Kit for quantitative analysis of glycated albumin

Abstract

A kit for quantitative analysis of glycated albumin (GA) according to an embodiment of the present application comprises: a first composition comprising a reaction buffer for inducing binding to albumin present in a blood sample; a second composition containing a decomposition reagent for a decomposition reaction to decompose glycated albumin (GA) contained in the blood sample into glycated amino acids; a third composition comprising a reaction reagent for the enzymatic reaction of glycated amino acids present in the blood sample; and a fourth composition containing a color developing agent oxidized by hydrogen peroxide (H2O2) generated by an enzymatic reaction of the saccharified amino acid. The glycated albumin quantitative analysis kit of the present application can provide an effect of performing both a BCP reaction for quantifying the total amount of albumin and an enzymatic reaction for quantifying the amount of glycated albumin on a single kit by an optimal combination of a reaction buffer and a surfactant.

Description

[0001] [CROSS-REFERENCE TO RELATED APPLICATIONS]

[0002] This application claims priority to and the benefit of Korean Patent Application No. 10-2024-0029436, filed on February 29, 2024, the disclosure of which is incorporated herein by reference in its entirety. Technical Field

[0003] The present application relates to quantitative analysis of biological samples, and more particularly to a kit for quantitative analysis of glycated albumin (GA), a GA quantitative analysis method, and / or a quantitative analysis device for performing the method. Background Art

[0004] In the diagnosis of diabetes, in addition to blood sugar measurement, the demand for testing glycated albumin (hereinafter referred to as GA) is also increasing. Glycated albumin is a glycated substance that forms a ketoamine structure at the lysine site of albumin through a non-enzymatic oxidation reaction. The concentration of glycated albumin is related to blood sugar levels and will increase or decrease accordingly. Compared with HbA1c, which is widely used as an indicator for the diagnosis of diabetes, glycated albumin has the advantage of sensitively reflecting changes in blood sugar levels. Therefore, the development and research of technologies for quantitative analysis of glycated albumin to diagnose diabetes are attracting significant attention.

[0005] The quantitative analysis of glycated albumin is quantified as the ratio of glycated albumin to total albumin in the blood (GA%). In conventional techniques for the 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 separate and measured in separate spaces. This is necessary because the reaction environments for measuring the amount of total albumin and the reaction for measuring the amount of glycated albumin are different. According to conventional techniques, separating the two reactions brings limitations to miniaturization, and therefore the quantitative analysis of glycated albumin must be mainly carried out on large-scale biochemical equipment. In addition, according to conventional techniques, separating the two reactions causes the quantitative analysis of glycated albumin to take a relatively long time, thereby causing inconvenience to the user.

[0006] Therefore, there is a need to develop and study a technology for quantitative analysis of glycated albumin that can be miniaturized and improve user convenience. Summary of the Invention

[0007] 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 an apparatus for carrying out the method, wherein the apparatus performs a reaction for measuring the amount of total albumin and a reaction for measuring the amount of glycated albumin on a single kit, thereby enabling miniaturization.

[0008] 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 an apparatus for implementing the method, wherein the apparatus performs a reaction for measuring the amount of total albumin and a reaction for measuring the amount of glycated albumin on a single kit, thereby reducing analysis time and improving user convenience.

[0009] The objects of the present invention are not limited to the above objects, and those skilled in the art can clearly understand other objects not described based on this specification and the accompanying drawings.

[0010] According to an embodiment of the present application, a kit for quantitative analysis of glycated albumin (GA) may include: 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 to decompose 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 oxidized by hydrogen peroxide (H2O2) generated by the enzymatic reaction of the glycated amino acids, wherein the GA quantitative analysis kit may include at least one reagent fixing portion, and the second to fourth compositions may each be independently fixed to the reagent fixing portion.

[0011] The solutions of the present invention are not limited to the above solutions, and those skilled in the art can clearly understand other solutions not described based on this specification and the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The above and other objects, features and advantages of the present invention will become more apparent to those skilled in the art by describing in detail exemplary embodiments of the present invention with reference to the accompanying drawings, in which:

[0013] Figure 1 Schematic diagram of a glycated albumin quantitative analysis kit according to an embodiment of the present application.

[0014] Figure 2 Schematic diagram of a sample collector of a glycated albumin quantitative analysis kit according to an embodiment of the present application.

[0015] Figure 3 Schematic diagram of the main box of the glycated albumin quantitative analysis kit according to an embodiment of the present application.

[0016] Figure 4 Schematic diagram of the solution pool of the glycated albumin quantitative analysis kit according to an embodiment of the present application.

[0017] Figure 5 is a diagram illustrating a situation in which a composition stored in a solution reservoir flows into a mixing area of ​​the main cartridge when a sample collector is inserted into the main cartridge according to an embodiment of the present application.

[0018] Figure 6 An example of an analysis method for quantifying GA according to an embodiment of the present application is shown.

[0019] Figure 7 and Figure 8 3 is a diagram showing the specific structure of a kit for quantitative analysis of glycated albumin according to an embodiment of the present application.

[0020] Figure 9 FIG. 4 is a diagram illustrating an analysis process for quantifying glycated albumin in a blood sample according to an embodiment of the present application.

[0021] Figure 10 1 and 2 are tables and graphs showing the results of evaluating the albumin measurement performance based on different reaction buffers according to the examples of the present application.

[0022] Figure 11 1 and 2 are tables and graphs showing the results of evaluating the measurement performance of glycated albumin based on different reaction buffers according to the examples of the present application.

[0023] Figure 12 This is a table showing the results of evaluating the measurement performance of albumin and / or glycated albumin based on the surfactant added to the reaction buffer according to Examples of the present application.

[0024] Figure 13 This is a table showing the results of evaluating the accelerated stability of reaction reagents, oxidases, and / or decomposition reagents based on stabilizers added thereto according to Examples of the present application.

[0025] Figure 14 This is a graph showing the results of evaluating the accelerated stability of a reaction reagent, an oxidase, and / or a decomposition reagent based on a stabilizer added thereto according to an example of the present application.

[0026] Figure 15 This is a table showing measured values ​​and accuracy based on the type of disaccharide contained in the stabilizer added to the reaction reagent, oxidase, and / or decomposition reagent according to Examples of the present application.

[0027] Figure 16These are a table and a 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 decomposition reagent according to Examples of the present application.

[0028] Figure 17 1 is a diagram showing identification information included in a glycated albumin quantitative analysis kit according to an embodiment of the present application. DETAILED DESCRIPTION

[0029] The above-mentioned purpose, features and advantages of the present invention will be clearly understood by reading the following detailed description in conjunction with the accompanying drawings. However, although the present invention can be modified in various ways and take various alternative forms, its specific embodiments are shown in the accompanying drawings and are described in detail below as examples.

[0030] In the entire specification, the same reference numerals refer to the same elements in principle. In addition, the same reference numerals will be used to describe elements with the same functions within the scope of the same concept shown in the drawings of each embodiment, and they will not be described in detail.

[0031] When it is determined that the detailed description of related known functions or configurations may unnecessarily obscure the main points of the present invention, they will not be described in detail.In addition, the ordinal numbers (eg, first, second, etc.) used in the description of the specification are only used to distinguish each element.

[0032] In addition, the terms “module,” “unit,” “section,” or “part” of the elements used herein are designated or incorporated for convenience of description, and the terms themselves do not have precise meanings or roles.

[0033] As used herein, the singular forms "a" and "an" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0034] It will also be understood that the terms “comprise,” “comprising,” and “include,” “including” as used herein specify the presence of stated features or elements, but do not preclude the presence or addition of one or more other features or elements.

[0035] For the convenience of explanation, the size of the elements in the drawings may be exaggerated. In other words, since the size and thickness of the elements in the drawings are arbitrarily shown for the convenience of explanation, the following embodiments are not limited thereto.

[0036] When a certain embodiment can be implemented in different ways, the specific process order can be performed in a different order than described. For example, two processes described in succession can be performed substantially simultaneously, or in a reverse order to the described order.

[0037] In the following embodiments, when a first element is referred to as being “connected” to a second element, this includes not only a case where the two elements are “directly connected” but also a case where the two elements are “indirectly connected” using a third element interposed between the two elements. For example, in this specification, when a first element is referred to as being “electrically connected” to a second element, this includes not only a case where the two elements are “directly electrically connected” but also a case where the two elements are “indirectly electrically connected” using a third element interposed between the two elements.

[0038] According to an embodiment of the present application, a kit for quantitative analysis of glycated albumin (GA) may include: 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 to decompose 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 oxidized by hydrogen peroxide (H2O2) generated by the enzymatic reaction of the glycated amino acids, wherein the GA quantitative analysis kit may include at least one reagent fixing portion, and the second to fourth compositions may each be independently fixed to the reagent fixing portion.

[0039] According to an embodiment of the present application, the first composition can be selected from the group consisting of bromocresol purple (BCP) and bromocresol green (BCG).

[0040] According to an embodiment of the present application, the reaction buffer can be selected from the group consisting of 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES), phosphate buffer (PB), Tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl), Tris(hydroxymethyl)aminomethane (Tris-Base) and phosphate-buffered saline (PBS).

[0041] 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.

[0042] 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 performed under weakly alkaline pH conditions.

[0043] According to an embodiment of the present application, the first composition may further include a surfactant selected from the group consisting of 3-[(3-cholamidopropyl]dimethylammonio]-2-hydroxy-1-propanesulfonate (CHAPSO), Triton X-100, and Tween 20.

[0044] According to an embodiment of the present application, the first composition may further comprise a Triton X-100 surfactant at a concentration greater than 0% and at most 0.1%.

[0045] According to an embodiment of the present application, the reaction buffer may contain Tris-HCl, the first composition may further contain a Triton X-100 surfactant at a concentration greater than 0% and at most 0.1%, and the decomposition reaction of glycated albumin and the enzymatic reaction of glycated amino acids may be carried out under weakly alkaline pH conditions.

[0046] According to an embodiment of the present application, the decomposition agent may be proteinase.

