A protective agent for proprotein convertase subtilisin 9 standard product, a detection kit for determining proprotein convertase subtilisin 9 and its application

Abstract

The present application relates to the field of biomedical testing technology and specifically discloses a protective agent for proprotein convertase subtilisin 9 standard sample, a detection kit for measuring proprotein convertase subtilisin 9, and its application. The protective agent comprises the following components in weight percentage: 1 wt% to 3 wt% protein, 0.5 wt% to 2 wt% amino acid, 1 wt% to 5 wt% surfactant, 0.01 wt% to 0.5 wt% antioxidant, 0.01 wt% to 0.2 wt% defoaming agent, 0.01 wt% to 0.2 wt% EDTA, 0.05 wt% to 0.2 wt% preservative, and the remainder being buffer. The protective agent for proprotein convertase subtilisin 9 standard sample provided herein can significantly improve the stability of proprotein convertase subtilisin 9, extend the shelf life of the detection reagent, and ensure the accuracy and reproducibility of the test results. The detection kit provided herein also has a short detection time, good precision, high sensitivity, and good reagent uniformity.

Description

Technical Field

[0001] The present application relates to the field of biomedical testing technology, and in particular to a protective agent for a proprotein convertase subtilisin 9 standard product, a detection kit for determining proprotein convertase subtilisin 9, and applications thereof. Background Art

[0002] Proprotein convertase subtilisin-9 plays a role in maintaining cholesterol homeostasis in the human body. During metabolism, low-density lipoprotein cholesterol (LDL-C) first binds to the low-density lipoprotein cholesterol receptor (LDL-R), then enters the cell through endocytosis. Once inside the cell, the LDL-R and LDL-C separate, returning to the cell surface for repeated use. Proprotein convertase subtilisin-9 binds to the LDL-R and then to LDL-C, forming a new complex that enters the lysosome for degradation. This ultimately leads to a decrease in LDL-R and, consequently, decreased LDL-C degradation, resulting in elevated LDL-C levels. Elevated LDL-C levels can lead to a range of cardiovascular diseases. Proprotein convertase subtilisin-9 has also become an emerging target in lipid-lowering drug development in recent years, with several successful drug developments both domestically and internationally. Detection of proprotein convertase subtilisin-9 is essential for both drug development and treatment monitoring.

[0003] Research testing reagents are already available from multiple manufacturers both domestically and internationally, but the shelf life of most kits is limited to six months. While the ELISA method is well established, the primary factor impacting the shelf life of the kit is the stability of the standard. The standard in existing research kits is typically a lyophilized powder containing a high concentration of proprotein convertase subtilisin 9 antigen. During use, the standard must first be reconstituted with diluent and then diluted to a series of concentration gradients. The reconstituted standard must be stored at -20°C and has a shelf life of no more than one month.

[0004] In general, the standards used in current research kits have the following drawbacks: a) They require lyophilization, increasing production costs for manufacturers and requiring reconstitution for use, which increases the uncertainty of the standards. b) They are not ready-to-use and require dilution to different concentrations, which can lead to deviations in test results between users and poor reproducibility. c) After reconstitution, storage conditions are demanding, and the freeze-thaw process can disrupt the structure of proprotein convertase subtilisin 9, necessitating aliquots for storage, which is inconvenient for the operator. d) The shelf life of the kits is short. Considering shipping time, the test reagents are nearing their expiration date by the time they reach the user, significantly increasing the cost of use.

[0005] Furthermore, the current detection reagents for proprotein convertase subtilisin 9 are all enzyme-linked immunosorbent assay (ELISA), which has the disadvantages of low sensitivity, long detection time, and inability to achieve fully automatic detection. Summary of the Invention

[0006] The present application aims to overcome the shortcomings of the aforementioned prior art by providing a protective agent for a proprotein convertase subtilisin 9 standard, a detection kit for measuring proprotein convertase subtilisin 9, and its use. The protective agent for the standard effectively protects the proprotein convertase subtilisin 9 antigen, addressing the short shelf life of the test kit and ensuring high accuracy, good reproducibility, simple operation, and cost savings. The test kit provided herein offers a short detection time, excellent precision, and high sensitivity.

[0007] To achieve the above objectives, the technical solutions adopted in this application are:

[0008] In a first aspect, the present application provides a protective agent for a standard product of proprotein convertase subtilisin 9, the protective agent comprising the following components in percentage by weight:

[0009] 1wt% to 3wt% of protein, 0.5wt% to 2wt% of amino acid, 1wt% to 5wt% of surfactant, 0.01wt% to 0.5wt% of antioxidant, 0.01wt% to 0.2wt% of defoaming agent, 0.01wt% to 0.2wt% of EDTA, 0.05wt% to 0.2wt% of preservative, and the balance is buffer solution.

