A kit for quantitative analysis of c-reactive protein, a method for quantitative analysis of c-reactive protein, and a device for performing the same

Abstract

A method for quantitative analysis of C-Reactive Protein according to one embodiment of the present application comprises: a hemolysis step of producing a hemolyzed sample by hemolyzing blood cells in a blood sample using a hemolysis reagent; a reaction step of performing an antigen-antibody reaction between the CRP contained in the hemolyzed sample and the Anti-CRP antibody by contacting the hemolyzed sample with an Anti-CRP antibody; and a quantification step of quantifying the CRP contained in the blood sample based on the antigen-antibody reaction, wherein the Anti-CRP antibody is in contact with the hemolyzed sample while fixed to a reagent fixation part by a stabilizer.

Description

Technology Field

[0001] The present application relates to the quantitative analysis of biological samples, and more specifically, to a kit for the quantitative analysis of C-Reactive Protein (CRP), a method for the quantitative analysis of CRP, and / or a quantitative analysis apparatus for performing the same. Background Technology

[0003] C-Reactive Protein (CRP) is a substance produced by the liver and secreted into the bloodstream within hours of infection or inflammation; blood CRP levels have been utilized as a useful indicator for monitoring therapeutic effects in infectious and autoimmune diseases. Meanwhile, with the publication of papers indicating that chronic inflammation acts as a mediator in the development of type 2 diabetes, research and the development of technologies to predict the onset of type 2 diabetes by measuring CRP, an indicator related to inflammation, are garnering attention. (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 type 2 diabetes, 2013)

[0004] The prior art according to the patent literature below for CRP quantitative analysis quantified CRP contained in a blood sample without hemolyzing the red blood cells contained in the blood sample. However, since the blood cells were not hemolyzed, the hematocrit, which is the proportion of red blood cells in the blood sample, had to be determined separately, and additional modifications to the substrate were required to prevent hemolysis of red blood cells, which had the limitation that CRP quantitative analysis was relatively complex. Furthermore, there was a lack of research on stabilizers to increase the storage stability of reagents included in CRP quantitative analysis kits in the past.

[0005] Therefore, there is a need for technological development and research on CRP quantitative analysis that can improve usability and increase the storage stability of reagents. Prior art literature

[0007] 1. Korean Published Patent Application, No. 10-2023-0038489 (March 20, 2023) The problem to be solved

[0008] One problem that the present invention aims to solve is to provide a kit for the quantitative analysis of C-reactive protein with improved usability using a hemolytic reagent that does not affect the quantitative analysis of CRP, a method for the quantitative analysis of C-reactive protein, and / or an apparatus for performing the same.

[0009] One problem that the present invention aims to solve is to provide a kit for quantitative analysis of C-reactive proteins to increase the storage stability of reagents, a method for quantitative analysis of C-reactive proteins, and / or an apparatus for performing the same.

[0010] The problems that the present invention aims to solve are not limited to those described above, and problems not mentioned will be clearly understood by those skilled in the art from this specification and the attached drawings. means of solving the problem

[0012] A method for quantitative analysis of C-Reactive Protein according to one embodiment of the present application comprises: a hemolysis step of producing a hemolyzed sample by hemolyzing blood cells in a blood sample using a hemolysis reagent; a reaction step of performing an antigen-antibody reaction between the CRP contained in the hemolyzed sample and the Anti-CRP antibody by contacting the hemolyzed sample with an Anti-CRP antibody; and a quantification step of quantifying the CRP contained in the blood sample based on the antigen-antibody reaction, wherein the Anti-CRP antibody may be characterized by contacting the hemolyzed sample while fixed to a reagent fixation part by a stabilizer.

[0013] The means for solving the problem of the present invention are not limited to the means for solving the problem described above, and unmentioned means for solving the problem will be clearly understood by those skilled in the art from this specification and the attached drawings. Effects of the invention

[0015] According to a kit for quantitative analysis of C-reactive protein, a method for quantitative analysis of C-reactive protein, and / or an apparatus for performing the same according to one embodiment of the present application, a hemolytic reagent capable of performing quantitative analysis of CRP with high precision over a wide range of CRP concentrations without affecting the immune response can be provided.

[0016] According to a kit for quantitative analysis of C-reactive protein, a method for quantitative analysis of C-reactive protein, and / or an apparatus for performing the same according to one embodiment of the present application, a stabilizer can be provided that allows solid-dried Anti-CRP antibodies to sufficiently re-dissolve to participate in an immune response, enables quantitative analysis of CRP with high precision and accuracy over a wide range of CRP concentrations, and can increase storage stability.

[0017] The effects of the present invention are not limited to the effects described above, and unmentioned effects will be clearly understood by those skilled in the art from this specification and the accompanying drawings. Brief explanation of the drawing

[0019] FIG. 1 is a schematic diagram of a kit for quantitative analysis of C-reactive protein according to one embodiment of the present application. FIG. 2 is a schematic diagram of a sample collector of a kit for quantitative analysis of C-reactive protein according to one embodiment of the present application. FIG. 3 is a schematic diagram of the main cartridge of a kit for quantitative analysis of C-reactive protein according to one embodiment of the present application. FIG. 4 is a schematic diagram of a solution cell of a kit for quantitative analysis of C-reactive protein according to one embodiment of the present application. FIG. 5 is a diagram illustrating the manner in which a reaction buffer stored in a solution cell flows out into the mixing area of ​​the main cartridge as a sample collector according to one embodiment of the present application is inserted into the main cartridge. FIGS. 6 and 7 are drawings showing the specific structure of a kit for quantitative analysis of C-reactive protein according to one embodiment of the present application. FIG. 8 is a diagram illustrating an analysis aspect for quantifying C-reactive protein in a blood sample according to one embodiment of the present application. FIG. 9 is a table showing the results of evaluating the hemolytic effect of each hemolytic reagent according to one embodiment of the present application. FIG. 10 is a graph showing the results of evaluating antigen-antibody reactivity for each hemolytic reagent according to one embodiment of the present application. FIG. 11 is a graph showing the results of evaluating antigen-antibody reactivity for each hemolytic reagent according to one embodiment of the present application. FIG. 12 is a graph and diagram showing the results of evaluating the accelerated stability according to the stabilizer added to the Anti-CRP antibody according to one embodiment of the present application. FIG. 13 is a graph showing the results of evaluating the accelerated stability according to the stabilizer added to the Anti-CRP antibody according to one embodiment of the present application. FIG. 14 is a graph showing the results of evaluating the accelerated stability according to the stabilizer added to the Anti-CRP antibody according to one embodiment of the present application. FIG. 15 is a graph showing the results of evaluating the accelerated stability according to the stabilizer added to the Anti-CRP antibody according to one embodiment of the present application. FIG. 16 is a diagram showing identification information included in a kit for quantitative analysis of C-reactive proteins according to one embodiment of the present application. Specific details for implementing the invention

[0020] The aforementioned objectives, features, and advantages of the present application will become more apparent from the following detailed description in conjunction with the accompanying drawings. However, as the present application is subject to various modifications and may have various embodiments, specific embodiments are illustrated in the drawings and described in detail below.

[0021] Throughout the specification, identical reference numbers generally represent identical components. Additionally, components with identical functions within the same scope of concept appearing in the drawings of each embodiment are described using the same reference numeral, and redundant descriptions thereof are omitted.

[0022] If it is determined that a detailed description of known functions or configurations related to this application could unnecessarily obscure the essence of this application, such detailed description is omitted. Furthermore, numbers used in the description of this specification (e.g., First, Second, etc.) are merely identifiers to distinguish one component from another.

[0023] Furthermore, the suffix "bu" for components used in the following embodiments is assigned or used interchangeably solely for the ease of drafting the specification, and does not inherently possess a distinct meaning or role.

[0024] In the following examples, singular expressions include plural expressions unless the context clearly indicates otherwise.

[0025] In the following embodiments, terms such as "include" or "have" mean that the features or components described in the specification are present, and do not preclude the possibility that one or more other features or components may be added.

[0026] In the drawings, the size of components may be exaggerated or reduced for convenience of explanation. For example, the size and thickness of each component shown in the drawings are arbitrarily depicted for convenience of explanation, and the present invention is not necessarily limited to what is illustrated.

[0027] Where an embodiment can be implemented differently, the order of a particular process may be performed differently from the order described. For example, two processes described consecutively may be performed substantially simultaneously or proceed in the reverse order of the description.

[0028] In the following embodiments, when components are described as being connected, it includes not only cases where the components are directly connected but also cases where components are interposed between other components and are indirectly connected. For example, when components are described as being electrically connected in this specification, it includes not only cases where the components are directly electrically connected but also cases where components are interposed between other components and are indirectly electrically connected.