[0047] According to an embodiment of the present application, the reaction reagents may include ketoamine oxidase (KAO) and peroxidase (POD).

[0048] According to an embodiment of the present application, the developer may be DA-67.

[0049] 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 in a blood sample, and the oxidase may include ascorbate oxidase (ASOx).

[0050] According to an embodiment of the present application, the second composition may further include a stabilizer for immobilizing the decomposition reagent in a solid form on the at least one reagent fixing portion, and the stabilizer may include disaccharide, diethylaminoethyl-dextran (DEAE-Dextran) and neo protein preservative (Neo Protein Saver, NPS).

[0051] 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 the at least one reagent immobilization portion, and the stabilizer may include disaccharide, DEAE-dextran, and NPS.

[0052] According to an embodiment of the present application, the fourth composition may further include a stabilizer for immobilizing the color developing reagent in a solid form on the at least one reagent immobilizing portion, and the stabilizer may include a disaccharide.

[0053] According to an embodiment of the present application, the fifth composition may further include a stabilizer for immobilizing the ascorbate oxidase in a solid form on the at least one reagent immobilization portion, and the stabilizer may include disaccharide, DEAE-dextran, and NPS.

[0054] According to an embodiment of the present application, the third composition can be immobilized in a solid state on a first reagent fixing portion located in a first zone of the GA quantitative analysis kit, and the second composition can be immobilized in a solid state on a second reagent fixing portion located in a second zone separated from the first zone of the GA quantitative analysis kit.

[0055] According to an embodiment of the present application, the GA quantitative analysis kit can 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 can be characterized in that an enzymatic reaction with the glycated amino acid is carried out from the moment when the glycated amino acid is produced by the decomposition reaction caused by the decomposition reagent.

[0056] According to an embodiment of the present application, the GA quantitative analysis kit may include a main body, which includes an upper plate and a lower plate configured in a facing arrangement, the third composition can be fixed to the first reagent fixing portion located in the first area of ​​the upper plate, and the fifth composition can be fixed to the third reagent fixing portion located in the first area of ​​the lower plate, and the first reagent fixing portion and the third reagent fixing portion are configured to face each other.

[0057] According to an embodiment of the present application, the second composition may be fixed to the second reagent fixing portion located in the second area of ​​the upper plate, and the fourth composition may be fixed to the fourth reagent fixing portion located in the second area of ​​the lower plate, and the second reagent fixing portion and the fourth reagent fixing portion are configured to face each other.

[0058] In the following we will refer 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 are described in more detail.

[0059] Figure 1Schematic diagram of a glycated albumin quantitative analysis kit according to an embodiment of the present application.

[0060] 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 may be inserted and received.

[0061] Figure 2 Schematic diagram of a sample collector 100 of a glycated albumin quantitative analysis kit according to an embodiment of the present application.

[0062] The sample collector 100 according to an embodiment of the present application can 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 sample inlet 101, and may collect a biological sample (e.g., a blood sample) to be analyzed via a capillary disposed in the sample inlet 101, thereby injecting the collected biological sample into the main cartridge 200.

[0063] In addition, the sample collector 100 may include a protrusion 102 designed to contact the solution pool 301 inside the main box 200 and push the solution pool 301 inward when the sample collector 100 is inserted into the receiving portion 201 of the main box 200, which will be explained later. Specifically, the protrusion 102 may be positioned on the sample collector 100 to contact the solution pool 301 when the sample collector 100 is inserted into the receiving portion 201 of the main box 200. Therefore, when the sample collector 100 is inserted into the receiving portion 201 of the main box 200, the solution pool 301 may move inward toward the interior of the main box 200, thereby causing the cover tape 302 of the solution pool 301 to be removed or broken. This will be referred to Figure 5 Elaborate in more detail.

[0064] 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 Figure 2. A locking clip 104 may be provided on one side of the sample collector 100 to secure the sample collector 100 to the main box 200 when the sample collector 100 is inserted into the main box 200. The locking clip 104 is designed to match the size and shape of the locking groove 211 provided in the receiving portion 201 of the main box 200. The locking clip 104 of the sample collector 100 and the locking groove 211 of the main box 200 can engage with each other. Therefore, the inserted sample collector 100 can be fixed to the main box 200, and the sample collector 100 can be prevented from moving or detaching even if the GA quantitative analysis kit is rotated during analysis.

[0065] Figure 3 Schematic diagram of the main box 200 of the glycated albumin quantitative analysis kit according to an embodiment of the present application.

[0066] The main box 200 according to an embodiment of the present application may include a receiving portion 201 into which the sample collector 100 may be inserted. In addition, the receiving portion 201 may include a locking groove 211 configured to engage with the locking clip 104 of the sample collector 100 as described above to retain the sample collector 100.

[0067] The main cartridge 200 also includes a moving frame 203 configured to secure and move a solution reservoir 301 disposed within the main cartridge. Specifically, the moving frame 203 is configured to secure the solution reservoir 301 prior to insertion of the sample collector 100. When the sample collector 100 is inserted, the solution reservoir 301, pushed by the protrusion 102 of the sample collector 100, moves along a movement path within the moving frame 203.

[0068] The main box 200 may further include a cover tape rupture portion 202 configured to remove or rupture the cover tape 302 of the solution pool 301 when the solution pool 301 moves along the moving frame 203. Figure 5 Elaborate in more detail.

[0069] The main box 200 may include a mixing portion 204 (or mixing area) in which the biological sample (e.g., blood sample) discharged from the sample collector 100 through the receiving portion 201 is mixed with the composition (hereinafter referred to as the first composition) discharged from the solution reservoir 301.

[0070] The main box 200 may also include at least one reagent fixing part 205 (referred to as a sample fixing part), into which a chemical reagent is introduced. The chemical reagent can react with the first composition and the biological sample mixed in the mixing part 204 to induce an enzymatic reaction and / or an antigen-antibody reaction.

[0071] The main cartridge 200 may further include a flow path 206 through which the first composition and the biological sample mixed in the mixing section 204 can move. Specifically, the analysis sample can travel between the mixing section 204, the reagent fixing section 205, and / or the measurement unit 207 that optically measures the analysis sample via the flow path 206. The structure of the flow path 206 is not limited, provided that the flow path 206 is designed to enable the analysis sample to move by gravity when the main cartridge 200 is tilted.

[0072] The main cartridge 200 according to an embodiment of the present application may include a measurement unit 207 for measuring a reaction result performed at the reagent fixing portion 205. The GA quantitative analysis apparatus according to an embodiment of the present application may quantify an analysis sample (e.g., GA) through optical analysis (e.g., ultraviolet / visible (UV / VIS)) via the measurement unit 207.

[0073] The main box 200 may include a waste liquid treatment unit 208 for collecting waste liquid analyzed by the measurement unit 207. The waste liquid treatment unit 208 enables the collection and separate disposal of waste liquid (which is a type of medical waste). Collection of waste liquid by the waste liquid treatment unit 208 can be achieved by absorbing the waste liquid into highly absorbent cotton, an absorption filter, or a polymer-based absorbent material placed in the waste liquid treatment unit 208.

[0074] The main box 200 may further include an air vent 209 to facilitate smooth movement of the waste liquid and absorption into the absorbent material. Through the air vent 209, the transfer of the waste liquid to the waste liquid treatment unit 208 and its subsequent collection can be more efficiently implemented.

[0075] The main box 200 may also include a handle 210 to facilitate transportation and use of the main box 200. The structure of the handle 210 is not limited to Figure 3 The form shown in .

[0076] In the following we will refer to Figure 4 and Figure 5 A more detailed description is provided of a case in which the first composition of the solution reservoir 301 flows into the mixing portion 204 of the main cartridge 200 when the sample collector 100 is inserted into the receiving portion 201 of the main cartridge 200 . Figure 4 Schematic diagram of the solution pool 301 of the GA quantitative analysis kit according to an embodiment of the present application. Figure 5 3 is a diagram illustrating a situation in which a composition stored in the solution reservoir 301 flows into the mixing area 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.

[0077] The master cartridge 200 according to an embodiment of the present application may include a solution reservoir 301 that stores a first composition for reacting with a biological sample from the sample collector 100. The solution reservoir 301 includes an opening at one end, which is sealed with a cover tape 302 to prevent leakage of the first composition stored therein. The opening sealed with the cover tape 302 of the solution reservoir 301 is positioned to face the cover tape rupture portion 202 of the master cartridge 200.

[0078] Before the sample collector 100 is inserted into the receiving portion 201 of the main cartridge 200, the opening of the solution reservoir 301 sealed with the cover tape 302 can be positioned so as to be spaced apart from the cover tape rupture portion 202. Before the sample collector 100 is inserted into the receiving portion 201 of the main cartridge 200, the opening of the solution reservoir 301 sealed with the cover tape 302 can be positioned so as to be spaced apart from the cover tape rupture portion 202. When the sample collector 100 is inserted into the receiving portion 201 of the main cartridge 200, the protrusion 102 of the sample collector 100 applies pressure to the opposite end of the solution reservoir 301 spaced apart from the opening sealed with the cover tape 302. This applied pressure causes the solution reservoir 301 to move along the movement path of the moving frame 203.

[0079] Therefore, when the cover tape 302 of the solution reservoir 301 comes into contact with the cover tape rupture portion 202, the cover tape 302 of the solution reservoir 301 is removed or ruptured. Then, the first composition stored in the solution reservoir 301 is transferred to the mixing portion 204 of the main cartridge 200 through the flow path or hollow structure formed in the cover tape rupture portion 202. In the mixing portion 204, the reaction buffer is mixed with the biological sample (e.g., blood sample) supplied from the sample collector 100.

[0080] 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 enables the biological sample contained in the capillary sample inlet 101 to move through the sample inlet 101 into the mixing section 204 of the main cartridge 200. Therefore, in the mixing section 204 of the main cartridge 200, the first composition stored in the solution reservoir 301 and the biological sample collected by the sample collector 100 can be mixed.