[0010] After extensive research and testing, the inventors of the present application have discovered that the addition of protein and amino acids in a synergistic ratio to the standard protective agent can effectively prevent the aggregation and degradation of proprotein convertase subtilisin 9 and improve the thermal stability of proprotein convertase subtilisin 9.

[0011] The addition of surfactants in this application can increase the solubility of the target protein, act as a protective layer, and improve thermal stability. This application also adds EDTA and antioxidants. EDTA can complex metal ions in the solution to prevent metal ions from oxidizing amino acids, and antioxidants can prevent some oxygen free radicals from oxidizing amino acids and causing structural changes. In addition, this application analyzed the amino acid sequence of the proprotein convertase subtilisin 9 protein and found that up to 22% of the amino acids in the protein are easily oxidized. It was unexpectedly discovered that the synergistic ratio of adding EDTA and antioxidants can prevent the oxidation of amino acids in the proprotein convertase subtilisin 9 protein from multiple angles.

[0012] In addition, a defoaming agent is added to the standard protective agent of this application to eliminate bubbles generated by proteins, amino acids, and surfactants, ensuring that the formulated proprotein convertase subtilisin 9 is evenly distributed in the solution and ensuring the reliability of the test results. The added buffer can provide a stable pH environment.

[0013] As a preferred embodiment of the protective agent for the proprotein convertase subtilisin 9 standard product described in the present application, the protective agent comprises the following components in percentage by weight:

[0014] 2wt% protein, 1wt% amino acid, 5wt% surfactant, 0.1wt% antioxidant, 0.04wt% defoaming agent, 0.2wt% EDTA, 0.1wt% preservative, and the balance is buffer.

[0015] The protective agent of the present application adopts the above-mentioned preferred weight percentage components, which can better protect the proprotein convertase subtilisin 9 antigen, increase the shelf life of the detection product, and improve the accuracy and sensitivity of the detection product.

[0016] The use of the preferred weight percentages of EDTA and antioxidants in a synergistic ratio can better prevent oxidation of amino acids in the proprotein convertase subtilisin 9 protein, thereby enhancing the protective effect of the proprotein convertase subtilisin 9 standard. The use of the preferred weight percentages of defoaming agents can improve the uniformity of proprotein convertase subtilisin 9 distribution in the solution, thereby increasing the reliability of the test results.

[0017] As a preferred embodiment of the proprotein convertase subtilisin 9 standard protective agent described in the present application, the protein includes at least one of bovine serum albumin, gelatin and acid-hydrolyzed casein; and the amino acid includes at least one of glycine, lysine and arginine.

[0018] More preferably, the protein is acid-hydrolyzed casein; and the amino acid is arginine.

[0019] The present application adopts the above-mentioned preferred types of synergistic ratios of proteins and amino acids, which can better and more effectively prevent the aggregation and degradation of proprotein convertase subtilisin 9, and can also better improve the thermal stability of proprotein convertase subtilisin 9.

[0020] As a preferred embodiment of the protective agent for the proprotein convertase subtilisin 9 standard product described in the present application, the surfactant includes at least one of PEG400, glycerol and Tween 20.

[0021] More preferably, the surfactant is glycerol.

[0022] As a preferred embodiment of the protective agent for the proprotein convertase subtilisin 9 standard described in the present application, the oxidant includes tea polyphenols and / or sodium thiosulfate.

[0023] More preferably, the antioxidant is tea polyphenols.

[0024] Preferably, the buffer in the protective agent includes phosphate buffer or PBS buffer, the concentration of the buffer is 5mM-30mM (preferably 10mM), and the pH is 7.0-7.4.

[0025] The present application adopts the above preferred types of antioxidants and EDTA in synergistic combination, which can better prevent the oxidation of amino acids in the proprotein convertase subtilisin 9 protein and is beneficial to protecting the proprotein convertase subtilisin 9 protein.

[0026] As a preferred embodiment of the protective agent for the proprotein convertase subtilisin 9 standard product described in the present application, the defoaming agent includes at least one of BLOTTO-defoaming agent TBS solution, SE-15 defoaming agent and 204 defoaming agent.

[0027] Preferably, the defoaming agent is BLOTTO-defoaming agent TBS solution.

[0028] The present application uses the above-mentioned preferred type of defoaming agent to better eliminate bubbles generated by proteins, amino acids and surfactants, improve the uniformity of the prepared solution of proprotein convertase subtilisin 9, and improve the reliability of the detection results.

[0029] In a second aspect, the present application provides the use of the above-mentioned proprotein convertase subtilisin 9 standard protective agent in the preparation of a detection product for measuring proprotein convertase subtilisin 9.

