyzer (RX Imola, RANDOX).

[0159] Furthermore, using the i-SENS A1Care analyzer, absorbance measurements for the color development of the color former (DA-67) in the glycated albumin enzymatic assay were performed for each sample at 660 nm, where the maximum peak of DA-67 was observed. Based on the absorbance-related measurements for each candidate surfactant at various concentrations, the linearity (RQS) and slope were calculated. For each sample, if the linearity (RQS) was measured to be greater than 0.98 and the slope was 0.0003 or greater, the corresponding surfactant and its concentration were deemed to provide sufficient measurement performance for the glycated albumin enzymatic assay.

[0160] The three candidate surfactants and their concentration conditions are shown in Table 3 below. Table 3 shows the candidate surfactants and their concentration conditions.

[0161] Table 3: Candidate surfactants and surfactant concentration conditions. [Table 3]

[0162] 2. Experimental Results FIG. 12 is a table showing the results of evaluating the performance of measuring albumin and / or glycated albumin based on the surfactant added to the reaction buffer, according to an embodiment of the present application. 1) Measurement performance of albumin-BCP analysis For CHAPSO surfactant, the linearity (RQS) was measured to be greater than 0.98 and the slope was measured to be 0.003 or greater at 0.01% and 0.001% concentrations, but the linearity (RQS) was measured to be less than 0.98 and the slope was measured to be less than 0.003 at 0.1% concentration.

[0163] For Triton X-100 surfactant, the linearity (RQS) was measured to be greater than 0.98 and the slope was measured to be 0.003 or greater under the concentration conditions of 0.1%, 0.01%, and 0.001%.

[0164] For Tween 20 surfactant, the linearity (RQS) was measured to be greater than 0.98 and the slope was measured to be 0.003 or greater under the concentration conditions of 0.1%, 0.01% and 0.001%. 2) Measurement performance of glycated albumin enzyme analysis For CHAPSO surfactant, the linearity (RQS) was determined to be greater than 0.98 and the slope was determined to be 0.0003 or greater under the concentration conditions of 0.1%, 0.01%, and 0.001%.

[0165] For Triton X-100 surfactant, the linearity (RQS) was measured to be greater than 0.98 and the slope was measured to be 0.0003 or greater under the concentration conditions of 0.1%, 0.01%, and 0.001%.

[0166] For Tween 20 surfactant, the linearity (RQS) was measured to be greater than 0.98 and the slope was measured to be 0.0003 or greater at 0.1% and 0.01% concentrations, but the linearity (RQS) was measured to be less than 0.98 and the slope was measured to be less than 0.0003 at 0.1% concentration.

[0167] Through Experimental Example 3, it was confirmed that the surfactant in the embodiment of the present application can be selected from the group consisting of CHAPSO, Triton X-100, and Tween 20.

[0168] Meanwhile, referring to Experimental Example 3, Triton X-100 was confirmed to satisfy the evaluation criteria under all concentration conditions for both 1) the albumin-BCP assay and 2) the glycated albumin enzyme assay. Therefore, in a preferred embodiment of the present application, the surfactant may be Triton X-100, and the concentration of Triton X-100 may be greater than 0% and up to 0.1% in the first composition.

[0169] Furthermore, referring to Experimental Examples 1 to 3, in a preferred embodiment of the present application, the first composition may contain Triton X-100 at a concentration greater than 0% and up to 0.1% together with a Tris-HCl buffer solution. This ensures that both the glycated albumin degradation reaction and the enzymatic reaction of glycated amino acids are carried out in the optimal analytical environment (weakly alkaline). More preferably, the first composition may contain Triton X-100 at a concentration of approximately 0.01% and a Tris-HCl buffer solution.

[0170] The second, third, and fifth compositions according to embodiments of the present application may each include a stabilizer for immobilizing the reagent in the "solid state," as described above. Each stabilizer may include a disaccharide, DEAE dextran, and NPS.

