A stable protective agent for low-concentration enzyme-labeled streptococcal protein G

Abstract

The present invention relates to the field of biological detection, and specifically discloses a stable protective agent for low-concentration enzyme-labeled streptococcal protein G. In order to solve the problem of poor stability of enzyme-labeled streptococcal protein G at low concentration, the protective agent is composed of the following raw materials: 0.1-0.5% fish gelatin, 0.1%-0.5% chicken egg white albumin, 0.5% polyvinyl pyrrolidone, 0.5% triethanolamine, 0.2% glycerol, 0.02% Tween 20, and 0.1% Proclin 300. The present invention adopts inert protein, high molecular polymer, and polyhydroxy compound, and can maintain the structure and activity stability of enzyme-labeled streptococcal protein G in a low-concentration liquid state for a long time; adopts a new bactericidal preservative inhibition system, can effectively prevent microbial growth, is safe and environmentally friendly, and effectively prolongs the shelf life; adopts inert protective protein to avoid interference with enzyme-labeled streptococcal protein G.

Description

Technical Field

[0001] The invention relates to the technical field of biological detection, in particular to a stable protective agent for low-concentration enzyme-labeled streptococcal protein G. Background Art

[0002] Enzyme-linked immunosorbent assay (ELISA) is an immunodiagnostic technique that uses enzyme-labeled complexes as secondary antibodies. It has the characteristics of simple operation, rapid reaction, and high-throughput detection of multiple samples. It is widely used in many fields such as biology and medicine. As one of the key components in ELISA kits, the stability of enzyme-labeled complexes is also a key indicator for measuring the quality of kit products. As a protein, enzyme-labeled complexes need to be stored under low temperature conditions. Therefore, under normal circumstances, the enzyme-labeled complex stock solution needs to be stored at -20°C or below. However, in ELISA kits, enzyme-labeled complexes need to be diluted more than thousands of times before use, and stored at 2°C-8°C together with the ELISA kit. At this time, low-concentration enzyme-labeled complexes are often unstable and easily degraded and lose biological activity. Therefore, it is urgent to provide a stable protective agent for low-concentration enzyme-labeled complexes so that they can be stored at 2°C-8°C with stable properties and no significant reduction in biological activity.

[0003] Streptococcal protein G (G protein for short) is a streptococcal cell wall protein that can bind to a variety of mammalian antibodies IgG. Because of its strong binding force and wide binding spectrum, it is often used in the field of antibody detection of various mammals instead of secondary antibodies. At present, there are invention patents CN115993460A involving alkaline phosphatase labeled myoglobin antibody dilution, CN109900900A involving alkaline phosphatase labeled procalcitonin antigen or antibody dilution, etc., but so far, there is no patent report involving enzyme labeled streptococcal protein G stabilizing protective agent. In view of the unique biological characteristics of enzyme labeled streptococcal protein G and its wide application prospects in detection kits, it is necessary to further improve and enhance the stability of enzyme labeled streptococcal protein G at low concentrations. Summary of the invention

[0004] The purpose of the present invention is to solve the shortcomings of the prior art and to propose a low-concentration enzyme-labeled streptococcal protein G stabilizing protectant, which can effectively protect the working concentration enzyme-labeled streptococcal protein G and keep it stable at 2°C-8°C for 12 months without affecting its biological activity.

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

[0006] A stabilizing protective agent for low-concentration enzyme-labeled streptococcal protein G is composed of the following raw materials: 0.1-0.5% fish gelatin, 0.1%-0.5% chicken egg white albumin, 0.5% polyvinyl pyrrolidone, 0.5% triethanolamine, 0.2% glycerol, 0.02% Tween 20, and 0.1% Proclin 300. The percentages of the raw materials are all W / V, i.e., mass volume percentage. The buffer used for the stabilizing protective agent is a 0.02 mol / L tris(hydroxymethyl)aminomethane-hydrochloric acid buffer containing 0.2 mol / L sodium chloride, and the pH of the buffer is 7.2±0.2.

[0007] As a further technical solution of the present invention, the present invention is composed of the following raw materials: 0.3-0.5% fish gelatin, 0.3%-0.5% chicken egg white albumin, 0.5% polyvinyl pyrrolidone, 0.5% triethanolamine, 0.2% glycerol, 0.02% Tween 20, and 0.1% Proclin300. The percentages of the raw materials are all W / V, i.e., mass volume percentage. The buffer used for the stabilizing protective agent is a 0.02 mol / L tris(hydroxymethyl)aminomethane-hydrochloric acid buffer containing 0.2 mol / L sodium chloride, and the pH of the buffer is 7.2±0.2.