[0081] exist Figure 4 and Figure 5 , the shape of solution reservoir 301 is shown as a specific example. However, this is merely an example, and the shape of solution reservoir 301 is not limited as long as solution reservoir 301 can provide a structure suitable for storing the first composition. Similarly, the material of cover tape 302 is not particularly limited as long as cover tape 302 prevents leakage of the first composition while enabling easy removal or rupture.

[0082] In addition, for illustrative purposes, Figure 5 A specific shape of the cover tape rupture portion 202 is shown. However, this is merely an example, and the shape of the cover tape rupture portion 202 is not limited as long as the cover tape rupture portion 202 facilitates removal or rupturing of the cover tape. Suitable structures include, but are not limited to, a needle-shaped configuration or an edge-shaped configuration.

[0083] The GA quantitative analysis kit 10 according to an embodiment of the present application can be used to quantitatively analyze GA present in blood (e.g., plasma, serum). According to one embodiment, the GA quantitative analysis kit 10 can be configured to quantitatively analyze GA using a BCP method and / or an enzymatic method. Figures 7 to 9 A more detailed description is provided of the case where GA is quantitatively analyzed using the GA quantitative analysis kit 10 according to an embodiment of the present application.

[0084] The analytical method for quantifying GA can be roughly divided into four steps, as follows Figure 6 As shown in . Figure 6 An example of an analysis method for quantifying GA according to an embodiment of the present application is shown.

[0085] The first step involves quantifying the total amount (or concentration) of albumin present in the 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. By measuring the absorbance of a sample containing the BCP complex, the total amount of albumin can be quantified.

[0086] The second step involves decomposing albumin (including glycated albumin) having a tertiary structure. Specifically, a decomposing enzyme (e.g., a protease or proteinase) can be used to decompose the tertiary structure of albumin into at least one glycated amino acid (e.g., fructosyl-lysine).

[0087] The third step involves generating hydrogen peroxide through an enzymatic reaction of glycated amino acids. According to one embodiment, glycated amino acids can be decomposed into amino acids and glucose by a reaction enzyme (eg, ketoamine oxidase (KAO)), during which hydrogen peroxide is generated.

[0088] The fourth step involves oxidizing a color developing agent (e.g., DA-67) using the generated hydrogen peroxide in the presence of a reactive enzyme (e.g., peroxidase (POD)). As the color developing agent undergoes oxidation, a color change occurs. By measuring absorbance at a specific wavelength corresponding to this color change, the amount of glycated albumin can be quantified.

[0089] However, the aforementioned analysis mechanism for quantifying GA is merely an example, and any suitable GA quantitative analysis method may be employed. The GA quantitative analysis kit according to the embodiments of the present application may be appropriately modified according to the GA quantitative analysis method.

[0090] Figure 7 and Figure 8 1 is a diagram showing a specific structure of a GA quantitative analysis kit 10 according to an embodiment of the present application.

[0091] The solution reservoir 301 of the GA quantitative analysis kit 10 according to an embodiment of the present application can store a composition (hereinafter referred to as a first composition) containing a reagent for inducing binding to albumin present in a blood sample. According to one embodiment, the first composition may include bromocresol purple (BCP) for forming a complex by binding to albumin present in a blood sample and a reaction buffer (B) for establishing a reaction environment. Bromocresol purple (BCP) is exemplified as a substance for forming a complex by binding to albumin, but any suitable substance having similar properties to BCP (including bromocresol green) can be used to replace bromocresol purple.

[0092] According to one embodiment, reaction buffer B can 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 can be implemented within the pH range of 4.1 to 10.0. More preferably, the pH of the first composition can be implemented within the pH range of 5.8 to 8.0.

[0093] According to one embodiment, reaction buffer B can 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 can be implemented within the pH range of 4.1 to 10.0. More preferably, the pH of the first composition can be implemented within the pH range of 5.8 to 8.0.

[0094] According to one embodiment, the reaction buffer B may be Tris-HCl. In this case, the pH of the first composition containing the reaction buffer B may be implemented within a pH range of 4.1 to 10.0. More preferably, the pH of the first composition may be implemented within a pH range of 4.1 to 8.0. Preferably, the pH of the first composition may be implemented within a pH range of 5.8 to 8.0.

[0095] More preferably, the analytical environment established by the reaction buffer B (e.g., the analytical environment for the decomposition reaction of glycated albumin and the enzymatic reaction of glycated amino acids described later) can be maintained in a weakly alkaline state (e.g., pH 8). Specifically, the decomposition reaction of glycated albumin and the enzymatic reaction of glycated amino acids described later can be carried out under weakly alkaline conditions (e.g., pH 8).

[0096] According to the glycated albumin quantitative analysis kit, the glycated albumin quantitative analysis method and / or the device for implementing the method based on the embodiments of the present application, the pH of the analysis environment is maintained at a weak alkaline level (which is the optimal reaction environment for the enzyme ketoamine oxidase (KAO)), which can provide an effect of improving the accuracy of glycated albumin quantification by enzymatic methods.

[0097] The first composition according to one embodiment may further comprise a surfactant to create an optimal analysis environment. More specifically, the first composition may further comprise a surfactant to prevent protein precipitation.

[0098] According to 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 greater than 0% and at most 0.1% in the first composition.

[0099] According to the glycated albumin quantitative analysis kit, glycated albumin quantitative analysis method, and / or apparatus for performing the method according to embodiments of the present application, the combination of an optimized reaction buffer and a surfactant allows both the BCP reaction for quantifying the total amount of albumin and the enzymatic reaction for quantifying the amount of glycated albumin to be performed in a single kit. Furthermore, this allows for miniaturization of the glycated albumin quantitative analysis kit. Furthermore, this reduces glycated albumin analysis time and improves user convenience.

[0100] According to an embodiment of the present application, the GA quantitative analysis kit 10 may include a composition (hereinafter referred to as a second composition) containing a decomposition reagent (R2) for performing a decomposition reaction to decompose glycated albumin (GA) contained in a blood sample into glycated amino acids. The decomposition reagent (R2) may be a protease. The decomposition reagent (R2) decomposes the tertiary protein structure of glycated albumin, thereby converting it into glycated amino acids in a form capable of participating in the enzymatic reaction described below.

[0101] According to an embodiment of the present application, the GA quantitative analysis kit 10 may include a composition (hereinafter referred to as a third composition) containing a reaction reagent (R1), and the reaction reagent (R1) is used for the enzymatic reaction of glycated amino acids contained in the 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 produced by the decomposition reagent (R2) and oxidizes the result of the enzymatic reaction. Specifically, the ketoamine oxidase (KAO) in the reaction reagent (R1) produces hydrogen peroxide through an enzymatic reaction with the glycated amino acids, and the peroxidase (POD) reacts with the hydrogen peroxide to oxidize the color developer, which will be explained later.

[0102] According to an embodiment of the present application, the GA quantitative analysis kit 10 may include a composition containing a color developing agent (hereinafter referred to as a fourth composition), wherein the color developing agent is oxidized by hydrogen peroxide generated by the enzymatic reaction of glycated amino acids. The color developing agent may be DA-67.

[0103] 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) for preventing interference from ascorbic acid present in a blood sample. The oxidase (E) may include ascorbic acid oxidase (ASOx).

[0104] 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 the at least one reagent fixing portion 205 .

[0105] According to one embodiment, the second composition may further include a stabilizer for immobilizing the decomposition agent (R2) 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 of the second composition may include trehalose, DEAE-dextran, and NPS.

[0106] According to one embodiment, the third composition may further include 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 of the third composition may include trehalose, DEAE-dextran, and NPS.

[0107] According to one embodiment, the fourth composition may further include a stabilizer for immobilizing the developer (D) in a solid state, wherein the stabilizer may be a disaccharide (eg, trehalose, sucrose). More preferably, the stabilizer of the fourth composition may be trehalose.

[0108] According to one embodiment, the fifth composition may further include 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 of the fifth composition may include trehalose, DEAE-dextran, and NPS.

[0109] As described above, the GA quantitative analysis kit 10 may include at least one reagent fixing portion 205. According to one embodiment, the second to fifth compositions may be fixed to the at least one reagent fixing portion 205 independently.

[0110] According to one embodiment, a third composition containing a reaction reagent (R1) may be fixed to a first reagent fixing portion 205-1 located in a first zone of the GA quantitative analysis kit 10. The first zone may be an area positioned on the mixing portion 204. In contrast, a second composition containing a decomposition reagent (R2) may be fixed to a second reagent fixing portion 205-2 located in a second zone of the GA quantitative analysis kit 10. The second zone is a separate area separated from the first zone. The first zone and the second zone may be connected via a flow path 206, and the GA quantitative analysis kit 10 may be configured such that the measuring unit 207 is positioned midway along the path through which the blood sample moves from the first zone to the second zone.

[0111] Reference Figure 8 The GA quantitative analysis kit 10 may include a body including an upper plate and a lower plate configured in a facing arrangement.

[0112] According to one embodiment, the third composition containing the reaction reagent (R1) can be dispensed on the first reagent fixing portion 205-1 provided in the first zone of the upper plate of the body and dried. In contrast, the fifth composition containing the oxidase (E) can be dispensed on the third reagent fixing portion 205-3 provided in the first zone of the lower plate of the body and dried. In addition, the first reagent fixing portion 205-1 provided on the upper plate of the body and the third reagent fixing portion 205-3 provided on the lower plate can be configured in a facing arrangement. Figure 8 , the third composition containing the reaction reagent (R1) is disposed on the upper plate of the body and the fifth composition containing the oxidase (E) is disposed on the lower plate. However, this is merely an example, and the compositions may be disposed on the plurality of reagent-fixing portions 205 in any suitable configuration, such as the third composition containing the reaction reagent (R1) being disposed on the lower plate and the fifth composition containing the oxidase (E) being disposed on the upper plate.