[0029] A kit for quantitative analysis of C-reactive protein according to one embodiment of the present application may comprise: 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 C-reactive protein (CRP) contained in the hemolytic sample, wherein the kit for quantitative analysis of CRP comprises at least one reagent fixing part, the first composition and the second composition are each independently fixed to the reagent fixing part, and the second composition may further comprise a stabilizer for the stability of the anti-CRP antibody.

[0030] According to one embodiment of the present application, the hemolytic reagent may be selected from the group consisting of Sodium Deoxycholate (SDO) and Saponin.

[0031] According to one embodiment of the present application, the hemolytic reagent may be Sodium Deoxycholate (SDO).

[0032] According to one embodiment of the present application, the stabilizer may be selected from the group consisting of trehalose and sucrose.

[0033] According to one embodiment of the present application, the stabilizer may include both trehalose and sucrose.

[0034] According to one embodiment of the present application, the stabilizer may comprise trehalose and sucrose in substantially the same concentration ratio.

[0035] According to one embodiment of the present application, the at least one reagent fixing part may include a first reagent fixing part located in a first compartment area of ​​the CRP quantitative analysis kit and a second reagent fixing part located in a second compartment area partitioned from the first compartment area, wherein the first composition is fixed to the first reagent fixing part and the second composition is fixed to the second reagent fixing part.

[0036] According to one embodiment of the present application, the CRP quantitative analysis kit may be configured such that, when a sample collector containing a blood sample is inserted into a receiving portion of the CRP quantitative analysis kit, the reaction buffer contained in the solution cell and the blood sample contained in the sample collector are introduced into a mixing portion in which a first reagent fixing portion is placed within the first compartment, and at least a portion of the blood cells in the blood sample are hemolyzed on the mixing portion.

[0037] According to one embodiment of the present application, the reaction buffer may comprise glycine, sodium chloride, sodium azide, ethylenediaminetetraacetic acid disodium salt dihydrate, and bovine serum albumin (BSA).

[0038] According to one embodiment of the present application, the CRP quantitative analysis kit further comprises a channel connecting the first compartment region and the second compartment region, wherein a sample in which at least a portion of the blood cells have been hemolyzed is introduced from the mixing region within the first compartment region to the second compartment region through the channel as the CRP quantitative analysis kit is rotated by a predetermined angle by an external force applied to the CRP quantitative analysis kit.

[0039] According to one embodiment of the present application, the CRP quantitative analysis kit further comprises a measuring unit for quantifying CRP contained in the blood sample, wherein the sample in which at least a portion of the blood cells have been hemolyzed may be configured to pass through the measuring unit while moving from the mixing area within the first compartment area to the second reagent fixation unit within the second compartment area through the flow path.

[0040] According to one embodiment of the present application, the quantification of the CRP may be characterized by being performed based on a first turbidity measured through the measuring unit prior to the antigen-antibody reaction and a second turbidity measured through the measuring unit after the antigen-antibody reaction performed on the second reagent fixation unit.

[0041] According to one embodiment of the present application, the second composition may be characterized by including the stabilizer to fix the Anti-CRP antibody in a solid state to the reagent fixation portion.

[0042] According to one embodiment of the present application, the Anti-CRP antibody may be provided in a form in which the Anti-CRP antibody is coated on a Latex particle.

[0044] Hereinafter, with reference to FIGS. 1 to 16, the structure of a kit for quantitative analysis of C-reactive protein, a method for quantitative analysis of C-reactive protein, and / or a device for quantitative analysis of C-reactive protein according to one embodiment of the present application will be described in more detail.

[0045] FIG. 1 is a schematic diagram of a kit for quantitative analysis of C-reactive protein according to one embodiment of the present application.

[0046] A kit (10) for quantitative analysis of C-reactive protein (hereinafter referred to as CRP) according to one embodiment of the present application may include a sample collector (100) for supplying a biological sample and / or a main cartridge (200) into which the sample collector (100) can be inserted and received.

[0047] FIG. 2 is a schematic diagram of a sample collector (100) of a kit for quantitative analysis of C-reactive protein according to one embodiment of the present application.

[0048] A sample collector (100) according to one embodiment of the present application may be configured to collect a predetermined amount of a biological sample (e.g., a blood sample) to be analyzed. Specifically, the sample collector (100) may include a sample injection part (101) in the form of a capillary, and the sample collector (100) may collect a biological sample (e.g., a blood sample) to be analyzed through a capillary provided in the sample injection part (101) and inject the collected biological sample into a main cartridge (200).

[0049] Furthermore, a sample collector (100) according to one embodiment of the present application may include a protrusion (102) that contacts a solution cell (301) provided within the main cartridge (200) and pushes the solution cell (301) into the main cartridge when the sample collector (100) is inserted into the receiving portion (201) of the main cartridge (200) to be described later. Specifically, the protrusion (102) may be provided on the sample collector (100) at a position where it can contact the solution cell (301) when the sample collector (100) is inserted into the receiving portion (201) of the main cartridge (200). Accordingly, as the sample collector (100) is inserted into the receiving portion (201) of the main cartridge (200), the solution cell (301) may be moved toward the inside of the main cartridge (200), thereby causing the cover tape of the solution cell (301) to be removed or ruptured. This will be described in more detail in relation to Fig. 5.

[0050] Furthermore, a sample collector (100) according to one embodiment of the present application may further include a handle (103) and / or a fastener (104). Specifically, the handle (103) is a component for facilitating the transport or use of the sample collector (100) and is not limited to the structure shown in FIG. 2. A fastener (104) may be provided on one side of the sample collector (103) to secure the sample collector (100) onto the main cartridge (200) when the sample collector (100) is inserted into the main cartridge (200). The fastener (104) is provided with a size and shape corresponding to a fixing groove (211) provided in the receiving portion (201) of the main cartridge (200) to be described later, so that the fastener (104) of the sample collector (100) and the fixing groove (211) of the main cartridge (200) can interlock with each other. Accordingly, the inserted sample collector (100) is fixed to the main cartridge (200), so that even if the CRP quantitative analysis kit (10) rotates while the analysis is performed, the sample collector (100) may not move or become detached.

[0051] FIG. 3 is a schematic diagram of a main cartridge (200) of a kit for quantitative analysis of C-reactive protein according to one embodiment of the present application.

[0052] A main cartridge (200) according to one embodiment of the present application may include a receiving portion (201) into which a sample collector (100) can be inserted. Furthermore, the receiving portion (201) may include a fixing groove (211) configured to engage with a fixing bracket (104) for fixing the sample collector (100) as described above.

[0053] A main cartridge (200) according to one embodiment of the present application may include a moving frame (203) for fixing and moving a solution cell (301) provided inside the main cartridge. Specifically, the moving frame (203) may include a moving path configured to fix the solution cell (301) before insertion of the sample collector (100), and configured so that when the sample collector (100) is inserted, the solution cell (301) pushed by the protrusion (102) of the sample collector (100) moves.

[0054] A main cartridge (200) according to one embodiment of the present application may include a cover tape rupture portion (202) configured to remove or rupture the cover tape (302) of a solution cell (301) to be described later by moving the solution cell (302) through a moving frame (203). This will be explained in more detail in relation to FIG. 5.

[0055] A main cartridge (200) according to one embodiment of the present application may include a mixing section (204, or mixing area) in which a biological sample (e.g., blood sample) flowing out from a sample collector (100) inserted through a receiving section (201) and a reaction buffer flowing out from a solution cell (301) are mixed.

[0056] A main cartridge (200) according to one embodiment of the present application may include at least one sample fixation part (205, or referred to as a reagent fixation part) into which a chemical sample capable of inducing an enzymatic reaction and / or an antigen-antibody reaction by reacting with a reaction buffer mixed in a mixing part (204) and a biological sample is introduced.

[0057] A main cartridge (200) according to one embodiment of the present application may include a flow path (206) through which a reaction buffer mixed in a mixing section (204) and a biological sample can move. Specifically, an analysis sample may move through the flow path (206) between a mixing section (204), a sample fixing section (205), and / or a measuring section (207) that optically measures the analysis sample, and the structure of the flow path (206) is not limited as long as it is designed so that the analysis sample can move by gravity when the reaction cartridge (200) is tilted.