[0030] In a third aspect, the present application provides a detection kit for determining proprotein convertase subtilisin 9, the detection kit comprising a standard, reagent 1, reagent 2, and the proprotein convertase subtilisin 9 standard protective agent;

[0031] The components and final concentrations of the reagent R1 are:

[0032] Buffer 5mM~100mM;

[0033] Acridinium ester-labeled anti-proprotein convertase subtilisin 9 monoclonal antibody 1 mg / L to 4 mg / L;

[0034] Stabilizer 1 1w / v%~10w / v%;

[0035] The components and final concentrations of the reagent R2 are:

[0036] Buffer 5mM~100mM;

[0037] Horseradish peroxidase-labeled proprotein convertase subtilisin-9 monoclonal antibody 0.1mg / L~1mg / L;

[0038] Stabilizer 2 1 w / v% to 10 w / v%.

[0039] As a preferred embodiment of the detection kit described in the present application, when the anti-proprotein convertase subtilisin 9 monoclonal antibody is labeled with an acridinium ester, Tween 20 is further added at a mass concentration of 1 wt% to 5 wt% (preferably, the mass concentration of Tween 20 is 5 wt%);

[0040] After the anti-proprotein convertase subtilisin 9 monoclonal antibody is labeled with acridinium ester, glycerol with a mass concentration of 20wt% to 50wt% is added, preferably with a mass concentration of 30wt%.

[0041] Conventional acridinium ester labeling methods suffer from low labeling efficiency, resulting in high costs and reduced test sensitivity. Preservation of labeled acridinium ester antibodies is also crucial. Currently, long-term storage is primarily achieved through freezing, but freeze-thawed acridinium ester-labeled antibodies significantly affect the homogeneity of reagents after preparation.

[0042] In the present application, the addition of Tween 20 during acridinium ester labeling can increase the solubility of acridinium ester and have a certain protective effect on acridinium ester, thereby improving the labeling efficiency, increasing the sensitivity of the detection reagent, and reducing the cost of the kit.

[0043] The anti-proprotein convertase subtilisin 9 monoclonal antibody of the present application is labeled with an acridinium ester, and then glycerol with a mass concentration of 20wt% to 50wt% is added as a surfactant. Glycerol has a protective effect on the acridinium ester-labeled monoclonal antibody during the freeze-thaw process, reduces aggregation caused by the freeze-thaw process, and improves the uniformity of the prepared reagent.

[0044] As a preferred embodiment of the detection kit described in the present application, the preparation method of the standard comprises diluting the antigen using the proprotein convertase subtilisin 9 protective agent.

[0045] In the technical solution of the present application, the standard is prepared using a preservative for the proprotein subtilisin 9 standard, which can significantly improve the stability of the standard and ensure the stability and reliability of the test results.

[0046] High and low concentration standards prepared using the preservative for the preprotein subtilisin 9 standard of the present invention remain stable when stored at 37°C for 15 days, at room temperature at 25°C for 3 months, and at 2-8°C for 18 months. The preservative does not require lyophilization or dilution to different concentration gradients for standard testing, allowing for immediate use. This reduces errors caused by manual dissolution and dilution processes and ensures accurate and reliable test results. This extends the shelf life of the test kit, reduces production and storage costs for manufacturers, and reduces user costs.

[0047] As a preferred embodiment of the detection kit described in the present application, the buffer in the reagent 1 or reagent 2 includes at least one of phosphate buffer, Tris buffer and MOPS buffer; preferably, the buffer is MOPS buffer with a mass concentration of 25mM.

[0048] The pH of the buffer solution in the reagent 1 or reagent 2 is 6.5 to 8;

[0049] The stabilizer 1 or the stabilizer 2 comprises at least one of bovine serum albumin, polyethylene glycol 20000, arginine, sodium thiosulfate, KY100 and horseradish peroxidase stabilizer.

[0050] Preferably, stabilizer 1 is at least one of bovine serum albumin, sodium thiosulfate, and polyethylene glycol 20000; stabilizer 2 is at least one of bovine serum albumin, arginine, and horseradish peroxidase stabilizer.

[0051] The detection kit of the present application utilizes an acridinium ester-labeled anti-proprotein convertase subtilisin 9 monoclonal antibody, an antigen, and a horseradish peroxidase-labeled anti-proprotein convertase subtilisin 9 monoclonal antibody to form an antibody-antigen-antibody complex. No washing process is required. After the substrate is added, continuous detection is performed for a period of time, each time at intervals of 0.02 to 0.05 seconds. The peak area is calculated, and a dose-response curve is drawn using a known concentration of proprotein convertase subtilisin 9 and the calculated peak area. The proprotein convertase subtilisin 9 content in the test sample is inferred from the curve.