[0171] A drying process is required to fix the reaction reagent (R1), decomposition reagent (R2), and oxidase (E) in a "solid state." During this drying process, rapid removal of water can cause structural damage to the proteins that make up the reagents. To prevent such structural damage, a disaccharide was selected as one of the stabilizers. Specifically, trehalose, a disaccharide, was selected as one of the stabilizers.

[0172] In a dry state, the reaction reagent (R1), degradation reagent (R2), and oxidase (E) must remain stable for months or even years. To ensure storage stability, NPS (Neo Protein Saver) was selected as one of the stabilizers because it interacts with protein molecules in the reagents to protect their structure and prevent protein denaturation caused by oxidation.

[0173] In the dry state, the reaction reagent (R1), degradation reagent (R2), and oxidase (E) also need to be able to redissolve and participate in the reaction when mixed with a blood sample. To facilitate this, DEAE-dextran was selected as one of the stabilizers because it protects the protein structure through ion exchange and helps the dried reagents to redissolve.

[0174] <Experimental Example 4: Evaluation of enhanced stability by stabilizers> 1. Experimental Method The A1Care cartridge of i-SENS (in vitro diagnostic medical device approval number 21-4641, 3 lots) in which the present invention was implemented was used.

[0175] A stabilizer consisting of 250 mM trehalose, 1.25 (w / v%) DEAE dextran, and 1.5 (w / v%) NPS (Toyobo) was added to the reagent immobilization section of the A1Care cartridge, on which the reaction reagent (R1) consisting of KAO and POD was immobilized.

[0176] A stabilizer consisting of 250 mM trehalose, 1.25 (w / v%) DEAE dextran, and 1.5 (w / v%) NPS (Toyobo) was added to the reagent immobilization section of the A1Care cartridge, on which the oxidase (E) consisting of ASOx was immobilized.

[0177] A stabilizer consisting of 250 mM trehalose, 1.25 (w / v%) DEAE dextran, and 1.5 (w / v%) NPS (Toyobo) was added to the reagent immobilization section of the A1Care cartridge, on which a degradation reagent (R2) consisting of protease was immobilized.

[0178] On the other hand, 100 mM trehalose was added to the reagent immobilization part of the A1Care cartridge on which the color developer (D) consisting of DA-67 was immobilized.

[0179] Test samples were prepared using Asahi Kasei Pharma's standard solution (Lucica GA-L Control), which serves as the reference reagent for i-SENS. The albumin (g / L) and glycated albumin (g / L and %) values ​​of low-concentration (4.51 g / L) and high-concentration (17.59 g / L) solutions were verified prior to use using the reference reagent (Lucica GA-L, Asahi Kasei Pharma) and a reference device, an automated biochemistry analyzer (RX Imola, RANDOX). The glycated albumin ratio (GA%) was calculated using two lots of i-SENS A1Care analyzers.

[0180] For one of the two lots of A1Care analyzers, Lusica GA-L control solution (low) was used to perform 10 replicate measurements on the evaluation day for three lots of cartridges. For the remaining lot of A1Care analyzers, Lusica GA-L control solution (high) was used to perform 10 replicate measurements on the evaluation day for three lots of cartridges to measure the glycated albumin ratio (GA%).

[0181] In addition, the bias (%) and precision (CV%) of the measured glycated albumin ratio relative to the initial glycated albumin ratio (GA%) were calculated.

[0182] The above analytical method was performed for each of the following storage conditions in Table 4 below, which shows the storage temperatures and storage periods for the cartridges.

[0183] Table 4: Cartridge storage temperature and storage period [Table 4]

[0184] Furthermore, a stabilizer was evaluated as suitable for quantitative analysis of glycated albumin ratio (GA%) if it met all of the following evaluation criteria: 1) At least two of the three cartridge lots must meet the following conditions for the initial glycated albumin ratio measurement (i.e., GA% on day 0): The bias must be within ±10% and the CV must be within 10%. 2) Additionally, for each evaluation period, the average measured value of the glycated albumin ratio for the three cartridge lots must meet the following conditions: bias must be within ±10% and CV must be within 10%.