[0113] According to one embodiment, the second composition containing the decomposition reagent (R2) can be distributed on the second reagent fixing portion 205-2 provided in the second zone of the upper plate of the body and dried. In contrast, the fourth composition containing the developer (D) can be distributed on the fourth reagent fixing portion 205-4 provided in the second zone of the lower plate of the body and dried. In addition, the second reagent fixing portion 205-2 provided on the upper plate of the body and the fourth reagent fixing portion 205-4 provided on the lower plate can be configured in a facing arrangement. Figure 8 , the second composition containing the decomposition reagent (R2) is disposed on the upper plate of the body, while the fourth composition containing the developer (D) is disposed on the lower plate. However, this is merely an example, and the compositions may be disposed on the plurality of reagent holding portions 205 in any suitable configuration, such as the second composition containing the decomposition reagent (R2) being disposed on the lower plate of the body, and the fourth composition containing the developer (D) being disposed on the upper plate of the body.

[0114] According to an embodiment of the present application, a third composition containing a reaction reagent (R1) is fixed to a first reagent fixing portion 205-1 provided on the mixing portion 204, and a second composition containing a decomposition reagent (R2) is fixed to a second reagent fixing portion 205-2 separated from the mixing portion 204. This configuration ensures that the reaction buffer released from the solution reservoir 301 (and / or the blood sample released from the sample collector 100) mixes with the reaction reagent (R1) fixed to the first reagent fixing portion 205-1 before coming into contact with the decomposition reagent (R2) fixed to the second reagent fixing portion 205-2. However, the reaction reagent (R1) does not perform an enzymatic reaction from the moment the reaction buffer and the blood sample are mixed. In contrast, the reaction reagent (R1) is characterized in that an enzymatic reaction with the glycated amino acid is carried out from the moment when the glycated amino acid is produced by the decomposition reaction performed by the decomposition reagent (R2) (i.e., from the moment when the glycated albumin contained in the analysis sample is decomposed by the decomposition reagent (R2) of the second reagent fixing part (205-2) to produce the glycated amino acid. According to the glycated albumin quantitative analysis kit, the glycated albumin quantitative analysis method and / or the device for implementing the method described in the embodiments of the present application, the second composition containing the decomposition reagent (R2) is fixed to the second reagent fixing part 205-2 instead of the first reagent fixing part 205-1, thereby preventing the loss of the BCP binding site present in the albumin, thereby improving the accuracy of the total albumin quantification using the BCP analysis method.

[0115] Figure 9 FIG. 4 is a diagram illustrating an analysis process for quantifying glycated albumin in a blood sample according to an embodiment of the present application.

[0116] Reference Figure 9The quantitative analysis method of glycated albumin according to the embodiment of the present application can be implemented as follows.

[0117] 1) Prepare the master box 200.

[0118] 2) Position the sample collector 100 on the receiving portion 201 of the main box 200 .

[0119] 3) Apply pressure to the sample collector 100 in a direction toward the receiving portion 201 of the main cartridge 200 .

[0120] 4) When the protrusion 102 of the sample collector 100 applies pressure to the solution reservoir 301, the solution reservoir 301 moves in the direction of the cover tape rupture portion 202 by the movable frame 203 as described above. The cover tape rupture portion 202 contacts the cover tape 302 of the solution reservoir 301, causing the solution (first composition) stored in the solution reservoir 301 to flow into the mixing portion 204 of the main cartridge 200.

[0121] 5) When the first composition flows into the mixing portion 204 , the first composition comes into contact with the blood sample from the sample collector 100 , causing the blood sample (eg, plasma, serum) to also flow into the mixing portion 204 of the main cartridge 200 .

[0122] 6) In this stage, a first composition containing a reaction buffer (B) and bromocresol purple (BCP) is mixed with the blood sample in the mixing section 204. During this process, albumin (including both non-glycated and glycated albumin) in the blood sample binds to the BCP, forming a BCP complex.

[0123] Simultaneously, in mixing section 204, the reaction reagent (R1) and oxidase (E) immobilized on reagent immobilization sections 205-1 and 205-2 disposed therein 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 only occurs when the blood sample reaches reagent immobilization sections 205-3 and 205-4, where the decomposition reagent (R2) is immobilized, and the tertiary structure of albumin and / or glycated albumin is decomposed by the decomposition reagent (R2), producing glycated amino acids.

[0124] At the same time, according to an embodiment of the present application, the GA quantitative analysis kit 10 is rotated by a predetermined angle due to an external force (e.g., a rotational force applied by a quantitative analysis device 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.

[0125] 7) The blood sample is moved to the measurement unit 207 by an external force applied to the GA quantitative analysis kit 10. When the sample is positioned on the measurement unit 207, the absorbance corresponding to the total amount (or concentration) of albumin (total albumin) in the blood sample can be measured (hereinafter referred to as the first absorbance).

[0126] As described above, the GA quantitative analysis kit 10 may include a measurement unit 207 for quantifying GA contained in a blood sample. According to one embodiment, the GA quantitative analysis kit 10 may be configured such that the blood sample passes through the measurement unit 207 while traveling from the mixing unit 204 via the flow path 206 to the reagent fixing units 205-3 and 205-4, where the enzymatic reaction is performed. In this embodiment, both the absorbance before the enzymatic reaction corresponding to the concentration of total albumin and the absorbance after the enzymatic reaction corresponding to the concentration of glycated albumin can be easily measured. This allows for more convenient quantification of the glycated albumin ratio.

[0127] 8) External force applied to the GA quantitative analysis kit 10 causes the analyte sample containing the blood sample to move from the measurement unit 207 to the reagent fixing portion 205-3 and / or 205-4, where a decomposition reagent (R2, e.g., a proteolytic enzyme) is fixed. In the reagent fixing portion 205-3 and / or 205-4, the glycated albumin contained in the blood sample is decomposed into glycated amino acids by the decomposition reagent (R2).

[0128] Furthermore, an enzymatic reaction (eg, Figure 9 By this enzymatic reaction, hydrogen peroxide is generated, and when the generated hydrogen peroxide is oxidized by a reaction reagent (R1, such as POD), electrons are released, causing the color of the color developer (D) to change, thereby changing the absorbance.

[0129] 9) The sample after the enzymatic reaction is moved from the reagent fixing portion 205-3 and / or 205-4 to the measurement unit 207 by an external force applied to the GA quantitative analysis kit 10. When the sample is positioned on the measurement unit 207, a measurement value related to the absorbance (second absorbance) of the sample after the enzymatic reaction can be measured. The second absorbance can correspond to the amount (or concentration) of glycated albumin present in the blood sample.

[0130] Furthermore, based on the first absorbance correlated with the total albumin concentration measured before the enzymatic reaction at the measurement unit 207 and the second absorbance correlated with the glycated albumin concentration measured after the enzymatic reaction at the measurement unit 207, the ratio of glycated albumin to total albumin in the blood sample can be quantified.

[0131] 10) Due to the external force applied to the GA quantitative analysis kit 10 , wastes after the quantitative analysis are moved from the measurement unit 207 to the waste liquid treatment section 208 , where they are collected.

[0132] In the following we will refer to Figures 10 to 16 The present invention is described in detail by means of experimental examples. However, the following experimental examples are merely illustrative and should not be construed as limiting.

[0133] <Experimental Example 1: Evaluation of Albumin Measurement Performance Based on Reaction Buffer>

[0134] 1. Experimental Methods

[0135] To determine the optimal reaction buffer and reaction conditions (pH) for albumin analysis, five candidate buffers were selected (HEPES, phosphate buffer, Tris-HCl, Tris-Base, and phosphate-buffered saline). For each candidate buffer, five pH conditions (pH 4.1, pH 5.8, pH 6.5, pH 8.0, and pH 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.

[0136] Albumin powder (albumin from human serum, lyophilized powder, Sigma Aldrich; A3782) was used to prepare the test samples. 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 sample with the high-concentration sample at a 1:1 ratio.

[0137] In addition, a low-medium concentration sample (28.4 g / L) was prepared by mixing a low-concentration sample with a medium-concentration sample at a ratio of 1:1, and a medium-high concentration sample (52.6 g / L) was prepared by mixing a medium-concentration sample with a high-concentration sample at a ratio of 1:1, thereby obtaining 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 automatic biochemical analyzer (RX Imola, RANDOX). Each candidate buffer was applied to an albumin sample (Sigma-Aldrich) under five different pH conditions. In addition, the 25 candidate buffers were mixed with five test samples (specifically, 195 μL candidate buffer and 5 μL test sample) in a 96-microwell plate at a ratio of 20:1. After reacting for 1 minute, 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.

[0138] The linearity (RQS) and slope were calculated based on the absorbance correlation measurements of each candidate buffer at the five pH conditions.

[0139] The five candidate buffers and the five pH conditions are shown in Table 1 below. Table 1 shows the five candidate reaction buffers and pH conditions used to evaluate albumin measurement performance. For each sample, if the linearity (RQS) was greater than 0.98 and the slope was 0.003 or higher, the corresponding candidate buffer and pH condition were evaluated as providing sufficient measurement performance for albumin-BCP analysis.

[0140] Table 1: Candidate reaction buffers and pH conditions evaluated for albumin measurement performance.

[0141]

[0142] 2. Experimental Results

[0143] Figure 10 1 and 2 are tables and graphs showing the results of evaluating the albumin measurement performance based on different reaction buffers according to the examples of the present application.

[0144] For HEPES buffer:

[0145] 1) Under the conditions of pH 5.8, pH 6.5, and pH 8.0, the measured linearity (RQS) was greater than 0.98 and the measured slope was 0.003 or higher.

[0146] 2) At pH 4.1 and pH 10.0, the slope was measured to be less than 0.003.

[0147] For PB buffer:

[0148] 1) Under the conditions of pH 5.8, pH 6.5, and pH 8.0, the measured linearity (RQS) was greater than 0.98 and the measured slope was 0.003 or higher.

[0149] 2) At pH 4.1 and pH 10.0, the slope was measured to be less than 0.003.

[0150] For Tris-HCl buffer:

[0151] 1) Under the conditions of pH 5.8, pH 6.5, and pH 8.0, the measured linearity (RQS) was greater than 0.98 and the measured slope was 0.003 or higher.

[0152] 2) At pH 4.1, the linearity (RQS) was measured to be less than 0.98, and at pH 10.0, the slope was measured to be less than 0.003.

