Kit for quantitative analysis of C-reactive protein

Abstract

A kit for quantitative analysis of C-reactive protein (CRP) according to an embodiment of the present application may comprise: a first composition comprising a hemolytic agent for hemolyzing at least a portion of blood cells in a blood sample; and a second composition comprising an anti-CRP antibody for the antigen-antibody reaction of the CRP contained in the hemolyzed sample. The CRP quantitative analysis kit of the present application can provide a stabilizer which enables a solid-phase dry anti-CRP antibody to be sufficiently redissolved to participate in an immune reaction, so that quantitative analysis of CRP can be highly accurately and accurately performed in a wide concentration range, and storage stability is improved.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to and the benefit of Korean Patent Application No. 10-2024-0027250, filed on February 26, 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 C-reactive protein (CRP), a method for quantitative analysis of CRP, and / or a quantitative analysis device for performing the method. Background Art

[0004] C-reactive protein (CRP) is a substance produced in the liver and secreted into the bloodstream within hours of infection or inflammation. CRP levels in the blood have been widely used as a valuable marker for monitoring the effectiveness of treatments for infectious and autoimmune diseases. Furthermore, with the publication of studies implicating chronic inflammation as a mediator of type 2 diabetes, measuring CRP (an inflammation-related marker) has attracted attention for the development and research of technologies to predict the development of type 2 diabetes. (Reference 1: The clinical significance and potential role of C-reactive protein in chronic inflammatory and neurodegenerative diseases, 2018; Reference 2: Inflammatory markers and risk of type 2 diabetes, 2013).

[0005] As described in the following patent literature for quantitative analysis of CRP, the prior art quantifies CRP in a blood sample without hemolyzing the red blood cells contained in the blood sample. However, without hemolysis, it is necessary to separately measure the hematocrit (HCT), which is the proportion of red blood cells in a blood sample. In addition, in order to prevent hemolysis of red blood cells, the substrate needs to be further modified, which makes the CRP quantitative analysis process relatively complicated. In addition, previous studies lack a sufficient investigation of stabilizers, which are intended to improve the storage stability of the reagents included in the CRP quantitative analysis kit.

[0006] Therefore, there is a need to develop and research CRP quantitative analysis technology for improving the usability of reagents and enhancing the storage stability of reagents.

[0007] [refer to]

[0008] [Patent Document]

[0009] [Patent Document 1] Korean Published Patent Application No. 10-2023-0038489 (March 20, 2023) Summary of the Invention

[0010] An object of the present invention is to provide a kit for quantitative analysis of C-reactive protein (CRP) with improved usability by utilizing a hemolytic reagent that does not affect the quantitative analysis of CRP, a method for quantitative analysis of CRP, and / or an apparatus for carrying out the method.

[0011] The object of the present invention is to provide a kit for quantitative analysis of C-reactive protein (CRP) with improved storage stability of the reagent, a method for quantitative analysis of CRP and / or an apparatus for carrying out the method.

[0012] 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.

[0013] According to an embodiment of the present application, a kit for quantitative analysis of C-reactive protein (CRP) may include: a first composition comprising a hemolytic reagent for hemolyzing at least a portion of blood cells in a blood sample; and a second composition comprising an anti-CRP antibody for an antigen-antibody reaction of CRP contained in the hemolyzed sample, wherein the kit for quantitative analysis of CRP includes at least one reagent fixing portion, the first composition and the second composition are independently fixed to the reagent fixing portion, and the second composition further comprises a stabilizer for the stability of the anti-CRP antibody.

[0014] 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

[0015] 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:

[0016] Figure 1 Schematic diagram of a kit for quantitative analysis of C-reactive protein (CRP) according to an embodiment of the present application.

[0017] Figure 2 Schematic diagram of a sample collector of a CRP quantitative analysis kit according to an embodiment of the present application.

[0018] Figure 3 Schematic diagram of the main box of the CRP quantitative analysis kit according to an embodiment of the present application.

[0019] Figure 4 Schematic diagram of the solution pool of the CRP quantitative analysis kit according to an embodiment of the present application.

[0020] Figure 5 3 is a diagram illustrating a situation in which a reaction buffer 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.

[0021] Figure 6 : is a diagram showing the state of antigen-antibody reaction used for quantitative analysis according to an embodiment of the present application.

[0022] Figure 7 and Figure 8 is a diagram showing the detailed structure of a CRP quantitative analysis kit according to an embodiment of the present application.

[0023] Figure 9 FIG. 4 is a diagram illustrating an analysis process for quantifying CRP in a blood sample according to an embodiment of the present application.

[0024] Figure 10 is a table showing the results of evaluating the hemolytic effects of different hemolytic reagents according to the examples of the present application.

[0025] Figure 11 This is a graph showing the results of evaluating the antigen-antibody reactivity of various hemolytic reagents according to Examples of the present application.

[0026] Figure 12 This is a graph showing the results of evaluating the antigen-antibody reactivity of various hemolytic reagents according to Examples of the present application.

[0027] Figure 13 These are graphs and diagrams showing the results of accelerated stability evaluation of stabilizers added to anti-CRP antibodies according to Examples of the present application.

[0028] Figure 14 This is a graph showing the results of accelerated stability evaluation of stabilizers added to anti-CRP antibodies according to Examples of the present application.

[0029] Figure 15 This is a graph showing the results of accelerated stability evaluation of stabilizers added to anti-CRP antibodies according to Examples of the present application.

[0030] Figure 16 This is a graph showing the results of accelerated stability evaluation of stabilizers added to anti-CRP antibodies according to Examples of the present application.

[0031] Figure 17 : is a diagram showing identification information included in the CRP quantitative analysis kit according to an example of the present application. DETAILED DESCRIPTION

[0032] 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.

[0033] 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.

[0034] 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.

[0035] 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.

[0036] As used herein, the singular forms "a" and "an" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] According to an embodiment of the present application, a kit for quantitative analysis of C-reactive protein (CRP) may include: a first composition comprising a hemolytic reagent for hemolyzing at least a portion of blood cells in a blood sample; and a second composition comprising an anti-CRP antibody for an antigen-antibody reaction of CRP contained in the hemolyzed sample, wherein the CRP quantitative analysis kit for quantitative analysis of CRP includes at least one reagent fixing portion, the first composition and the second composition are independently fixed to the reagent fixing portion, and the second composition further comprises a stabilizer for the stability of the anti-CRP antibody.

[0042] According to an embodiment of the present application, the hemolytic agent can be selected from the group consisting of sodium deoxycholate (SDO) and saponin.

[0043] According to an embodiment of the present application, the hemolytic agent may be sodium deoxycholate (SDO).

[0044] According to an embodiment of the present application, the stabilizer can be selected from the group consisting of trehalose and sucrose.

[0045] According to an embodiment of the present application, the stabilizer may include both trehalose and sucrose.

[0046] According to an embodiment of the present application, the stabilizer may include trehalose and sucrose at substantially the same concentration ratio.

