A cysteine-rich protein 61 antigen preservative solution

Abstract

The application discloses a cysteine-rich protein 61 antigen storage solution, which comprises a protein protective agent, a non-ionic surfactant, a preservative, a soluble metal salt, a sugar substance and a buffer solution, wherein the buffer solution is a citric acid-citrate buffer solution, and the storage solution further comprises polyethylene glycol and polyvinylpyrrolidone; the pH value of the storage solution is in the range of 5.0-5.5; the added mass of the polyvinylpyrrolidone is 1.0-1.5 times of the mass of the polyethylene glycol; and the protein protective agent at least contains serum. The storage solution can stably store the cysteine-rich protein 61 antigen, and the cysteine-rich protein 61 antigen can be stably stored for 12 months at 2-8 DEG C and for 7 days at 37 DEG C, thereby solving the inconvenience caused by the freeze-drying storage of Cyr61 protein due to the stability and providing important support for Cyr61 as a new marker for subsequent clinical application.

Description

Technical Field

[0001] This invention belongs to the field of immunoassay technology, and more specifically, relates to a preservation solution rich in cysteine ​​protein 61 antigen. Background Technology

[0002] The CCN (Cysteine-rich 61, Connective tissue growth factor, and Nephroblastoma overexpressed) protein family was discovered in the 1990s and consists of six secreted proteins, named CCN1 through CCN6. The first protein discovered in the CCN family was named CCN1, and because it is rich in cysteine, it is also known as Cyr61 (Cysteine-rich protein 61), with a molecular weight of approximately 42 kDa and a pI of 8.64. Many studies have shown that Cyr61 is a multifunctional protein, crucial 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, driving myofibroblast maturation and ceasing proliferation, and secreting matrix-degrading enzymes. In inflammatory responses, Cyr61 can enhance the apoptotic effects of inflammatory factors (TNFα, FasL, and TRAIL). In vascular diseases, Cyr61 is highly expressed in vascular smooth muscle and newly formed endothelial tissue at plaque lesions; inhibiting Cyr61 expression can reduce endothelial hyperplasia. Related studies have identified CCN family proteins involved in the pathogenesis and progression of malignant and non-malignant lung diseases, with Cyr61 playing a crucial role in acute lung injury, chronic obstructive pulmonary disease, pulmonary fibrosis, pulmonary hypertension, pulmonary infection, and lung cancer. Blood Cyr61 levels are significantly elevated in patients with ST-segment elevation myocardial infarction (STEMI), and also significantly elevated in patients with HOCM (hypertrophic ventricular septal disease) who undergo coronary ablation for ventricular septal hypertrophy after ventricular septal branch occlusion. Other studies have shown that incorporating the Cyr61 index into the reference GRACE risk score at 30 days improves risk stratification for all-cause mortality.

[0003] Currently, the only Cyr61 protein detection ELISA kits on the market are for research use. Most of their calibrator antigens are in high-concentration lyophilized form, requiring users to perform serial dilutions using the provided diluents. Furthermore, their stability is poor, lasting only one month at 2–8°C. Additionally, commercially available Cyr61 proteins are all lyophilized due to stability issues, making them extremely inconvenient to use. Moreover, existing liquid antigen preservation solutions only meet the preservation time requirements at 37°C, failing to do so at 2–8°C. Therefore, solving the stability problem of Cyr61 antigen is crucial for Cyr61 to be used clinically as a novel biomarker. Summary of the Invention

[0004] In view of this, it is necessary to provide a preservation solution for cysteine-rich protein 61 antigen that can simultaneously meet the conditions for stable preservation time at both low temperature (2-8℃) and relatively high temperature (37℃), so as to provide some assistance for the subsequent development of Cyr61 clinical diagnostic kits.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] A preservation solution rich in cysteine ​​protein 61 antigen, the preservation solution comprising a protein protectant, a nonionic surfactant, a preservative, a soluble metal salt, a sugar substance and a buffer solution, wherein the buffer solution is a citrate-citrate buffer solution, and further comprises polyethylene glycol and polyvinylpyrrolidone.

