Protection liquid for protecting immunocompetence of antigen as well as preparation method and application of protection liquid

Abstract

The invention provides a protective solution for protecting antigen immunocompetence as well as a preparation method and application of the protective solution. The protective solution comprises the following components: soybean protein, sucrose, trehalose, thiourea, glycerol and Proclin-300. The method has the beneficial effects that 1, the cost is low: as the components in the protection liquid are common reagents, the price is low, and the cost of a product is not increased; 2, the processing and the use are convenient: only mixing with the antigen according to the proportion is needed, and no instrument or equipment is needed to be added; 3, the storage cost and the transportation cost are saved: compared with the storage cost at the temperature of-20 DEG C, the storage cost is reduced at the temperature of 2-8 DEG C, and the transportation cost is also reduced; and 4, the kit is convenient for a user to use, the antigen does not need to be independently stored in the use process by the user due to the characteristic of good antigen stability at 2-8 DEG C and normal temperature, the antigen activity cannot be reduced even if the kit is repeatedly used, and the accuracy of a detection result is ensured.

Description

Technical Field

[0001] The present invention belongs to the technical field of immunology, and in particular relates to a protective solution for protecting the immune activity of an antigen and a preparation method thereof, which is suitable for the long-term stable storage of antigens in immunological diagnostic reagents. Background Art

[0002] The specificity of an antigen is determined by specific chemical groups on its surface. These groups, known as antigenic determinants, are the material basis for the specificity of the immune response and immune reaction, and play a crucial role in antigenic specificity. Antigenic determinants primarily exist on the surface of the antigen, with a smaller number potentially present internally, requiring enzymatic or other treatment to expose them. A single antigen can possess multiple specific chemical groups.

[0003] Antigen specificity primarily refers to the compatibility, targeting, and specificity between substances, manifested in immunogenicity and reactogenicity. The former refers to the ability of an antigen to stimulate the body's immune cells to produce corresponding specific antibodies and sensitized lymphocytes; the latter refers to the ability of a specific antigen to bind specifically only to the corresponding antibodies or sensitized lymphocytes stimulated by it. Substances that possess both immunogenicity and reactogenicity are called complete antigens; substances that possess only reactogenicity are called haptens.

[0004] The specific reaction between antigens and antibodies is the foundation of immunological testing. If the determinants that determine antigen specificity change, the antigen's specificity will disappear or weaken, leading to abnormal or failed immune responses. Antigen determinants are susceptible to change at room temperature or 2-8°C, a problem that can pose challenges for corresponding immunological testing products. Immunological products are typically stored at 2-8°C and used at room temperature, and antigens are a component of antibody-blocking products.

[0005] To ensure product stability, there are currently two commonly used methods: 1. Freeze-drying the antigen, which can maintain stability at 2-8°C, but the user needs to re-dissolve it when using it, and after re-dissolution, it still needs to be stored at -20°C, which causes inconvenience in use, and freeze-drying increases product costs. 2. Separate the reagents and store them separately at -20°C, which is different from other components stored at 2-8°C. This causes inconvenience to the user and causes inconvenience in packaging and transportation. In addition, the freeze-dried antigen needs to be dissolved when used for the first time, and the dissolved antigen still needs to be stored at -20°C. It cannot be stored at 2-8°C with other components. The use process requires repeated freezing and thawing, which seriously damages the activity of the antigen.

[0006] In immunological reactions, the activity of antigens is extremely unstable and is greatly affected by storage conditions. Generally, they need to be stored at -20°C, which conflicts with the storage conditions of 2-8°C for immunological products. In addition, repeated freeze-thaw cycles during use will accelerate the loss of antigen activity, significantly affecting experimental results. Therefore, maintaining the stability of antigens is an issue that must be addressed. Summary of the Invention

[0007] The present invention aims to solve the problem that antigens easily lose their activity when stored at 2-8°C, and provides a low-cost, easy-to-operate protective solution so that antigens and supporting reagents can be stored at the same temperature, avoiding activity loss caused by repeated freezing and thawing.

[0008] The technical solution adopted by the present invention is: a protective liquid for protecting the immune activity of an antigen, comprising the components of soybean protein, sucrose, trehalose, thiourea, glycerol and Proclin-300.