[0185] 2. Experimental Results Figure 13 is a table showing results of evaluating the enhanced stability of a reaction reagent, an oxidase, and / or a degradation reagent based on stabilizers added thereto, according to an embodiment of the present application. Figure 14 is a graph showing results of evaluating the enhanced stability of a reaction reagent, an oxidase, and / or a degradation reagent based on stabilizers added thereto, according to an embodiment of the present application.

[0186] Under storage conditions at 40°C, the mean measured glycated albumin ratios for the three cartridge lots were determined to remain within ±10% bias and 10% CV up to 14 days of storage.

[0187] Under storage conditions of 20-25°C, the mean measured glycated albumin ratio for the three cartridge lots was confirmed to remain within ±10% bias and 10% CV up to 24 weeks of storage.

[0188] Under storage conditions of 2-8°C, the mean measured glycated albumin ratio for the three cartridge lots was confirmed to remain within ±10% bias and 10% CV up to 52 weeks of storage.

[0189] Through Experimental Example 4, it was determined that the stabilizers containing disaccharides, DEAE dextran, and NPS, which were added to Compositions 2, 3, and 5, respectively, were suitable for quantitative analysis of the glycated albumin ratio.

[0190] Meanwhile, trehalose was used as the disaccharide contained in the stabilizer in Experimental Example 4. However, any suitable disaccharide having similar characteristics to trehalose, such as sucrose, may also be contained in the stabilizer.

[0191] Experimental Example 5: Equivalence of trehalose and sucrose as stabilizers To verify that disaccharides other than trehalose can be used as stabilizers, the equivalence of representative disaccharides trehalose and sucrose as stabilizers was additionally examined.

[0192] 1. Experimental Method To verify the use of disaccharides as stabilizers, a comparative evaluation was performed, as shown in Table 5 below. Table 5 shows the compositions of the two stabilizers used in the comparative evaluation. This evaluation compared two stabilizer formulations: 1) stabilizer condition 1, which consisted of trehalose, 1.25% w / v DEAE dextran, and 1.5% w / v NPS, and 2) stabilizer condition 2, which consisted of sucrose, 1.25% w / v DEAE dextran, and 1.5% w / v NPS.

[0193] Table 5: Compositions of the two stabilizers used in the comparative evaluation [Table 5]

[0194] First, the measurements of glycated albumin, total albumin, and the glycated albumin ratio when using the stabilizer in condition 1 containing trehalose were compared with the measurements of glycated albumin, total albumin, and the glycated albumin ratio when using the stabilizer in condition 2 containing sucrose. Furthermore, the accuracy of the measurements (i.e., the measurements of glycated albumin, total albumin, and the glycated albumin ratio) when using the stabilizer in condition 1 containing trehalose was measured and compared with the accuracy of the measurements (i.e., the measurements of glycated albumin, total albumin, and the glycated albumin ratio) when using the stabilizer in condition 2 containing sucrose.

[0195] Samples used for comparison of measurements and precision evaluation were prepared using low- and high-concentration standard solutions (Lucica GA-L Control) from Asahi Kasei Pharma. The glycated albumin (g / L) and total albumin (g / L) values ​​of the prepared samples were confirmed before use using a reference reagent (Lucica GA-L, Asahi Kasei Pharma) and a reference device, an automated biochemistry analyzer (RX Imola, RANDOX). The final glycated albumin ratio (%) was calculated based on these two values. Furthermore, one lot of the i-SENS A1Care analyzer and one lot of A1Care GA cartridges containing each of the stabilizers in Condition 1 and Condition 2 were used to perform 33 replicate measurements of the standard solution samples at each concentration. Based on these measurements, the glycated albumin (GA g / L), total albumin (ALB g / L), glycated albumin ratio (GA%), and precision of each measurement were calculated.