[0047] According to an embodiment of the present application, the at least one reagent fixing portion may include: a first reagent fixing portion, located in a first compartment area of ​​the CRP quantitative analysis kit; and a second reagent fixing portion, located in a second compartment area separated from the first compartment area, wherein the first composition can be fixed to the first reagent fixing portion, and the second composition can be fixed to the second reagent fixing portion.

[0048] According to an embodiment of the present application, the CRP quantitative analysis kit may include a solution cell arranged in a first compartment area, the solution cell may store a reaction buffer, and when a sample collector containing a blood sample is inserted into the receiving part of the CRP quantitative analysis kit, the reaction buffer stored in the solution cell and the blood sample contained in the sample collector may flow into a mixing area in which a first reagent fixing part in the first compartment area is arranged, so that at least a portion of the blood cells in the blood sample can be hemolyzed in the mixing area.

[0049] According to an embodiment of the present application, the reaction buffer may include glycine, sodium chloride, sodium azide, ethylenediaminetetraacetic acid disodium salt dihydrate, and bovine serum albumin (BSA).

[0050] According to an embodiment of the present application, the CRP quantitative analysis kit may further include a flow path connecting the first compartment area with the second compartment area, and when the CRP quantitative analysis kit is rotated by a predetermined angle due to an applied external force, the sample in which at least a portion of the blood cells are hemolyzed can flow from the mixing area in the first compartment area to the second compartment area through the flow path.

[0051] According to an embodiment of the present application, the CRP quantitative analysis kit may further include a measuring unit for quantifying CRP contained in a blood sample, and the sample in which at least a portion of the blood cells are hemolyzed may pass through the measuring unit via a flow path while moving from the mixing zone in the first compartment area to the second reagent fixing portion in the second compartment area.

[0052] According to an embodiment of the present application, CRP can be quantified based on a first turbidity measured by a measuring unit before an antigen-antibody reaction and a second turbidity measured by a measuring unit after an antigen-antibody reaction at a second reagent immobilization unit.

[0053] According to an embodiment of the present application, the second composition may include a stabilizer for immobilizing the anti-CRP antibody in a solid state on the reagent-immobilizing portion.

[0054] According to an embodiment of the present application, the anti-CRP antibody may be provided in the form of being coated on latex particles.

[0055] In the following we will refer to Figures 1 to 17 The structure of a kit for quantitative analysis of C-reactive protein (CRP), a method for quantitative analysis of CRP, and / or an apparatus for quantitative analysis of CRP according to embodiments of the present application are described in more detail.

[0056] Figure 1Schematic diagram of a kit for quantitative analysis of C-reactive protein (CRP) according to an embodiment of the present application.

[0057] The kit 10 for quantitative analysis of C-reactive protein (hereinafter referred to as "CRP") according to an embodiment of the present application may include: a sample collector 100 for supplying a biological sample and / or a main box 200 into which the sample collector 100 can be inserted and received.

[0058] Figure 2 FIG. 1 is a schematic diagram of a sample collector 100 of a CRP quantitative analysis kit according to an embodiment of the present application.

[0059] 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.

[0060] In addition, the sample collector 100 may include a protrusion 102, which is 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 can 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 can move inward toward the interior of the main box 200, thereby causing the cover tape of the solution pool 301 to be removed or broken. This will be referred to Figure 5 Elaborate in more detail.

[0061] 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 CRP quantitative analysis kit is rotated during analysis.

[0062] Figure 3 Schematic diagram of the main box 200 of the CRP quantitative analysis kit according to an embodiment of the present application.

[0063] 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.

[0064] 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.

[0065] 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.

[0066] The main cartridge 200 may include a mixing portion 204 (or a mixing zone) in which the biological sample (eg, blood sample) discharged from the sample collector 100 through the receiving portion 201 is mixed with a reaction buffer discharged from the solution reservoir 301 .

[0067] The main box 200 may also include at least one reagent fixing part 205 (referred to as a sample fixing part), into which chemical reagents are introduced. The chemical reagents can react with the reaction buffer and biological sample mixed in the mixing part 204 to induce enzyme reaction and / or antigen-antibody reaction.

[0068] The main cartridge 200 may further include a flow path 206 through which the reaction buffer and biological sample mixed in the mixing section 204 can be moved. Specifically, the analysis sample can travel between the mixing section 204, the reagent holding 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.

[0069] The master cartridge 200 according to an embodiment of the present application may include a measurement unit 207 for measuring the reaction results performed at the reagent fixing portion 205. The CRP quantitative analysis apparatus according to an embodiment of the present application may quantify the analysis sample (e.g., CRP) through optical analysis (e.g., ultraviolet / visible light (UV / VIS)) via the measurement unit 207. For example, the measurement unit 207 of the master cartridge 200 may quantify the analysis sample (e.g., CRP) by measuring changes in turbidity (or absorbance) based on immunoturbidimetry.

[0070] 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.

[0071] 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.

[0072] 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 .

[0073] In the following we will refer to Figure 4 and Figure 5 A more detailed description is provided of a case in which the reaction buffer 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 CRP quantitative analysis kit according to an embodiment of the present application. Figure 53 is a diagram illustrating a situation in which a reaction buffer 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.

[0074] The main cartridge 200 according to an embodiment of the present application may include a solution reservoir 301 that stores a reaction buffer for reacting with a biological sample from the sample collector 100. The solution reservoir 301 includes an opening at one end that is sealed with a cover tape 302 to prevent leakage of the reaction buffer 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 main cartridge 200.

[0075] 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.

[0076] 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 reaction buffer 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.

[0077] Specifically, the reaction buffer 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 reaction buffer stored in the solution reservoir 301 and the biological sample collected by the sample collector 100 can be mixed.

[0078] In addition, at least a portion of the blood cells contained in the biological sample (eg, blood sample) can be hemolyzed by the composition fixed on the reagent fixing portion 205 provided in the mixing portion 204. Figure 7 and Figure 8 Explain this process in more detail.

[0079] 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 provides a structure suitable for storing reaction buffer. Similarly, the material of cover tape 302 is not particularly limited as long as cover tape 302 prevents leakage of the reaction buffer while allowing easy removal or rupture.

[0080] 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.

[0081] The CRP quantitative analysis kit 10 according to an embodiment of the present application can be used to quantitatively analyze CRP present in blood (e.g., plasma, serum, and / or whole blood). According to one embodiment, the CRP quantitative analysis kit 10 can be configured to quantitatively analyze CRP using an immunoassay. Specifically, the CRP quantitative analysis kit 10 can be configured to quantitatively analyze CRP using a turbidimetric immunoassay. Figures 7 to 9 A more detailed description is provided of the case of quantitatively analyzing CRP using the CRP quantitative analysis kit 10 according to an embodiment of the present application.

[0082] The quantitative analysis of CRP according to the embodiment of the present application may utilize turbidimetric immunoassay.