[0058] A main cartridge (200) according to one embodiment of the present application may include a measuring unit (207) for measuring the reaction results performed in a sample fixing unit (205). A CRP quantitative analysis device according to one embodiment of the present application may quantify an analysis sample (e.g., CRP) through optical analysis such as UV / VIS via the measuring unit (207). For example, the measuring unit (207) of the main cartridge (200) may quantify an analysis sample (e.g., CRP) by measuring a change in turbidity (or absorbance) by an immunoturbidity method.

[0059] A main cartridge (200) according to one embodiment of the present application may include a waste liquid treatment unit (208) for collecting waste liquid that has been analyzed through a measuring unit (207). Waste liquid, which is a type of medical waste, can be collected and processed separately through the waste liquid treatment unit (208). The collection of waste liquid through the waste liquid treatment unit (208) can be performed by absorbing waste liquid by placing highly absorbent cotton, an absorbent filter, and an absorbent raw material made of a polymer material into the waste liquid treatment unit (208).

[0060] A main cartridge (200) according to one embodiment of the present application may include an air outlet (209) for the smooth movement of waste liquid and absorption into an absorption material. Through the air outlet (209), the movement of waste liquid to a waste liquid treatment unit (208) and the absorption for waste liquid collection can be performed more smoothly.

[0061] A main cartridge (200) according to one embodiment of the present application may include a handle (210) to facilitate carrying or using the main cartridge (200), and the handle (210) is not limited to the structure shown in FIG. 3.

[0063] Hereinafter, with reference to FIGS. 4 and 5, the manner in which the reaction buffer of the solution cell (301) flows into the mixing section (204) of the main cartridge (200) when the sample collector (100) is inserted into the receiving section (201) of the main cartridge (200) will be explained in more detail. FIG. 4 is a schematic diagram of the solution cell (301) of a kit for quantitative analysis of C-reactive protein according to one embodiment of the present application. FIG. 5 is a diagram for explaining the manner in which the reaction buffer stored in the solution cell (301) flows out into the mixing section of the main cartridge as the sample collector (100) according to one embodiment of the present application is inserted into the main cartridge (200).

[0064] A main cartridge (200) according to one embodiment of the present application may include a solution cell (301) in which a reaction buffer that participates in a reaction with a biological sample of a sample collector (100) is stored internally. The solution cell (301) has an opening at one end, and a cover tape (302) is attached to the opening to prevent the reaction buffer stored in the solution cell (301) from leaking out. Meanwhile, the opening of the solution cell (301) to which the cover tape (302) is attached may be provided to face the cover tape rupture portion (203) of the main cartridge (200). Before the sample collector (100) is inserted into the receiving portion (201) of the main cartridge (200), the opening of the solution cell (301) to which the cover tape (302) is attached may be provided to be spaced apart from the cover tape rupture portion (203). At this time, as described above, when the sample collector (100) is inserted into the receiving portion (201) of the main cartridge (200), as described above, the protrusion (102) of the sample collector (100) applies pressure to the other end provided on the opposite side of the opening to which the cover tape (302) of the solution cell (301) is attached, and the solution cell (301) moves through the path of the moving frame (203) due to the applied pressure. Accordingly, when the cover tape (302) of the solution cell (301) comes into contact with the cover tape rupture portion (203), the cover tape (302) of the solution cell (301) is removed or ruptured. At this time, the reaction buffer stored inside the solution cell (301) is moved to the mixing section (204) of the main cartridge (200) through the flow path or hollow formed in the cover tape rupture section (203), and is mixed with a biological sample (e.g., blood sample) supplied from the sample collector (100) on the mixing section (204). More specifically, the reaction buffer introduced into the mixing section (204) can be configured to come into contact with the sample injection section (101) of the sample collector (100), and upon contact, the biological sample within the sample injection section (101), which is in the form of a capillary tube, moves to the mixing section (204) of the main cartridge (200) through the sample injection section (101).Accordingly, on the mixing section (204) of the main cartridge (200), the reaction buffer stored in the solution cell (301) and the biological sample collected through the sample collector (100) can be mixed. Furthermore, at least some of the blood cells contained in the biological sample (e.g., blood sample) can be hemolyzed through a composition fixed on the sample fixing section (205) provided on the mixing section (204). This will be explained in more detail in relation to FIGS. 6 and FIGS. 7.

[0065] Meanwhile, in FIGS. 4 and 5, the shape of the solution cell (301) is illustrated and described in a specific form. However, this is merely an example, and the shape of the solution cell (301) may be any structure that facilitates the storage of the reaction buffer, and should not be interpreted as being limited to the shape illustrated in FIGS. 4 and 5. Furthermore, the material of the cover tape (302) is not particularly limited as long as it is a material that prevents the reaction buffer from leaking out but is easy to remove or rupture.

[0066] Furthermore, in FIG. 5, the cover tape rupture portion (302) is illustrated and described in a specific shape. However, this is merely an example, and the shape of the cover tape rupture portion (302) is not particularly limited as long as it is a structure that makes it easy to remove or rupture the cover tape (e.g., needle shape or edge shape).

[0068] A CRP quantitative analysis kit (10) according to one embodiment of the present application may be used to quantitatively analyze CRP present in blood (e.g., plasma, serum, and / or white blood). According to one embodiment, the CRP quantitative analysis kit (10) may be configured to quantitatively analyze CRP using an immunoassay. Specifically, the CRP quantitative analysis kit (10) may be configured to quantitatively analyze CRP using a turbidimetric immunoassay. Hereinafter, with reference to FIGS. 6 to 8, a more specific description will be provided regarding the quantitative analysis of CRP using the CRP quantitative analysis kit (10) according to one embodiment of the present application.

[0069] Quantitative analysis of CRP according to one embodiment of the present application may utilize a turbidimetric immunoassay.

[0070] Immunoturbidity utilizes antigen-antibody reactions. Specifically, antibodies corresponding to the target substance associated with the analysis sample are added to the sample, causing an antigen-antibody complex to form through the antigen-antibody reaction. The principle of immunoturbidity is to determine the concentration of the target substance by measuring the turbidity, based on the fact that the turbidity of the antigen-antibody complex is proportional to the amount of antigen of the test substance.

[0071] According to one embodiment of the present application, a kit (10) for quantitative analysis of CRP comprises a composition containing an Anti-CRP antibody corresponding to CRP (antigen), which is the target substance for measurement. At this time, a complex is formed through agglutination between the CRP antigen and the Anti-CRP antibody, and as the complex is formed, the turbidity changes. Therefore, based on the change in turbidity, the concentration of CRP, which is the target substance for measurement, contained in a blood sample can be quantified.

[0072] According to one embodiment of the present application, CRP can be quantitatively analyzed using the following antigen-antibody reaction.

[0073]

[0075] FIG. 6 is a diagram showing the specific structure of a kit (10) for quantitative analysis of C-reactive protein according to one embodiment of the present application. FIG. 7 is a diagram showing the specific structure of a kit (10) for quantitative analysis of C-reactive protein according to one embodiment of the present application.

[0076] A reaction buffer (B) may be stored in the solution cell (301) of the CRP quantitative analysis kit (10) according to one embodiment of the present application. As described above, the reaction buffer (B) stored in the solution cell (301) is moved to the mixing section (204) of the main cartridge (200) as the sample collector (100) is inserted. At this time, the reaction buffer (B) is mixed on the mixing section (204) with a composition (hereinafter referred to as the first composition) comprising a blood sample collected through the sample collector (100) and a hemolytic reagent (R1) fixed to at least one sample fixing section (205). During the mixing process, at least a portion of the blood cells contained in the blood sample may be lysed through the hemolytic reagent (R1). A reaction buffer (B) according to one embodiment may include glycine, sodium chloride, sodium azide, ethylenediaminetetraacetic acid disodium salt dihydrate, and bovine serum albumin (BSA).

[0077] A hemolytic reagent (R1) according to one embodiment may be selected from the group consisting of Sodium Deoxycholate (SDO), ASB-14, Saponin, and Triton X-100 (Tx-100). A hemolytic reagent (R1) according to one embodiment may be selected from the group consisting of Sodium Deoxycholate (SDO) and Saponin. According to a preferred embodiment, the hemolytic reagent (R1) may be Sodium Deoxycholate (SDO). According to a kit for quantitative analysis of C-reactive protein, a method for quantitative analysis of C-reactive protein, and / or an apparatus for performing the same according to a preferred embodiment of the present application, by using Sodium Deoxycholate (SDO) as the hemolytic reagent (R1), the effect of performing quantitative analysis of CRP with high precision over a wide range of CRP concentrations without affecting the immune response may be provided. However, this is merely an example, and any composition or any surfactant (Detergent) may be used as the hemolytic agent (R1) to achieve the purpose of hemolyzing blood cells without affecting the immune response (antigen-antibody reaction).