[0008] As a further technical solution of the present invention, it is composed of the following raw materials: 0.5% fish gelatin, 0.5% chicken egg white albumin, 0.5% polyvinyl pyrrolidone, 0.5% triethanolamine, 0.2% glycerol, 0.02% Tween 20, and 0.1% Proclin300. The percentages of the raw materials are all W / V, that is, mass volume percentage. The buffer used for the stabilizing protective agent is 0.02 mol / L tris(hydroxymethyl)aminomethane-hydrochloric acid buffer containing 0.2 mol / L sodium chloride, and the pH of the buffer is 7.2±0.2.

[0009] Polyvinyl pyrrolidone, triethanolamine, glycerol, Tween 20 and Proclin 300 are synergistically compounded in a specific ratio so that the enzyme-labeled streptococcal protein G can maintain high activity and stability at low concentrations.

[0010] Fish gelatin and chicken egg white albumin are both inert proteins that can replace fetal bovine serum, bovine serum albumin, etc. commonly used in immunological testing as low-concentration protein supplementary protective agents, and will not specifically react with the target protective protein.

[0011] Polyvinyl pyrrolidone, as a non-ionic polymer compound, can be used as a dispersant for liquid preparations to improve the stability of enzymes and G proteins in liquid environments.

[0012] Triethanolamine is an antioxidant, which can prevent enzyme-labeled streptococcal protein G from being oxidized and degraded, thereby improving the stability of enzyme-labeled streptococcal protein G.

[0013] Glycerol, as a polyhydroxy compound, can form hydrogen bonds with enzyme-labeled streptococcal protein G and form a water film around the enzyme-labeled streptococcal protein G to prevent the enzyme-labeled streptococcal protein G from being hydrolyzed and denatured, and can prevent the denaturant from approaching the active center of the enzyme.

[0014] As a nonionic surfactant, Tween 20 can react with the hydrophobic groups on the surface of enzyme-labeled streptococcal protein G, reduce the risk of local aggregation and precipitation of enzyme-labeled streptococcal protein G, and reduce the adsorption loss of enzyme-labeled streptococcal protein G in the container.

[0015] Proclin300, as a heterocyclic bactericidal preservative, can effectively inhibit the growth of microorganisms in the enzyme-labeled dilution protection solution system to extend the shelf life of the preparation.

[0016] As a further technical solution of the present invention, the preparation steps of the tris(hydroxymethyl)aminomethane-hydrochloric acid buffer solution are as follows:

[0017] S1: Weigh tris(hydroxymethyl)aminomethane) and sodium chloride into an appropriate amount of pure water and stir to dissolve;

[0018] S2: Adjust the pH to 7.2±0.2 with hydrochloric acid, and add purified water to a final concentration of 0.02 mol / L of tris(hydroxymethyl)aminomethane and 0.2 mol / L of sodium chloride;

[0019] S3: Stir and mix well, filter and sterilize with a 0.22 μm filter membrane to obtain tris(hydroxymethylaminomethane)-hydrochloride buffer solution.

[0020] The beneficial effects of the present invention are:

[0021] 1. By using inert protein, high molecular polymer and polyhydroxy compound in a certain proportion, the structure and activity stability of enzyme-labeled streptococcal protein G in a low concentration liquid state can be maintained for a long time.

[0022] 2. The new bactericidal preservative inhibition system is adopted, which not only does not produce biochemical reaction with enzyme-labeled streptococcal protein G, but also can effectively prevent the growth of microorganisms, is safe and environmentally friendly, and effectively extends the shelf life.

[0023] 3. Take full account of the high affinity and broad spectrum of enzyme-labeled streptococcal protein G, avoid using protective proteins such as fetal bovine serum, BSA, casein, etc. that may react specifically with protein G, and instead use inert protective proteins such as fish gelatin and chicken egg white albumin from non-mammalian systems to avoid interference with enzyme-labeled streptococcal protein G. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1The present invention proposes a stable protective agent for low-concentration enzyme-labeled streptococcal protein G. In Example 3, the detection OD of enzyme-labeled streptococcal protein G stored at 37°C ± 2°C is 450 Value change trend chart;

[0025] Figure 2 The OD value of the enzyme-labeled streptococcal protein G stored at 2°C-8°C in Example 3 is 0.0447 W / m2 / L. 450 Value change trend chart. DETAILED DESCRIPTION

[0026] In order to make the technical means, creative features, objectives and effects achieved by the present invention easy to understand, the present invention is further explained below in conjunction with specific implementation methods.