[0083] Turbidimetric immunoassays utilize an antigen-antibody reaction. Specifically, an antibody specific for the target analyte in the analytical sample is added to the analytical sample, forming an antigen-antibody complex through the antigen-antibody reaction. The principle of turbidimetric immunoassays is to measure the turbidity of the antigen-antibody complex, as turbidity is proportional to the amount of antigen in the test substance. This measurement is used to determine the concentration of the target analyte.

[0084] According to an embodiment of the present application, the CRP quantitative analysis kit 10 includes a composition containing an anti-CRP antibody corresponding to a target analyte (CRP (antigen)). In this case, a complex is formed by agglutination between the CRP antigen and the anti-CRP antibody, and the formation of this complex causes a change in turbidity. Therefore, the concentration of the target analyte (CRP) in the blood sample can be quantified based on the change in turbidity.

[0085] According to the embodiment of the present application, by implementing Figure 6CRP is quantitatively analyzed by the antigen-antibody reaction. Figure 6 : is a diagram showing the state of antigen-antibody reaction for quantitative analysis according to an embodiment of the present application.

[0086] Figure 7 and Figure 8 is a diagram showing the detailed structure of a CRP quantitative analysis kit 10 according to an embodiment of the present application.

[0087] The solution pool 301 of the CRP quantitative analysis kit 10 according to an embodiment of the present application can store a reaction buffer (B). As described above, when the sample collector 100 is inserted, the reaction buffer (B) stored in the solution pool 301 moves to the mixing portion 204 of the main box 200. At this time, the reaction buffer (B) is mixed with the blood sample collected by the sample collector 100 in the mixing portion 204 and mixed with a composition (hereinafter referred to as the first composition) containing a hemolytic reagent (R1) fixed on at least one reagent fixing portion 205. During the mixing process, the hemolytic reagent (R1) can cause at least a portion of the blood cells contained in the blood sample to hemolyze. The reaction buffer (B) according to one embodiment may include glycine, sodium chloride, sodium azide, disodium ethylenediaminetetraacetic acid dihydrate and bovine serum albumin (BSA).

[0088] According to one embodiment, the hemolytic reagent (R1) can be selected from the group consisting of sodium deoxycholate (SDO), ASB-14, saponin, and Triton X-100 (Tx-100). In another embodiment, the hemolytic reagent (R1) can be selected from the group consisting of sodium deoxycholate (SDO) and saponin. In a preferred embodiment, the hemolytic reagent (R1) can be sodium deoxycholate (SDO). According to the CRP quantitative analysis kit, the method for quantitative analysis of CRP, and / or the device for implementing the method in the preferred embodiment of the present application, the use of sodium deoxycholate (SDO) as the hemolytic reagent (R1) enables quantitative analysis of CRP with high accuracy over a wide range of CRP concentrations without affecting the immune response. However, this is merely an example, and the hemolytic reagent (R1) is not limited thereto. Any suitable composition or surfactant (detergent) that can achieve the purpose of hemolyzing blood cells without affecting the immune response (antigen-antibody reaction) can be used as the hemolytic reagent (R1).

[0089] The sample in which at least a portion of the blood cells present in the blood sample have been hemolyzed by the hemolytic reagent (R1) can be moved to the measurement unit 207 and / or at least one reagent fixing portion 205 through the flow path 206. The at least one reagent fixing portion 205 may have a composition (hereinafter referred to as the second composition) containing an immune reagent (R2), which is used for an antigen-antibody reaction (or immune reaction) of CRP present in the hemolyzed sample. According to one embodiment, the immune reagent (R2) may include an anti-CRP antibody. In a preferred embodiment, the anti-CRP antibody of the immune reagent (R2) can be provided in the form of a coating on latex particles.

[0090] According to an embodiment of the present application, a first composition containing a hemolytic reagent (R1) and a second composition containing an immune reagent (R2) can each be independently fixed to at least one reagent fixing portion 205. In one embodiment, the first composition containing a hemolytic reagent (R1) can be fixed to the first reagent fixing portion 205 located in a first compartment area (e.g., an area present in the mixing portion 204). At the same time, the second composition containing an immune reagent (R2) can be fixed to the second reagent fixing portion 205 located in a second compartment area separated from the first compartment area.

[0091] Reference Figure 8 , the CRP quantitative analysis kit 10 may include a body, wherein the body includes an upper plate and a lower plate configured to face each other. In this configuration, the first composition containing the hemolytic reagent (R1) can be distributed on the first reagent fixing portion 205-1 arranged in the first compartment area of ​​the upper plate of the body and / or the first reagent fixing portion 205-2 arranged in the first compartment area of ​​the lower plate of the body and dried. In addition, the first reagent fixing portion 205-1 arranged on the upper plate of the body and the first reagent fixing portion 205-2 arranged on the lower plate of the body can be configured to face each other.

[0092] At the same time, the second composition containing the immune reagent (R2) can be distributed on the second reagent fixing portion 205-3 in the second compartment area of ​​the upper plate of the body and / or the second reagent fixing portion 205-4 in the second compartment area of ​​the lower plate of the body and dried. In addition, the second reagent fixing portion 205-3 arranged on the upper plate of the body and the second reagent fixing portion 205-4 arranged on the lower plate of the body can be configured to face each other.

[0093] As described above, the first compartment region where the first reagent fixing portion 205-1 and / or 205-2 for holding the hemolytic reagent (R1) is located can be separated from the second compartment region where the second reagent fixing portion 205-3 and / or 205-4 for holding the immunological reagent (R2) is located. The first compartment region and the second compartment region can be connected via a flow path 206.

[0094] According to an embodiment of the present application, the first composition containing the hemolytic reagent (R1) and / or the second composition containing the immunological reagent (R2) can be fixed in a solid state on the reagent fixing part 205. Specifically, the first composition containing the hemolytic reagent (R1) and / or the second composition containing the immunological reagent (R2) can be dispensed onto and fixed on at least one reagent fixing part 205 in a dry state.

[0095] According to an embodiment of the present application, the second composition containing the immune reagent (R2) (and / or the first composition containing the hemolytic reagent (R1)) may also include a stabilizer to improve the stability of the immune reagent (R2) (and / or the hemolytic reagent (R1)) and fix the reagent in a solid state on the reagent fixing portion 205.

[0096] According to an embodiment of the present application, the stabilizer may be selected from the group consisting of trehalose and sucrose. In a preferred embodiment, the stabilizer may include both trehalose and sucrose. In a more preferred embodiment, the stabilizer may include trehalose and sucrose at substantially the same concentration ratio.

[0097] According to the CRP quantitative analysis kit, the method for quantitative analysis of CRP, and / or the device for implementing the method in the examples of the present application, using a stabilizer containing sucrose and trehalose at an optimal concentration ratio provides the following benefits:

[0098] 1) Anti-CRP antibodies dried in a solid state can be fully rehydrated and participate in immune reactions.