[0052] The kit of the present application has a good correlation with the commercial comparison reagent in determining the proprotein convertase subtilisin 9 in the sample and has good comparability.

[0053] In a fourth aspect, the present application further provides a method for determining proprotein convertase subtilisin 9 by chemiluminescence, comprising the following steps:

[0054] (1) Add the reagents at a ratio of sample / standard: reagent R1: reagent R2 = 1:8-12:8-12, incubate for 30 min, and immediately after adding the substrate solution, continuously measure the luminescence value within 2 s, each time at an interval of 0.02 s to 0.05 s, and calculate the peak area.

[0055] (3) Detect the peak areas of the standard and sample, draw a dose-response curve with the concentration as the X coordinate and the peak area as the Y coordinate, and calculate the concentration of proprotein convertase subtilisin-9 in the sample based on the curve.

[0056] The detection kit uses chemiluminescence to detect proprotein convertase subtilisin 9, and has the advantages of high sensitivity and fast detection speed. At the same time, it can be equipped with supporting instruments to realize fully automatic detection, which is convenient for use and promotion.

[0057] Furthermore, the preferred ratio of sample / standard:reagent R1:reagent R2 is 1:10:10.

[0058] Compared with the prior art, this application has the following beneficial effects:

[0059] The present application provides a preservative for a proprotein convertase subtilisin 9 standard, a detection kit for measuring proprotein convertase subtilisin 9, and its application. The standard prepared using the preservative for a proprotein convertase subtilisin 9 standard formulated in the present application remains stable after being placed at 37°C for 15 days, at room temperature at 25°C for 3 months, and at 2-8°C for 18 months. The preservative for a proprotein convertase subtilisin 9 standard prepared using the present application does not require freeze-drying or dilution to different concentration gradients for standard testing, and can be used immediately after opening, thereby reducing errors caused by manual dissolution and dilution processes and ensuring that the test results are accurate and reliable. The shelf life of the test kit is extended, the manufacturer's production and storage costs are reduced, and the user's usage costs are reduced. In addition, the detection kit provided in the present application has the advantages of high sensitivity and fast detection speed, and can be equipped with supporting instruments to achieve fully automatic detection, which is convenient for promotion and use. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 This is a standard curve obtained by measuring the detection kit prepared in Example 10, wherein the X-axis represents the peak area and the Y-axis represents the concentration of proprotein convertase subtilisin 9;

[0061] Figure 2 This is a correlation comparison chart of the detection kit prepared in Example 10 and the proprotein convertase subtilisin 9 detection kit (enzyme-linked immunosorbent assay) produced by R&D, a comparative reagent, wherein the X-axis represents the serum results measured by the R&D kit and the Y-axis represents the serum results measured by the kit of the present application.

[0062] Figure 3This is the linear evaluation result of the detection kit prepared in Example 10, the X-axis indicates the predicted concentration, and the Y-axis indicates the concentration measured by the kit of this application. DETAILED DESCRIPTION

[0063] In order to better illustrate the purpose, technical solutions and advantages of this application, this application will be further described below with reference to the accompanying drawings and specific embodiments.

[0064] In the following examples and comparative examples, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used are all commercially available unless otherwise specified, and the components and raw materials used in each parallel experiment are all of the same kind.

[0065] Example 1

[0066] As an embodiment of the protective agent for the proprotein convertase subtilisin 9 standard product described in the present application, the protective agent for the proprotein convertase subtilisin 9 standard product of this embodiment includes the following components in percentage by weight:

[0067] Acid hydrolyzed casein 2wt%, arginine 1wt%, glycerol 5wt%, tea polyphenols 0.1wt%, BLOTTO-antifoaming agent TBS solution 0.04wt%, EDTA 0.2wt%, KY100 0.1wt%, the balance is phosphate buffer 10mM (pH 7.4).

[0068] Example 2

[0069] As an embodiment of the protective agent for the proprotein convertase subtilisin 9 standard product described in the present application, the protective agent for the proprotein convertase subtilisin 9 standard product of this embodiment includes the following components in percentage by weight:

[0070] Bovine serum albumin 3wt%, glycine 2wt%, Tween 20 1wt%, sodium thiosulfate 0.5wt%, BLOTTO-antifoaming agent TBS solution 0.2wt%, EDTA 0.2wt%, KY100 0.1wt%, and the balance is phosphate buffer 5mM (pH 7.4).

[0071] Example 3

[0072] As an embodiment of the protective agent for the proprotein convertase subtilisin 9 standard product described in the present application, the protective agent for the proprotein convertase subtilisin 9 standard product of this embodiment includes the following components in percentage by weight:

[0073] Gelatin 1wt%, lysine 0.5wt%, PEG400 2wt%, tea polyphenols 0.01wt%, SE-15 defoamer 0.1wt%, EDTA 0.2wt%, KY 100 0.2wt%, and the balance is PBS buffer 30mM (pH 7.0).