[0153] For Tris-Base buffer:

[0154] 1) Under the conditions of pH 4.1, pH 5.8, pH 6.5, and pH 8.0, the measured linearity (RQS) was greater than 0.98 and the measured slope was 0.003 or higher.

[0155] 2) At pH 10.0, the slope was measured to be less than 0.003.

[0156] For PBS buffer:

[0157] 1) At pH 5.8 and pH 6.5, the measured linearity (RQS) was greater than 0.98 and the measured slope was 0.003 or higher.

[0158] 2) At pH 4.1, pH 8.0, and pH 10.0, the slope was measured to be less than 0.003.

[0159] Experimental Example 1 demonstrates that the reaction buffer (B) according to the present invention can be selected from the group consisting of HEPES, PB, Tris-HCl, Tris-Base, and PBS, and that the pH of the first composition containing the reaction buffer (B) can be within a range of 4.1 to 10.0. Preferably, the pH of the first composition containing the reaction buffer (B) can be within a range of 5.8 to 8.0.

[0160] <Experimental Example 2: Evaluation of Glycated Albumin Measurement Performance Based on Reaction Buffer>

[0161] 1. Experimental Methods

[0162] To select the reaction buffer and reaction conditions (pH) that create the optimal environment 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. The measurement performance of the albumin-BCP assay was evaluated using these five candidate buffers at five different pH conditions (pH 4.1, pH 5.8, pH 6.5, pH 8.0, and pH 10.0), resulting in a total of 25 buffer-pH combinations. Specifically, each candidate buffer was supplemented with an appropriate concentration of bromocresol purple (BCP) reagent and the nonionic surfactant Triton X-100.

[0163] The test samples were prepared by mixing a low-concentration (4.51 g / L) standard solution of Lucica GA-L control (a standard solution from Asahi Kasei Pharmaceuticals used as a reference reagent by i-SENS) with a high-concentration (17.59 g / L) standard solution. Specifically, a medium-concentration sample (11.37 g / L) was prepared by mixing the low-concentration sample and the high-concentration sample of the standard solution at a ratio of 1:1, a medium-low concentration sample (8.01 g / L) was prepared by mixing the low-concentration sample and the medium-concentration sample at a ratio of 1:1, and a medium-high concentration sample (14.66 g / L) was prepared by mixing the medium-concentration sample and the high-concentration sample at a ratio of 1:1.

[0164] Before use, the glycated albumin (g / L) content of the prepared samples was verified using a reference reagent (Lucica GA-L, Asahi Kasei Pharmaceuticals) and a reference equipment (automatic biochemical analyzer (RX Imola, Landau)).

[0165] In addition, the absorbance measurement values ​​associated with the color development of the color developing agent (DA-67) for the glycated albumin-enzymatic assay were analyzed using an Al Care Analyzer from Essence (In Vitro Diagnostic Approval No. 21-1020) at a wavelength of 660 nm where the maximum peak of the color developing agent (DA-67) was observed.

[0166] In addition, the linearity (RQS) and slope were calculated based on the absorbance correlation measurements of each candidate buffer at different pH conditions.

[0167] The five candidate buffers and the five pH conditions are shown in the following Table 2. Table 2 shows five candidate reaction buffers and pH conditions used to evaluate the glycated albumin measurement performance.

[0168] For each sample, if the linearity (RQS) was measured to be greater than 0.98 and the slope was 0.0003 or higher, the corresponding candidate buffer and pH conditions were evaluated as providing sufficient measurement performance for the glycated albumin-enzymatic analysis.

[0169] Table 2: Candidate reaction buffers and pH conditions for evaluation of glycated albumin measurement performance.

[0170]

[0171] 2. Experimental Results

[0172] Figure 11 1 and 2 are tables and graphs showing the results of evaluating the measurement performance of glycated albumin based on different reaction buffers according to the examples of the present application.

[0173] For HEPES buffer:

[0174] 1) At pH 6.5 and pH 8.0, the measured linearity (RQS) was greater than 0.98 and the measured slope was 0.0003 or higher.

[0175] 2) At pH 4.1, pH 5.8, and pH 10.0, the slope was measured to be less than 0.0003.

[0176] 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 at pH 4.1, pH 5.8, pH 6.5, pH 8.0, and pH 10.0. It should be noted that for PB buffer, the slope was actually 0 at pH 5.8, and the slope was actually negative at pH 6.5 and pH 8.0. This confirmed that the enzymatic reaction did not proceed with PB buffer, or that the signal intensity decreased with increasing glycated albumin (or glycated amino acid) concentration, indicating that PB buffer is not suitable for glycated albumin-enzymatic analysis.

[0177] For Tris-HCl buffer:

[0178] 1) Under the conditions of pH 4.1, pH 5.8, pH 6.5, and pH 8.0, the measured linearity (RQS) was greater than 0.98 and the measured slope was 0.0003 or higher.

[0179] 2) At pH 10.0, the slope was measured to be less than 0.0003.

[0180] For Tris-Base buffer:

[0181] 1) Under the conditions of pH 4.1, pH 5.8, pH 6.5, and pH 8.0, the measured linearity (RQS) was greater than 0.98 and the measured slope was 0.0003 or higher.

[0182] 2) At 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.

[0183] For PBS buffer:

[0184] At 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.

[0185] Experimental Example 2 demonstrates that the reaction buffer (B) according to an embodiment of the present application can be selected from the group consisting of Tris-HCl and Tris-Base, and the pH of the first composition containing the reaction buffer (B) can fall within the range of 4.1 to 10.0. Preferably, the pH of the first composition containing the reaction buffer (B) can be implemented within the range of 4.1 to 8.0. More preferably, the pH of the first composition containing the reaction buffer (B) can be implemented within the range of 5.8 to 8.0.

[0186] Meanwhile, it is observed that under the conditions of pH 6.5 and pH 8.0, the slope of the Tris-HCl buffer is relatively greater than the slope of the Tris-Base buffer. Therefore, the reaction buffer (B) in the preferred embodiment of the present application can be Tris-HCl. In addition, the pH of the first composition containing Tris-HCl as the reaction buffer (B) can be implemented in the range of 4.1 to 10.0. More preferably, the pH of the first composition containing Tris-HCl as the reaction buffer (B) can be implemented in the range of 4.1 to 8.0, and even more preferably in the range of 5.8 to 8.0.

[0187] Meanwhile, considering the optimal reaction environment of ketoamine oxidase (KAO) of the reaction reagent (R1), the pH of the first composition containing Tris-HCl as the reaction buffer (B) may be preferably provided in a weakly alkaline pH range.

[0188] <Experimental Example 3: Evaluation of Measurement Performance of Surfactants>

[0189] 1. Experimental Methods

[0190] The following evaluations were performed to select surfactants that provide the best analytical environment for both the BCP and enzymatic methods:

[0191] 1) Measurement performance of the albumin-BCP assay at different concentrations for each candidate surfactant.

[0192] 2) Measurement performance of the glycated albumin-enzymatic assay at different concentrations of each candidate surfactant.

[0193] Specifically, regarding 1) 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%.

[0194] Albumin powder (albumin from human serum, lyophilized powder, Sigma-Aldrich; A3782) was used to prepare the measurement sample.

[0195] 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), and a medium concentration sample (40.4 g / L) was prepared by mixing the low concentration sample with the high concentration sample at a ratio of 1: 1. In addition, a medium-low concentration sample (28.4 g / L) was prepared by mixing the low concentration sample with the medium concentration sample at a ratio of 1: 1, and a medium-high concentration sample (52.6 g / L) was prepared by mixing the medium concentration sample with the high concentration sample at a ratio of 1: 1, thereby creating a total of five sample concentrations.

[0196] Before use, the albumin (g / L) values ​​of the prepared samples were verified using a reference reagent (Lucica GA-L, Asahi Kasei Pharmaceuticals) and a reference device (automatic biochemical analyzer (RX Imola, Landau)).

[0197] In addition, absorbance-related measurements for each sample were analyzed using an Al Kehl analyzer from Essence at 610 nm, where the maximum peak of the BCP complex is observed. 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 greater than 0.98 and the slope was 0.003 or higher, the corresponding surfactant and its concentration were evaluated as providing sufficient measurement performance for albumin-BCP analysis.

[0198] Furthermore, regarding the performance of the 2) 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 a low-concentration (4.51 g / L) standard solution of Lucica GA-L control (a standard solution from Asahi Kasei Pharmaceuticals used as a reference reagent by Essens) with a high-concentration (17.59 g / L) standard solution. Specifically, a medium-concentration sample (11.37 g / L) was prepared by mixing the low-concentration standard solution with the high-concentration standard solution at a 1:1 ratio, a medium-low-concentration sample (8.01 g / L) was prepared by mixing the low-concentration sample with the medium-concentration sample at a 1:1 ratio, and a medium-high-concentration sample (14.66 g / L) was prepared by mixing the medium-concentration sample with the high-concentration sample at a 1:1 ratio.

[0199] Before use, the glycated albumin (g / L) values ​​of the prepared samples were verified using a reference reagent (Lucica GA-L, Asahi Kasei Pharmaceuticals) and a reference device (automatic biochemical analyzer (RX Imola, Landau)).

[0200] In addition, absorbance measurements related to the color development of DA-67 for the glycated albumin enzymatic analysis were analyzed for each sample using an Al Kehl analyzer from Essex at 660 nm, where the maximum peak of the color developer (DA-67) is observed. 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 greater than 0.98 and the slope was 0.0003 or higher, the corresponding surfactant and its concentration were evaluated as providing sufficient measurement performance for the glycated albumin enzymatic analysis.

[0201] The three candidate surfactants and their concentration conditions are shown in the following Table 3. Table 3 shows the candidate surfactants and the concentration conditions of the surfactants.

[0202] Table 3: Candidate surfactants and surfactant concentration conditions.

[0203]

[0204] 2. Experimental Results

[0205] Figure 12 This is a table showing the results of evaluating the measurement performance of albumin and / or glycated albumin based on the surfactant added to the reaction buffer according to Examples of the present application.