[0099] 2) CRP can be quantitatively analyzed with high precision and accuracy over a wide range of CRP concentrations.

[0100] 3) It can improve the storage stability of the reagent.

[0101] However, this is merely an example, and any suitable material that can enhance the stability of the immunoreagent while improving the precision and accuracy of CRP quantitative analysis may be used as the stabilizer.

[0102] Figure 9 FIG. 4 is a diagram illustrating an analysis process for quantifying CRP in a blood sample according to an embodiment of the present application.

[0103] Reference Figure 9 The CRP quantitative analysis method according to the embodiment of the present application can be implemented as follows:

[0104] 1) Prepare the master box 200.

[0105] 2) Position the sample collector 100 on the receiving portion 201 of the main box 200 .

[0106] 3) Apply pressure to the sample collector 100 in a direction toward the receiving portion 201 of the main cartridge 200 .

[0107] 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 (reaction buffer) stored in the solution reservoir 301 to flow into the mixing portion 204 of the main cartridge 200.

[0108] 5) When the reaction buffer flows into the mixing portion 204 , the reaction buffer contacts the blood sample from the sample collector 100 , causing the blood sample (eg, plasma, serum, and / or whole blood) to also flow into the mixing portion 204 of the main cartridge 200 .

[0109] 6) In the mixing section 204, the hemolytic reagent (R1), the reaction buffer (B), and the blood sample immobilized on the first reagent immobilization section 205-1 and / or 205-2 in the mixing section 204 are mixed. Furthermore, the hemolytic reagent (R1) hemolyzes at least a portion of the blood cells in the blood sample.

[0110] At the same time, according to an embodiment of the present application, the CRP 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 blood sample inside the main cartridge 200 moves through the flow path 206 of the main cartridge 200 under the influence of gravity.

[0111] 7) Due to the external force applied to the CRP quantitative analysis kit 10, the sample in which at least a portion of the blood cells have been hemolyzed moves to the measurement unit 207. When the sample is positioned on the measurement unit 207, a background measurement value (first turbidity) related to turbidity (or absorbance) before the antigen-antibody reaction is performed can be measured.

[0112] As described above, the CRP quantitative analysis kit 10 may include a measurement unit 207 for quantifying CRP contained in a blood sample. According to one embodiment, the CRP quantitative analysis kit 10 may be configured so that the hemolyzed sample passes through the measurement unit 207 while moving from the mixing section 204, where the first reagent fixing section for fixing the hemolytic reagent (R1) is located, to the second reagent fixing section for fixing the immunological reagent (R2) via the flow path 206. In this embodiment, the turbidity before and after the antigen-antibody reaction can be easily measured, thereby making it easier to quantify the CRP concentration associated with the turbidity change.

[0113] 8) Due to the external force applied to the CRP quantitative analysis kit 10, the hemolyzed sample moves from the measurement unit 207 to the second reagent fixing portion 205-3 and / or 205-4 in which the immune reagent (R2) is fixed. On the second reagent fixing portion 205-3 and / or 205-4, an antigen-antibody reaction occurs between the CRP (antigen) contained in the sample and the anti-CRP antibody contained in the immune reagent (R2). Figure 8 Through this antigen-antibody reaction, CRP antigen and anti-CRP antibody form an immune complex (Immuno-Complex), which causes the turbidity of the sample to change.

[0114] 9) Due to the external force applied to the CRP quantitative analysis kit 10, the sample in which the antigen-antibody reaction occurs moves from the second reagent fixing portion 205-3 and / or 205-4 to the measurement unit 207. When the sample is positioned on the measurement unit 207, the turbidity (or absorbance) of the sample after the antigen-antibody reaction can be measured (referred to as the second turbidity). In addition, CRP can be quantified based on the first turbidity measured by the measurement unit 207 before the antigen-antibody reaction and the second turbidity measured by the measurement unit 207 after the antigen-antibody reaction.

[0115] 10) Due to the external force applied to the CRP 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.

[0116] 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.

[0117] <Experimental Example 1: Evaluation of Hemolytic Effect of Hemolytic Reagents>

[0118] 1. Experimental Methods

[0119] To select a hemolytic agent capable of lysing blood cells in a blood sample, five candidate hemolytic agents were selected. Each candidate hemolytic agent was applied to a fresh venous whole blood sample (HCT 48%). The background measurement value (i.e., first turbidity or first absorbance) of each sample treated with the candidate hemolytic agent was measured. Specifically, the first absorbance of the sample was measured at a wavelength of 750 nm using an AlCare Analyzer from i-SENS Inc.

[0120] The five candidate hemolytic agents are listed in Table 1 below. Table 1 shows the five candidate hemolytic agents and their estimated concentrations. If the first turbidity of the sample is lower than 0.05, the candidate hemolytic agent is evaluated as having a hemolytic effect on the blood sample.

[0121] Table 1: Candidate hemolytic agents and their evaluated concentrations.

[0122]

[0123] 2. Experimental Results

[0124] Figure 10 This is a table showing the results of evaluating the hemolytic effects of various hemolytic reagents according to Examples of the present application.

[0125] For the SDC hemolytic reagent, the first absorbance was measured to be less than 0.05 within the concentration range of 0.12 to 0.20 (w / v%), and greater than 0.05 within the concentration range of 0.10 to 0.11 (w / v%).

[0126] For the ASB-14 hemolytic reagent, the first absorbance was measured to be less than 0.05 within the concentration range of 0.04 to 0.20 (w / v%), and greater than 0.05 within the concentration range of 0.01 to 0.03 (w / v%).

[0127] For the saponin hemolytic reagent, the first absorbance was measured to be less than 0.05 in the concentration range of 0.02 to 0.20 (w / v%), and greater than 0.05 at a concentration of 0.01 (w / v%).

[0128] For the Tween 20 hemolytic reagent, the first absorbance measured was greater than 0.05 within a concentration range lower than 1.00 (w / v%).

[0129] For the Tx-100 hemolytic reagent, the first absorbance was measured to be less than 0.05 in the concentration range of 0.04 to 0.20 (w / v%), and greater than 0.05 in the concentration range of 0.02 to 0.03 (w / v%).

[0130] This experimental example demonstrates that the Tween 20 hemolytic reagent exhibits relatively low hemolytic activity against blood cells in a blood sample, even at a high concentration (1.00 w / v%). In contrast, the SDC, ASB-14, saponin, and Tx-100 hemolytic reagents exhibit hemolytic activity against blood cells across most concentration ranges. Therefore, the hemolytic reagent (R1) according to one embodiment of the present application can be selected from the group consisting of sodium deoxycholate (SDO), ASB-14, saponin, and Triton X-100 (Tx-100).

