Preservation solution rich in cysteine protein 61 antigen

Abstract

The invention discloses a cysteine-rich protein 61 antigen preserving fluid, which comprises a protein protective agent, a nonionic surfactant, a preservative, a soluble metal salt, a carbohydrate and a buffer solution, the buffer solution is a citric acid-citrate buffer solution, and the preserving fluid also comprises polyethylene glycol and polyvinylpyrrolidone; the pH (Potential of Hydrogen) value of the preservation solution is in a range of 5.0 to 5.5; the adding mass of the polyvinylpyrrolidone is 1.0-1.5 times of the mass of the polyethylene glycol; the protein protective agent at least contains serum. The preservation solution can stably preserve the cysteine-rich protein 61 antigen, can be stably preserved for 12 months at the temperature of 2-8 DEG C and can be stably preserved for 7 days at the temperature of 37 DEG C, the inconvenience caused by freeze-drying preservation of Cyr61 protein sold in the market due to stability is solved, and an important support is provided for clinical application of Cyr61 as a novel marker subsequently.

Description

Technical Field

[0001] The invention belongs to the technical field of immunoassay, and in particular relates to a cysteine-rich protein 61 antigen preservation solution. Background Art

[0002] The CCN (Cysteine-rich 61, Connective tissue growth factor, and Nephroblastoma overexpressed) protein family was discovered in the 1990s. There are six secreted proteins, named CCN1-CCN6. The first protein discovered in the CCN protein family was named CCN1. Because it is rich in cysteine, it is also called Cyr61 (Cysteine-rich protein 61, cysteine-rich protein 61). The molecular weight is about 42kDa, and pI = 8.64. Many studies have shown that Cyr61 protein is a multifunctional protein that is essential for physiological processes such as embryonic development, tissue damage repair, aging and angiogenesis. Studies have found that during tissue repair, myofibroblasts proliferate and secrete Cyr61, which drives myofibroblasts to mature and stop proliferation, and secrete matrix-degrading enzymes; in inflammatory responses, Cyr61 can enhance the apoptotic effect of inflammatory factors (TNFα, FasL and TRAIL); in vascular diseases, Cyr61 is highly expressed in vascular smooth muscle and stenotic neoendothelium at plaque lesions, and inhibiting the expression of Cyr61 can reduce endothelial proliferation. Related studies have determined that CCN family proteins are involved in the pathogenesis and progression of malignant and non-malignant lung diseases, among which Cyr61 plays an important role in lung diseases such as acute lung injury, chronic obstructive pulmonary disease, pulmonary fibrosis, pulmonary hypertension, lung infection, and lung cancer. Cyr61 in the blood is significantly increased in patients with ST-segment elevation myocardial infarction, and Cyr61 can also be significantly increased after septal branch occlusion in HOCM patients who undergo transcoronary artery ablation for ventricular septal hypertrophy. Other studies have shown that adding the Cyr61 index to the reference GRACE risk score at 30 days improved the risk stratification of all-cause mortality.

[0003] Currently, there are only Cyr61 protein detection ELISA kits for scientific research on the market. Most of the calibrator antigens are in high-concentration freeze-dried form, requiring users to use matching diluents for gradient dilution, and have poor stability, and can only be stored for 1 month at 2-8°C. In addition, due to stability issues, the Cyr61 proteins sold on the market are all stored in freeze-dried form, which is extremely inconvenient during use. In addition, the existing liquid antigen preservation solution can only meet the storage time conditions at 37°C, and cannot meet the storage time conditions at 2-8°C. Therefore, solving the stability of the Cyr61 antigen is extremely important for Cyr61 to move towards clinical application as a new marker. Summary of the invention

[0004] In view of this, it is necessary to provide a cysteine-rich protein 61 antigen preservation solution that can meet the stable storage time conditions at both low temperature (2-8°C) and relatively high temperature (37°C) to address the above problems, which is used to stably preserve the cysteine-rich protein 61 antigen and provide certain help for the subsequent development of Cyr61 clinical diagnostic kits.