[0206] 1) Measurement performance of albumin-BCP analysis

[0207] For CHAPSO surfactant, at concentrations of 0.01% and 0.001%, the linearity (RQS) was measured to be greater than 0.98 and the slope was measured to be 0.003 or higher. However, at a concentration of 0.1%, the linearity (RQS) was measured to be less than 0.98 and the slope was measured to be less than 0.003.

[0208] 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 higher at concentrations of 0.1%, 0.01%, and 0.001%.

[0209] 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 higher at concentrations of 0.1%, 0.01%, and 0.001%.

[0210] 2) Regarding the measurement performance of glycated albumin-enzymatic analysis

[0211] For CHAPSO surfactant, the linearity (RQS) was measured to be greater than 0.98 and the slope was measured to be 0.0003 or higher at concentrations of 0.1%, 0.01%, and 0.001%.

[0212] 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 higher at concentrations of 0.1%, 0.01%, and 0.001%.

[0213] For Tween 20 surfactant, at concentrations of 0.1% and 0.01%, the linearity (RQS) was measured to be greater than 0.98 and the slope was measured to be 0.0003 or higher. However, at a concentration of 0.1%, the linearity (RQS) was measured to be less than 0.98 and the slope was measured to be less than 0.0003.

[0214] Experimental Example 3 confirmed that the surfactant in the examples of the present application can be selected from the group consisting of CHAPSO, Triton X-100 and Tween 20.

[0215] Meanwhile, referring to Experimental Example 3, it was confirmed that Triton X-100 met the evaluation criteria for both 1) the albumin-BCP analysis and 2) the glycated albumin-enzymatic analysis under all concentration conditions. Therefore, in a preferred embodiment of the present application, the surfactant may be Triton X-100, and the concentration of Triton X-100 may exceed 0% and be included in the first composition at a concentration of up to 0.1%.

[0216] Furthermore, referring to Experimental Examples 1 to 3, in a preferred embodiment of the present application, the first composition may include Triton X-100 and Tris-HCl buffer at a concentration exceeding 0% and up to 0.1%. This ensures that both the glycated albumin decomposition reaction and the enzymatic reaction of glycated amino acids are performed in an optimal analytical environment (weakly alkaline). More preferably, the first composition may include Triton X-100 and Tris-HCl buffer at a concentration of approximately 0.01%.

[0217] As described above, the second composition, the third composition, and the fifth composition according to the embodiments of the present application may each include a stabilizer for fixing the reagent in a “solid state.” Each stabilizer may include a disaccharide, DEAE-dextran, and NPS.

[0218] A drying process is required to immobilize the reaction reagent (R1), decomposition reagent (R2), and oxidase (E) in a "solid state." Rapid water removal during this drying process can cause structural damage to the proteins that make up the reagents. To prevent this structural damage, a disaccharide is selected as one of the stabilizers. Specifically, trehalose, a disaccharide, is selected as one of the stabilizers.

[0219] In a dry state, the reaction reagents (R1), decomposition reagents (R2), and oxidase (E) must maintain stability for months or even years. To ensure storage stability, Neo Protein Saver (NPS) is selected as one of the stabilizers. NPS interacts with the protein molecules in the reagents to protect their structure and prevent protein denaturation caused by oxidation.

[0220] Even in their dried state, the reaction reagent (R1), decomposition reagent (R2), and oxidase (E) must be able to resolubilize and participate in the reaction when mixed with the blood sample. To this end, DEAE-dextran was selected as one of the stabilizers because it protects the protein structure through ion exchange and facilitates the resolubilization of the dried reagents.

[0221] <Experimental Example 4: Evaluation of Accelerated Stability of Stabilizer>

[0222] 1. Experimental Methods

[0223] The A1 Care Cartridge from Essence (In Vitro Diagnostic Approval No. 21-4641, Batch 3) was used in which the present invention was implemented.

[0224] A stabilizer composed of 250 mM trehalose, 1.25 (w / v%) DEAE-dextran, and 1.5 (w / v%) NPS (TOYOBO) was added to the reagent immobilization portion of the A1 Kell cell where the reaction reagent (R1) composed of KAO and POD was immobilized.

[0225] A stabilizer composed of 250 mM trehalose, 1.25 (w / v%) DEAE-dextran, and 1.5 (w / v%) NPS (Toyobo) was added to the reagent-immobilizing portion of the A1 Kell cell where the oxidase (E) composed of ASOx was immobilized.

[0226] 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 portion of the A1 Kell cell where the degradation reagent (R2) consisting of a proteolytic enzyme was immobilized.

[0227] At the same time, 100 mM trehalose was added to the reagent-immobilizing portion of the A1 Kell cell where the color developing reagent (D) composed of DA-67 was immobilized.

[0228] Test samples were prepared using a standard solution (Lucica GA-L control) from Asahi Kasei Pharmaceuticals, which served as a reference reagent for Essence. Prior to use, albumin (g / L) and glycated albumin (g / L and %) values ​​for the low (4.51 g / L) and high (17.59 g / L) concentrations were verified using a reference reagent (Lucica GA-L, Asahi Kasei Pharmaceuticals) and a reference instrument (automatic biochemical analyzer (RX Imola, Landau)). The glycated albumin ratio (GA%) was calculated using an Al Kehl analyzer (2 batches) from Essence.

[0229] For one batch of A1 Kell analyzers (2 batches), 10 replicate measurements were performed on 3 batches of cartridges using the Lucica GA-L control solution (Low) on the evaluation day. For the remaining batch of A1 Kell analyzers, 10 replicate measurements were performed on 3 batches of cartridges using the Lucica GA-L control solution (High) on the evaluation day to measure the glycated albumin ratio (GA%).

[0230] In addition, the skewness (%) and precision (CV%) of the measured glycated albumin ratio relative to the initial glycated albumin ratio (GA%) were calculated.

[0231] The above analysis method was performed for each of the following storage conditions in the following Table 4. Table 4 shows the storage temperature and storage time period of the cartridges.

[0232] Table 4: Storage temperature and storage time of the box

[0233]

[0234] In addition, stabilizers were evaluated as suitable for quantitative analysis of glycated albumin ratio (GA%) if they met all of the following evaluation criteria:

[0235] 1) The measured values ​​of the initial glycated albumin ratio (ie, GA% on day 0) for at least 2 of the 3 cartridge batches must meet the following conditions: The skew must be within ±10%, and the CV must be within 10%.

[0236] 2) In addition, at each evaluation period, the average measured value of the glycated albumin ratio of the three cartridge batches must meet the following conditions: The skew must be within ±10%, and the CV must be within 10%.

[0237] 2. Experimental Results

[0238] Figure 13This is a table showing the results of evaluating the accelerated stability of reaction reagents, oxidases, and / or decomposition reagents based on stabilizers added thereto according to Examples of the present application. Figure 14 This is a graph showing the results of evaluating the accelerated stability of a reaction reagent, an oxidase, and / or a decomposition reagent based on a stabilizer added thereto according to an example of the present application.

[0239] Under storage conditions at 40°C, it was confirmed that the average measured value of the glycated albumin ratio of the three box batches remained within a skewness of ±10% and a CV of 10% until 14 days of storage.

[0240] Under storage conditions of 20°C to 25°C, it was confirmed that the average measured value of the glycated albumin ratio of the three box batches remained within ±10% skewness and 10% CV until 24 weeks of storage.

[0241] Under storage conditions of 2°C to 8°C, it was confirmed that the average measured values ​​of the glycated albumin ratios of the three box batches remained within ±10% skewness and 10% CV until 52 weeks of storage.

[0242] According to Experimental Example 4, the stabilizers containing disaccharide, DEAE-dextran, and NPS, each added to the second composition, the third composition, and the fifth composition, were evaluated as being suitable for quantitative analysis of the glycated albumin ratio.

[0243] Meanwhile, trehalose was used as the disaccharide contained in the stabilizer in Experimental Example 4. However, any suitable disaccharide (eg, sucrose) having properties similar to those of trehalose may also be included in the stabilizer.

[0244] <Experimental Example 5: Equivalence of Trehalose and Sucrose as Stabilizers>

[0245] To verify that disaccharides other than trehalose can be used as stabilizers, the equivalence of trehalose and sucrose (a representative disaccharide) as stabilizers was also tested.

[0246] 1. Experimental Methods

[0247] To verify that disaccharides can be used as stabilizers, a comparative evaluation was conducted as shown in Table 5 below. Table 5 shows the composition of the two stabilizers used in the comparative evaluation. Two stabilizer formulations were evaluated: 1) Stabilizer Condition 1, consisting of trehalose, 1.25% w / v DEAE-dextran, and 1.5% w / v NPS; and 2) Stabilizer Condition 2, consisting of sucrose, 1.25% w / v DEAE-dextran, and 1.5% w / v NPS.

[0248] Table 5: Composition of the two stabilizers used in the comparative evaluation

[0249] Reagent stabilizer Condition #1 Condition #2 Trehalose 250mM - sucrose - 250mM DEAE-dextran 1.25% w / v 1.25% w / v NPS 1.50% w / v 1.50% w / v

[0250] First, the measured values ​​of glycated albumin, total albumin, and the glycated albumin ratio when using a stabilizer containing trehalose under condition 1 were compared with the measured values ​​of glycated albumin, total albumin, and the glycated albumin ratio when using a stabilizer containing sucrose under condition 2. Furthermore, the accuracy of the measured values ​​when using a stabilizer containing trehalose under condition 1 (i.e., the measured values ​​of glycated albumin, total albumin, and the glycated albumin ratio) was measured and compared with the accuracy of the measured values ​​when using a stabilizer containing sucrose under condition 2 (i.e., the measured values ​​of glycated albumin, total albumin, and the glycated albumin ratio).