[0131] <Experimental Example 2: Evaluation of Antigen-Antibody Reactivity Using Hemolytic Reagents>

[0132] 1. Experimental Methods

[0133] To select a hemolytic agent that does not interfere with the immune reaction between CRP and anti-CRP antibodies, four candidate hemolytic agents (SDC, ASB-14, saponin, and Tx-100) with proven hemolytic effects, as identified in Experimental Example 1, were prepared. Taking into account sample influences (including hematocrit), the concentration of each candidate hemolytic agent was determined as shown in Table 2 below. Table 2 shows experimental conditions based on the type and concentration of the hemolytic agent.

[0134] Table 2: Experimental conditions according to the type and concentration of hemolytic reagent.

[0135]

[0136] In addition, each candidate hemolytic agent was applied to a 7-level CRP serum sample, followed by an immune reaction between the hemolyzed sample and latex particles coated with anti-CRP antibodies. Specifically, the latex particles coated with anti-CRP antibodies supplied by RANDOX were concentrated and stabilized by the addition of a stabilizer. The immune reaction was configured to occur between the hemolyzed sample and the latex particles coated with anti-CRP antibodies.

[0137] After the immune reaction, the absorbance of each sample after the immune reaction was measured (referred to as the second absorbance or the second turbidity). Specifically, the second absorbance of the sample was measured at a wavelength of 750 nm using an A1 Kell Analyzer manufactured by Essence.

[0138] As a control group, the experiment was designed to measure the secondary absorbance after the immune reaction between the 7-level CRP serum sample without the addition of any hemolytic agent and the latex particles coated with anti-CRP antibodies. In addition, the concentration of the 7-level CRP serum sample was varied from 0 to 195.8 mg / L, and the secondary absorbance of each sample was measured.

[0139] 2. Experimental Results

[0140] Figure 11 This is a graph showing the results of evaluating the antigen-antibody reactivity of various hemolytic reagents according to Examples of the present application.

[0141] The absorbance of the SDC hemolytic reagent was confirmed to be similar to that of the control group to which the hemolytic reagent was not added (i.e., the immune reaction was not affected by the hemolytic reagent). In other words, the SDC hemolytic reagent confirmed that the hemolytic reaction did not affect the antigen-antibody reaction between CRP and anti-CRP antibodies.

[0142] For the ASB-14 hemolytic reagent, it was confirmed that the absorbance corresponding to the CRP concentration decreased by approximately 50% or more compared to a control group to which no hemolytic reagent was added (ie, immune response was not affected by the hemolytic reagent).

[0143] The saponin hemolytic reagent demonstrated that the absorbance corresponding to the CRP concentration was relatively high compared to the control group to which the hemolytic reagent was not added (i.e., the immune response was not affected by the hemolytic reagent). In other words, the saponin hemolytic reagent demonstrated that the hemolytic reaction did not affect the antigen-antibody reaction between CRP and anti-CRP antibodies.

[0144] For the Tx-100 hemolytic reagent, it was observed that the measured absorbance did not show any correlation with the CRP concentration.

[0145] This experimental example demonstrates that the hemolytic reagent (R1) according to the preferred embodiment of the present application can be selected from the group consisting of sodium deoxycholate (SDO) and saponin.

[0146] <Experimental Example 3: Evaluation of Antigen-Antibody Reactivity by Hemolytic Reagents (2)>

[0147] 1. Experimental Methods

[0148] In order to select a hemolytic agent that exhibits excellent accuracy while not interfering with the immune reaction between CRP and anti-CRP antibodies, two candidate hemolytic agents (SDC and saponin) that were demonstrated in Experimental Example 2 not to interfere with the immune reaction through a hemolytic reaction were prepared as shown in Table 3 below. Table 3 shows the experimental conditions depending on the type and concentration of the selected hemolytic agent.

[0149] Table 3: Experimental conditions according to the type and concentration of the selected hemolytic reagent.

[0150]

[0151] In addition, each candidate hemolytic agent was applied to 1) a CRP control solution (low & high) and 2) a three-level CRP venous whole blood sample (low, medium, and high). After application, an immune reaction was performed between the hemolyzed sample and latex particles coated with anti-CRP antibodies. The absorbance of each sample after the immune reaction (i.e., the second absorbance) was measured. Specifically, the second absorbance of the sample was measured at a wavelength of 750 nm using an Al Kell analyzer from Essence.

[0152] Regarding the control group, the experiment was designed to measure the second absorbance after performing an immunoreaction between a CRP control solution (Low & High) to which no hemolytic agent was added and latex particles coated with anti-CRP antibodies.

[0153] In addition, for each sample, at each concentration of each sample and for each hemolytic reagent, the second absorbance was measured 20 times, and the average (AVG), standard deviation (SD), and precision (CV) of the measured second absorbance were calculated.

[0154] 2. Experimental Results

[0155] Figure 12 This is a graph showing the results of evaluating the antigen-antibody reactivity of various hemolytic reagents according to Examples of the present application.

[0156] For the CRP control solution (low) sample, the precision (CV) of the SDC hemolytic reagent was measured to be 2.8%, the precision (CV) of the saponin hemolytic reagent was measured to be 3.4%, and the precision (CV) of the control group was measured to be 3.0%. It was confirmed that both the SDC and saponin hemolytic reagents showed similar levels of precision compared to the control group.

[0157] For the CRP control solution (high) sample, the precision (CV) of the SDC hemolytic reagent was measured to be 3.9%, the precision (CV) of the saponin hemolytic reagent was measured to be 4.3%, and the precision (CV) of the control group was measured to be 3.9%. It was confirmed that both the SDC and saponin hemolytic reagents showed similar levels of precision compared to the control group.

[0158] For CRP venous whole blood (low) samples, the precision (CV) of the SDC hemolytic reagent was measured to be 5.3%, while the precision (CV) of the saponin hemolytic reagent was measured to be 20.7%.

[0159] For CRP venous whole blood (medium) samples, the precision (CV) of the SDC hemolytic reagent was measured to be 5.8%, while the precision (CV) of the saponin hemolytic reagent was measured to be 12.2%.

[0160] For CRP venous whole blood (high) samples, the precision (CV) of the SDC hemolytic reagent was measured to be 4.0%, while the precision (CV) of the saponin hemolytic reagent was measured to be 4.9%.

[0161] Therefore, for grade 3 CRP whole blood samples, the precision of the SDC hemolytic reagent was confirmed to be superior to that of the saponin hemolytic reagent over all concentration ranges of grade 3 CRP whole blood samples.

[0162] According to this experimental example, in a preferred embodiment of the present application, the hemolytic reagent (R1) may be sodium deoxycholate (SDO).

[0163] Furthermore, the CRP quantitative analysis kit 10 according to one embodiment of the present application can exhibit a precision (CV) of less than 10%. For example, the CRP quantitative analysis kit 10 can exhibit a precision (CV) of less than 8%. In another example, the CRP quantitative analysis kit 10 can exhibit a precision (CV) of less than 6%.

