Proprotein convertase subtilisin / kexin type 9 standard product protective agent, detection kit for determining proprotein convertase subtilisin / kexin type 9 and application of proprotein convertase subtilisin / kexin type 9 standard product protective agent

Abstract

The invention relates to the technical field of biomedical examination, and particularly discloses a proprotein convertase subtilisin / kexin type 9 standard product protective agent, a detection kit for determining proprotein convertase subtilisin / kexin type 9 and application of the detection kit. The protective agent is prepared from the following components in percentage by weight: 1 percent to 3 percent of protein, 0.5 percent to 2 percent of amino acid, 1 percent to 5 percent of a surfactant, 0.01 percent to 0.5 percent of an antioxidant, 0.01 percent to 0.2 percent of a de-foaming agent, 0.01 percent to 0.2 percent of EDTA (Ethylene Diamine Tetraacetic Acid), 0.05 percent to 0.2 percent of a preservative and the balance of a buffer solution. The proprotein convertase subtilisin / kexin type 9 standard product protective agent provided by the invention can significantly improve the stability of the proprotein convertase subtilisin / kexin type 9, prolong the effective period of the detection reagent, and ensure the accuracy and repeatability of the detection result. The detection kit provided by the invention is short in detection time, good in precision, high in sensitivity and good in 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 stability in the human body. Low-density lipoprotein cholesterol (LDL-C) first binds to the low-density lipoprotein cholesterol receptor (LDL-R) during metabolism, and then enters the cell through endocytosis. After entering the cell, LDL-R and LDL-C separate and return to the cell surface again, which can be reused repeatedly. Proprotein convertase subtilisin 9 binds to LDL-R and then to LDL-C to form a new complex that enters the lysosome and is degraded together, eventually leading to a decrease in LDL-R and a decrease in the degradation of LDL-C, thereby increasing the level of LDL-C. Increased LDL-C may lead to the occurrence of a series of cardiovascular diseases. Proprotein convertase subtilisin 9 is also an emerging target in the field of lipid-lowering drug development in recent years, and many drugs have been successfully developed at home and abroad. Whether it is the drug development process or the monitoring of the treatment process, the detection of proprotein convertase subtilisin 9 is indispensable.

[0003] There are already many manufacturers of scientific research testing reagents at home and abroad, but the shelf life of the test kits is mostly 6 months. The ELISA test method is already very mature, and the main factor affecting the shelf life of the test kit is the stability of the standard. The standard products in the existing scientific research kits are basically lyophilized powders, and they are high-concentration proprotein convertase subtilisin 9 antigens. During use, they need to be reconstituted with diluent first, and then diluted to a series of concentration gradient standards. The reconstituted standard needs to be stored at -20°C, and the shelf life does not exceed 1 month.

[0004] In general, the current standard products in scientific research kits have the following defects: a: Standard products need to be freeze-dried, which increases the production cost of manufacturers, and need to be reconstituted when used, which increases the uncertainty of the standard products; b: Standard products are not ready-to-use, and need to be diluted to different concentrations. Different people may have deviations in operation, resulting in differences in test results and poor repeatability of test results; c: The storage conditions after reconstitution are harsh and the freeze-thaw process may destroy the structure of proprotein convertase subtilisin 9, so it needs to be packaged and stored, which brings inconvenience to the operator. d: The shelf life of the kit is short. Considering the transportation time, the test reagents are close to the expiration date when they reach the user, which greatly increases the user's cost of use.

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

[0006] The purpose of the present application is to overcome the deficiencies of the above-mentioned prior art and provide a proprotein convertase subtilisin 9 standard product protective agent, a detection kit for determining proprotein convertase subtilisin 9 and its application. The standard product protective agent of the present application can effectively protect the proprotein convertase subtilisin 9 antigen, solve the problem of short validity period of the kit, and provide guarantee for the kit to achieve high accuracy, good repeatability, simple operation and cost saving. The detection kit provided by the present application has short detection time, good precision and high sensitivity.

[0007] To achieve the above purpose, the technical solution adopted by this application is:

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

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

[0010] The inventors of the present application have found through extensive research and experiments 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 surfactant added in this application can increase the solubility of the target protein, act as a protective layer, and improve thermal stability. The 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, the 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 found that the addition of EDTA and antioxidants in a synergistic ratio 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 the present application to eliminate bubbles generated by proteins, amino acids and surfactants, ensure that the prepared proprotein convertase subtilisin 9 can be evenly distributed in the solution, and ensure 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] Among them, the use of the preferred weight percentage of EDTA and antioxidant synergistic ratio can better prevent the oxidation of amino acids in the proprotein convertase subtilisin 9 protein and improve the protection of the proprotein convertase subtilisin 9 standard. The use of the preferred weight percentage of the defoaming agent can improve the uniformity of the distribution of proprotein convertase subtilisin 9 in the solution, thereby improving the reliability of the detection 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 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 substance 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 a phosphate buffer or a 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 a coordinated manner, 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 proprotein convertase subtilisin 9 standard protective agent 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 defoamer is BLOTTO-defoamer TBS solution.

