Preparation method of high-coverage-rate polyclonal antibody and obtained polyclonal antibody
Through the compromise and separation of the primary material, the immunization is suitable for young and middle -aged animals, which obtains antibodies of different components, and mix it in proportion to solve the problem of low coverage of the dopon anti -system. The high -coverage of the Dopolon anti -system preparation is achieved, which significantly reduces the risk of exogenous protein detection in biological products.
Patent Information
- Application Number
- CN202411508082.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-28
- Publication Date
- 2025-01-28
AI Technical Summary
In the existing polyclonal antibody preparation methods, the coverage rate of antibodies is relatively low, which leads to a greater risk of detection of residual exogenous proteins in biological products, which may lead to serious adverse reactions or shock.
Through the compromise and separation of the primary substance, according to the differences of molecular weight, polarity, antigen tables, and dissolving degree in water in the antigen substances, they are immune to young and young animals that are suitable for menstrual age, obtain antibodies of different components, and these these and these will be used. The antibody is mixed according to a certain proportion to obtain a highly covered polyxon antibody.
The high coverage of Dopolon antibodies can be achieved, and the coverage rate can reach 75 ~ 93%, which significantly reduces the risk of residual exogenous protein detection in biological products.
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Figure CN119371519A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of biotechnology, and particularly relates to a method for preparing a high-coverage polyclonal antibody and the obtained polyclonal antibody. Background Art
[0002] In the quality control of residual exogenous proteins in biological products, ELISA kits are often used for sample detection. In sample detection, the polyclonal antibodies used in the kits need to have a high coverage rate to meet the needs of biological residue monitoring. Low antibody coverage rates lead to a large risk of missed detection of risk proteins in the established biological residue detection methods. Biological products released using such methods will cause serious adverse reactions in patients, and in severe cases may even cause shock in people with allergic constitutions.
[0003] The conventional method for preparing polyclonal antibodies is to actively immunize animals (such as rabbits, goats, etc.) multiple times, collect serum, and purify it to obtain polyclonal antibodies. There is also a method of re-immunizing animals with the immunized serum as an antigen to provide a coverage level.
[0004] At present, the coverage rate of polyclonal antibodies obtained by purifying serum collected from animals through multiple active immunizations still does not meet the requirements. Once the antigen is injected into the animal, the immunization process is completely uncontrollable, which easily leads to a waste of animal resources. Summary of the invention
[0005] The purpose of the present invention is to provide a method for preparing a high-coverage polyclonal antibody and the obtained polyclonal antibody, so as to solve the technical problem of low coverage in the current polyclonal antibody preparation.
[0006] The present invention adopts the following technical solution: A method for preparing a high-coverage polyclonal antibody comprises the following steps: (S1) Fully analyzing the antigenic substances, distinguishing and separating them according to one or more characteristics, and preparing antigens of different components; (S2) using the antigens of different components obtained in step (S1) to immunize young and middle-aged animals of appropriate age, respectively, to obtain serum antibodies, collect and purify them, and obtain antibodies of different components from corresponding sources; (S3) Mix the different component antibodies in a certain proportion to obtain a polyclonal antibody product with high coverage.
[0007] Because there may be up to thousands of proteins of different properties in the antigen, it is necessary to fully analyze the antigen components before animal immunization to understand the protein mixture of the antigen chemical and biological characteristics in the antigen components, including the differences in molecular weight, polarity, antigenic epitopes and solubility in water. Therefore, in the above technical scheme of the present invention, by fully analyzing and separating the antigenic substances, according to the differences in molecular weight, polarity, antigenic epitopes and solubility in water of the antigenic substances, the prepared antigenic substances of different components are respectively immunized with young and middle-aged animals of suitable months of age, and the serum antibodies obtained are respectively collected, purified and analyzed, and the antibodies from these different sources are mixed according to a certain ratio to obtain a polyclonal antibody product with a specific coverage rate.
[0008] The recognition rate or coverage of the polyclonal antibody raw materials used in the enzyme-linked immunosorbent assay of biological detection technology for the antigen of the test sample plays a decisive role in the successful development of subsequent detection methods. The present invention can obtain polyclonal antibody raw materials with a coverage rate of up to 75-93% by fully analyzing the antigen and combining it with a unique animal immunization strategy.