[0196] Second, the linearity of each measurement value (i.e., glycated albumin measurement value, total albumin measurement value, and glycated albumin ratio measurement value) obtained using the stabilizer containing trehalose under condition 1 was evaluated. Similarly, the linearity of each measurement value (i.e., glycated albumin measurement value, total albumin measurement value, and glycated albumin ratio measurement value) obtained using the stabilizer containing sucrose under condition 2 was also evaluated.

[0197] Measurement samples for evaluating the linearity of total albumin were prepared using albumin powder (lyophilized human serum albumin powder, Sigma-Aldrich; albumin A3782). Specifically, a low-concentration sample (16.6 g / L) was prepared by dissolving 83 mg of albumin powder in 5 mL of deionized water (DIW), and a high-concentration sample (64.8 g / L) was prepared by dissolving 130 mg of albumin powder in 5 mL of DIW. A medium-concentration sample (40.4 g / L) was prepared by mixing the low- and high-concentration samples in a 1:1 ratio. A low-medium-concentration sample (28.4 g / L) was prepared by mixing the low- and medium-concentration samples in a 1:1 ratio, and a medium-high-concentration sample (52.6 g / L) was prepared by mixing the medium- and high-concentration samples in a 1:1 ratio, resulting in a total of five sample concentrations. The albumin (g / L) values ​​of the prepared samples were verified before use using a reference reagent (Lucica GA-L, Asahi Kasei Pharma) and a reference device, an automatic biochemistry analyzer (RX Imola, RANDOX).

[0198] Measurement samples for evaluating the linearity of glycated albumin were prepared by mixing low-concentration (5.96 g / L) and high-concentration (15.41 g / L) standard solutions (Lucica GA-L control, Asahi Kasei Pharma). Specifically, a medium-concentration sample (10.836 g / L) was prepared by mixing the low-concentration and high-concentration standard solutions in a 1:1 ratio. Furthermore, a low-medium-concentration sample (8.41 g / L) was prepared by mixing the low-concentration and medium-concentration samples in a 1:1 ratio, and a medium-high-concentration sample (15.41 g / L) was prepared by mixing the medium-concentration and high-concentration samples in a 1:1 ratio. The glycated albumin (g / L) values ​​of the prepared samples were verified before use using a reference reagent (Lucica GA-L, Asahi Kasei Pharma) and a reference device, an automated biochemistry analyzer (RX Imola, RANDOX).

[0199] Furthermore, using the i-SENS A1Care analyzer (in vitro diagnostic medical device approval number 21-1020), measurements of absorbance for the color development of the color development agent (DA-67) resulting from the glycated albumin-enzyme assay were analyzed at a wavelength of 660 nm, where the maximum peak of the color development agent (DA-67) was observed. Additionally, measurements of the change in absorbance from the total albumin-BCP assay were analyzed at a wavelength of 610 nm, where the maximum peaks of the total albumin and BCP reagents were observed.

[0200] Furthermore, for each of the stabilizers under condition 1 and condition 2, the RSQ value indicating the linearity of the measured values ​​depending on the sample concentration (i.e., the measured values ​​of glycated albumin, total albumin, and glycated albumin ratio) was calculated.

[0201] In addition, when all of the following evaluation criteria are met, the stabilizer of condition 1 containing trehalose and the stabilizer of condition 2 containing sucrose are evaluated as exhibiting equivalent performance as stabilizers for the quantitative analysis of glycated albumin. 1) The measured values ​​for the stabilizer in condition 1 containing trehalose and the stabilizer in condition 2 containing sucrose must satisfy a bias % within the range of -10% to 10%. 2) The precision (CV%) of the measurements for the stabilizer in condition 1 containing trehalose and the stabilizer in condition 2 containing sucrose must be within 5%. 3) The linearity (RSQ) of the measurements for the stabilizer in condition 1 containing trehalose and the stabilizer in condition 2 containing sucrose must be 0.98 or greater.