[0074] Example 4

[0075] As an embodiment of the protective agent for the proprotein convertase subtilisin 9 standard product described in the present application, the protective agent for the proprotein convertase subtilisin 9 standard product of this embodiment includes the following components in percentage by weight:

[0076] Acid hydrolyzed casein 1wt%, arginine 2wt%, glycerol 3wt%, sodium thiosulfate 0.2%, 204 defoaming agent 0.01wt%, EDTA 0.01wt%, KY100 0.05wt%, and the balance is PBS buffer 20mM (pH 7.0).

[0077] Example 5

[0078] As an embodiment of the protective agent for the proprotein convertase subtilisin 9 standard product described in the present application, the protective agent for the proprotein convertase subtilisin 9 standard product of this embodiment includes the following components in percentage by weight:

[0079] Bovine serum albumin 2wt%, lysine 0.5wt%, glycerol 5wt%, EDTA 0.02wt%, tea polyphenols 0.02wt%, KY100 0.1wt%, BLOTTO-antifoaming agent TBS solution 0.04wt%, KY100 0.1wt%, and the balance is PBS buffer 10mM (pH 7.2).

[0080] Comparative Example 1

[0081] Physiological saline containing 5% BSA was used as the diluent for proprotein convertase subtilisin-9.

[0082] Comparative Example 2

[0083] Compared with Example 1, the difference is that the protective agent does not contain a surfactant, and the other components and their contents are the same as those in Example 1.

[0084] Comparative Example 3

[0085] Compared with Example 1, the difference is that the protective agent does not contain protein and amino acid, and the other components and their contents are the same as those in Example 1.

[0086] Comparative Example 4

[0087] Compared with Example 1, the difference is that the protective agent does not contain a defoaming agent, and the other components and their contents are the same as those in Example 1.

[0088] Comparative Example 5

[0089] Compared with Example 1, the difference is that the protective agent does not contain an antioxidant, and the other components and their contents are the same as those in Example 1.

[0090] Comparative Example 6

[0091] Compared with Example 1, the difference is that the protective agent does not contain EDTA, and the other components and their contents are the same as those in Example 1.

[0092] Comparative Example 7

[0093] Compared with Example 1, the difference is that the defoamer PE-M is used to replace the BLOTTO-defoamer TBS solution, and the other components and their contents are the same as those in Example 1.

[0094] Comparative Example 8

[0095] Compared with Example 2, the difference is that the amount of the defoaming agent is 0.5%wt, and the other components and their contents are the same as those in Example 2.

[0096] Comparative Example 9

[0097] Compared with Example 2, the difference is that the amount of the antioxidant is 1% by weight, and the other components and their contents are the same as those in Example 2.

[0098] Example 6

[0099] The above examples and comparative examples were evaluated for appearance, uniformity and stability of the prepared standards.

[0100] The proprotein convertase subtilisin 9 antigen was diluted using the formulations of Examples 1 to 5 and Comparative Examples 1 to 9 to obtain three concentrations of 50 ng / mL, 200 ng / mL, and 800 ng / mL, respectively.

[0101] Appearance evaluation: The freshly prepared standard products were observed. The standard products prepared with the protective agents of Examples 1 to 5 and Comparative Examples 1 to 9 were clear and transparent, but Comparative Example 4 had obvious bubbles, indicating that the defoaming agent had a significant effect on eliminating bubbles.

[0102] Uniformity evaluation: The high and low standards prepared in Examples 1 to 5 and Comparative Examples 4 and 7 to 8 were tested 10 times each, and the CV and range (R) were calculated. The results are shown in Table 1.

[0103] Table 1 Uniformity evaluation of the prepared standards of Examples 1 to 5 and Comparative Examples 4, 7 to 8 Concentration unit: ng / mL

[0104]

[0105] The in-bottle uniformity (CV) of Examples 1-5 and Comparative Examples 7-8 was within 2%, and the relative range (R) was within 5%. The in-bottle uniformity (CV) of Comparative Example 4 was approximately 4%, and the relative range (R) exceeded 10%. The in-bottle uniformity (CV) and relative range (R) of Comparative Example 4 were significantly worse than those of Examples 1-5 and Comparative Example 8, indicating that the addition of a defoamer can eliminate bubbles in the standard product and improve its uniformity. Comparative Example 8 differs from Comparative Example 4 in that the defoamer concentration is increased, but there is no significant difference in the in-bottle uniformity (CV) and relative range (R), indicating that the defoamer has fully achieved its defoaming effect in Example 2, and further increasing the defoamer concentration would only increase costs.