[0164] <Experimental Example 4: Accelerated Stability Evaluation of Stabilizers (1)>

[0165] 1. Experimental Methods

[0166] In order to select a stabilizer that ensures the storage stability of the immune reagent (R2) containing anti-CRP antibodies, five candidate stabilizers were selected. Each candidate stabilizer was added to latex particles coated with anti-CRP antibodies and dried. Reagent solution and level 1 CRP serum sample (CRP 60 mg / L) were added to the dried composition to facilitate the immune reaction between CRP and anti-CRP antibodies. After the immune reaction, the absorbance was measured to obtain a baseline measurement value for storage day 0. The reagent solution was configured to include glycine, sodium chloride, sodium azide, ethylenediaminetetraacetic acid disodium salt dihydrate and bovine serum albumin (BSA).

[0167] Furthermore, the dried composition containing each candidate stabilizer added to the latex particles coated with anti-CRP antibodies was stored in an oven at 50°C. The reagent solution was stored separately under refrigeration. The reagent solution and the first-level CRP serum sample were added to the dried composition containing each candidate stabilizer at one-day intervals over a period of 7 days. An immune reaction between CRP and the anti-CRP antibodies was performed, and the absorbance after the immune reaction was measured to obtain measurement values ​​on the first, second, third, fourth, fifth, sixth, and seventh storage days.

[0168] As a control group, the experiment was configured to measure absorbance after performing an immunoreaction between a level 1 CRP serum sample (CRP 60 mg / L) to which no stabilizer was added and latex particles coated with anti-CRP antibodies.

[0169] Additionally, a deviation (Bias) is calculated based on the baseline measurement and the measurements obtained during the storage period.

[0170] The five candidate stabilizers are shown in Table 4 below, and stabilizers whose deviation (skew) measured during the entire storage period was within 20% were evaluated as effectively ensuring the storage stability of the immunoreagent (R2). Table 4 shows experimental conditions according to the type and concentration of the stabilizer.

[0171] Table 4: Experimental conditions according to stabilizer type and concentration

[0172]

[0173] 2. Experimental Results

[0174] Figure 13 This is a graph showing the results of accelerated stability evaluation of stabilizers added to anti-CRP antibodies according to Examples of the present application.

[0175] In the case of the control group (ie, no stabilizer added), a greater than 90% decrease in the measured value was observed starting from storage day 1 compared to the baseline measured value on day 0. This demonstrates that the stabilizer is crucial for ensuring the storage stability of the immunoreagent (R2).

[0176] For the diethylaminoethyl (DEAE)-dextran stabilizer, visual inspection confirmed that the dried anti-CRP antibody was not properly dissolved. In addition, starting from storage day 1, the measured value increased by more than 42% compared to the baseline value at day 0.

[0177] Regarding the polyacrylic acid (PAA) stabilizer, visual inspection confirmed that the dried anti-CRP antibody did not dissolve properly. Furthermore, the immune reaction did not proceed properly, making it impossible to measure absorbance.

[0178] For the Neo Protein Saver (NPS) stabilizer, starting from day 1 of storage, deviations (skews) in the measured values ​​exceeding 20% ​​compared to the baseline values ​​at day 0 were observed.

[0179] For both trehalose and sucrose stabilizers, visual inspection confirmed that the dried anti-CRP antibody dissolved appropriately.In addition, the measured values ​​remained within a 20% deviation (skew) compared to the baseline measured values ​​on day 0 even up to day 7 of storage.

[0180] According to this experimental example, the stabilizer in one embodiment of the present application can be selected from the group consisting of trehalose and sucrose.

[0181] <Experimental Example 5: Accelerated Stability Evaluation of Stabilizers (2)>

[0182] 1. Experimental Methods

[0183] In order to select a stabilizer for ensuring the storage stability of the immune reagent (R2) containing anti-CRP antibodies, two candidate stabilizers (trehalose and sucrose) whose storage stability effect was confirmed by Experimental Example 4 were prepared. Each candidate stabilizer was added to latex particles coated with anti-CRP antibodies and dried. Reagent solution and 3-level CRP serum samples (CRP 10 mg / L, 50 mg / L, 150 mg / L) were added to the dried composition, and the immune reaction between CRP and anti-CRP antibodies was performed. After the immune reaction, the absorbance was measured to obtain a baseline measurement value for storage day 0.

[0184] Each candidate stabilizer was added to latex particles coated with anti-CRP antibodies, and the dried composition was stored in an oven at 50 ° C. The reagent solution was stored separately under refrigeration. Over a period of 7 days, the reagent solution and 3-level CRP serum samples (CRP 10 mg / L, 50 mg / L, 150 mg / L) were added to the dried composition containing each candidate stabilizer at intervals of 1 day. The immune reaction between CRP and anti-CRP antibodies was performed, and the absorbance after the immune reaction was measured to obtain the measured values ​​on the 1st, 2nd, 3rd, 4th, 5th, 6th and 7th days of storage.

[0185] The deviation (skew) is calculated based on the baseline measurement and the measurements obtained during the storage period.

[0186] In addition, for each condition, the experiment was repeated five times, and the precision (CV) was calculated.

[0187] Table 5 below lists the two candidate stabilizers, and stabilizers whose deviation (skew) measured during the entire storage period was within 20% were evaluated as effectively ensuring the storage stability of the immunoreagent (R2). Stabilizers whose measured precision (CV) was within 10% were evaluated as having excellent precision. Table 5 shows the experimental conditions according to the type and concentration of the selected stabilizer.

[0188] Table 5: Experimental conditions according to the type and concentration of the selected stabilizer

[0189]

[0190] 2. Experimental Results

[0191] Figure 14 This is a graph showing the results of accelerated stability evaluation of stabilizers added to anti-CRP antibodies according to Examples of the present application.

[0192] For the stabilizer trehalose and CRP serum (10 mg / L, low) samples, a deviation (skewness) exceeding 20% ​​was observed for the measurements on storage days 5 and 7 compared to the baseline measurement on day 0. In addition, for the trehalose and CRP serum (10 mg / L, low) samples, the precision (CV) was observed to remain below 10% throughout the storage period.

[0193] For the stabilizer trehalose and CRP serum (50 mg / L, medium) samples, it was confirmed that the measured values ​​remained within a deviation (skew) range of 20% compared to the baseline measurement value on day 0 until storage day 7. In addition, for the trehalose and CRP serum (50 mg / L, medium) samples, it was observed that the precision (CV) remained below 10% throughout the storage period.

[0194] For the stabilizer trehalose and CRP serum (150 mg / L, high) samples, it was confirmed that the measured values ​​remained within a deviation (skew) range of 20% compared to the baseline measurement value on day 0 until storage day 7. In addition, for the trehalose and CRP serum (150 mg / L, high) samples, it was observed that the precision (CV) remained below 10% throughout the storage period.

[0195] For the stabilizer sucrose and CRP serum (10 mg / L, low) samples, it was confirmed that the measured values ​​remained within a deviation (skewness) of 20% compared to the baseline measurement value on Day 0 until storage day 7. However, for the stabilizer sucrose and CRP serum (10 mg / L, low) samples, the precision (CV) was observed to be greater than 10% during the entire storage period.