[0202] 2. Experimental Results FIG. 15 is a table showing measurement results and accuracy based on the type of disaccharide contained in the reaction reagent, the oxidase, and / or the stabilizer added to the degradation reagent according to an embodiment of the present application.

[0203] For the low-concentration (low) standard solution samples, the measured glycated albumin (GA) was 6.5 for the stabilizer containing trehalose (Condition 1), and 6.4 for the stabilizer containing sucrose (Condition 2), with a bias (%) of -1.5%. The measured total albumin (ALB) was 39.6 for the stabilizer containing trehalose (Condition 1), and 39.6 for the stabilizer containing sucrose (Condition 2), with a bias (%) of 0%. The measured glycated albumin ratio (GA (%)) for the stabilizer containing trehalose (Condition 1), and 17.0 for the stabilizer containing sucrose (Condition 2), with a bias (%) of -1.2%.

[0204] For the high-concentration (high) standard solution samples, the measured glycated albumin (GA) was 13.6 for the stabilizer containing trehalose (Condition 1), and 13.6 for the stabilizer containing sucrose (Condition 2), with a bias of 0%. The measured total albumin (ALB) was 38.8 for the stabilizer containing trehalose (Condition 1), and 39.1 for the stabilizer containing sucrose (Condition 2), with a bias of 0.8%. The measured glycated albumin ratio (GA (%)) was 33.7 for the stabilizer containing trehalose (Condition 1), and 33.5 for the stabilizer containing sucrose (Condition 2), with a bias of -0.6%.

[0205] Thus, it was confirmed that the measurements for the stabilizers under Condition 1 and Condition 2 met the evaluation criteria requiring that the bias (%) in all cases be within the range of -10% to 10%.

[0206] For the low-concentration (low) standard solution samples, when using stabilizer condition 1 containing trehalose, the precision (CV%) of glycated albumin (GA) measurements was 4.3, the precision (CV%) of total albumin (ALB) measurements was 2.7, and the precision (CV%) of the glycated albumin ratio (GA (%)) was 3.5. When using stabilizer condition 2 containing sucrose, the precision (CV%) of glycated albumin (GA) measurements was 4.2, the precision (CV%) of total albumin (ALB) measurements was 3.3, and the precision (CV%) of the glycated albumin ratio (GA (%)) was 2.8.

[0207] For the high-concentration (high) standard solution samples, when using stabilizer condition 1 containing trehalose, the precision (CV%) of glycated albumin (GA) measurements was 3.9, the precision (CV%) of total albumin (ALB) measurements was 4.3, and the precision (CV%) of the glycated albumin ratio (GA (%)) was 3.3. When using stabilizer condition 2 containing sucrose, the precision (CV%) of glycated albumin (GA) measurements was 4.1, the precision (CV%) of total albumin (ALB) measurements was 4.2, and the precision (CV%) of the glycated albumin ratio (GA (%)) was 2.9.

[0208] Thus, it was confirmed that the precision (CV) of the measurement values ​​for the stabilizers under condition 1 and condition 2 met the evaluation criterion of being within 5% in all cases.

[0209] FIG. 16 is a table and graph showing the results of evaluating linearity based on the type of disaccharide contained in the stabilizer added to the reaction reagent, oxidase, and / or degradation reagent according to an embodiment of the present application.

[0210] As a result of the linearity evaluation, the index (RSQ) representing the linearity of the glycated albumin (GA) measurement value when using the stabilizer containing trehalose under Condition 1 was measured to be 0.988, the index (RSQ) for the linearity of the total albumin (ALB) measurement value was measured to be 0.988, and the index (RSQ) for the linearity of the glycated albumin ratio (GA%) measurement value was measured to be 0.980.