[0078] A sample in which at least a portion of the blood cells present in the blood sample have been hemolyzed by a hemolyzing reagent (R1) may be moved through a flow path (206) to a measuring section (207) and / or at least one sample fixing section (205). A composition (hereinafter referred to as the second composition) comprising an immune reaction reagent (R2) for an antigen-antibody reaction (or immune reaction) of CRP present in the hemolyzed sample may be fixed in at least one reagent fixing section (205). According to one embodiment, the immune reaction reagent (R2) may include an Anti-CRP antibody. According to a preferred embodiment, the Anti-CRP antibody of the immune reaction reagent (R2) may be provided in a form in which the Anti-CRP antibody is coated on a latex particle.

[0079] According to one embodiment of the present application, a first composition comprising a hemolytic reagent (R1) and a second composition comprising an immune response reagent (R2) may each be independently fixed on at least one reagent fixing part (205). According to one embodiment, the first composition comprising a hemolytic reagent (R1) may be fixed on a first reagent fixing part (205) located in a first compartment area (e.g., an area existing on the mixing part (204)). Meanwhile, the second composition comprising an immune response reagent (R2) may be fixed on a second reagent fixing part (205) located in a second compartment area partitioned from the first compartment area.

[0080] Referring to FIG. 7, the CRP quantitative analysis kit (10) may include a main body comprising an upper plate and a lower plate configured to face each other. At this time, a first composition containing a hemolytic reagent (R1) may be dispensed and dried into a first reagent fixing part (205-1) provided in a first compartment area of ​​the upper plate of the main body and / or a first reagent fixing part (205-2) provided in a first compartment area of ​​the lower plate of the main body. Furthermore, the first reagent fixing part (205-1) provided in the upper plate of the main body and the first reagent fixing part (205-2) provided in the lower plate of the main body may be configured to face each other.

[0081] Meanwhile, the second composition containing the immune reaction reagent (R2) can be dispensed and dried into the second reagent fixing part (205-3) located in the second compartment area of ​​the upper plate of the main body and / or the (205-4) located in the second compartment area of ​​the upper plate of the main body. Furthermore, the second reagent fixing part (205-3) provided on the upper plate of the main body and the second reagent fixing part (205-4) provided on the lower plate of the main body may be configured to face each other.

[0082] Meanwhile, as described above, the first compartment region where the first reagent fixation part (205-1 and / or 205-2) where the hemolytic reagent (R1) is fixed is located may be partitioned with the second compartment region where the second reagent fixation part (205-3 and / or 205-4) where the immune reaction reagent (R2) is fixed is fixed, and the first compartment region and the second compartment region may be connected through a channel (206).

[0083] According to one embodiment of the present application, a first composition comprising a hemolytic reagent (R1) and / or a second composition comprising an immunoreaction reagent (R2) may be fixed in a solid state on a reagent fixing part (205). Specifically, the first composition comprising a hemolytic reagent (R1) and / or the second composition comprising an immunoreaction reagent (R2) may be dispensed onto at least one reagent fixing part (205) and fixed in a dried state.

[0084] According to one embodiment of the present application, the second composition (and / or the first composition including the hemolytic reagent (R1)) comprising an immune response reagent (R2) may further include a stabilizer to increase the stability of the immune response reagent (R2, and / or the hemolytic reagent (R1)) and to fix the reagent in a solid state to the reagent fixing part (205).

[0085] A stabilizer according to one embodiment of the present application may be selected from the group consisting of trehalose and sucrose. According to a preferred embodiment of the present application, the stabilizer may include both trehalose and sucrose. According to a more preferred embodiment, the stabilizer may include trehalose and sucrose in substantially the same concentration ratio. According to a kit for quantitative analysis of C-reactive protein, a method for quantitative analysis of C-reactive protein, and / or an apparatus for performing the same according to one embodiment of the present application, by using a stabilizer comprising sucrose and trehalose configured in an optimal concentration ratio, 1)Allows the solid-dried Anti-CRP antibodies to sufficiently re-dissolve to participate in the immune response, 2) Quantitative analysis of CRP can be performed with high precision and accuracy over a wide range of CRP concentrations, and 3) The effect of increasing storage stability may be provided. However, this is merely an example, and any suitable substance may be used as a stabilizer to achieve the purpose of increasing the precision and accuracy of CRP quantitative analysis while enhancing the stability of immunoreaction reagents, etc.

[0087] FIG. 8 is a diagram illustrating an analysis aspect for quantifying C-reactive protein in a blood sample according to one embodiment of the present application.

[0088] Referring to FIG. 8, a CRP quantitative analysis method according to one embodiment of the present application can be implemented as follows.

[0089] 1) Prepare the main cartridge (200).

[0090] 2) Place the sample collector (100) on the receiving portion (201) of the main cartridge (200).

[0091] 3) Apply pressure to the sample collector (100) in a direction toward the receiving portion (201) of the main cartridge (200).

[0092] 4) As the protrusion (102) of the sample collector (100) applies pressure to the solution cell (301), the solution cell (301) moves in the direction of the cover tape rupture part (203) through the moving frame (203) as described above, and the cover tape rupture part (203) and the cover tape (301) of the solution cell (301) come into contact, so that the solution (Solution, i.e., reaction buffer) stored in the solution cell (301) flows into the mixing part (204) of the main cartridge (200).

[0093] 5) As the reaction buffer is introduced into the mixing section (204), it comes into contact with the blood sample of the sample collector (100), and the blood sample (e.g., in the form of plasma, serum, and / or white blood) is also introduced into the mixing section (204) of the main cartridge (200).

[0094] 6) At this time, on the mixing section (204), the hemolytic agent (R1), the reaction buffer (B), and the blood sample fixed in the first sample fixing section (205-1, and / or 205-2) provided in the mixing section (204) are mixed. Furthermore, at least a portion of the blood cells contained in the blood sample are hemolytically lysed by the hemolytic agent (R1).

[0095] Meanwhile, according to one embodiment of the present application, the CRP quantitative analysis kit (10) is rotated by a predetermined angle by an external force applied to the CRP quantitative analysis kit (10) (e.g., rotational force applied by a quantitative analysis device to be described later), and accordingly, a blood sample in the main cartridge (200) can be moved through the flow path (206) of the main cartridge (200) by gravity.

[0096] 7) A sample in which at least a portion of the blood cells have been hemolyzed by an external force applied to the CRP quantitative analysis kit (10) is moved to the measuring unit (207), and a background measurement value (first turbidity) related to the turbidity (or absorbance) before the antigen-antibody reaction is performed can be measured while the sample is positioned on the measuring unit (207).

[0097] Meanwhile, as described above, the CRP quantitative analysis kit (10) may include a measuring 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 measuring unit (207) while moving from the mixing unit (204), where the first sample fixing unit (R1) fixed with the hemolyzing reagent (R1) is located, to the second sample fixing unit (R2) fixed with the immune reaction reagent (R2) through the flow path (206). According to this embodiment, the turbidity before and after the antigen-antibody reaction can be easily measured, thereby providing the effect of more easily quantifying the CRP concentration, which is correlated with the amount of change in turbidity.

[0098] 8) A sample in which at least a portion of the blood cells have been hemolyzed by an external force applied to the CRP quantitative analysis kit (10) is moved from the measuring unit (207) to a second reagent fixing unit (205-3, and / or 205-4) in which an immune reaction reagent (R2) is fixed, and an antigen-antibody reaction (Ab-CRP Reaction of FIG. 8) between the CRP (Antibody) contained in the sample and the Anti-CRP antibody contained in the immune reaction reagent (R2) is carried out on the second reagent fixing unit (205-3 and / or 205-4). Through the antigen-antibody reaction, the CRP antigen forms an immune complex with the Anti-CRP antibody, thereby changing the turbidity of the sample.

[0099] 9) A sample in which an antigen-antibody reaction is performed by an external force applied to the CRP quantitative analysis kit (10) is moved from the second reagent fixing unit (205-3 and / or 205-4) to the measuring unit (207), and a measurement value (second turbidity) related to the turbidity (or absorbance) of the sample after the antigen-antibody reaction is performed can be measured while the sample is positioned on the measuring unit (207). Furthermore, CRP can be quantified based on the first turbidity measured through the measuring unit (207) before the antigen-antibody reaction and the second turbidity measured through the measuring unit (207) after the antigen-antibody reaction.

[0100] 10) The waste liquid (waste) for which quantitative analysis has been completed is moved from the measuring unit (207) to the waste liquid treatment unit (208) and collected by an external force applied to the CRP quantitative analysis kit (10).