[0027] A stabilizing protective agent for low-concentration enzyme-labeled streptococcal protein G is composed of the following raw materials: 0.1-0.5% fish gelatin, 0.1%-0.5% chicken egg white albumin, 0.5% polyvinyl pyrrolidone, 0.5% triethanolamine, 0.2% glycerol, 0.02% Tween 20, and 0.1% Proclin 300. The percentages of the raw materials are all W / V, i.e., mass volume percentage. The buffer used for the stabilizing protective agent is a 0.02 mol / L tris(hydroxymethyl)aminomethane-hydrochloric acid buffer containing 0.2 mol / L sodium chloride, and the pH of the buffer is 7.2±0.2.

[0028] In a preferred embodiment, the preparation steps of Tris-HCl buffer are:

[0029] S1: Weigh tris(hydroxymethyl)aminomethane) and sodium chloride into an appropriate amount of pure water and stir to dissolve;

[0030] S2: Adjust the pH to 7.2±0.2 with hydrochloric acid, and add purified water to a final concentration of 0.02 mol / L of tris(hydroxymethyl)aminomethane and 0.2 mol / L of sodium chloride;

[0031] S3: Stir and mix well, filter and sterilize with a 0.22 μm filter membrane to obtain tris(hydroxymethylaminomethane)-hydrochloride buffer solution.

[0032] Example 1

[0033] To every 100 ml of tris(hydroxymethyl)aminomethane-hydrochloride buffer, add 0.3 g of fish gelatin, 0.3 g of chicken egg white albumin, 0.02 ml of Tween 20, and 0.1 ml of Proclin 300, respectively. After fully dissolved, filter and sterilize with a 0.22 μm filter membrane to obtain the enzyme-labeled streptococcal protein G stabilizing protective agent 1 (EDB1).

[0034] Example 2

[0035] To every 100 ml of tris(hydroxymethyl)aminomethane-hydrochloride buffer, add 0.3 g of fish gelatin, 0.3 g of chicken egg white albumin, 0.5 g of polyvinyl pyrrolidone, 0.2 ml of glycerol, 0.02 ml of Tween 20, and 0.1 ml of Proclin 300 respectively. After fully dissolved, filter and sterilize with a 0.22 μm filter membrane to obtain the enzyme-labeled streptococcal protein G stabilizing protective agent 2 (EDB2).

[0036] Example 3

[0037] To every 100 ml of tris(hydroxymethylaminomethane)-hydrochloride buffer, add 0.1 g of fish gelatin, 0.1 g of chicken egg white albumin, 0.5 g of polyvinyl pyrrolidone, 0.2 ml of glycerol, 0.02 ml of Tween 20, and 0.1 ml of Proclin 300 respectively. After fully dissolved, filter and sterilize with a 0.22 μm filter membrane to obtain the enzyme-labeled streptococcal protein G stabilizing protective agent 3 (EDB3).

[0038] Example 4

[0039] To every 100 ml of tris(hydroxymethylaminomethane)-hydrochloride buffer, add 0.1 g of fish gelatin, 0.5 g of chicken egg white albumin, 0.5 g of polyvinyl pyrrolidone, 0.2 ml of glycerol, 0.02 ml of Tween 20, and 0.1 ml of Proclin 300 respectively. After fully dissolved, filter and sterilize with a 0.22 μm filter membrane to obtain the enzyme-labeled streptococcal protein G stabilizing protective agent 4 (EDB4).

[0040] Example 5

[0041] To every 100 ml of tris(hydroxymethylaminomethane)-hydrochloride buffer, add 0.5 g of fish gelatin, 0.1 g of chicken egg white albumin, 0.5 g of polyvinyl pyrrolidone, 0.5 g of triethanolamine, 0.2 ml of glycerol, 0.02 ml of Tween 20, and 0.1 ml of Proclin 300 respectively. After fully dissolved, filter and sterilize with a 0.22 μm filter membrane to obtain the enzyme-labeled streptococcal protein G stabilizing protective agent 5 (EDB5).