[0211] Furthermore, when using the stabilizer of condition 2 containing sucrose, the index for linearity (RSQ) of glycated albumin (GA) measurements was measured as 0.986, the index for linearity (RSQ) of total albumin (ALB) measurements was measured as 0.995, and the index for linearity (RSQ) of glycated albumin ratio (GA%) measurements was measured as 0.991.

[0212] Thus, the linearity of measurements (RSQ) for the stabilizers in both Condition 1 and Condition 2 was confirmed to meet the criteria of at least 0.98 in all cases.

[0213] Experimental Example 5 confirmed that both the stabilizer containing trehalose under Condition 1 and the stabilizer containing sucrose under Condition 2 can be used as stabilizers for the quantitative analysis of glycated albumin. Furthermore, Experimental Example 5 additionally confirmed that disaccharides exhibiting similar characteristics to trehalose and sucrose can exhibit sufficient performance as stabilizers for the quantitative analysis of glycated albumin.

[0214] FIG. 17 is a diagram showing identification information included in a glycated albumin quantitative analysis kit according to an embodiment of the present application.

[0215] The GA quantitative analysis kit (10) according to one embodiment of the present application may include identification information for recognizing a biological sample. For example, the GA quantitative analysis kit (10) may include identification information (e.g., information in the form of a barcode) on one surface of the main cartridge (200) to identify the type of biological sample.

[0216] The quantitative analysis device described below can acquire identification information for recognizing the type of biological sample to be analyzed by the GA quantitative analysis kit (10). For example, the quantitative analysis device can determine that the biological sample for analysis is a GA based on the barcode. In this case, the quantitative analysis device can be configured to perform quantitative analysis of the biological sample by executing a pre-stored analysis protocol associated with the recognized type based on the recognized type of biological sample. For example, the quantitative analysis device can be implemented to execute a pre-stored GA analysis protocol based on the recognition that the type of biological sample to be analyzed is a GA, thereby performing quantitative analysis of the GA.

[0217] 17 illustrates, as an example, the identification information in the form of a barcode, however, this is merely an example, and any suitable form of identification information may be provided in any suitable location on the GA quantitative analysis kit 10.

[0218] A quantitative analysis device (or quantitative analysis apparatus) according to embodiments of the present disclosure may be configured to perform quantitative analysis of a biological sample (eg, 1,5-AG, glycated albumin (GA), CRP, etc.).

[0219] Additionally, the quantitative analysis device may include a communication module (which may also be referred to as a transceiver), a memory, and / or a processor.

[0220] The communication module of the quantitative analysis device may communicate with any external device or server, for example, the quantitative analysis device may transmit the quantitative analysis results to an external device or server via the communication module.

[0221] The quantitative analysis device may access a network through a communication module and transmit or receive various types of data. The communication module may primarily include a wired type communication module and a wireless type communication module. Because the wired type communication module and the wireless type communication module have their own advantages and disadvantages, in some cases, both the wired type communication module and the wireless type communication module may be provided as a quantitative analysis device. Here, for the wireless type communication module, a wireless local area network (WLAN) type communication method such as Wi-Fi (registered trademark) may be primarily used. Alternatively, for the wireless type communication module, cellular communication such as Long Term Evolution (LTE) or 5G communication method may be used. However, the wireless communication protocol is not limited to the examples described above, and any suitable wireless type communication method may be used. For the wired type communication module, local area network (LAN) or universal serial bus (USB) communication is typical, but other methods may also be used.

[0222] Various types of information can be stored in the memory of the quantitative analysis device. Various types of data can be stored in the memory temporarily or semi-permanently. Examples of memory can include a hard disk drive (HDD), a solid-state drive (SSD), flash memory, read-only memory (ROM), random access memory (RAM), etc. The memory can be built into the quantitative analysis device or can be provided in a removable form. Various types of data necessary for the operation of the quantitative analysis device can be stored in the memory, including an operating program (OS) for driving the quantitative analysis device or programs for operating each component of the quantitative analysis device.