[0106] Stability Assessment: Standard samples of each protective agent were stored at 37°C, 25°C, and 2-8°C for 15 days, 3 months, and 18 months, respectively, at 37°C, and then tested. Recovery rates under different conditions were calculated, with a recovery rate of 90%-110% being the standard. The results are shown in Table 2.

[0107] Table 2 Stability evaluation of standard products prepared in Examples and Comparative Examples

[0108]

[0109]

[0110] The recovery of the standard products prepared in Examples 1 to 5 after being placed at 37°C for 15 days, 25°C for 3 months, and 2-8°C for 18 months was between 90% and 110%, and the stability met the requirements. The recovery of Example 1 fluctuated around 100%, which can be considered to be caused by systematic error. The recovery of Examples 2 to 5 was slightly lower than 100%, indicating that the activity had decreased slightly, but the decrease was within an acceptable range. Therefore, Example 1 is the optimal formula for the protective agent of this application.

[0111] Comparative Example 1, prepared with a conventional buffer, showed a significant decrease in the concentration of the prepared standard under different storage conditions, demonstrating that the protective agents in this application have a significant protective effect on proprotein convertase subtilisin 9. The standard prepared in Comparative Example 2 showed an increasing concentration under different storage conditions, suspected to be due to hydrolysis of proprotein convertase subtilisin 9 under the protective agent, exposing some active sites capable of antibody binding. While this is related to the specificity of the antibody, the fundamental cause is the instability of the standard.

[0112] The difference between Comparative Example 2 and Example 1 is that the surfactant is missing, which indicates that the surfactant in the present application has a certain protective effect on the hydrolysis of the standard.

[0113] The standards prepared in Comparative Examples 3 and 5 showed a significant decrease in storage under different conditions, especially under high temperature conditions. The difference between Comparative Example 3 and Example 1 is that no protein and amino acids were added, indicating that the protein and amino acids in this application have a protective effect on proprotein convertase subtilisin 9, preventing its degradation, and also have a certain effect on the thermal stability of the standard.

[0114] The difference between Comparative Example 5 and Example 1 is that the antioxidant is absent, indicating that the antioxidant can effectively prevent the oxidation of amino acids in proprotein convertase subtilisin 9 and has a strong protective effect on the protein structure.

[0115] The protective effects of Comparative Example 9 and Example 2 on proprotein convertase subtilisin 9 protein are basically the same, indicating that increasing the concentration of the antioxidant no longer improves the protective ability, and the screened concentration has met the protective effect.

[0116] Comparative Example 6 differs from Example 1 in that it lacks EDTA, but its protective effect on proprotein convertase subtilisin 9 is significantly reduced. EDTA can prevent protein oxidation by complexing metal ions, but it cannot prevent free radical oxidation of proteins. Combining Comparative Examples 1 and 5 reveals that neither an antioxidant nor EDTA alone can effectively prevent protein oxidation. Only the combined use of an antioxidant and EDTA effectively prevents oxidation of proprotein convertase subtilisin 9.

[0117] The protective effects of Comparative Examples 4 and 8 are basically consistent with those of Examples 1 and 2, indicating that the selected defoaming agent has no significant protective effect on proprotein convertase subtilisin 9. Comprehensive analysis of uniformity data shows that the role of the defoaming agent is to maintain good uniformity of the prepared standard.

[0118] Comparative Example 7 differs from Comparative Example 1 in that a different defoamer is used, but the protective effect of Comparative Example 7 is significantly reduced. The defoamer PE-M itself is a surfactant and may have a certain effect on the protein structure of proprotein convertase subtilisin 9. Therefore, not all defoamers are suitable as protective agents for proprotein convertase subtilisin 9.

[0119] Example 7

[0120] The reactivity of the anti-proprotein convertase subtilisin 9 monoclonal antibody after labeling with acridinium ester was investigated by adding different concentrations of Tween 20 (0%, 1%, 3%, 5%, and 7%). The results are shown in Table 3.

[0121] Table 3 Luminescence values after labeling with different concentrations of Tween 20 when acridinium ester is labeled

[0122]

[0123] During the acridinium ester labeling of an anti-proprotein convertase subtilisin-9 monoclonal antibody, the addition of different concentrations of Tween 20 standard resulted in an initial increase in luminescence followed by a plateau. Maximum luminescence was achieved at a 5% Tween 20 concentration, with the increase exceeding 100% compared to the control. The addition of Tween 20 increased the solubility of the acridinium ester and antibody, enhancing their binding efficiency and labeling efficiency, leading to a significant increase in luminescence. However, at a certain Tween 20 concentration, the luminescence value stopped increasing. This may be because the amounts of acridinium ester and antibody were low, and 5% Tween 20 was sufficient, while excess Tween 20 was ineffective.