[0042] Example 6

[0043] To every 100 ml of tris(hydroxymethylaminomethane)-hydrochloride buffer, add 0.5 g of fish gelatin, 0.5 g of chicken egg white albumin, 0.5 g of polyvinyl pyrrolidone, 0.5 g of triethanolamine, 0.2 ml of glycerol, 0.02 ml of Tween 20, and 0.1 ml of Proclin 300 respectively. After fully dissolved, filter and sterilize with a 0.22 μm filter membrane to obtain the enzyme-labeled streptococcal protein G stabilizing protective agent 6 (EDB6).

[0044] Comparative Example 1

[0045] To every 100 ml of tris(hydroxymethylaminomethane)-hydrochloride buffer, add 0.6 g BSA, 0.5 g polyvinyl pyrrolidone, 0.2 ml glycerol, 0.02 ml Tween 20, and 0.1 ml Proclin 300 respectively. After fully dissolved, filter and sterilize with a 0.22 μm filter membrane to obtain a stable protective agent (EDB) for comparison enzyme-labeled streptococcal protein G.

[0046] The effects of the stabilizing protective agents for enzyme-labeled streptococcal protein G prepared in Examples 1 to 6 were evaluated, and the specific implementation steps were carried out according to the following application examples:

[0047] Application Example 1

[0048] The enzyme-labeled streptococcal protein G stabilizing protective agents prepared in Examples 1 to 6 and Comparative Example 1 were used to dilute the enzyme-labeled streptococcal protein G by 1:4000 times to a working concentration.

[0049] The diluted enzyme-labeled streptococcal protein G was stored in a constant temperature and humidity chamber at 37°C±2°C for 1 week and in a refrigerator at 2°C-8°C for 12 months, and the changes in physical properties and the decrease in absorbance were observed.

[0050] Application Example 2

[0051] The working concentration of enzyme-labeled streptococcal protein G stored at 37°C ± 2°C in Application Example 1 was sampled and tested for OD450 every day. nm The absorbance value of the working concentration enzyme-labeled streptococcal protein G stored at 2℃-8℃ in Application Example 1 was sampled and tested for OD450 at 0, 6, and 12 months. nm The absorbance value was calculated and the decrease in absorbance value was calculated.

[0052] The test results show that the enzyme-labeled streptococcal protein G diluted with a stabilizing protective agent is stored at 37°C±2°C for 7 days and at 2°C-8°C for 12 months. Its physical properties are clear and transparent liquid without precipitation or turbidity, and remain unchanged.

[0053] Application Example 3

[0054] The ELISA plate coated with the E2 protein of classical swine fever virus was added with the working concentration of the positive serum and negative serum of classical swine fever virus antibody, incubated at 37°C for 45 minutes, washed 4 times with PBST, and then the enzyme-labeled streptococcal protein G prepared in the above-mentioned comparative example 1 and examples 1-6 was added, incubated at 37°C for 45 minutes, washed 4 times with PBST, and the substrate solution was added for color development. After 10 minutes, the stop solution was added and the plate was set to read the OD450. nm The absorbance values ​​of the test results are shown in Table 1 and Table 2, and the stability change trend is shown in Figure 1 and Figure 2 .

[0055] From Table 1 and Figure 1 It can be seen that after being stored at 37℃±2℃ for 7 days, the OD 450 The value decreased by 32.95%. The OD of Example 1 to Example 6 450 The values ​​decreased by 14.05%, 8.06%, 14.85%, 11.85%, 11.23% and 4.98% respectively, indicating that Example 6 has the best effect.

[0056] From Table 2 and Figure 2 It can be seen that after being stored at 2°C-8°C for 12 months, the OD 450 The value decreased by 36.70%, and the OD of Example 1 to Example 6 450 The values ​​decreased by 21.57%, 6.03%, 22.34%, 16.51%, 15.17% and 4.71% respectively, indicating that Example 6 has the best effect.

[0057] The above results show that the stabilizing protective agent in Example 6 has the best protective effect on enzyme-labeled Streptococcus protein G. The enzyme-labeled Streptococcus protein G diluted with the stabilizing protective agent in Example 6 has no significant change in biological activity when stored at 37°C±2°C for 7 days and 2°C-8°C for 12 months. 450 The value variation range is less than 10%, indicating that the present invention has excellent effect of preserving and stabilizing enzyme-labeled streptococcal protein G.