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

[0008] The protein protectant contains at least serum.

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

[0010] The molecular weight of the polyvinylpyrrolidone 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] Further, the polyvinylpyrrolidone is at least one of polyvinylpyrrolidone K13-18, polyvinylpyrrolidone K16-18, polyvinylpyrrolidone K23-27, and polyethylene polyvinylpyrrolidone K30, or a mixture of polyvinylpyrrolidone K29-32 and low molecular weight polyvinylpyrrolidone, such that its average molecular weight does not exceed 40,000.

[0013] Polyethylene glycol and polyvinylpyrrolidone are added by mass.

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

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

[0016] Further, the serum is at least one of commercially available hormone-free human serum or commercially available horse serum, preferably 5% commercially available 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 at a concentration of 0.3%.

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

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

[0020] Citrate-citrate buffer solution 8-15 mmol / L;

[0021] Non-serum protein protectant 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 100-150 mmol / L;

[0027] Nonionic surfactant 0.1-0.5%;

[0028] Preservative: 0.08-0.2%.

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

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

[0031] Bovine serum albumin 2-4%;

[0032] Commercially available hormone-free human serum contains 4-6%;

[0033] Polyethylene glycol 0.8-1.5%;

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

[0035] Sucrose 1-5%;

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

[0037] Nonionic surfactant 0.2-0.4%;

[0038] Preservative: 0.08-0.15%.

[0039] More preferably, the components and their contents of the preservation solution are as follows: 10 mmol / L citrate-citrate buffer, 1% polyethylene glycol, 1% polyvinylpyrrolidone, 1% sucrose, 100-150 mmol / L sodium chloride, 3% bovine serum albumin, 5% commercially available hormone-free human serum, 0.1-0.5% nonionic 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 polyvinylpyrrolidone is 8000-20000.

[0040] The preservation solution can stably preserve cysteine-rich protein 61 antigen. It can be stably preserved at 37°C for 7 days and at 2-8°C for at least 12 months. Within 12 months, the CV decrease is about 10%.

[0041] This invention also protects the application of the above-mentioned preservation solution rich in cysteine ​​protein 61 antigen.

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

[0043] The preservation solution can be used to prepare calibrators for reagent kits rich in cysteine ​​protein 61.

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

[0045] Compared with the prior art, the beneficial effects of the present invention are:

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

[0047] In addition, the preservation solution of the present invention can also be used to prepare calibrators rich in cysteine ​​protein 61, which can significantly improve the stability of the calibrators and facilitate the use of cysteine ​​protein 61-rich kits in clinical testing. Detailed Implementation

[0048] To enable those skilled in the art to better understand the solutions of this invention, they can refer to the content herein and implement them by appropriately modifying the process parameters. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included within the scope of the technical solutions protected by this invention. The products and methods of this invention have been described through preferred embodiments, and those skilled in the art should clearly not deviate from the content, spirit, and scope of this invention to modify or appropriately combine the methods and applications described herein to implement and apply the technology of this invention.

[0049] The reagents and materials used in this invention are all commercially available products and can be obtained from the market.

[0050] The present invention will be further illustrated below with reference to embodiments. Unless otherwise specified, the raw materials used in the embodiments of the present invention are all commonly used in the art, and the methods used in the embodiments are all conventional methods in the art.