[0102] Hereinafter, the present invention will be described in detail by experimental examples with reference to FIGS. 9 to 15. However, the experimental examples described below are merely illustrative and should not be interpreted as being limited thereto.

[0103] <Experimental Example 1: Evaluation of Hemolytic Effects by Hemolytic Reagent>

[0104] 1. Experimental Method

[0105] To select a hemolytic reagent capable of hemolytically lysing blood cells contained in blood samples—that is, a hemolytic reagent having a hemolytic effect—five candidate hemolytic reagents were selected. Furthermore, each candidate hemolytic reagent was applied to a Fresh Venous whole blood (HCT 48%) sample. Additionally, the background measurements (i.e., first turbidity or first absorbance) of the samples to which each candidate hemolytic reagent was applied were measured. Specifically, the first absorbance of the samples to which each candidate hemolytic reagent was applied was measured at a wavelength of 750 nm using an A1Care Analyzer from i-Sens.

[0106] The five candidate hemolytic reagents are as follows, and for each sample, if the first turbidity is less than 0.05, the candidate hemolytic reagent is evaluated to have a hemolytic effect on the blood sample.

[0107]

[0108] 2. Experimental Results

[0109] FIG. 9 is a table showing the results of evaluating the hemolytic effect of each hemolytic reagent according to one embodiment of the present application.

[0110] In the case of the SDC hemolytic reagent, the first absorbance was measured to be less than 0.05 in the concentration range of 0.12 - 0.20 (w / v%), and the first absorbance was measured to be greater than 0.05 in the concentration range of 0.10 - 0.11 (w / v%).

[0111] In the case of the ASB-14 hemolytic reagent, the first absorbance was measured to be less than 0.05 in the concentration range of 0.04 - 0.20 (w / v%), and the first absorbance was measured to be greater than 0.05 in the concentration range of 0.01 - 0.03 (w / v%).

[0112] In the case of the saponin hemolytic reagent, the first absorbance was measured to be less than 0.05 in the concentration range of 0.02 - 0.20 (w / v%), and the first absorbance was measured to be greater than 0.05 at a concentration of 0.01 (w / v%).

[0113] In the case of Tween20 hemolytic reagent, the first absorbance was measured to be greater than 0.05 in the concentration range of less than 1.00 (w / v%).

[0114] In the case of Tx-100 hemolytic reagent, the first absorbance was measured to be less than 0.05 in the concentration range of 0.04 - 0.20 (w / v%), and the first absorbance was measured to be greater than 0.05 in the concentration range of 0.02 - 0.03 (w / v%).

[0115] Through this experimental example, it was confirmed that the hemolytic effect on blood cells contained in blood samples was relatively low for the Tween 20 hemolytic reagent even at high concentrations (1.00 w / v %). On the other hand, it was confirmed that the hemolytic effect on blood cells contained in blood samples existed for most concentration ranges for the SDC, ASB-14, Saponin, and Tx-100 hemolytic reagents. 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).

[0117] <Experimental Example 2: Evaluation of Antigen-Antibody Reactivity by Hemolytic Reagent>

[0118] 1. Experimental Method

[0119] In order to select a hemolytic reagent that does not affect the immune response between CRP and Anti-CRP antibodies, four candidate hemolytic reagents (SDC, ASB-14, Saponin, Tx-100) with hemolytic effects were prepared through Experimental Example 1. The concentrations of each candidate hemolytic reagent were determined as follows, taking into account the effects on the sample, such as hematocrit.

[0120]

[0121] Furthermore, after applying each candidate hemolytic reagent to 7-Level CRP Serum Samples, an immune reaction was conducted between the hemolytic samples and latex particles coated with anti-CRP antibodies. Specifically, latex particles coated with anti-CRP antibodies supplied by RANDOX were concentrated and a stabilizer was added, and the process was configured to allow an immune reaction to proceed between the latex particles coated with anti-CRP antibodies and the hemolytic samples. After the immune reaction was conducted, the post-immune absorbance (referred to as second absorbance or second turbidity) of each sample was measured. Specifically, the second absorbance of the samples was measured at a wavelength of 750 nm using an A1Care Analyzer from i-Sens. Meanwhile, regarding the control group, the experiment was configured to measure the second absorbance after an immune reaction between samples without any hemolytic agent added (i.e., 7-Level CRP Serum Samples) and latex particles coated with anti-CRP antibodies. Furthermore, the second absorbance was measured for each sample while varying the concentration of 7-Level CRP Serum Samples from 0 to 195.8 mg / L.

[0122] 2. Experimental Results

[0123] FIG. 10 is a graph showing the results of evaluating antigen-antibody reactivity for each hemolytic reagent according to one embodiment of the present application.

[0124] In the case of the SDC hemolytic reagent, it was confirmed that it exhibited absorbance similar to the control group without added hemolytic agents (i.e., cases where the immune response is not affected by the hemolytic agent). In other words, it was confirmed that with the SDC hemolytic reagent, the antigen-antibody reaction between CRP and Anti-CRP antibodies is not affected by the hemolytic reaction.

[0125] In the case of the ASB-14 hemolytic reagent, it was confirmed that the absorbance according to CRP concentration decreased by more than 50% compared to the control group without the addition of the hemolytic agent (i.e., when the immune response is not affected by the hemolytic agent).

[0126] In the case of the saponin hemolytic reagent, it was confirmed that the absorbance according to CRP concentration was relatively high compared to the control group without the addition of the hemolytic agent (i.e., when the immune response is not affected by the hemolytic agent). In other words, it was confirmed that in the case of the saponin hemolytic reagent, the antigen-antibody reaction between CRP and anti-CRP antibodies is not affected by the hemolytic reaction.

[0127] In the case of the Tx-100 hemolytic reagent, no trend was observed between the measured absorbance and the CRP concentration.

[0128] In this experimental example, the hemolytic reagent (R1) according to a preferred embodiment of the present application may be selected from the group consisting of Sodium Deoxycholate (SDO) and Saponin.

[0130] <Experimental Example 3: Evaluation of Antigen-Antibody Reactivity by Hemolytic Reagent 2>

[0131] 1. Experimental Method

[0132] In order to select a hemolytic reagent that exhibits excellent precision without affecting the immune response between CRP and Anti-CRP antibodies, two candidate hemolytic reagents (SDC, Saponin) that do not affect the immune response through the hemolytic reaction were prepared as follows through Experimental Example 2.

[0133]

[0134] Furthermore, each candidate hemolytic reagent was applied to 1) CRP Control Solution (Low & High) and 2) 3 Level CRP Venous Whole Blood Sample (Low, Mild, High), respectively, and an immune reaction was conducted between the hemolytic samples and latex particles coated with anti-CRP antibodies. After the immune reaction was conducted, the absorbance following the immune reaction (i.e., secondary absorbance) of each sample was measured. Specifically, the secondary absorbance of the samples was measured at a wavelength of 750 nm using an A1Care Analyzer from i-Sens. Meanwhile, regarding the control group, the experiment was designed to measure the secondary absorbance following the immune reaction between a sample without any hemolytic agent added (i.e., CRP Control Solution (Low & High)) and latex particles coated with anti-CRP antibodies. Furthermore, the secondary absorbance was measured 20 times for each sample, each sample concentration, and each hemolytic reagent, and the mean (AVG), standard deviation (SD), and precision (CV) of the measured secondary absorbances were calculated.

[0135] 2. Experimental Results

[0136] FIG. 11 is a graph showing the results of evaluating antigen-antibody reactivity for each hemolytic reagent according to one embodiment of the present application.

[0137] 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 3.4%, and the precision (CV) of the control group was 3.0%, confirming that both the SDC hemolytic reagent and the Saponin hemolytic reagent showed a similar level of precision compared to the control group.

[0138] 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 4.3%, and the precision (CV) of the control group was 3.9%, confirming that both the SDC hemolytic reagent and the Saponin hemolytic reagent showed a similar level of precision compared to the control group.

[0139] For CRP Venous Whole Blood (Low) samples, the CV of the SDC hemolytic reagent was measured at 5.3%, and the CV of the Saponin hemolytic reagent was measured at 20.7%. For CRP Venous Whole Blood (Mid) samples, the CV of the SDC hemolytic reagent was measured at 5.8%, and the CV of the Saponin hemolytic reagent was measured at 12.2%. For CRP Venous Whole Blood (High) samples, the CV of the SDC hemolytic reagent was measured at 4.0%, and the CV of the Saponin hemolytic reagent was measured at 4.9%. In other words, for 3 Level CRP Whole Blood samples, it was confirmed that the CV of the SDC hemolytic reagent was superior to that of the Saponin hemolytic reagent across all concentration ranges of the 3 Level CRP Whole Blood samples.