[0009] Furthermore, the invention comprises the following components by volume percentage: 1-3% soy protein, 8-12% sucrose, 3-7% trehalose, 0.5-2% thiourea, 2-5% glycerol, 0.01-0.1% Proclin-300, and the balance is deionized water.

[0010] Furthermore, the following components are included by volume percentage: 2% soy protein, 10% sucrose, 5% trehalose, 1% thiourea, 3% glycerol, 0.05% Proclin-300, and the balance is deionized water.

[0011] A method for preparing a protective solution for protecting the immune activity of an antigen comprises the following steps: weighing the components according to the ratio of claim 2 or 3; mixing and then fixing the volume, filtering with a 0.45u microporous filter and storing at 2-8°C.

[0012] The protective liquid is used in an immunoassay reagent, wherein the mixing volume ratio of the protective liquid to the antigen is 1:3.

[0013] Furthermore, the antigen is a foot-and-mouth disease type A liquid-phase blocking ELISA reagent.

[0014] The beneficial effects achieved by the present invention are as follows: 1. Low cost: Since the components in the protective solution are all common reagents, they are inexpensive and will not increase the cost of the product; 2. Easy processing and use: It only needs to be mixed with the antigen in proportion, and no additional instruments or equipment are required; 3. Savings in storage and transportation costs: Storage at 2-8°C reduces storage costs compared to storage at -20°C, and transportation costs are also reduced; 4. Convenient use for users: The good stability of the antigen at 2-8°C and room temperature means that users do not need to store the antigen separately during use, and repeated use will not lead to a decrease in antigen activity, thereby ensuring the accuracy of the test results.

[0015] Principle Description

[0016] Soy protein (especially soybean trypsin inhibitor, Soybean Trypsin Inhibitor, STI) can specifically inhibit the activity of serine proteases such as trypsin and chymotrypsin. These proteases may be present due to sample contamination or cell lysis, resulting in hydrolysis and destruction of antibodies. By inhibiting proteases, the risk of antibody degradation during storage or experiments is reduced. Soy protein can be used as an inert protein (similar to BSA or casein), competitively binding to the container surface (such as the wall of a plastic tube), reducing the nonspecific adsorption of antibodies and avoiding antibody loss due to adsorption. High-concentration proteins (such as soy protein or BSA) can provide a stable colloidal environment and reduce the probability of antibody aggregation or denaturation. Soy protein is low in cost and is a common substitute for bovine serum albumin (BSA). It is particularly suitable for experiments that are sensitive to animal-derived components (avoiding the risk of cross-reaction of exogenous antibodies or viral contamination).

[0017] Certain soy protein ingredients may have antioxidant properties, helping to reduce oxidative stress damage to antibodies. They also work synergistically with other buffer components (such as Tris and PBS) to maintain pH stability. Applicability: Not all antibody buffers contain soy protein; the choice depends on the antibody type (e.g., enzyme-labeled or fluorescently labeled) and experimental requirements.

[0018] Potential interference: In immunoassays, soy protein may cross-react with certain experimental systems (e.g., soy protein-based ELISA blocking agents), and compatibility needs to be evaluated.

[0019] In summary, soy protein in antibody protection solution mainly extends the shelf life by inhibiting proteases and stabilizing the antibody structure, while reducing costs and reducing nonspecific adsorption.

[0020] In immunoassays (e.g., ELISA), glycine may affect certain antigen-antibody bindings, requiring experimental verification of compatibility. It is often used in combination with sugars (e.g., sucrose, trehalose), BSA, or surfactants (e.g., Tween-20) to enhance protective effects.

[0021] Glycine extends the shelf life and maintains the activity of antibodies through multiple mechanisms, including stabilizing antibody structure, inhibiting aggregation, regulating pH, and freeze-drying protection. It is particularly widely used in freeze-dried antibody formulations. DETAILED DESCRIPTION

[0022] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments.

[0023] Example

[0024] This protective solution is composed of a variety of reagents, prepared in scientific proportions. The proportions and configuration process are as follows:

[0025] 1. Reagent composition

[0026]

[0027] 2. Preparation process of protective solution

[0028] Weigh each reagent → adjust volume → mix → filter

[0029] 3. Preparation method:

[0030] 3.1. Calculate the required quantity of each ingredient according to the above ratio based on the volume to be prepared.