[0005] In order to achieve the above-mentioned invention object, the present invention provides the following technical solutions:

[0006] A cysteine-rich protein 61 antigen preservation solution, the preservation solution comprising a protein protective agent, a nonionic surfactant, a preservative, a soluble metal salt, a sugar substance and a buffer solution, the buffer solution being a citric acid-citrate buffer solution, and further comprising polyethylene glycol and polyvinyl pyrrolidone;

[0007] The pH value of the preservation solution is in the range of 5.0 to 5.5; the mass of the added polyvinyl pyrrolidone is 1.0 to 1.5 times the mass of the polyethylene glycol;

[0008] The protein protective agent contains at least serum.

[0009] Furthermore, the polyethylene glycol is polyethylene glycol 2000.

[0010] The molecular weight of the polyvinyl pyrrolidone is not greater than 40,000, preferably not greater than 20,000.

[0011] The soluble metal salt is at least one of sodium chloride and potassium chloride, preferably sodium chloride.

[0012] Furthermore, the polyvinyl pyrrolidone is at least one of polyvinyl pyrrolidone K13-18, polyvinyl pyrrolidone K16-18, polyvinyl pyrrolidone K23-27, and polyvinyl pyrrolidone K30, or is a mixture of polyvinyl pyrrolidone K29-32 and low molecular weight polyvinyl pyrrolidone, so that its average molecular weight does not exceed 40,000.

[0013] Polyethylene glycol and polyvinyl pyrrolidone were added in equal amounts.

[0014] Furthermore, the polyvinyl pyrrolidone is at least one of polyvinyl pyrrolidone K13-18 (molecular weight of about 10,000), polyvinyl pyrrolidone K16-18 (average molecular weight of 8,000), polyvinyl pyrrolidone K23-27 (average molecular weight of 24,000), and polyvinyl pyrrolidone K30 (average molecular weight of 40,000), or a mixture of polyvinyl pyrrolidone K29-32 (average molecular weight of 58,000) and the above-mentioned low molecular weight polyvinyl pyrrolidone; preferably, polyvinyl pyrrolidone K13-18 (molecular weight of about 10,000).

[0015] Furthermore, the protein protective agent also includes one or more of bovine serum albumin, casein, and casein sodium salt; preferably, 3% bovine serum albumin.

[0016] Furthermore, the serum is at least one of commercial hormone-free human serum or commercial horse serum, preferably 5% commercial hormone-free human serum.

[0017] Furthermore, the nonionic surfactant comprises at least one of Tween 20, Triton X-100, Tween 40, Tween 80, and Brij 35; preferably, Tween 20, with a concentration of 0.3%.

[0018] Furthermore, the preservative comprises at least one of Proclin-300 or Proclin-950. Preferably, Proclin-300.

[0019] Furthermore, the components and contents of the preservation solution are as follows:

[0020] Citric acid-citrate buffer 8-15mmol / L;

[0021] Non-serum protein protective agent 1-5%;

[0022] Serum 3-10%;

[0023] Polyethylene glycol 0.8-1.5%;

[0024] Polyvinylpyrrolidone 0.8-1.5%;

[0025] Sucrose 1-5%;

[0026] Sodium chloride 100-150mmol / L;

[0027] Nonionic surfactant 0.1-0.5%;

[0028] Preservatives 0.08-0.2%.

[0029] Further preferably, the components and contents of the preservation solution are:

[0030] Citric acid-citrate buffer 10-13mmol / L;

[0031] Bovine serum albumin 2-4%;

[0032] Commercial hormone-free human serum 4-6%;

[0033] Polyethylene glycol 0.8-1.5%;

[0034] Polyvinylpyrrolidone K13-18 0.8-1.5%;

[0035] Sucrose 1-5%;

[0036] Sodium chloride 100-150mmol / L;

[0037] Nonionic surfactant 0.2-0.4%;

[0038] Preservatives 0.08-0.15%.

[0039] More preferably, the components and contents of the preservation solution are: 10 mmol / L citric acid-citrate buffer, 1% polyethylene glycol, 1% polyvinyl pyrrolidone, 1% sucrose, 100-150 mmol / L sodium chloride, 3% bovine serum albumin, 5% commercial hormone-free human serum, 0.1-0.5% non-ionic surfactant and 0.1% preservative; wherein the pH value of the preservation solution is in the range of 5.0 to 5.5; and the average molecular weight of polyvinyl pyrrolidone is 8000-20000.

[0040] The storage solution can stably store the cysteine-rich protein 61 antigen at 37° C. for 7 days and can stably store it at 2-8° C. for not less than 12 months, with a CV reduction of about 10% within 12 months.