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

[0052]

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

[0054]

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

[0056]

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

[0058]

[0059]

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

[0061]

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

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

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

[0065] Comparative Example 4: Preparation of a preservation solution rich in cysteine ​​protein 61 antigen, the preparation method is the same as in 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 preservation solution rich in cysteine ​​protein 61 antigen. The preparation method is the same as in Example 1, except that polyvinylpyrrolidone K13-18 in Example 1 is removed, while the remaining 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. The samples were stored at 2–8°C and 37°C, respectively. Stability was detected and monitored using a Cyr61 magnetic microparticle chemiluminescence reagent and a Leadman CI1000 instrument. The Cyr61 magnetic microparticle chemiluminescence reagent mainly consisted of carboxyl magnetic beads conjugated with Cyr61 monoclonal antibody and alkaline phosphatase conjugated with Cyr61 monoclonal antibody. The reaction mode was as follows: 60 μL of sample and 50 μL of Cyr61 monoclonal antibody-conjugated alkaline phosphatase were reacted at 37°C for 20 min to form an immune complex. Then, 50 μL of Cyr61 monoclonal antibody-conjugated carboxyl magnetic beads were added, and the reaction was carried out at 37°C for 20 min. After washing three times, substrate was added, and the luminescence value (RLU) was detected. The specific test results of the examples are shown below. Tables 1-5 show the results of Cyr61 antigen prepared with the preservation solution formula of Examples 1-5 being placed at 2-8°C, and Tables 6-10 show the results of Cyr61 antigen prepared with the preservation solution formula of Examples 1-5 being placed at 37°C.

[0069]

[0070]

[0071]

[0072]

[0073] The results of the comparative experiments are shown in the following tables. Tables 11-15 show the results of Cyr61 antigen prepared according to the formulations of comparative examples 1-5 when placed at 2-8℃, and Tables 16-20 show the results of Cyr61 antigen prepared according to the formulations of comparative examples 1-5 when placed at 37℃.

[0074]

[0075]

[0076]

[0077]

[0078] In summary, as shown in Tables 1 and 6, the Cyr61 antigen prepared using the formulation of Example 1 showed a degradation rate of no more than 10% after 13 months at 2–8°C and 7 days at 37°C, proving that the antigen preservation solution of the present invention can provide long-term stable protection for the Cyr61 antigen. Analysis of Tables 1, 6, 11, 12, 13, 16, 17, and 18 (i.e., Example 1, Comparative Example 1, Comparative Example 2, and Comparative Example 3) reveals that the citrate-citrate buffer solution of the present invention has significant advantages over MES, MOPS, and HEPES buffers. Under the same acidic pH conditions, the Cyr61 antigen prepared using MES, MOPS, and HEPES buffers showed a degradation rate exceeding 10% after 11 months at 2–8°C, exceeding 20% ​​after 13 months, and exceeding 10% after 5 days at 37°C, far inferior to the solution of the present invention. Analysis of Tables 1, 3, 6, 8, 14, and 19 (i.e., Example 1, Example 3, and Comparative Example 4) shows that an acidic environment plays a crucial role in the stability of Cyr61. When the pH value is 5.0 or 5.5, the Cyr61 antigen decreases by less than 10% after being placed at 2–8°C for 13 months and at 37°C for 7 days. However, when the solution is weakly alkaline (pH value 7.2), the Cyr61 antigen decreases by more than 10% after being placed at 2–8°C for 11 months, by more than 20% after 13 months, and by more than 10% after 3 days at 37°C. Analysis of Tables 1, 2, 6, 7, 15, and 20 (i.e., Example 1, Example 2, and Comparative Example 5) shows that polyvinylpyrrolidone (PVP) is an important substance for protecting the stability of Cyr61 antigen in this invention. 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 formulation of Example 2 decreased by more than 10% after being placed at 2-8°C for 12 months and by more than 10% after being placed at 37°C for 7 days. However, after removing PVP from the formulation of Comparative Example 5, the Cyr61 antigen prepared by it decreased by more than 10% after being placed at 2-8°C for 11 months and by more than 10% after being placed at 37°C for 5 days. Therefore, PVP K13-18 is preferred in this invention. Analysis of Tables 1, 4, 6, and 9 (i.e., Examples 1 and 4) shows that the two methods of using preservatives have no significant effect on the stability of Cyr61 antigen. The Cyr61 antigen prepared by the formulation in Example 4 decreased by less than 10% after being stored at 2–8°C for 12 months, while the decrease slightly exceeded 10% after 13 months. Therefore, Proclin-300 is preferred to be used alone in this invention.Analysis of Tables 1, 5, 6, and 10 (i.e., Examples 1 and 5) shows that there is no significant difference in the protective effect of buffer salt concentrations of 10-15 mmol / L on Cyr61 antigen. The Cyr61 antigen prepared by the formulation in Example 5 decreased by less than 10% after 12 months of storage at 2-8°C, and slightly exceeded 10% after 13 months. Therefore, the preferred concentration of citrate-citrate buffer in this invention is 10 mmol / L.