[0124] Example 8

[0125] The anti-proprotein convertase subtilisin-9 monoclonal antibody from Example 7 was labeled with an acridinium ester and then incubated at -20°C for one month with varying concentrations of glycerol. The reagent was then prepared and the difference in luminescence values before and after mixing after 48 hours of stabilization was evaluated. The glycerol concentrations were 0%, 10%, 20%, 30%, 40%, and 50%, with the criterion for determining a decrease within 10%. The results are shown in Table 4.

[0126] Table 4 Uniformity of acridinium ester labeled antibodies prepared with different glycerol concentrations

[0127]

[0128] Acridinium ester-labeled antibodies need to be stored at low temperatures for long-term storage. Glycerol, as a surfactant, can increase the solubility of acridinium ester-labeled antibodies and reduce aggregation of acridinium ester-labeled antibodies during the freeze-thaw process, thereby improving the uniformity of the reagent.

[0129] As can be seen from Table 4, when the amount of glycerol added is above 20%, the uniformity of the reagent meets the requirements. As the amount of glycerol increases from 30% to 50%, the uniformity is basically consistent. Considering that excessive glycerol concentration and high viscosity can easily affect the accuracy of the preparation and sample addition process, the optimal concentration of glycerol is set at 30%.

[0130] Example 9

[0131] The concentration selection criteria of acridinium ester-labeled anti-proprotein convertase subtilisin-9 monoclonal antibody and horseradish peroxidase-labeled anti-proprotein convertase subtilisin-9 monoclonal antibody are as follows:

[0132] Acridinium ester-labeled anti-proprotein convertase subtilisin 9 monoclonal antibody and horseradish peroxidase-labeled anti-proprotein convertase subtilisin 9 monoclonal antibody were paired at different dilutions using the matrix method. The lowest cost pairing was selected based on the lowest signal-to-noise ratio greater than 5 and the highest signal-to-noise ratio greater than 100.

[0133] A square array assay was performed using acridinium ester-labeled anti-proprotein convertase subtilisin-9 monoclonal antibody at different dilutions of 1 / 50, 1 / 100, 1 / 250, 1 / 500, 1 / 1000, and 1 / 2000, and horseradish peroxidase-labeled anti-proprotein convertase subtilisin-9 monoclonal antibody at different dilutions of 1 / 1000, 1 / 2000, 1 / 4000, 1 / 8000, 1 / 16000, and 1 / 32000. The two ratios were cross-matched, and the optimal ratio was ultimately selected as 1:500 for acridinium ester-labeled anti-proprotein convertase subtilisin-9 monoclonal antibody and 1:4000 for horseradish peroxidase-labeled anti-proprotein convertase subtilisin-9 monoclonal antibody.

[0134] Example 10

[0135] As an embodiment of the proprotein convertase subtilisin 9 detection kit described in the present application, the detection reagents include a standard, reagent R1 (as shown in Table 5), and reagent R2 (as shown in Table 6). The standard is prepared according to the formula of Example 1.

[0136] Table 5 Reagent R1 (pH 7.5)

[0137]

[0138] Table 6 Reagent R2 (pH 7.5)

[0139]

[0140] Example 11

[0141] The proprotein convertase subtilisin 9 detection kit described in this application requires a fully automatic chemiluminescence analyzer (model HomoG 100) and a fully automatic immunoassay system substrate solution (Yuesui Medical No. 20190832) for detection, and its parameters are shown in Table 7.

[0142] Analytical method: The instrument is tested according to the process in Table 5. After the determination, the concentration of the standard is used as the X coordinate and the peak area is used as the Y coordinate to draw a dose-response curve as shown in the following figure: Figure 1 As shown, the equation is Y=776.99+52.851X, R^2=0.9988. The concentration of proprotein convertase subtilisin 9 in the sample was calculated based on the dose-response curve.

[0143] Table 7 Reaction Scheme

[0144] Sample size 5μL Reagent 1 volume 50μL Reagent 2 volume 50μL Incubation time 20min Substrate A amount 20 μL Substrate B amount 60μL Reading time 2S Interval time 0.05S

[0145] Test Example 1: Accuracy Test

[0146] Using the kit of the present application (the specific formula is the same as that of Example 10) and the proprotein convertase subtilisin 9 assay kit (enzyme-linked immunosorbent assay) of R&D, 40 fresh human sera were simultaneously assayed according to their respective parameters, and correlation regression analysis was performed on the measured values. The assay results are shown in FIG. Figure 2 .