[0041] The present invention also protects a use of the above-mentioned cysteine-rich protein 61 antigen preservation solution,

[0042] The storage solution is used for the storage of cysteine-rich protein 61 antigen;

[0043] The preservation solution can be used to prepare a cysteine-rich protein 61 test kit calibrator.

[0044] The concentration of the cysteine-rich protein 61 antigen is 600-2500 pg / mL.

[0045] Compared with the prior art, the present invention has the following beneficial effects:

[0046] The invention provides a cysteine-rich protein 61 antigen preservation solution, which can significantly improve the stability of the cysteine-rich protein 61 antigen when it is stored at 2-8°C and 37°C in a liquid state.

[0047] In addition, the preservation solution of the present invention can also be used for the preparation of cysteine-rich protein 61 calibrants, which can significantly improve the stability of the calibrants and facilitate the use of the cysteine-rich protein 61 kit in clinical testing. DETAILED DESCRIPTION

[0048] In order to enable those skilled in the art to better understand the solutions of the present invention, those skilled in the art can refer to the contents of this article and implement them by appropriately improving the process parameters. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the technical solutions protected by the present invention. The products and methods of the present invention have been described through preferred embodiments, and relevant personnel obviously cannot change or appropriately change the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0049] The reagent materials used in the present invention are all common commercial products and can be obtained in the market.

[0050] The present invention is further described below in conjunction with the examples. Unless otherwise specified, the raw materials used in the examples of the present invention are all commonly used raw materials in the art, and the methods used in the examples are all conventional methods in the art.

[0051] Example 1: Preparation of a cysteine-rich protein 61 antigen preservation solution, which is prepared from the following components:

[0052]

[0053] Example 2: Preparation of a cysteine-rich protein 61 antigen preservation solution, which is prepared from the following components:

[0054]

[0055] Example 3: Preparation of a cysteine-rich protein 61 antigen preservation solution, which is prepared from the following components:

[0056]

[0057] Example 4: Preparation of a cysteine-rich protein 61 antigen preservation solution, which is prepared from the following components:

[0058]

[0059]

[0060] Example 5: Preparation of a cysteine-rich protein 61 antigen preservation solution, which is prepared from the following components:

[0061]

[0062] Comparative Example 1: Preparation of a cysteine-rich protein 61 antigen preservation solution, the preparation method of which is the same as that of Example 1, except that the citric acid-citrate buffer in Example 1 is replaced with a 10 mmol / LMES buffer, and the other components and pH values ​​are the same.

[0063] Comparative Example 2: Preparation of a cysteine-rich protein 61 antigen preservation solution, the preparation method of which is the same as that of Example 1, except that the citric acid-citrate buffer in Example 1 is replaced with a 10 mmol / L MOPS buffer, and the other components and pH values ​​are the same.

[0064] Comparative Example 3: Preparation of a cysteine-rich protein 61 antigen preservation solution, the preparation method of which is the same as that of Example 1, except that the citric acid-citrate buffer in Example 1 is replaced with a 10 mmol / L HEPES buffer, and the other components and pH values ​​are the same.

[0065] Comparative Example 4: Preparation of a cysteine-rich protein 61 antigen preservation solution, the preparation method of which is the same as that of Example 1, except that the pH in Example 1 is changed to 7.2, and the other components are the same.

[0066] Comparative Example 5: Preparation of a cysteine-rich protein 61 antigen preservation solution, the preparation method of which is the same as that of Example 1, except that the polyvinyl pyrrolidone K13-18 in Example 1 is removed, and the other components and pH value are the same.

[0067] Stability test

[0068] Cysteine-rich protein 61 antigen (Cyr61) was prepared using Examples 1-5 and Comparative Examples 1-5 at concentrations of 625, 1250, and 2500 pg / mL, respectively, and stored at 2-8°C and 37°C, respectively. Cyr61 magnetic particle chemiluminescence reagent and Leadman CI1000 instrument were used to detect and track its stability, wherein the Cyr61 magnetic particle chemiluminescence reagent mainly includes carboxyl magnetic beads coupled to Cyr61 monoclonal antibodies and alkaline phosphatase coupled to Cyr61 monoclonal antibodies, and the reaction mode is that 60 μL of sample and 50 μL of alkaline phosphatase coupled to Cyr61 monoclonal antibodies react at 37°C for 20 minutes to form an immune complex, and then 50 μL of carboxyl magnetic beads coupled to Cyr61 monoclonal antibodies are added to react at 37°C for 20 minutes, washed three times, and the luminescence value (RLU) was detected after adding the substrate. The specific test results of the embodiments are shown below. Table 1-5 shows the results of Cyr61 antigen prepared by the preservation solution formula of Example 1-5 at 2-8°C, and Table 6-10 shows the results of Cyr61 antigen prepared by the preservation solution formula of Example 1-5 at 37°C.