[0028] The present application adopts the above-mentioned preferred type of defoaming agent, which can better eliminate the 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 product 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 1mg / L~4mg / 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 acridinium ester, Tween 20 with a mass concentration of 1wt%-5wt% is also added (preferably, the mass concentration of Tween 20 is 5wt%);

[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 30wt% of glycerol.

[0041] The conventional acridinium ester labeling method has a low labeling efficiency, resulting in high costs and low sensitivity of the test results. The storage of labeled acridinium ester antibodies is also crucial. Currently, long-term storage is mainly achieved through freezing, but the uniformity of the frozen and thawed acridinium ester labeled antibodies after preparation into reagents is greatly affected.

[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, improving 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 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 monoclonal antibody labeled with acridinium ester 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 method for preparing 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 pre-protein subtilisin 9 standard protective agent, which can significantly improve the stability of the standard and ensure the stability and reliability of the test results.

[0046] The high and low concentration standards prepared with the preservative of the preprotein subtilisin 9 standard of the present application formula remain stable at 37°C for 15 days, 25°C for 3 months, and 2-8°C for 18 months. The preservative does not require freeze-drying or dilution to different concentration gradients for standard testing, and can be used immediately, thereby reducing errors caused by artificial 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.

[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 a phosphate buffer, a Tris buffer and a MOPS buffer; preferably, the buffer is a MOPS buffer with a mass concentration of 25 mM.

[0048] The pH of the buffer 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 acridinium ester-labeled anti-proprotein convertase subtilisin 9 monoclonal antibody, antigen, and horseradish peroxidase-labeled anti-proprotein convertase subtilisin 9 monoclonal antibody to form an antibody-antigen-antibody complex, and no washing process is required. After the substrate is added, continuous detection is performed for a period of time immediately, each time at an interval of 0.02S to 0.05S, and the peak area is calculated. A dose-response curve is made using a known concentration of proprotein convertase subtilisin 9 and the calculated peak area, and the proprotein convertase subtilisin 9 content in the sample to be tested is inferred from the curve.

[0052] The kit of the present application has 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 also provides a method for determining proprotein convertase subtilisin 9 by chemiluminescence, comprising the following steps:

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

[0055] (3) Detecting the peak areas of the standard and the sample, making a dose-response curve with the concentration as the X coordinate and the peak area as the Y coordinate, and calculating 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 product, a detection kit for determining proprotein convertase subtilisin 9, and its application. The standard product prepared with the preservative for a proprotein convertase subtilisin 9 standard product of 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 standard product of proprotein convertase subtilisin 9 prepared with the preservative of the present application does not need to be lyophilized or diluted to different concentration gradients for standard product testing, and can be used immediately after opening, thereby reducing the errors caused by the artificial dissolution and dilution process, and ensuring that the test results are accurate and reliable. The validity period of the test kit is extended, the production and storage costs of the manufacturer are reduced, and the use costs of the user are reduced. In addition, the detection kit provided by the present application has the advantages of high sensitivity and fast detection speed, and can be equipped with instruments to realize fully automatic detection, which is convenient for promotion of 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 value of proprotein convertase subtilisin 9;

[0061] Figure 2 This is a correlation comparison chart between the detection kit prepared in Example 10 and the proprotein convertase subtilisin 9 detection kit (enzyme-linked immunosorbent assay) produced by R&D, 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 the present application. DETAILED DESCRIPTION

[0063] In order to better illustrate the purpose, technical solutions and advantages of the present application, the present application will be further described below in conjunction with 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 of the same type.

[0065] Example 1

[0066] As an embodiment of the proprotein convertase subtilisin 9 standard product protective agent described in the present application, the proprotein convertase subtilisin 9 standard product protective agent 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 (pH7.4).

[0068] Example 2

[0069] As an embodiment of the proprotein convertase subtilisin 9 standard product protective agent described in the present application, the proprotein convertase subtilisin 9 standard product protective agent 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 proprotein convertase subtilisin 9 standard product protective agent described in the present application, the proprotein convertase subtilisin 9 standard product protective agent 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 (pH7.0).