[0251] Samples for measurement value comparison and precision evaluation were prepared using a low-concentration standard solution and a high-concentration standard solution (Lucica GA-L control) from Asahi Kasei Pharmaceuticals. Prior to use, the glycated albumin (g / L) and total albumin (g / L) values ​​of the prepared samples were confirmed using a reference reagent (Lucica GA-L, Asahi Kasei Pharmaceuticals) and a reference device (automatic biochemical analyzer (RX Imola, Landau)), and the final glycated albumin ratio (%) value was calculated based on these two values. In addition, 33 repeated measurements of the standard solution samples at each concentration were performed using an A1 Kell analyzer (1 batch) and an A1 Kell GA cartridge (1 batch each) from Essence containing each of the stabilizers for Condition 1 and Condition 2, respectively. Based on these measurements, the glycated albumin (GA g / L) value, the total albumin (ALB g / L) value, the glycated albumin ratio (GA%) value, and the precision of each measurement value were calculated.

[0252] Next, the linearity of each measurement value obtained using the stabilizer containing trehalose in Condition 1 (i.e., glycated albumin measurement value, total albumin measurement value, and glycated albumin ratio measurement value) was evaluated. Similarly, the linearity of each measurement value obtained using the stabilizer containing sucrose in Condition 2 (i.e., glycated albumin measurement value, total albumin measurement value, and glycated albumin ratio measurement value) was also evaluated.

[0253] Albumin powder (albumin from human serum albumin lyophilized powder, Sigma-Aldrich; A3782) is used to prepare a measurement sample for evaluating the linearity of total albumin. Specifically, 83 mg of albumin powder is dissolved in 5 mL of deionized water (DIW) to prepare a low concentration sample (16.6 g / L), and 130 mg of albumin powder is dissolved in 5 ml of DIW to prepare a high concentration sample (64.8 g / L). And a medium concentration sample (40.4 g / L) is prepared by mixing a low concentration sample with a high concentration sample at a ratio of 1: 1. In addition, a low-medium concentration sample (28.4 g / L) is prepared by mixing a low concentration sample with a medium concentration sample at a ratio of 1: 1, and a medium-high concentration sample (52.6 g / L) is prepared by mixing a medium concentration sample with a high concentration sample at a ratio of 1: 1, thereby obtaining a total of five concentration samples. Before use, the albumin (g / L) values ​​of the prepared samples were verified using a reference reagent (Lucica GA-L, Asahi Kasei Pharmaceuticals) and a reference device (automatic biochemical analyzer (RX Imola, Landau)).

[0254] The measurement samples for evaluating the linearity of glycated albumin were prepared by mixing a low-concentration solution (5.96 g / L) of a standard solution (Lucica GA-L control) from Asahi Kasei Pharmaceuticals with a high-concentration solution (15.41 g / L). Specifically, a medium-concentration sample (10.836 g / L) was prepared by mixing the low-concentration standard solution with the high-concentration standard solution at a ratio of 1:1. In addition, a low-medium-concentration sample (8.41 g / L) was prepared by mixing the low-concentration sample with the medium-concentration sample at a ratio of 1:1, and a medium-high-concentration sample (15.41 g / L) was prepared by mixing the medium-concentration sample with the high-concentration sample at a ratio of 1:1. Prior to use, the glycated albumin (g / L) values ​​of the prepared samples were verified using a reference reagent (Lucica GA-L, Asahi Kasei Pharmaceuticals) and a reference device (automatic biochemical analyzer (RX Imola, Landau)).

[0255] In addition, absorbance-related measurements related to the color of the color developer (DA-67) obtained from the glycated albumin-enzyme assay were analyzed using an Essence Al Kehl analyzer (In Vitro Diagnostic Approval No. 21-1020) at a wavelength of 660 nm, where the maximum peak of the color developer (DA-67) is observed. Furthermore, absorbance-related measurements related to changes in the total albumin-BCP assay were analyzed at a wavelength of 610 nm, where the maximum peaks of the total albumin and BCP reagent are observed.

[0256] Furthermore, for each of the stabilizer of Condition 1 and the stabilizer of Condition 2, an RSQ value indicating the linearity of the measured values ​​(ie, the measured values ​​of glycated albumin, total albumin, and the glycated albumin ratio) according to the sample concentration was calculated.

[0257] In addition, if all of the following evaluation criteria were satisfied, the stabilizer under condition 1 containing trehalose and the stabilizer under condition 2 containing sucrose were evaluated as exhibiting performance equivalent to that of the stabilizer used for quantitative analysis of glycated albumin.

[0258] 1) The measured values ​​of the stabilizer under condition 1 containing trehalose and the stabilizer under condition 2 containing sucrose must satisfy the % skewness within the range of -10% to 10%.

[0259] 2) The precision (CV%) of the measured values ​​of the stabilizer of condition 1 containing trehalose and the stabilizer of condition 2 containing sucrose must be within 5%.

[0260] 3) The linearity (RSQ) of the measured values ​​of the stabilizer under condition 1 containing trehalose and the stabilizer under condition 2 containing sucrose must be 0.98 or greater.

[0261] 2. Experimental Results

[0262] Figure 15 This is a table showing the measurement and accuracy based on the type of disaccharide contained in the stabilizer added to the reaction reagent, oxidase, and / or decomposition reagent according to Examples of the present application.

[0263] For the low-concentration (low) standard solution samples, the measured value of glycated albumin (GA) was 6.5 for the stabilizer in Condition 1 containing trehalose, and 6.4 for the stabilizer in Condition 2 containing sucrose, with a skewness (%) of -1.5%. Furthermore, the measured value of total albumin (ALB) was 39.6 for the stabilizer in Condition 1 containing trehalose, and also 39.6 for the stabilizer in Condition 2 containing sucrose, with a skewness (%) of 0%. Furthermore, the measured value of the glycated albumin ratio (GA (%)) was 17.2 for the stabilizer in Condition 1 containing trehalose, and 17.0 for the stabilizer in Condition 2 containing sucrose, with a skewness (%) of -1.2%.

[0264] For the high-concentration (high) standard solution samples, the measured values ​​for glycated albumin (GA) were 13.6 for the stabilizer in Condition 1 containing trehalose, and also 13.6 for the stabilizer in Condition 2 containing sucrose, with a skewness (%) of 0%. Furthermore, the measured values ​​for total albumin (ALB) were 38.8 for the stabilizer in Condition 1 containing trehalose, and 39.1 for the stabilizer in Condition 2 containing sucrose, with a skewness (%) of 0.8%. Furthermore, the measured values ​​for the glycated albumin ratio (GA (%)) were 33.7 for the stabilizer in Condition 1 containing trehalose, and 33.5 for the stabilizer in Condition 2 containing sucrose, with a skewness (%) of -0.6%.

[0265] It was thus confirmed that the measured values ​​of the stabilizer of Condition 1 and the stabilizer of Condition 2 satisfied the evaluation criterion requiring that the skewness (%) fall within the range of -10% to 10% in all cases.

[0266] For low-concentration (low) standard solution samples, when using the stabilizer in condition 1 containing trehalose, the precision (CV%) of the glycated albumin (GA) measurement value was 4.3, the precision (CV%) of the total albumin (ALB) measurement value was 2.7, and the precision (CV%) of the glycated albumin ratio (GA (%)) was 3.5. When using the stabilizer in condition 2 containing sucrose, the precision (CV%) of the glycated albumin (GA) measurement value was 4.2, the precision (CV%) of the total albumin (ALB) measurement value was 3.3, and the precision (CV%) of the glycated albumin ratio (GA (%)) was 2.8.

[0267] For high-concentration (high) standard solution samples, when using the stabilizer in condition 1 containing trehalose, the precision (CV%) of the glycated albumin (GA) measurement value was 3.9, the precision (CV%) of the total albumin (ALB) measurement value was 4.3, and the precision (CV%) of the glycated albumin ratio (GA (%)) was 3.3. When using the stabilizer in condition 2 containing sucrose, the precision (CV%) of the glycated albumin (GA) measurement value was 4.1, the precision (CV%) of the total albumin (ALB) measurement value was 4.2, and the precision (CV%) of the glycated albumin ratio (GA (%)) was 2.9.

[0268] It was thus confirmed that the precision (CV) of the measured values ​​of the stabilizer of Condition 1 and the stabilizer of Condition 2 satisfied the evaluation criterion of being within 5% in all cases.

[0269] Figure 16These are a table and a 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 decomposition reagent according to Examples of the present application.

[0270] As a result of the linearity evaluation, the linearity index (RSQ) of the glycated albumin (GA) measurement value when the stabilizer of Condition 1 containing trehalose was used was measured to be 0.988. The linearity index (RSQ) of the total albumin (ALB) measurement value was measured to be 0.988, and the linearity index (RSQ) of the glycated albumin ratio (GA%) measurement value was measured to be 0.980.

[0271] In addition, when the stabilizer of Condition 2 containing sucrose was used, the linearity index (RSQ) of the glycated albumin (GA) measurement value was measured to be 0.986, the linearity index (RSQ) of the total albumin (ALB) measurement value was measured to be 0.995, and the linearity index (RSQ) of the glycated albumin ratio (GA%) measurement value was measured to be 0.991.

[0272] It was thus confirmed that the linearity (RSQ) of the measured values ​​of the stabilizer in both Condition 1 and Condition 2 satisfied the evaluation criterion of at least 0.98 in all cases.

[0273] Experimental Example 5 confirmed that both the stabilizer under Condition 1, which contained trehalose, and the stabilizer under Condition 2, which contained sucrose, could be used as stabilizers for the quantitative analysis of glycated albumin. Furthermore, Experimental Example 5 also confirmed that a disaccharide exhibiting properties similar to those of trehalose and sucrose could effectively serve as a stabilizer for the quantitative analysis of glycated albumin.

[0274] Figure 17 1 is a diagram showing identification information included in a glycated albumin quantitative analysis kit according to an embodiment of the present application.

[0275] According to one embodiment of the present application, the GA quantitative analysis kit (10) may include identification information for distinguishing the biological sample. For example, the GA quantitative analysis kit (10) may include identification information (e.g., information in the form of a barcode) located on a surface of the main box (200) to identify the type of the biological sample.