[0069]

[0070]

[0071]

[0072]

[0073] The comparative test results are shown in the following table, wherein Tables 11-15 are the results of Cyr61 antigens prepared by the formulations of comparative examples 1-5 placed at 2-8°C, and Tables 16-20 are the results of Cyr61 antigens prepared by the formulations of comparative examples 1-5 placed at 37°C.

[0074]

[0075]

[0076]

[0077]

[0078] In summary, it can be seen from Table 1 and Table 6 that the Cyr61 antigen prepared using the formula of Example 1 is placed at 2-8°C for 13 months and 37°C for 7 days, and the decrease is no more than 10%, proving that the antigen preservation solution of the present invention can play a long-term stable protective role for the Cyr61 antigen. Combining Tables 1, 6, 11, 12, 13, 16, 17, and 18 (i.e., Example 1, Comparative Example 1, Comparative Example 2, and Comparative Example 3), it can be seen that the citric acid-citrate buffer in the present invention has obvious advantages over MES, MOPS, and HEPES buffers. When the Cyr61 antigen prepared using MES, MOPS, and HEPES buffers is placed at 2-8°C for 11 months, the decrease has exceeded 10%, and the decrease in 13 months exceeds 20%, and the decrease in 5 days at 37°C exceeds 10%, which is far inferior to the present invention. Combining Tables 1, 3, 6, 8, 14, and 19 (i.e., Example 1, Example 3, and Comparative Example 4), it can be seen that the acidic environment plays a crucial role in the stability of Cyr61. When the pH value is 5.0 or 5.5, the Cyr61 antigen is stored at 2-8°C for 13 months and at 37°C for 7 days, and the decrease is within 10%. When the solution is weakly alkaline (pH value is 7.2), the Cyr61 antigen decreases by more than 10% when stored at 2-8°C for 11 months, more than 20% when stored for 13 months, and more than 10% when stored at 37°C for 3 days. Combined with Tables 1, 2, 6, 7, 15, and 20 (i.e., Example 1, Example 2, and Comparative Example 5), it can be seen that polyvinyl pyrrolidone (PVP) in the present invention is an important substance for protecting the stability of the Cyr61 antigen. Different types of PVP can play a protective role, and the protective effect of PVP is related to its molecular weight. The protective effect of PVP K30 (average molecular weight 40,000) is not as good as that of PVP K13-18 (average molecular weight 10,000). The Cyr61 antigen prepared by the formula of Example 2 decreases by more than 10% when placed at 2-8°C for 12 months, and decreases by more than 10% when placed at 37°C for 7 days. After removing PVP from the formula of Comparative Example 5, the prepared Cyr61 antigen decreases by more than 10% when placed at 2-8°C for 11 months, and decreases by more than 10% when placed at 37°C for 5 days. Therefore, PVP K13-18 is preferred in the present invention. Combining the analysis of Tables 1, 4, 6, and 9 (i.e., Example 1 and Example 4), it can be seen that the use of the two preservatives has no significant effect on the stability of the Cyr61 antigen. The Cyr61 antigen prepared by the formula of Example 4 is stored at 2-8°C for 12 months with a decrease of less than 10%, and the decrease after 13 months is slightly more than 10%. Therefore, the present invention preferably uses Proclin-300 alone.Combining Tables 1, 5, 6, and 10 (i.e., Example 1 and Example 5), it can be seen that there is no significant difference in the protective effect of the buffer solution salt ion concentration of 10-15 mmol / L on the Cyr61 antigen. The Cyr61 antigen prepared by the formula of Example 5 is stored at 2-8°C for 12 months and decreases by less than 10%, and slightly exceeds 10% after 13 months. Therefore, the preferred citric acid-citrate buffer concentration of the present invention is 10 mmol / L.