[0140] According to the present experimental example, the hemolytic reagent (R1) according to a preferred embodiment of the present application may be Sodium Deoxycholate (SDO).

[0141] Furthermore, the CRP quantitative analysis kit (10) according to one embodiment of the present application may exhibit a precision (CV) of less than 10%. For example, the CRP quantitative analysis kit (10) may exhibit a precision (CV) of less than 8%. For example, the CRP quantitative analysis kit (10) may exhibit a precision (CV) of less than 6%.

[0143] <Experimental Example 4: Evaluation of Accelerated Stability by Stabilizer 1>

[0144] 1. Experimental Method

[0145] To select a stabilizer for ensuring the storage stability of an immune reaction reagent (R2) containing an anti-CRP antibody, five candidate stabilizers were selected. Furthermore, each candidate stabilizer was added to latex particles coated with anti-CRP antibodies and dried. An analytical reagent solution and a Level 1 CRP Serum Sample (CRP 60 mg / L) were added to the dried composition to proceed with an immune reaction between CRP and the anti-CRP antibody. Furthermore, the absorbance after the immune reaction was performed was measured to obtain a reference measurement value for Day 0 of storage. The analytical reagent solution was composed to include glycine, sodium chloride, sodium azide, ethylenediaminetetraacetic acid disodium salt dihydrate, and bovine serum albumin (BSA).

[0146] Furthermore, each candidate stabilizer was added to latex particles coated with anti-CRP antibodies, and the dried compositions were stored in an oven at 50°C. The analytical reagent solutions were stored separately under refrigeration. At 1-day intervals for 7 days, the analytical reagent solution and a 1-Level CRP Serum Sample were added to the dried compositions containing each candidate stabilizer to induce an immune reaction between CRP and anti-CRP antibodies. The absorbance after the immune reaction was measured, and measurements were obtained for days 1, 2, 3, 4, 5, 6, and 7 of storage. Meanwhile, regarding the control group, the experiment was designed to measure the absorbance after the immune reaction between a sample without any stabilizer added (i.e., 1-Level CRP Serum Samples (CRP 60 mg / L)) and latex particles coated with anti-CRP antibodies.

[0147] Furthermore, the bias was calculated based on the reference measurement and the measurement according to the storage period.

[0148] The five candidate stabilizers are as follows, and the stabilizer with a bias of within 20% during all storage periods was evaluated as effective for the storage stability of the immune response reagent (R2).

[0149]

[0150] 2. Experimental Results

[0151] FIG. 12 is a graph and diagram showing the results of evaluating the accelerated stability according to the stabilizer added to the Anti-CRP antibody according to one embodiment of the present application.

[0152] In the case of the control group (i.e., when no stabilizer is added), it was confirmed that the measured value decreased by more than 90% compared to the reference measured value on day 0 starting from day 1 of storage. Through this, it was confirmed that a stabilizer is essential for the storage stability of the immunoreaction reagent (R2).

[0153] In the case of DEAE-Dextran stabilizer, it was visually confirmed that the Anti-CRP antibody stored in a dry state did not dissolve, and it was confirmed that the measured value increased by more than 42% compared to the reference measured value on day 0 starting from day 1 of storage.

[0154] In the case of the PAA stabilizer, it was visually confirmed that the Anti-CRP antibody stored in a dry state did not dissolve, and absorbance measurement was impossible due to the immune response not being properly performed.

[0155] In the case of NPS stabilizers, it was confirmed that from the first day of storage, the deviation (Bias) from the reference measurement value on day 0 exceeded the 20% range.

[0156] In the case of Trehalose stabilizers and Sucrose stabilizers, it was visually confirmed that the Anti-CRP antibodies stored in a dry state dissolved well, and it was confirmed that the deviation (Bias) from the reference measurement value on day 0 was within the 20% range up to day 7 of storage.

[0157] According to the present experimental example, the stabilizer according to one embodiment of the present application may be selected from the group consisting of trehalose and sucrose.

[0159] <Experimental Example 5: Evaluation of Accelerated Stability by Stabilizer 2>

[0160] 1. Experimental Method

[0161] In order to select a stabilizer to ensure the storage stability of an immune reaction reagent (R2) containing an anti-CRP antibody, two candidate stabilizers (Trehalose and Sucrose) with storage stability effects were prepared through Experimental Example 4. Furthermore, each candidate stabilizer was added to latex particles coated with anti-CRP antibodies and dried. An analytical reagent solution and a 3-level CRP Serum Sample (CRP 10 mg / L, 50 mg / L, and 150 mg / L) were each added to the dried composition to proceed with an immune reaction between CRP and anti-CRP antibodies. Furthermore, the absorbance after the immune reaction was carried out was measured to obtain a reference measurement value for Day 0 of storage.

[0162] Furthermore, each candidate stabilizer was added to latex particles coated with Anti-CRP antibodies, and the dried compositions were stored in an oven at 50°C. The analytical reagent solutions were stored separately under refrigeration. At 1-day intervals for 7 days, the analytical reagent solution and 3 Level CRP Serum Samples (CRP 10 mg / L, 50 mg / L, 150 mg / L) were added to the dried compositions containing each candidate stabilizer to induce an immune reaction between CRP and Anti-CRP antibodies. The absorbance after the immune reaction was in progress was measured to obtain the measurements for days 1, 2, 3, 4, 5, 6, and 7 of storage.

[0163] Furthermore, the bias was calculated based on the reference measurement and the measurement according to the storage period.

[0164] Furthermore, for each condition, the experiment was measured repeatedly five times to calculate the precision (CV).

[0165] Two candidate stabilizers are as follows, and the stabilizer with a bias of less than 20% over the entire storage period was evaluated as effective for the storage stability of the immunoreaction reagent (R2), and the stabilizer with a precision of less than 10% was evaluated as having excellent precision.

[0166]

[0167] 2. Experimental Results

[0168] FIG. 13 is a graph showing the results of evaluating the accelerated stability according to the stabilizer added to the Anti-CRP antibody according to one embodiment of the present application.

[0169] For the Trehalose stabilizer and CRP Serum (10 mg / L, Low) samples, it was confirmed that on days 5 and 7 of storage, the bias of the measured values ​​compared to the reference values ​​on day 0 exceeded the 20% range. Furthermore, for Trehalose and CRP Serum (10 mg / L, Low), the CV was observed to be less than 10% throughout all storage periods.

[0170] For the Trehalose stabilizer and CRP Serum (50 mg / L, Mid) samples, it was confirmed that the deviation (Bias) from the reference measurement value on day 0 was within the 20% range up to day 7 of storage. Furthermore, for Trehalose and CRP Serum (50 mg / L, Mid), the precision (CV) was observed to be less than 10% throughout the entire storage period.

[0171] For the Trehalose stabilizer and CRP Serum (150 mg / L, High) samples, it was confirmed that the deviation (Bias) from the reference measurement value on day 0 was within the 20% range up to day 7 of storage. Furthermore, for Trehalose and CRP Serum (50 mg / L, Mild), the precision (CV) was observed to be less than 10% throughout all storage periods.

[0172] For the sucrose stabilizer and CRP Serum (10 mg / L, Low) samples, it was confirmed that the deviation (Bias) from the reference measurement value on day 0 was within the 20% range up to day 7 of storage. Furthermore, for the sucrose stabilizer and CRP Serum (10 mg / L, Low), the precision (CV) was observed to exceed 10% at all storage periods.

[0173] For the sucrose stabilizer and CRP Serum (50 mg / L, Mid) samples, it was confirmed that the deviation (Bias) from the reference measurement value on day 0 was within the 20% range up to day 7 of storage. Furthermore, for the sucrose stabilizer and CRP Serum (50 mg / L, Mid), the precision (CV) was observed to exceed 10% on days 3, 6, and 7 of storage.

[0174] For the sucrose stabilizer and CRP Serum (150 mg / L, High) samples, it was confirmed that the deviation (Bias) from the reference measurement value on day 0 was within the 20% range up to day 7 of storage. Furthermore, for the sucrose stabilizer and CRP Serum (150 mg / L, High), the precision (CV) was observed to exceed 10% on day 7 of storage.

[0175] Through this experimental example, it was confirmed that both sucrose stabilizers and trehalose stabilizers can increase storage stability with high precision for most CRP concentration ranges. However, additional experiments were conducted to ensure storage stability and increase precision for some CRP concentration ranges as well.