[0223] The processor may control the overall operation of the quantitative analysis device. For example, the quantitative analysis device may control its overall operation, including actions such as recognizing identification information of a biological sample, executing a corresponding analysis protocol based on the recognized identification information, and / or performing quantitative analysis of the biological sample according to the analysis protocol. Specifically, the processor may load and execute a program for the overall operation of the quantitative analysis device from a memory. The processor may be implemented as an application processor (AP), a central processing unit (CPU), a microcontroller unit (MCU), or a similar device, depending on hardware, software, or a combination thereof. In this case, the processor may be provided in the form of electronic circuits that process electrical signals and perform control functions in hardware, or in the form of programs or codes that drive hardware circuits in software.

[0224] According to embodiments of the present application, the glycated albumin quantitative analysis kit, the glycated albumin quantitative analysis method, and / or the device for carrying out the method can provide the effect of implementing both the BCP reaction for quantifying the total amount of albumin and the enzymatic reaction for quantifying the amount of glycated albumin in a single kit through an optimal combination of reaction buffer and surfactant.

[0225] According to an embodiment of the present application, by implementing the BCP reaction and the enzyme reaction on a single kit, the glycated albumin quantitative analysis kit can be made smaller, the analysis time for glycated albumin can be reduced, and user convenience can be increased.

[0226] According to an embodiment of the present application, by immobilizing a composition containing a degradation reagent on a suitable reagent immobilization site, the loss of BCP binding sites present on albumin can be prevented, thereby improving the accuracy and quantification of total albumin using BCP analytical methods.

[0227] The effects of the present invention are not limited to the above-mentioned effects, and other effects not described can be clearly understood by those skilled in the art from the above detailed description.

[0228] The features, structures, and effects described in the exemplary embodiments described above may be included in at least one exemplary embodiment of the present invention, but are not necessarily limited to only one exemplary embodiment. Furthermore, the features, structures, and effects described in each embodiment may be combined or modified by those skilled in the art to which the embodiment belongs, and implemented in other embodiments. Therefore, the contents related to such combinations and modifications should be interpreted as being included in the scope of the present invention.

[0229] Furthermore, although the present invention has been particularly described with reference to the embodiments, the embodiments are merely exemplary embodiments of the present invention and are not intended to limit the present invention thereto. It will be understood by those skilled in the art that other forms of modification and application can be made without departing from the spirit and scope of the present invention. That is, each element specifically shown in the embodiments can be changed and embodied. In addition, differences regarding these modifications and applications should be understood to be within the scope of the present invention, as defined in the appended claims.

Claims

1. A glycated albumin (GA) quantitative analysis kit comprising: a first composition comprising a reaction buffer that induces binding with albumin present in a blood sample; a second composition containing a degradation reagent for a degradation reaction that decomposes glycated albumin (GA) contained in the blood sample into glycated amino acids; a third composition comprising a reaction reagent for an enzymatic reaction of the glycated amino acids present in the blood sample; and Hydrogen peroxide (H ) produced from the enzymatic reaction of the glycated amino acid ２ O ２ a fourth composition comprising a color former that is oxidized by Equipped with wherein the GA quantitative analysis kit includes at least one reagent immobilizing portion, the second composition to the fourth composition are each independently immobilized on the reagent immobilizing portion; GA quantitative analysis kit.

2. 2. The GA quantitative analysis kit of claim 1, wherein the first composition is selected from the group consisting of bromocresol purple (BCP) and bromocresol green (BCG).

3. 3. The GA quantitative analysis kit according to claim 2, wherein the reaction buffer is selected from the group consisting of HEPES, PB, Tris-HCl, Tris-Base, and PBS.

4. 3. The GA quantitative analysis kit according to claim 2, wherein the reaction buffer is selected from the group consisting of Tris-HCl and Tris-Base, and the pH of the first composition is in the range of 4.1 to 10.

0.