[0196] For the stabilizer sucrose and CRP serum (50 mg / L, medium) samples, it was confirmed that the measured values ​​remained within a deviation (skew) range of 20% compared to the baseline measurement value on day 0 until storage day 7. However, for the stabilizer sucrose and CRP serum (50 mg / L, medium) samples, the precision (CV) was observed to exceed 10% on storage days 3, 6, and 7.

[0197] For the stabilizer sucrose and CRP serum (150 mg / L, high) samples, it was confirmed that the measured values ​​remained within a deviation (skew) range of 20% compared to the baseline measurement value on day 0 until storage day 7. However, for the stabilizer sucrose and CRP serum (150 mg / L, high) samples, the precision (CV) was observed to exceed 10% on storage day 3.

[0198] Through this experimental example, it was confirmed that both sucrose stabilizer and trehalose stabilizer can improve storage stability with high accuracy over most CRP concentration ranges. However, additional experiments were performed to further ensure storage stability and improve accuracy over certain CRP concentration ranges.

[0199] <Experimental Example 6: Accelerated Stability Evaluation of Stabilizers (3)>

[0200] 1. Experimental Methods

[0201] The same experimental method as in Experimental Example 5 was used to evaluate the use of a stabilizer that was a mixture of sucrose and trehalose. Specifically, a stabilizer consisting of a mixture of sucrose and trehalose (hereinafter referred to as a "mixed stabilizer") was prepared. In addition, the mixed stabilizer was added to latex particles coated with anti-CRP antibodies and dried. Reagent solution and 3-level CRP serum samples (CRP 10 mg / L, 50 mg / L, 150 mg / L) were added to the dried composition, and an immune reaction between CRP and anti-CRP antibodies was performed. After the immune reaction, the absorbance was measured to obtain a baseline measurement value for storage day 0.

[0202] The dried composition containing the mixed stabilizer and the latex particles coated with the anti-CRP antibody was stored in an oven at 50°C. The reagent solution was stored separately under refrigeration. Over a period of 7 days, the reagent solution and the 3-level CRP serum samples (CRP 10 mg / L, 50 mg / L, 150 mg / L) were added to the dried composition containing the mixed stabilizer at intervals of 1 day. An immune reaction between CRP and the anti-CRP antibody was performed, and the absorbance after the immune reaction was measured to obtain measurement values ​​on the 1st, 2nd, 3rd, 4th, 5th, 6th and 7th days of storage.

[0203] The deviation (skew) is calculated based on the baseline measurement and the measurements obtained during the storage period.

[0204] In addition, for each CRP concentration condition, the experiment was repeated 5 times, and the precision (CV) was calculated.

[0205] The mixed stabilizers are described in the following Table 6. Table 6 shows the stabilizers used in Experimental Example 6.

[0206] Stabilizers whose deviation (skew) measured during the entire storage period was within 20% were evaluated as effectively ensuring the storage stability of the immunoreagent (R2). Stabilizers whose precision (CV) measured was within 10% were evaluated as having excellent precision.

[0207] Table 6: Stabilizers used in Experimental Example 6

[0208] condition 1 stabilizer 100mM trehalose + 100mM sucrose

[0209] 2. Experimental Results

[0210] Figure 15 This is a graph showing the results of accelerated stability evaluation of stabilizers added to anti-CRP antibodies according to Examples of the present application.

[0211] For the mixed stabilizer and CRP serum (10 mg / L, low) samples, it was confirmed that the measured values ​​remained within a deviation (skew) range of 20% compared to the baseline measurement value on Day 0 until storage day 7. In addition, for the mixed stabilizer and CRP serum (10 mg / L, low) samples, it was observed that the precision (CV) remained below 10% throughout the storage period.

[0212] For the mixed stabilizer and CRP serum (50 mg / L, medium) samples, it was confirmed that the measured values ​​remained within a deviation (skew) range of 20% compared to the baseline measurement value on day 0 until storage day 7. In addition, for the mixed stabilizer and CRP serum (50 mg / L, medium) samples, it was observed that the precision (CV) remained below 10% throughout the storage period.

[0213] For the mixed stabilizer and CRP serum (150 mg / L, high) samples, it was confirmed that the measured values ​​remained within a deviation (skew) range of 20% compared to the baseline measurement value on day 0 until storage day 7. In addition, for the mixed stabilizer and CRP serum (150 mg / L, high) samples, it was observed that the precision (CV) remained below 10% during the entire storage period.

[0214] This demonstrates that using a stabilizer consisting of a mixture of sucrose and trehalose can improve storage stability with high accuracy across all CRP concentration ranges. According to Experimental Example 6, the stabilizer in a preferred embodiment of the present application may contain both trehalose and sucrose.

[0215] 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.

[0216] <Experimental Example 7: Accelerated Stability Evaluation of Stabilizers (4)>

[0217] 1. Experimental Methods

[0218] Compared with Experimental Example 6, the storage stability of a stabilizer consisting of a mixture of sucrose and trehalose under modified storage temperature conditions was evaluated. Specifically, a stabilizer consisting of a mixture of sucrose and trehalose was prepared. In addition, the mixed stabilizer was added to latex particles coated with anti-CRP antibodies and dried. Reagent solution and CRP control solution (low, high) were each added to the dried composition to facilitate the immune reaction between CRP and anti-CRP antibodies. Subsequently, the absorbance after the immune reaction was measured to obtain a baseline measurement value for storage day 0.

[0219] In addition, a dried composition was prepared by adding a mixed stabilizer to latex particles coated with anti-CRP antibodies and stored in an oven at 37°C. In Experimental Example 7, the reagent solution was also stored in an oven at 37°C. Reagent solution and CRP control solution (low, high) were added to the dried composition containing the mixed stabilizer at regular intervals over 63 days to facilitate the immune reaction between CRP and anti-CRP antibodies. The absorbance was measured after the immune reaction to obtain measurement values ​​on the 4th, 7th, 14th, 21st, 28th, 35th, 42nd, 56th and 63rd days.

[0220] The deviation (skew) was calculated based on the baseline measurement value and the measurement value obtained during the storage period.In addition, for each CRP concentration condition, the experiment was repeated five times and the precision (CV) was calculated.

[0221] The mixed stabilizers are described in Table 7 below. Table 7 shows the stabilizers used in Experimental Example 7. Stabilizers whose deviation (skew) measured over the entire storage period was within 20% were evaluated as effectively ensuring the storage stability of the immunoreagent (R2). Stabilizers whose measured precision (CV) was within 10% were evaluated as having excellent precision.

[0222] Table 7: Stabilizers used in Experimental Example 7

[0223] condition 1 stabilizer 100mM trehalose + 100mM sucrose

[0224] 2. Experimental Results

[0225] Figure 16 This is a graph showing the results of accelerated stability evaluation of stabilizers added to anti-CRP antibodies according to Examples of the present application.