[0177] <Experimental Example 6: Evaluation of Accelerated Stability by Stabilizer 3>

[0178] 1. Experimental Method

[0179] Evaluation was conducted using the same experimental method as in Experimental Example 5, using a stabilizer that 'mixed' sucrose and trehalose. Specifically, a stabilizer that mixed sucrose and trehalose (hereinafter referred to as the mixed stabilizer) was prepared. Furthermore, the mixed stabilizer was added to latex particles coated with anti-CRP antibodies and dried. An analytical reagent solution and a 3-level CRP serum sample (CRP 10 mg / L, 50 mg / L, 150 mg / L) were added to the dried composition, respectively, to conduct an immune reaction between CRP and anti-CRP antibodies. Furthermore, the absorbance after the immune reaction was conducted was measured to obtain the reference measurement value for Day 0 of storage.

[0180] Furthermore, a mixed stabilizer was added to latex particles coated with Anti-CRP antibodies, and the dried composition was stored in an oven at 50°C. The analytical reagent solution was stored separately in a refrigerator. At 1-day intervals for 7 days, the analytical 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 the mixed stabilizer to induce an immune reaction between CRP and Anti-CRP antibodies. The absorbance was measured after the immune reaction was performed, and measurements were obtained for days 1, 2, 3, 4, 5, 6, and 7 of storage.

[0181] Furthermore, the bias was calculated based on the reference measurement and the measurement according to the storage period.

[0182] Furthermore, the precision (CV) was calculated by repeatedly measuring the experiment five times for each CRP concentration condition.

[0183] The mixed stabilizers are as follows, and the stabilizer with a bias of less than 20% over all storage periods was evaluated as effective for the storage stability of the immunoreaction reagent (R2), and the stabilizer with a precision of less than 10% was evaluated as having excellent precision.

[0184]

[0185] 2. Experimental Results

[0186] FIG. 14 is a graph showing the results of evaluating the accelerated stability according to the stabilizer added to the Anti-CRP antibody according to one embodiment of the present application.

[0187] For the mixed stabilizer and CRP Serum (10 mg / L, Low) samples, it was confirmed that the deviation (Bias) from the reference measurement value on day 0 was within the 20% range up to day 7 of storage. Furthermore, for the mixed stabilizer and CRP Serum (10 mg / L, Low) samples, the precision (CV) was observed to be less than 10% throughout the entire storage period.

[0188] For the mixed stabilizer and CRP Serum (50 mg / L, Mid) samples, it was confirmed that the deviation (Bias) from the reference measurement value on day 0 was within the 20% range up to day 7 of storage. Furthermore, for the mixed stabilizer and CRP Serum (50 mg / L, Mid) samples, the precision (CV) was observed to be less than 10% throughout all storage periods.

[0189] For the mixed stabilizer and CRP Serum (150 mg / L, High) samples, it was confirmed that the deviation (Bias) from the reference measurement value on day 0 was within the 20% range up to day 7 of storage. Furthermore, for the mixed stabilizer and CRP Serum (150 mg / L, High) samples, the precision (CV) was observed to be less than 10% throughout the entire storage period.

[0190] Through this, it was confirmed that when a stabilizer is used by mixing sucrose and trehalose, storage stability can be increased with high precision across all CRP concentration ranges. According to Experimental Example 6, the stabilizer according to a preferred embodiment of the present application may include both trehalose and sucrose.

[0192] <Experimental Example 7: Evaluation of Accelerated Stability by Stabilizer 4>

[0193] 1. Experimental Method

[0194] Using a stabilizer 'mixed' with sucrose and trehalose, the storage stability of the mixed stabilizer was evaluated by changing the storage temperature conditions of Experimental Example 6. Specifically, a stabilizer mixed with sucrose and trehalose was prepared. Furthermore, the mixed stabilizer was added to latex particles coated with anti-CRP antibodies and dried. The analytical reagent solution and CRP Control Solution (Low, High) were added to the dried composition, respectively, to conduct an immune reaction between CRP and anti-CRP antibodies. Furthermore, the absorbance after the immune reaction was conducted was measured to obtain the reference measurement value for Day 0 of storage.

[0195] Furthermore, a mixed stabilizer was added to latex particles coated with Anti-CRP antibodies, and the dried composition was stored in an oven at 37°C. In Experimental Example 7, the analytical reagent solution was also stored together in an oven at 37°C. At regular intervals for 63 days, the analytical reagent solution and CRP Control Solution (Low, High) were added to the dried composition containing the mixed stabilizer to carry out an immune reaction between CRP and Anti-CRP antibodies, and the absorbance after the immune reaction was carried out was measured to obtain measurements on days 4, 7, 14, 21, 28, 35, 42, 56, and 63 of storage.

[0196] Furthermore, the bias was calculated based on the reference measurement and the measurement according to the storage period.

[0197] Furthermore, the precision (CV) was calculated by repeatedly measuring the experiment five times for each CRP concentration condition.

[0198] The mixed stabilizers are as follows, and the stabilizer with a bias of less than 20% over all storage periods was evaluated as effective for the storage stability of the immunoreaction reagent (R2), and the stabilizer with a precision of less than 10% was evaluated as having excellent precision.

[0199]

[0200] 2. Experimental Results

[0201] FIG. 15 is a graph showing the results of evaluating the accelerated stability according to the stabilizer added to the Anti-CRP antibody according to one embodiment of the present application.

[0202] For the mixed stabilizer and CRP Control Solution (Low) samples, it was confirmed that the deviation (Bias) from the reference measurement value on day 0 was within the 20% range up to day 63 of storage. Furthermore, for the mixed stabilizer and CRP Control Solution (Low) samples, the precision (CV) was observed to be less than 10% throughout the entire storage period.

[0203] For the mixed stabilizer and CRP Control Solution (High) samples, it was confirmed that the deviation (Bias) from the reference measurement value on day 0 was within the 20% range up to day 63 of storage. Furthermore, for the mixed stabilizer and CRP Control Solution (High) samples, the precision (CV) was observed to be less than 10% throughout the entire storage period.

[0204] Through this, it was confirmed that when a stabilizer is used by mixing sucrose and trehalose, storage stability can be maintained with high precision even when stored for more than 63 days at a storage temperature of 37°C. According to Experimental Example 7, the stabilizer according to a preferred embodiment of the present application may include both trehalose and sucrose. More preferably, the stabilizer may include trehalose and sucrose in substantially the same concentration ratio.

[0206] FIG. 16 is a diagram showing identification information included in a kit for quantitative analysis of C-reactive proteins according to one embodiment of the present application.

[0207] A CRP quantitative analysis kit (10) according to one embodiment of the present application may include identification information for recognizing a biological sample. For example, one side of the main cartridge (200) of the CRP quantitative analysis kit (10) may include identification information (e.g., information in the form of a barcode) for identifying the type of biological sample.

[0208] The quantitative analysis device described below can acquire identification information to recognize the type of biological sample to be analyzed by the CRP quantitative analysis kit (10). For example, the quantitative analysis device can recognize that the biological sample to be analyzed is CRP based on a barcode. At this time, the quantitative analysis device may be configured to perform quantitative analysis of the biological sample by executing a pre-stored analysis protocol related to the recognized type based on the recognition of the type of biological sample. For example, the quantitative analysis device may be implemented to perform quantitative analysis of CRP by executing a pre-stored CRP analysis protocol based on the recognition that the type of biological sample to be analyzed is CRP.

[0209] Meanwhile, in FIG. 16, identification information is explained by exemplifying a barcode shape. However, this is merely an example, and identification information of any appropriate shape may be provided at any location on the CRP quantitative analysis kit (10).

[0210] A quantitative analysis device according to one embodiment of the present application may be implemented to perform quantitative analysis of a biological sample (e.g., 1,5-AG, glycated albumin, CRP, etc.). Furthermore, the quantitative analysis device may include a communication module (or may be referred to as a transceiver), memory, and / or a processor.

[0211] 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 quantitative analysis results to an external device or external server through the communication module.

[0212] A quantitative analysis device can connect to a network and transmit and receive various data through a communication module. The communication module can broadly include wired and wireless types. Since wired and wireless types each have their own advantages and disadvantages, in some cases, both wired and wireless types may be provided simultaneously in the electronic device. Here, for the wireless type, communication methods of the WLAN (Wireless Local Area Network) family, such as Wi-Fi, can be mainly used. Alternatively, for the wireless type, cellular communication methods, such as LTE or 5G, can be used. However, wireless communication protocols are not limited to the examples described above, and it is possible to use any appropriate wireless communication method. For the wired type, LAN (Local Area Network) or USB (Universal Serial Bus) communication are representative examples, and other methods are also possible.