[0058] Table 1 Detection results of enzyme-labeled streptococcal protein G stored at 37°C ± 2°C

[0059]

[0060] Table 2 Test results of enzyme-labeled streptococcal protein G stored at 2°C-8°C

[0061]

[0062] From the above description, it can be seen that the above embodiments of the present invention achieve the following technical effects: by using inert protein, high molecular polymer and polyhydroxy compound in a certain proportion, the structure and activity of enzyme-labeled streptococcal protein G in a low concentration liquid state can be maintained for a long time;

[0063] The new bactericidal preservative inhibition system is used, which not only does not react biochemically with enzyme-labeled streptococcal protein G, but also effectively prevents the growth of microorganisms, is safe and environmentally friendly, and effectively extends the shelf life;

[0064] Taking into full consideration the high affinity and broad spectrum of enzyme-labeled streptococcal protein G, we avoid using protective proteins such as fetal bovine serum, BSA, casein, etc. that may react specifically with protein G. Instead, we use inert protective proteins such as fish gelatin and chicken ovalbumin from non-mammalian systems to avoid interference with enzyme-labeled streptococcal protein G.

[0065] Those skilled in the art should understand that the discussion of any of the above embodiments is merely illustrative and is not intended to imply that the scope of the present invention (including the claims) is limited to these examples. Under the concept of the present invention, the technical features in the above embodiments or different embodiments may be combined, the steps may be implemented in any order, and there are many other variations of the different aspects of the present invention as described above, which are not provided in detail for the sake of simplicity.

[0066] The present invention is intended to cover all such substitutions, modifications and variations that fall within the broad scope of the claims. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A stable protective agent for low-concentration enzyme-labeled streptococcal protein G, characterized in that: The invention is composed of the following raw materials: 0.1%-0.5% fish gelatin, 0.1%-0.5% chicken egg white albumin, 0.5% polyvinyl pyrrolidone, 0.5% triethanolamine, 0.2% glycerol, 0.02% Tween 20 and 0.1% Proclin 300. The percentages of the raw materials are all W / V, i.e., mass volume percentage. The buffer used for stabilizing the protective agent is 0.02 mol / L tris(hydroxymethyl)aminomethane-hydrochloric acid buffer containing 0.2 mol / L sodium chloride, and the pH of the buffer is 7.2±0.

2.

2. The stabilizing protective agent for low-concentration enzyme-labeled streptococcal protein G according to claim 1, characterized in that: The invention is composed of the following raw materials: 0.3%-0.5% fish gelatin, 0.3%-0.5% chicken egg white albumin, 0.5% polyvinyl pyrrolidone, 0.5% triethanolamine, 0.2% glycerol, 0.02% Tween 20 and 0.1% Proclin 300. The percentages of the raw materials are all W / V, i.e., mass volume percentage. The buffer used for stabilizing the protective agent is 0.02 mol / L tris(hydroxymethyl)aminomethane-hydrochloric acid buffer containing 0.2 mol / L sodium chloride, and the pH of the buffer is 7.2±0.

2.

3. The stabilizing protective agent for low-concentration enzyme-labeled streptococcal protein G according to claim 1, characterized in that: The invention is composed of the following raw materials: 0.5% fish gelatin, 0.5% chicken egg white albumin, 0.5% polyvinyl pyrrolidone, 0.5% triethanolamine, 0.2% glycerol, 0.02% Tween 20 and 0.1% Proclin 300. The percentages of the raw materials are all W / V, i.e., mass volume percentage. The buffer used for stabilizing the protective agent is 0.02 mol / L tris(hydroxymethyl)aminomethane-hydrochloric acid buffer containing 0.2 mol / L sodium chloride, and the pH of the buffer is 7.2±0.

2.

4. The stabilizing protective agent for low-concentration enzyme-labeled streptococcal protein G according to claim 1, characterized in that: The preparation steps of the tris(hydroxymethyl)aminomethane-hydrochloric acid buffer solution are as follows: S1: Weigh tris(hydroxymethyl)aminomethane) and sodium chloride into an appropriate amount of pure water and stir to dissolve; S2: Adjust the pH to 7.2±0.2 with hydrochloric acid, and add purified water to a final concentration of 0.02 mol / L of tris(hydroxymethyl)aminomethane and 0.2 mol / L of sodium chloride; S3: Stir and mix well, filter and sterilize with a 0.22 μm filter membrane to obtain tris(hydroxymethylaminomethane)-hydrochloride buffer solution.

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