[0074] Example 4

[0075] As an embodiment of the proprotein convertase subtilisin 9 standard product protective agent described in the present application, the proprotein convertase subtilisin 9 standard product protective agent 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 proprotein convertase subtilisin 9 standard product protective agent described in the present application, the proprotein convertase subtilisin 9 standard product protective agent 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 (pH7.2).

[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 remaining 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 remaining 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%wt, 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 formulas 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 standard products just prepared 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 standard samples prepared in Examples 1 to 5 and Comparative Examples 4 and Comparative Examples 7 to 8 were selected and 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 standard products 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 to 5 and Comparative Examples 7 to 8 is within 2%, and the relative range (R) is within 5%. The in-bottle uniformity (CV) of Comparative Example 4 is about 4%, and the relative range (R) exceeds 10%. The in-bottle uniformity (CV) and relative range (R) of Comparative Example 4 are significantly worse than those of Examples 1 to 5 and Comparative Example 8, indicating that the addition of a defoamer can eliminate bubbles in the standard product and improve the uniformity of the standard product. The difference between Comparative Example 8 and Comparative Example 4 is that the concentration of the defoamer is increased, but there is no significant difference in the in-bottle uniformity (CV) and relative range (R), indicating that the defoamer has completely played a defoaming role in Example 2, and further increasing the concentration of the defoamer will only increase the cost.

[0106] Stability evaluation: The prepared standard products of each protective agent were placed at 37°C, 25°C and 2-8°C respectively. They were taken out for testing after being placed at 37°C for 15 days, 25°C for 3 months and 2-8°C for 18 months. The recovery under different conditions was calculated and judged based on the recovery rate of 90%-110%. 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 system error. The recovery of Examples 2 to 5 was slightly lower than 100%, indicating that the activity was slightly reduced, but the reduction was within an acceptable range. Therefore, Example 1 is the optimal formula of the protective agent of this application.

[0111] Comparative Example 1 is a conventional buffer, and the prepared standard product shows a significant decrease in storage under different conditions, indicating that the protective agents of each component in the present application have a significant protective effect on proprotein convertase subtilisin 9. The standard product prepared in Comparative Example 2 shows an upward trend when stored under different conditions. It is suspected that proprotein convertase subtilisin 9 is hydrolyzed under the protective agent, resulting in the exposure of some active sites that can bind to antibodies. Although this is related to the specificity of the antibody, the most fundamental reason is the instability of the standard product.

[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 product.

[0113] The standard products 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 acid are added, indicating that the protein and amino acid in the present 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 product.

[0114] The difference between Comparative Example 5 and Example 1 is that the antioxidant is missing, which indicates that the antioxidant can effectively prevent the oxidation of amino acids of 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 selected concentration has met the protective effect.

[0116] The difference between Comparative Example 6 and Example 1 is that EDTA is missing, but the protective effect on proprotein convertase subtilisin 9 is significantly reduced. EDTA can prevent protein oxidation by complexing metal ions, but cannot prevent free radicals from oxidizing proteins. Combining Comparative Examples 1 and 5, it is found that the presence of antioxidants or EDTA alone cannot effectively prevent protein oxidation. Only when antioxidants and EDTA are used in combination can the oxidation of proprotein convertase subtilisin 9 be effectively prevented.

[0117] The protective effects of Comparative Examples 4 and 8 are basically consistent with those of Examples 1 to 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 product.

[0118] The difference between Comparative Example 7 and Comparative Example 1 is that the defoamers used are different, but the protective effect of Comparative Example 7 is significantly reduced. The defoamer PE-M itself is also a surfactant, which 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] When the anti-proprotein convertase subtilisin 9 monoclonal antibody was labeled with acridinium ester, different concentrations of Tween 20 were added, namely 0%, 1%, 3%, 5% and 7%. The reactivity after labeling was investigated, and 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] In the process of acridinium ester labeling of anti-proprotein convertase subtilisin 9 monoclonal antibody, the luminescence value of the detection of adding different concentrations of Tween 20 standard showed a trend of first rising and then reaching a plateau. When the concentration of Tween 20 was 5%, the maximum luminescence value was reached, and the increase in luminescence value compared with the control exceeded 100%. The addition of Tween 20 increased the solubility of acridinium ester and antibody, making the binding rate of acridinium ester and antibody higher, improving the labeling efficiency, and thus significantly improving the detection luminescence value. However, when Tween 20 reached a certain concentration, the luminescence value no longer increased. It may be because the amount of acridinium ester and antibody is small, 5% Tween 20 can already meet the requirements, and excessive Tween 20 can no longer play a role.