[0276] The quantitative analysis device described later can obtain identification information to distinguish 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 to be analyzed is 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 distinguished type based on the distinguished type of the biological sample. For example, the quantitative analysis device can be implemented to execute a pre-stored GA analysis protocol based on distinguishing that the type of the biological sample to be analyzed is GA, thereby performing quantitative analysis of GA.

[0277] at the same time, Figure 17 Identification information in the form of a barcode is shown as an example. However, this is merely an example, and any suitable form of identification information may be provided at any suitable position on the GA quantitative analysis kit (10).

[0278] The quantitative analysis apparatus (or quantitative analysis device) according to an embodiment of the present disclosure may be configured to perform quantitative analysis of biological samples (eg, 1,5-anhydro-D-glucitol (1,5-AG), glycated albumin (GA), CRP, etc.).

[0279] Furthermore, the quantitative analysis device may include a communication module (which may also be referred to as a transceiver), a memory, and / or a processor.

[0280] The communication module of the quantitative analysis device can communicate with any external device or external server. For example, the quantitative analysis device can transmit the quantitative analysis result to the external device or external server via the communication module.

[0281] Quantitative analysis device can access the network through the communication module to transmit or admit various types of data.Communication module can mainly include wired communication module and wireless communication module.Because wired communication module and wireless communication module have their own advantages and disadvantages, therefore in some cases, wired communication module and wireless communication module can be provided together as quantitative analysis device.Herein, in the case of wireless communication module, wireless local area network (wireless local area network, WLAN) type communication methods such as wireless fidelity (wireless fidelity, Wi-Fi) can be mainly used.Alternatively, in the case of wireless communication module, cellular communication (such as long-term evolution (long-term evolution, LTE) or fifth generation (fifth generation, 5G) communication method) can be used.However, wireless communication protocol is not limited to the above examples, and any suitable wireless type communication method can be used.In the case of wired communication module, local area network (local area network, LAN) or universal serial bus (Universal Serial Bus, USB) communication is representative example, but other methods can also be used.

[0282] Various types of information can be stored in the memory of quantitative analysis device. Various types of data can be temporarily or semi-permanently stored in memory. Examples of memory can include hard disk drive (HDD), solid state drive (SSD), flash memory, read-only memory (ROM), random access memory (RAM), etc. Memory can be configured to be embedded in the quantitative analysis device or to be configured to be detachable. Various types of data required for the operation of the quantitative analysis device can be stored in the memory, including the operating program (OS) for driving the quantitative analysis device or the program for each component of the quantitative analysis device.

[0283] The processor can control the overall operation of the quantitative analysis device. For example, the quantitative analysis device can control its overall operation, including, for example, identifying the identification information of the biological sample, executing the corresponding analysis protocol based on the identified identification information and / or performing quantitative analysis of the biological sample according to the analysis protocol. Specifically, the processor can load and execute the program for the overall operation of the quantitative analysis device from the memory. Depending on hardware, software or a combination thereof, the processor can be implemented as an application processor (AP), a central processing unit (CPU), a microcontroller unit (MCU) or a similar device. In this case, the processor can be provided in the form of an electronic circuit that processes electrical signals and implements control functions in hardware, and can be provided in the form of a program or code that drives the hardware circuit in software.

[0284] According to embodiments of the present application, a kit for quantitative analysis of glycated albumin, a method for quantitative analysis of glycated albumin, and / or an apparatus for implementing the method can provide the effects of implementing both a BCP reaction for quantifying the total amount of albumin and an enzymatic reaction for quantifying the amount of glycated albumin on a single kit through an optimal combination of a reaction buffer and a surfactant.

[0285] According to the embodiments of the present application, by performing the BCP reaction and the enzymatic reaction on a single kit, the glycated albumin quantitative analysis kit can be miniaturized, the analysis time of glycated albumin can be reduced, and the user convenience can be improved.

[0286] According to the embodiments of the present application, by immobilizing a composition containing a decomposition reagent on a suitable reagent immobilization portion, the loss of BCP binding sites present in albumin can be prevented, thereby improving the accuracy of total albumin quantification using a BCP analysis method.

[0287] The effects of the present invention are not limited to the above-described effects, and other effects that are not described can be clearly understood by those skilled in the art from the above detailed description.

[0288] The features, structures, and effects described in the above exemplary embodiments are included in at least one exemplary embodiment of the present invention, but are not necessarily limited to one exemplary embodiment. In addition, the features, structures, and effects described in each embodiment can be combined or modified and implemented in other embodiments by a person skilled in the art to which the embodiment belongs. Therefore, it should be understood that the content related to such combinations and modifications is also included in the scope of the present invention.

[0289] Furthermore, although the present invention has been described in detail with reference to the embodiments, the embodiments are merely exemplary embodiments of the present invention, and the present invention is not intended to be limited thereto. Those skilled in the art will appreciate that other modifications and applications may be made without departing from the spirit and scope of the present invention. In other words, each element specifically shown in the embodiments may be modified and implemented. Furthermore, it should be understood that differences associated with such modifications and applications are also within the scope of the present invention as defined in the appended claims.

Claims

1. A kit for quantitative analysis of glycated albumin, comprising: a first composition comprising a reaction buffer for inducing binding to albumin present in a blood sample; a second composition comprising a decomposition reagent for a decomposition reaction to decompose glycated albumin 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; as well as a fourth composition comprising a color developing agent oxidized by hydrogen peroxide H2O2 produced by the enzymatic reaction of the glycated amino acid, The glycated albumin quantitative analysis kit comprises at least one reagent fixing part, and The second to fourth compositions are each independently fixed to the at least one reagent fixing portion.

2. The glycated albumin quantitative analysis kit according to claim 1, The first composition is selected from the group consisting of bromocresol purple BCP and bromocresol green BCG.

3. The glycated albumin quantitative analysis kit according to claim 2, The reaction buffer is selected from the group consisting of 4-hydroxyethylpiperazineethanesulfonic acid, phosphate buffer, tris(hydroxymethylaminomethane) hydrochloride, tris(hydroxymethylaminomethane) and phosphate buffered saline.

4. The glycated albumin quantitative analysis kit according to claim 2, wherein the reaction buffer is selected from the group consisting of tris(hydroxymethyl)aminomethane hydrochloride and tris(hydroxymethyl)aminomethane, And the pH of the first composition is in the range of 4.1 to 10.

0.

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

6. The glycated albumin quantitative analysis kit according to claim 3, The first composition further comprises a surfactant selected from the group consisting of 3-[(3-cholamidopropyl)dimethylamino]-2-hydroxy-1-propanesulfonic acid, Triton X-100 and Tween 20.

7. The glycated albumin quantitative analysis kit according to claim 3, The first composition further comprises a Triton X-100 surfactant at a concentration greater than 0% and at most 0.1%.

8. The glycated albumin quantitative analysis kit according to claim 2, wherein the reaction buffer comprises tris(hydroxymethyl)aminomethane hydrochloride, and the first composition further comprises a Triton X-100 surfactant at a concentration greater than 0% and at most 0.1%, Furthermore, the decomposition reaction of the glycated albumin and the enzymatic reaction of the glycated amino acids are carried out under weakly alkaline pH conditions.

9. The glycated albumin quantitative analysis kit according to claim 1, The decomposition agent is a protease.

10. The glycated albumin quantitative analysis kit according to claim 1, The reaction reagents include ketoamine oxidase KAO and peroxidase POD.

11. The glycated albumin quantitative analysis kit according to claim 1, The color developing agent is DA-67.

12. The glycated albumin quantitative analysis kit according to claim 1, The glycated albumin quantitative analysis kit further comprises a fifth composition, wherein the fifth composition comprises an oxidase to prevent interference from ascorbic acid present in the blood sample. And the oxidase includes ascorbic acid oxidase ASOx.

13. The glycated albumin quantitative analysis kit according to claim 1, wherein the second composition further comprises a stabilizer for immobilizing the decomposition reagent in a solid form on the at least one reagent fixing portion, The stabilizer comprises disaccharide, diethylaminoethyl-dextran and a new protein preservative.

14. The glycated albumin quantitative analysis kit according to claim 1, wherein 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 stabilizer comprises disaccharide, diethylaminoethyl-dextran and a new protein preservative.

15. The glycated albumin quantitative analysis kit according to claim 1, wherein the fourth composition further comprises a stabilizer for immobilizing the color developing agent in a solid form on the at least one reagent fixing portion, And the stabilizer comprises disaccharide.

16. The kit for quantitative analysis of glycated albumin according to claim 12, wherein the fifth composition further comprises a stabilizer for immobilizing the ascorbic acid oxidase in a solid form on the at least one reagent immobilization portion, The stabilizer comprises disaccharide, diethylaminoethyl-dextran and a new protein preservative.

17. The glycated albumin quantitative analysis kit according to claim 12, wherein the third composition is immobilized in a solid state on the first reagent fixing portion located in the first area of ​​the glycated albumin quantitative analysis kit, The second composition remains immobilized in a solid state on a second reagent fixing portion located in a second region of the glycated albumin quantitative analysis kit separated from the first region.

18. The glycated albumin quantitative analysis kit according to claim 17, wherein the glycated albumin quantitative analysis kit is configured such 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, The reaction reagent is characterized in that the enzymatic reaction with the glycated amino acid is carried out from the moment when the glycated amino acid is produced by the decomposition reaction caused by the decomposition reagent.

19. The kit for quantitative analysis of glycated albumin according to claim 17, The glycated albumin quantitative analysis kit comprises a body, wherein the body comprises an upper plate and a lower plate arranged in a facing manner, The third composition is fixed to the first reagent fixing portion located in the first area of ​​the upper plate, and the fifth composition is fixed to the third reagent fixing portion located in the first area of ​​the lower plate, The first reagent fixing portion and the third reagent fixing portion are arranged to face each other.

20. The glycated albumin quantitative analysis kit according to claim 19, wherein the second composition is fixed to the second reagent fixing portion located in the second area of ​​the upper plate, and the fourth composition is fixed to the fourth reagent fixing portion located in the second area of ​​the lower plate, The second reagent fixing portion and the fourth reagent fixing portion are arranged to face each other.

Images