[0226] It was confirmed that the deviation (skew) of the measured values ​​of the mixed stabilizer with the CRP control solution (low) sample from the baseline measured value on day 0 remained within 20% until storage day 63. In addition, for the mixed stabilizer with the CRP control solution (low) sample, the precision (CV) was observed to be less than 10% during the entire storage period.

[0227] It was confirmed that the deviation (skew) of the measured values ​​of the mixed stabilizer with the CRP control solution (high) sample from the baseline measured value on day 0 remained within 20% until storage day 63. In addition, for the mixed stabilizer with the CRP control solution (high) sample, the precision (CV) was observed to be less than 10% during the entire storage period.

[0228] This indicates that when a stabilizer consisting of a mixture of sucrose and trehalose is used, storage stability can be maintained with high accuracy even after storage at 37°C for more than 63 days. According to Experimental Example 7, the stabilizer in a preferred embodiment of the present application may contain both trehalose and sucrose. More preferably, the stabilizer may contain substantially equal concentrations of trehalose and sucrose.

[0229] Figure 17 : is a diagram showing identification information included in the CRP quantitative analysis kit according to an example of the present application.

[0230] The CRP quantitative analysis kit 10 according to one embodiment of the present application may include identification information for distinguishing the biological sample. For example, the CRP 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.

[0231] The quantitative analysis device (or quantitative analysis apparatus) described later can obtain identification information to identify the type of biological sample to be analyzed by the CRP quantitative analysis kit 10. For example, the quantitative analysis apparatus can determine that the biological sample to be analyzed is CRP based on the barcode. In this case, the quantitative analysis apparatus can be configured to perform quantitative analysis of the biological sample by executing a pre-stored analysis protocol associated with the identified type based on the identified type of the biological sample. For example, the quantitative analysis apparatus can be implemented to execute a pre-stored CRP analysis protocol based on the identification that the type of the biological sample to be analyzed is CRP, thereby performing quantitative analysis of CRP.

[0232] 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 location on the CRP quantitative analysis kit 10.

[0233] The quantitative analysis device according to an embodiment of the present disclosure can be configured to perform quantitative analysis of a biological sample (e.g., 1,5-anhydro-D-glucitol (1,5-AG), glycated albumin, CRP, etc.). Furthermore, the quantitative analysis device can include a communication module (which can also be referred to as a transceiver), a memory, and / or a processor.

[0234] 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.

[0235] 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.

[0236] 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.

[0237] 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.

[0238] According to embodiments of the present application, a CRP quantitative analysis kit, a CRP quantitative analysis method, and / or an apparatus for performing the method can provide a hemolytic reagent capable of performing quantitative analysis of CRP with high accuracy over a wide concentration range without affecting immune response.

[0239] According to an embodiment of the present application, a CRP quantitative analysis kit, a CRP quantitative analysis method, and / or an apparatus for implementing the method may provide a stabilizer, which enables the solid-phase dried anti-CRP antibody to be fully redissolved to participate in the immune reaction, thereby enabling highly precise and accurate quantitative analysis of CRP within a wide concentration range and improving storage stability.

[0240] 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.

[0241] 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.

[0242] 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 C-reactive protein, comprising: a first composition comprising a hemolytic agent for hemolyzing at least a portion of blood cells in a blood sample; and A second composition comprising an anti-C-reactive protein antibody for antigen-antibody reaction with the C-reactive protein contained in the hemolyzed sample, The kit for quantitative analysis of C-reactive protein comprises at least one reagent fixing unit, The first composition and the second composition are each independently fixed to the at least one reagent fixing portion, and The second composition further comprises a stabilizer for the stability of the anti-C-reactive protein antibody.

2. The kit for quantitative analysis of C-reactive protein according to claim 1, wherein the hemolytic agent is selected from the group consisting of sodium deoxycholate (SDO) and saponin. 3 . The kit for quantitative analysis of C-reactive protein according to claim 1 , wherein the hemolytic reagent is sodium deoxycholate (SDO). 4 . The kit for quantitative analysis of C-reactive protein according to claim 1 , wherein the stabilizer is selected from the group consisting of trehalose and sucrose. The kit for quantitative analysis of C-reactive protein according to claim 1 , wherein the stabilizer comprises both trehalose and sucrose. The kit for quantitative analysis of C-reactive protein according to claim 5 , wherein the stabilizer comprises trehalose and sucrose at substantially the same concentration ratio.

7. The kit for quantitative analysis of C-reactive protein according to claim 1, wherein the at least one reagent fixing portion comprises: a first reagent fixing portion, located in the first compartment area of ​​the reagent kit, and a second reagent fixing portion, located in a second compartment area separated from the first compartment area; The first composition is fixed to the first reagent fixing portion, and the second composition is fixed to the second reagent fixing portion.

8. The kit for quantitative analysis of C-reactive protein according to claim 7, wherein the kit comprises a solution reservoir disposed in the first compartment area, The solution reservoir stores the reaction buffer, and When the sample collector containing the blood sample is inserted into the receiving portion of the reagent cartridge, the reaction buffer stored in the solution reservoir and the blood sample contained in the sample collector flow into the mixing area in which the first reagent fixing portion in the first compartment area is provided. At least a portion of the blood cells in the blood sample are hemolyzed in the mixing zone. 9 . The kit for quantitative analysis of C-reactive protein according to claim 8 , wherein the reaction buffer comprises glycine, sodium chloride, sodium azide, ethylenediaminetetraacetic acid disodium salt dihydrate, and bovine serum albumin (BSA).

10. The kit for quantitative analysis of C-reactive protein according to claim 8, wherein the kit further comprises a flow path connecting the first compartment region with the second compartment region, and When the cartridge is rotated by a predetermined angle due to an applied external force, the hemolyzed sample in which at least a portion of the blood cells are hemolyzed flows from the mixing zone in the first compartment zone to the second compartment zone through the flow path.

11. The kit for quantitative analysis of C-reactive protein according to claim 10, The kit further comprises a measuring unit for quantifying the C-reactive protein contained in the blood sample, and The hemolyzed sample in which the at least a portion of the blood cells is hemolyzed passes through the measurement unit via the flow path while moving from the mixing region in the first compartment region to the second reagent fixing portion in the second compartment region.

12. The kit for quantitative analysis of C-reactive protein according to claim 11, The quantification of the C-reactive protein is performed based on a first turbidity measured by the measurement unit before the antigen-antibody reaction and a second turbidity measured by the measurement unit after the antigen-antibody reaction is performed at the second reagent immobilization section. 13 . The kit for quantitative analysis of C-reactive protein according to claim 1 , wherein the second composition comprises the stabilizer for immobilizing the anti-C-reactive protein antibody in a solid state on the at least one reagent immobilization portion. 14 . The kit for quantitative analysis of C-reactive protein according to claim 1 , wherein the anti-C-reactive protein antibody is provided in a form coated on latex particles.

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