[0124] Example 8

[0125] After the anti-proprotein convertase subtilisin 9 monoclonal antibody of Example 7 was labeled with acridinium ester, different concentrations of glycerol were added and placed at -20°C for one month, and then the reagent was prepared to evaluate the difference in luminescence value before and after mixing after the reagent was left standing for 48 hours. The glycerol concentrations were 0%, 10%, 20%, 30%, 40% and 50%, and the judgment was made based on the standard of a decrease of less than 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 the 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 added glycerol is above 20%, the uniformity of the reagent meets the requirements, and as the amount of glycerol increases from 30% to 50%, the uniformity is basically consistent. Considering that excessive glycerol concentration and high viscosity may 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 standard of acridinium ester labeled anti-proprotein convertase subtilisin 9 monoclonal antibody and horseradish peroxidase labeled anti-proprotein convertase subtilisin 9 monoclonal antibody is as follows:

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

[0133] The square array method was used to investigate the dilutions of 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 the dilutions of 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 groups were cross-matched, and the optimal ratio was finally 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, a reagent R1 (as shown in Table 5), and a 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] Embodiment 11

[0141] The proprotein convertase subtilisin 9 detection kit described in the present application needs to be equipped with a fully automatic chemiluminescence analyzer (model HomoG 100) and a substrate solution for a fully automatic immunoassay system (Guangdong Suixibei No. 20190832) for detection, and its parameters are shown in Table 7.

[0142] Analysis 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 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 is 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 20μL 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 in 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 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] Operation steps: Select 2 serum samples with different concentrations, test each sample 10 times, and calculate the mean, SD and CV of the test. 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%, and the precision was good.

[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 the results of Table 9, the kit of the present 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] Operation steps: Place the kit produced in Example 10 at 2-8°C for 15 months and then test the five samples with internal fixed values. Calculate the relative deviation between the test results and the fixed value samples, and make a judgment based on the relative deviation of ±10.00%. The test results are shown in Table 10.

[0167] Table 10 Reagent stability verification - accuracy assessment

[0168]

[0169] As shown in the results of Table 8=10, the relative deviations of the five fixed value samples detected after the preparation of the reagent kit of the present application was placed at 2-8°C for 15 months were all within ±10%, indicating that the reagent was stable after being placed 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 solution of the present application rather than 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, ordinary technicians in this field should understand that the technical solution of the present application can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present application.

Claims

1. A protective agent for a standard product of proprotein convertase subtilisin 9, characterized in that: The protective agent includes the following components in weight percentage: 1wt% to 3wt% protein, 0.5wt% to 2wt% amino acid, 1wt% to 5wt% surfactant, 0.01wt% to 0.5wt% antioxidant, 0.01wt% to 0.2wt% defoaming agent, 0.01wt% to 0.2wt% EDTA, 0.05wt% to 0.2wt% preservative, and the balance is buffer solution.

2. The proprotein convertase subtilisin 9 standard product protective agent according to claim 1, characterized in that: The protective agent includes the following components in weight percentage: 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.

3. The proprotein convertase subtilisin 9 standard product protective agent according to claim 1 or 2, characterized in that: 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.

4. The proprotein convertase subtilisin 9 standard product protective agent according to claim 1 or 2, characterized in that: The surfactant includes at least one of PEG400, glycerol and Tween 20.

5. The proprotein convertase subtilisin 9 standard product protective agent according to claim 1 or 2, characterized in that: The oxidant includes tea polyphenols and / or sodium thiosulfate; the buffer includes phosphate buffer or PBS buffer, and the concentration of the buffer is 5mM-30mM.

6. The proprotein convertase subtilisin 9 standard product protective agent according to claim 1 or 2, characterized in that: The defoamer comprises at least one of BLOTTO-defoamer TBS solution, SE-15 defoamer and 204 defoamer.

7. Use of the proprotein convertase subtilisin 9 standard product protective agent according to any one of claims 1 to 6 in the preparation of a detection product for measuring proprotein convertase subtilisin 9.

8. A detection kit for determining proprotein convertase subtilisin 9, characterized in that: The detection kit comprises a standard, reagent 1, reagent 2 and a protective agent for the proprotein convertase subtilisin 9 standard according to any one of claims 1 to 6; 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 1 w / v% to 10 w / v%.

9. The detection kit according to claim 8, characterized in that When the anti-proprotein convertase subtilisin 9 monoclonal antibody is labeled with acridinium ester, Tween 20 with a mass concentration of 1wt%-5wt% is also added; The anti-proprotein convertase subtilisin 9 monoclonal antibody is labeled with acridinium ester and then added with glycerol with a mass concentration of 20wt% to 50wt%.

10. The detection kit according to claim 8, characterized in that 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 in the reagent 1 or reagent 2 is 6.5 to 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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