[0079] Example 6

[0080] In this example, on the basis of Example 1, the content of polyethylene glycol was kept unchanged, and the amount of polyvinyl pyrrolidone added was changed. The mass of polyvinyl pyrrolidone added was 0.8, 1.2, and 1.5 times the mass of polyethylene glycol.

[0081] After testing, the preservation solution of this embodiment can also achieve the requirement of preserving Cyr61 antigen at 2-8°C for more than 12 months and at 37°C for 7 days.

[0082] For those skilled in the art, various other corresponding changes and modifications can be made according to the technical solutions and concepts described above, and all of these changes and modifications should fall within the protection scope of the claims of the present invention.

[0083] Any matters not described in the present invention are applicable to the prior art.

Claims

1. A cysteine-rich protein 61 antigen preservation solution, comprising a protein protective agent, a nonionic surfactant, a preservative, a soluble metal salt, a carbohydrate substance and a buffer, characterized in that: The buffer is a citric acid-citrate buffer, and also includes polyethylene glycol and polyvinyl pyrrolidone; The pH value of the preservation solution is in the range of 5.0 to 5.5; the mass of the added polyvinyl pyrrolidone is 1.0 to 1.5 times the mass of the polyethylene glycol; The protein protective agent contains at least serum.

2. The cysteine-rich protein 61 antigen storage solution according to claim 1, characterized in that: The polyethylene glycol is polyethylene glycol 2000; The molecular weight of the polyvinyl pyrrolidone is not more than 40000; The protein protective agent also includes one or more of bovine serum albumin, casein, and casein sodium salt; The soluble metal salt is at least one of sodium chloride and potassium chloride, preferably sodium chloride.

3. The cysteine-rich protein 61 antigen storage solution according to claim 1, characterized in that: The polyvinyl pyrrolidone is at least one of polyvinyl pyrrolidone K13-18, polyvinyl pyrrolidone K16-18, polyvinyl pyrrolidone K23-27, and polyvinyl pyrrolidone K30, or a mixture of polyvinyl pyrrolidone K29-32 and low molecular weight polyvinyl pyrrolidone, so that its average molecular weight does not exceed 40,000.

4. The cysteine-rich protein 61 antigen preservation solution according to claim 1, characterized in that: The serum is one of commercial hormone-free human serum and commercial horse serum, and the serum mass content is 3-10%; The nonionic surfactant comprises at least one of Tween 20, Triton X-100, Tween 40, Tween 80, and Brij 35; The preservative comprises at least one of Proclin-300 or Proclin-950.

5. The cysteine-rich protein 61 antigen storage solution according to claim 1, characterized in that: The components and contents of the preservation solution are as follows: Citric acid-citrate buffer 8-15mmol / L; Non-serum protein protective agent 1-5%; Serum 3-10%; Polyethylene glycol 0.8-1.5%; Polyvinylpyrrolidone 0.8-1.5%; Sucrose 1-5%; Sodium chloride 100-150mmol / L; Nonionic surfactant 0.1-0.5%; Preservatives 0.08-0.2%.

6. The cysteine-rich protein 61 antigen storage solution according to claim 5, characterized in that: The components and contents of the preservation solution are as follows: Citric acid-citrate buffer 10-13mmol / L; Bovine serum albumin 2-4%; Commercial hormone-free human serum 4-6%; Polyethylene glycol 0.8-1.5%; Polyvinylpyrrolidone K13-18 0.8-1.5%; Sucrose 1-5%; Sodium chloride 100-150mmol / L; Nonionic surfactant 0.2-0.4%; Preservatives 0.08-0.15%.

7. The cysteine-rich protein 61 antigen preservation solution according to any one of claims 1 to 6, characterized in that: The storage solution can stably store the cysteine-rich protein 61 antigen at 37° C. for 7 days, and can stably store it at 2-8° C. for not less than 12 months.

8. A use of the cysteine-rich protein 61 antigen preservation solution according to any one of claims 1 to 7, characterized in that: The storage solution is used for the storage of cysteine-rich protein 61 antigen; Alternatively, the storage solution is used to prepare a cysteine-rich protein 61 kit calibrator.

9. The use according to claim 8, characterized in that: The concentration of the cysteine-rich protein 61 antigen is 600-2500 pg / mL.

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