[0213] The memory of a quantitative analysis device can store various types of information. Various types of data can be stored in the memory temporarily or semi-permanently. Examples of memory include hard disk drives (HDDs), solid-state drives (SSDs), flash memory, read-only memory (ROM), and random access memory (RAM). The memory can be provided in a form that is embedded in the quantitative analysis device or is detachable. The memory can store various data required for the operation of the quantitative analysis device, including an operating system (OS) for running the quantitative analysis device and programs for operating each component of the quantitative analysis device.

[0214] The processor can control the overall operation of the quantitative analysis device. For example, the quantitative analysis device can control the overall operation of the device, including the operation of recognizing identification information of a biological sample, the operation of executing a corresponding analysis protocol based on the recognized identification information, and / or the operation of quantitatively analyzing the biological sample based on the analysis protocol. Specifically, the processor can load and execute a program for the overall operation of the quantitative analysis device from memory. The processor may be implemented as an Application Processor (AP), a Central Processing Unit (CPU), a Microcontroller Unit (MCU), or a similar device depending on hardware, software, or a combination thereof. In this case, hardware-wise, it may be provided in the form of an electronic circuit that processes electrical signals to perform control functions, and software-wise, it may be provided in the form of a program or code that drives the hardware circuit.

[0215] According to a kit for quantitative analysis of C-reactive protein, a method for quantitative analysis of C-reactive protein, and / or an apparatus for performing the same according to one embodiment of the present application, a hemolytic reagent capable of performing quantitative analysis of CRP with high precision over a wide range of CRP concentrations without affecting the immune response can be provided.

[0216] According to a kit for quantitative analysis of C-reactive protein, a method for quantitative analysis of C-reactive protein, and / or an apparatus for performing the same according to one embodiment of the present application, a stabilizer can be provided that allows solid-dried Anti-CRP antibodies to sufficiently re-dissolve to participate in an immune response, enables quantitative analysis of CRP with high precision and accuracy over a wide range of CRP concentrations, and can increase storage stability.

[0217] The features, structures, effects, etc. described in the embodiments above are included in at least one embodiment of the present invention and are not necessarily limited to only one embodiment. Furthermore, the features, structures, effects, etc. exemplified in each embodiment may be combined or modified and implemented in other embodiments by a person skilled in the art to which the embodiments belong. Accordingly, details regarding such combinations and modifications should be interpreted as being included within the scope of the present invention.

[0218] Furthermore, although the embodiments have been described above, this is merely illustrative and does not limit the invention. Those skilled in the art will understand that various modifications and applications not exemplified above are possible within the scope of the essential characteristics of the embodiments. In other words, each component specifically shown in the embodiments may be modified and implemented. Differences related to such modifications and applications should be interpreted as being included within the scope of the invention as defined in the appended claims. Explanation of the symbols

[0220] 10: C-Reactive Protein Quantification Kit

Claims

Claim 1 A method for quantitatively analyzing C-reactive protein (CRP) contained in a blood sample, comprising: a hemolysis step of producing a hemolyzed sample by hemolyzing blood cells in the blood sample using a hemolysis reagent; a reaction step of contacting the hemolyzed sample with an Anti-CRP antibody to perform an antigen-antibody reaction between the CRP contained in the hemolyzed sample and the Anti-CRP antibody; and a quantification step of quantifying the CRP contained in the blood sample based on the antigen-antibody reaction, wherein the Anti-CRP antibody contacts the hemolyzed sample while fixed to a reagent fixation part by a stabilizer. Claim 2 In claim 1, the hemolysis step comprises a step of contacting the blood sample with the hemolysis reagent fixed to a first reagent fixing part located in a first compartment area of ​​a CRP quantitative analysis kit, and the reaction step comprises a step of contacting the hemolyzed sample with the Anti-CRP antibody fixed to a second reagent fixing part located in a second compartment area partitioned from the first compartment area, and the CRP quantitative analysis method further comprises, between the hemolysis step and the reaction step, a first transfer step of moving the hemolyzed sample from the first compartment area to the second compartment area; and after the reaction step, a second transfer step of moving the sample on which the antigen-antibody reaction was performed from the second compartment area to a location where the quantification is performed. Claim 3 A method for quantitative analysis of CRP according to claim 2, wherein the first moving step and the second moving step each include the step of moving a sample through a channel connecting the first compartment area and the second compartment area by applying an external force to the CRP quantitative analysis kit to rotate the CRP quantitative analysis kit. Claim 4 A method for quantitative analysis of CRP according to claim 3, wherein the CRP quantitative analysis kit further comprises a measuring unit for quantifying CRP contained in the blood sample, and the measuring unit is positioned between the first reagent fixing unit, on which the hemolytic reagent is fixed, and the second reagent fixing unit, on which the Anti-CRP antibody is fixed, such that the sample passes through the measuring unit while moving between the first compartment area and the second compartment area during the first movement step and the second movement step. Claim 5 A method for quantitative analysis of CRP according to claim 4, wherein the quantification step comprises: a step of measuring a first turbidity before the antigen-antibody reaction is performed while the hemolyzed sample passes through the measuring unit in the first transfer step; a step of measuring a second turbidity after the antigen-antibody reaction is performed while the sample in which the antigen-antibody reaction is performed passes through the measuring unit in the second transfer step; and a step of quantifying the CRP contained in the blood sample based on the first turbidity and the second turbidity. Claim 6 A method for quantitative analysis of CRP according to claim 5, wherein the first moving step is performed by rotating the CRP quantitative analysis kit in a first direction by an external force to move the hemolyzed sample from the first compartment area through the measuring unit to the second compartment area, and the second moving step is performed by rotating the CRP quantitative analysis kit in a second direction opposite to the first direction by an external force to move the sample in which the antigen-antibody reaction was performed to the measuring unit. Claim 7 A method for quantitative analysis of CRP according to claim 2, wherein the hemolysis step further comprises: a step of inserting a collector containing the blood sample into a receiving portion formed in one area of ​​the CRP quantitative analysis kit; and a step of introducing the blood sample into a mixing portion located in the first compartment area by means of the insertion, and bringing it into contact with the hemolysis reagent fixed in the first reagent fixing portion located in the first compartment area to generate the hemolyzed sample. Claim 8 A method for quantitative analysis of CRP according to claim 7, wherein the step of inserting the collector into the receiving portion further comprises: a step of pressurizing a solution cell provided in the CRP quantitative analysis kit through a protrusion provided in the collector; a step of moving the solution cell through a moving frame of the CRP quantitative analysis kit according to the pressurization; a step of rupturing the cover tape of the solution cell through a cover tape rupture portion formed at one end of the moving frame; and a step of introducing a reaction buffer stored inside the solution cell into the mixing portion together with the blood sample. Claim 9 A method for quantitative analysis of CRP according to claim 1, wherein the hemolytic reagent is selected from the group consisting of Sodium Deoxycholate (SDO) and Saponin. Claim 10 A method for quantitative analysis of CRP according to claim 1, characterized in that the hemolytic reagent is Sodium Deoxycholate (SDO). Claim 11 A method for quantitative analysis of CRP according to claim 1, characterized in that the stabilizer is selected from the group consisting of trehalose and sucrose. Claim 12 A method for quantitative analysis of CRP according to claim 1, characterized in that the stabilizer comprises both trehalose and sucrose. Claim 13 A method for quantitative analysis of CRP according to claim 1, characterized in that the anti-CRP antibody is fixed to the reagent fixation part in a form coated on a latex particle. Claim 14 A method for quantitative analysis of CRP according to claim 8, wherein the reaction buffer comprises glycine, sodium chloride, sodium azide, ethylenediaminetetraacetic acid disodium salt dihydrate, and bovine serum albumin (BSA). Claim 15 A method for quantitative analysis of CRP according to claim 1, characterized in that the blood sample is at least one of plasma, serum, and whole blood. Claim 16 A method for quantitative analysis of CRP according to claim 4, further comprising, after the quantification step, a waste liquid treatment step for treating the waste liquid after the quantitative analysis is completed, wherein the waste liquid treatment step comprises the step of applying an external force to the CRP quantitative analysis kit to rotate the CRP quantitative analysis kit and moving the waste liquid from the measurement unit to the waste liquid treatment unit. Claim 17 A method for quantitative analysis of CRP according to claim 1, wherein the anti-CRP antibody is fixed in a solid state on the reagent fixation part, and the stabilizer is characterized by ensuring that the anti-CRP antibody is sufficiently redissolved during the antigen-antibody reaction even when the anti-CRP antibody is fixed in a solid state on the reagent fixation part.

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