5. the reaction buffer is Tris-HCl; The GA quantitative analysis kit according to claim 2 , wherein the decomposition reaction of glycated albumin and the enzymatic reaction of glycated amino acids are carried out under weakly alkaline pH conditions.

6. 4. The GA quantitative analysis kit according to claim 3, wherein the first composition further comprises a surfactant selected from the group consisting of CHAPSO, Triton X-100, and Tween 20.

7. 4. The GA quantitative analysis kit according to claim 3, wherein the first composition further comprises Triton X-100 surfactant at a concentration greater than 0% and up to 0.1%.

8. the reaction buffer comprises Tris-HCl, and the first composition further comprises Triton X-100 surfactant at a concentration greater than 0% and up to 0.1%; The decomposition reaction of glycated albumin and the enzymatic reaction of glycated amino acids are carried out under weakly alkaline pH conditions. The GA quantitative analysis kit according to claim 2.

9. The GA quantitative analysis kit according to claim 1, wherein the degradation reagent is a proteinase.

10. The GA quantitative analysis kit according to claim 1 , wherein the reaction reagent comprises ketoamine oxidase (KAO) and peroxidase (POD).

11. 2. The GA quantitative analysis kit according to claim 1, wherein the color developer is DA-67.

12. The GA quantitative analysis kit further comprises a fifth composition comprising an oxidase for preventing interference from ascorbic acid present in the blood sample; The oxidase includes ascorbate oxidase (ASOx). The GA quantitative analysis kit according to claim 1.

13. the second composition further comprises a stabilizer for immobilizing the decomposition reagent in a solid form on the at least one reagent immobilizing portion; The stabilizers include disaccharides, DEAE dextran, and NPS. The GA quantitative analysis kit according to claim 1.

14. the third composition further comprises a stabilizer for immobilizing the reaction reagent in a solid form on the at least one reagent immobilizing portion; The stabilizers include disaccharides, DEAE dextran, and NPS. The GA quantitative analysis kit according to claim 1.

15. The GA quantitative analysis kit of claim 1, wherein the fourth composition further comprises a stabilizer for immobilizing the color developer in a solid form on the at least one reagent immobilization portion, and the stabilizer comprises a disaccharide.

16. the fifth composition further comprises a stabilizer for immobilizing the ascorbic acid oxidase in a solid form on the at least one reagent-immobilizing portion; The stabilizers include disaccharides, DEAE dextran, and NPS. The GA quantitative analysis kit according to claim 12.

17. the third composition is immobilized in a solid state on a first reagent immobilizing portion disposed in a first region of the GA quantitative analysis kit; The GA quantitative analysis kit of claim 12, wherein the second composition is immobilized in a solid state on a second reagent immobilization portion disposed in a second region of the GA quantitative analysis kit, separated from the first region.

18. the GA quantitative analysis kit is configured so that the reaction buffer solution is mixed with the reaction reagent immobilized on the first reagent immobilizing portion before being mixed with the degradation reagent immobilized on the second reagent immobilizing portion; the reaction reagent performs the enzymatic reaction of the glycated amino acid from the time when the glycated amino acid is produced by the decomposition reaction caused by the decomposition reagent; The GA quantitative analysis kit according to claim 17.

19. The GA quantitative analysis kit comprises a main body including an upper plate and a lower plate arranged in an opposing arrangement, the third composition is immobilized on the first reagent immobilizing section disposed in the first region of the upper plate, the fifth composition is immobilized on a third reagent immobilizing portion disposed in the first region of the lower plate, the first reagent fixing portion and the third reagent fixing portion are configured to face each other; The GA quantitative analysis kit according to claim 17.

20. the second composition is immobilized on the second reagent immobilizing portion disposed in the second region of the upper plate, and the fourth composition is immobilized on the fourth reagent immobilizing portion disposed in the second region of the lower plate, the second reagent fixing portion and the fourth reagent fixing portion are configured to face each other. The GA quantitative analysis kit according to claim 19.

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