[0147] Depend on Figure 2 The results show that the correlation coefficient of the two reagents is R^2=0.9967, and the regression equation is y=1.6785+0.9864x. The results show that the present kit has a good correlation with the commercial comparison kit in the determination of proprotein convertase subtilisin 9 in the sample, and has good comparability.

[0148] Test Example 2: Precision Test

[0149] Reagents: Kit of this application (specific formula is the same as Example 10).

[0150] Instrument: Fully automatic chemiluminescence analyzer, model: HomoG 100.

[0151] Procedure: Two serum samples of different concentrations were selected and tested 10 times each. The mean, SD, and CV were calculated. The results are shown in Table 8.

[0152] Table 8 Precision test evaluation results

[0153]

[0154]

[0155] The results in Table 8 show that the CV values of the kit of the present application for detecting two serum samples were both less than 2%, indicating good precision.

[0156] Test Example 3: Linear Test

[0157] The kit of the present application (specific formula is the same as that of Example 10) is used for high-value samples and low-value samples.

[0158] Instrument: Fully automatic chemiluminescence analyzer, model: HomoG 100.

[0159] Operation steps: Prepare 6 linear samples according to 5H, 4H+1L, 3H+2L, 2H+3L, 1H+4L, and 5L for the high and low value samples. The results of repeated measurements of each sample are shown in Table 9.

[0160] Table 9 Linear evaluation results

[0161]

[0162] As shown in Table 9, the kit of this application has good linearity in the range of 50 to 1000 ng / mL and a wide linear range. Figure 3 .

[0163] Test 4: Stability Assessment

[0164] The kit of the present application (specific formula is the same as that of Example 10) is used for high-value samples and low-value samples.

[0165] Instrument: Fully automatic chemiluminescence analyzer, model: HomoG 100.

[0166] Procedure: The kit produced in Example 10 was stored at 2-8°C for 15 months, and then tested against five internally determined samples. The relative deviation between the test results and the determined samples was calculated, with a relative deviation of ±10.00% being the standard for determination. The results are shown in Table 10.

[0167] Table 10 Reagent Stability Verification-Accuracy Assessment

[0168]

[0169] As shown in the results in Table 8=10, the relative deviations of the five fixed-value samples detected after the reagent kit of the present application was stored at 2-8°C for 15 months after preparation were all within ±10%, indicating that the reagent was stable after being stored at 2-8°C for 15 months, which is a significant improvement over the 6-month shelf life currently on the market.

[0170] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application and are not intended to limit the scope of protection of the present application. Although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application may be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present application.

Claims

1. A protective agent for proprotein convertase subtilisin 9 standard product, characterized in that: The protective agent is the following components in percentage by weight: Acid hydrolyzed casein 2wt%, arginine 1wt%, glycerol 5wt%, tea polyphenols 0.1wt%, BLOTTO-defoaming agent TBS solution 0.04wt%, EDTA 0.2wt%, KY1000.1wt%, the balance is phosphate buffer 10mM, and the pH value of the phosphate buffer is 7.

4.

2. Use of the protective agent for the proprotein convertase subtilisin 9 standard product according to claim 1 in the preparation of a detection product for measuring proprotein convertase subtilisin 9.

3. A detection kit for measuring proprotein convertase subtilisin 9, characterized in that: The detection kit comprises a standard, reagent 1, reagent 2 and the proprotein convertase subtilisin 9 standard protective agent according to claim 1; The components and final concentrations of the reagent R1 are: Buffer 5mM~100mM; Acridinium ester-labeled anti-proprotein convertase subtilisin 9 monoclonal antibody 1mg / L~4mg / L; Stabilizer 1 1w / v%~10w / v%; The components and final concentrations of the reagent R2 are: Buffer 5mM~100mM; Horseradish peroxidase-labeled proprotein convertase subtilisin-9 monoclonal antibody 0.1mg / L~1mg / L; Stabilizer 2 1w / v%~10w / v%.

4. The detection kit according to claim 3, wherein When the anti-proprotein convertase subtilisin 9 monoclonal antibody is labeled with acridinium ester, Tween 20 is also added at a mass concentration of 1wt%-5wt%; Anti-proprotein convertase subtilisin 9 monoclonal antibody was labeled with acridinium ester and then added with glycerol at a mass concentration of 20wt% to 50wt%.

5. The detection kit according to claim 3, wherein The buffer in the reagent 1 or reagent 2 includes at least one of a phosphate buffer, a Tris buffer and a MOPS buffer; The pH of the buffer solution in reagent 1 or reagent 2 is 6.5-8; The stabilizer 1 or the stabilizer 2 comprises at least one of bovine serum albumin, polyethylene glycol 20000, arginine, sodium thiosulfate, KY100 and horseradish peroxidase stabilizer.

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