[0031] 3.2. Weigh each ingredient using a 1 / 10,000 balance to an accuracy of 0.001.

[0032] 3.3. Combine all components in a clean container and adjust the volume to the required volume.

[0033] 3.4. Dissolve, mix well, filter through a 0.45u microporous filter, and store at 2-8℃.

[0034] 4. How to use the protective solution

[0035] The protective solution and antigen were mixed in the ratio of antigen protective solution: antigen = 1:3.

[0036] 5. Protective effect test: Use the foot-and-mouth disease (FMD) type A liquid phase blocking ELISA kit as an example for testing. The prepared antigen is placed at 2-8°C, -20°C, and 37°C. Test every two months at 2-8°C and -20°C, and every day at 37°C. The degree of reduction in antigen activity is determined by the change in OD value. The testing process is as follows:

[0037]

[0038]

[0039] 6. Detection process

[0040] Return the components of the kit to room temperature.

[0041] 6.1. Before starting the experiment, please remove all components of the kit and equilibrate them to room temperature.

[0042] 6.2. Add 50 μl of the sample diluent provided in the kit to the wells indicated in the sample loading diagram. Seal the plate, shake, and incubate at 37°C for 30 minutes.

[0043] 6.3. Wash the plate three times with PBST, pat dry, add 50 μl / well of foot-and-mouth disease type A enzyme-labeled antibody working solution, seal the plate, and incubate at 37°C for 30 minutes.

[0044] 6.4. Wash the plate three times as above and pat dry. Mix substrate solution A and solution B in a 1:1 ratio, add 50 μl of the mixed substrate solution to each well, and incubate at 37°C for 15 minutes to develop color.

[0045] 6.5. After the color reaction is completed, add 50ul / well of stop solution to stop the reaction and read the D value on the microplate reader. 450nm value.

[0046] 7. Test results:

[0047]

[0048] Note: Antigen activity decline rate = (OD value on the day - OD value on day 0) / OD value on day 0

[0049] The above results indicate the following: 1. After 14 months of storage at 2-8°C, the OD value of the antigen decreased by less than 2%, and the activity remained essentially unchanged. 2. After 14 months of storage at -20°C, the OD value of the antigen decreased by less than 45%, and the activity decreased by nearly half, making this temperature unsuitable for storage. 3. After 7 days of storage at 37°C, equivalent to 14 months of storage at 2-8°C, the OD value decreased by less than 4%, and the activity remained essentially unchanged.

[0050] The above results indicate that the protective solution of the present invention has a good protective effect on the activity of antigens at a temperature of 2-8°C.

[0051] The above description is only a preferred specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any technician familiar with the technical field, within the technical scope disclosed by the present invention, who makes equivalent replacements or changes based on the technical solution and inventive concept of the present invention, should be covered by the scope of protection of the present invention.

Claims

1. A protective solution for protecting the immune activity of an antigen, characterized in that: The invention comprises the following ingredients: soy protein, sucrose, trehalose, thiourea, glycerol and Proclin-300.

2. A protective solution for protecting the immune activity of an antigen according to claim 1, characterized in that: The invention comprises the following components by volume percentage: 1-3% soybean protein, 8-12% sucrose, 3-7% trehalose, 0.5-2% thiourea, 2-5% glycerol, 0.01-0.1% Proclin-300 and the balance is deionized water.

3. A protective solution for protecting the immune activity of an antigen according to claim 2, characterized in that: The invention comprises the following components by volume percentage: 2% soy protein, 10% sucrose, 5% trehalose, 1% thiourea, 3% glycerol, 0.05% Proclin-300, and the balance is deionized water.

4. A method for preparing a protective solution for protecting the immune activity of an antigen, characterized in that: The following steps are involved: Weigh the components according to the ratio of claim 2 or 3; After mixing, the mixture was fixed to volume, filtered through a 0.45u microporous filter and stored at 2-8°C.

5. The application of the protective solution in immunoassay reagents is characterized by: The mixing volume ratio of the protective solution to the antigen is 1:

3.

6. The use of the protective solution in an immunoassay reagent according to claim 5, characterized in that: The antigen is a foot-and-mouth disease type A liquid phase blocking ELISA reagent.

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