[0079] Example 6

[0080] This embodiment is based on Example 1, keeping the content of polyethylene glycol unchanged, but changing the amount of polyvinylpyrrolidone added. The mass of polyvinylpyrrolidone added is 0.8, 1.2, and 1.5 times the mass of polyethylene glycol.

[0081] Tests have shown that the preservation solution in this embodiment can also meet the requirements 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 based on the technical solutions and concepts described above, and all such changes and modifications should fall within the protection scope of the claims of this invention.

[0083] Any aspects not covered in this invention are applicable to existing technologies.

Claims

1. A cysteine-rich protein 61 antigen storage solution comprising a protein protective agent, a non-ionic surfactant, a preservative, a soluble metal salt, a saccharide substance and a buffer, characterized in that, The buffer is citric acid-citrate buffer, and further comprises polyethylene glycol and polyvinylpyrrolidone; The pH value of the storage solution is in the range of 5.0-5.5; the added mass of the polyvinylpyrrolidone is 1.0-1.5 times of the mass of the polyethylene glycol; The polyethylene glycol is polyethylene glycol 2000; The protein protective agent at least contains serum; The polyvinylpyrrolidone is polyvinylpyrrolidone K13-18; The storage solution can stably store the cysteine-rich protein 61 antigen, and can stably store the cysteine-rich protein 61 antigen at 37°C for 7 days and at 2-8°C for not less than 12 months.

2. The cysteine-rich protein 61 antigen storage solution according to claim 1, characterized in that, The protein protective agent further comprises one or more of bovine serum albumin, casein and sodium caseinate; The soluble metal salt is at least one of sodium chloride and potassium chloride.

3. The cysteine-rich protein 61 antigen storage solution of claim 1, wherein, The serum is one of commercial de-gonad human serum and commercial horse serum, and the mass content of the serum is 3-10%; The non-ionic 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.

4. The cysteine-rich protein 61 antigen storage solution of claim 1, wherein, The components and contents of the storage solution are as follows: The citric acid-citrate buffer is 8-15 mmol / L; The non-serum protein protective agent is 1-5%; The serum is 3-10%; The polyethylene glycol is 0.8-1.5%; The polyvinylpyrrolidone is 0.8-1.5%; The sucrose is 1-5%; The sodium chloride is 100-150 mmol / L; The non-ionic surfactant is 0.1-0.5%; The preservative is 0.08-0.2%.

5. The cysteine-rich protein 61 antigen storage solution of claim 4, wherein, The components and contents of the storage solution are as follows: The citric acid-citrate buffer is 10-13 mmol / L; The bovine serum albumin is 2-4%; The commercial de-gonad human serum is 4-6%; The polyethylene glycol is 0.8-1.5%; The polyvinylpyrrolidone K13-18 is 0.8-1.5%; The sucrose is 1-5%; The sodium chloride is 100-150 mmol / L; The non-ionic surfactant is 0.2-0.4%; The preservative is 0.08-0.15%.

6. Use of the cysteine-rich protein 61 antigen storage solution according to any one of claims 1-5, characterized in that, The storage solution is used for storing the cysteine-rich protein 61 antigen; Or the storage solution is used for preparing the cysteine-rich protein 61 kit calibrator.

7. Use according to claim 6, characterized in that, The concentration of the cysteine-rich protein 61 antigen is 600-2500 pg / mL.

Images