[0009] Preferably, the characteristic is any one of the molecular weight, polarity, antigenic epitope, and solubility in water of the antigenic substance. The general principle is to first distinguish the different component ratios of high, medium, low molecular weight or mixed antigen samples according to the size of the molecular weight of the antigen protein. After the component separation is completed, the prepared antigen is used to immunize animals to obtain antibodies prepared from different antigen components; when the molecular weight cannot meet the performance requirements of the required antibody, it is necessary to further use the polarity of the protein and the difference in the antigenic epitope to distinguish the antigen components for the preparation of antigen components before animal immunization.
[0010] Preferably, the animals include but are not limited to goats, sheep, rabbits, alpacas, chickens, geese, and calves.
[0011] Preferably, in step (S2), the antigens of different components are used to immunize different types of animals.
[0012] Preferably, antibodies from different sources are reasonably proportioned with antibody coverage as an indicator, and the final proportion formula is determined based on the main performance indicators of the established method, such as limit of quantitation, linearity and range.
[0013] The present invention also provides a high-coverage polyclonal antibody obtained by the above preparation method.
[0014] Preferably, the coverage of the polyclonal antibody is 75.0-93.0%.
[0015] By implementing the above technical solution, the present invention has the following beneficial effects: The present invention conducts a thorough analysis and component separation of the antigenic substances, and then immunizes young and middle-aged animals (including goats, sheep, rabbits, alpacas, chickens, geese, calves, etc.) of appropriate ages with the prepared antigenic substances of different components according to the differences in molecular weight, polarity, antigenic epitope and solubility in water. The serum antibodies obtained are collected, purified and analyzed, and the antibodies from different sources are mixed in a certain proportion to obtain a polyclonal antibody product with a specific coverage rate. BRIEF DESCRIPTION OF THE DRAWINGS
[0016] Figure 1 The present invention is a flowchart of the method for preparing high-coverage polyclonal antibodies. DETAILED DESCRIPTION
[0017] The present invention is further described in detail below with reference to the accompanying drawings and specific embodiments.
[0018] This specific embodiment is merely an explanation of the present invention and is not a limitation of the present invention. After reading this specification, those skilled in the art may make non-creative modifications to the present embodiment as needed. However, as long as they are within the scope of the claims of the present invention, they are protected by the patent law.
[0019] Example 1 This embodiment provides a method for preparing a high-coverage polyclonal antibody, in which the obtained Escherichia coli cell lysis protein is fully dissolved in a buffer, and high molecular weight, medium molecular weight and low molecular weight antigen protein components are obtained by preparative chromatography. The antigen component is used to immunize three rabbits aged 9 to 12 months with the same adjuvant, and serum antibodies containing antigens of the corresponding components are obtained after immunization. These serum antibodies are purified by protein A column chromatography, and the purified antibodies are mixed in a ratio of 3:4:2. The obtained mixed antibodies and the original antigen are tested for coverage, and the coverage value is 75.1~89.2%.
[0020] Example 2 This embodiment provides a method for preparing a high-coverage polyclonal antibody, wherein the obtained CHO cell lysate protein is fully dissolved in a buffer, and the antigen protein components of high polarity, medium polarity and low polarity are obtained by preparative chromatography. The antigen components are used to immunize three sheep aged 12 to 18 months respectively using the same adjuvant, and serum antibodies containing antigens of the corresponding components are obtained after immunization. These serum antibodies are purified by protein A column chromatography, and the purified antibodies are mixed in a ratio of 4:3:3. The obtained mixed antibodies and the original antigens are tested for coverage, and the coverage value is 80.1~90.8%.
[0021] Example 3 This embodiment provides a method for preparing a high-coverage polyclonal antibody, wherein the obtained Vero cell lysate protein is fully dissolved in a buffer, and single-epitope, 2-5-epitope and multi-epitope antigen protein components are obtained by preparative chromatography. The antigen components are used to immunize three sheep aged 12 to 18 months with the same adjuvant, and serum antibodies containing antigens of the corresponding components are obtained after immunization. These serum antibodies are purified by protein A column chromatography, and the purified antibodies are mixed in a ratio of 2:1:1. The obtained mixed antibodies and the original antigens are tested for coverage, and the coverage value is 76.2~83.8%.
[0022] Example 4 This embodiment provides a method for preparing a high-coverage polyclonal antibody, wherein the obtained Vero cell lysate protein is fully dissolved in a buffer, and single-epitope, 2-5-epitope and multi-epitope antigen protein components are obtained by preparative chromatography. The single-epitope and 2-5-epitope antigen components are used with the same adjuvant to immunize sheep aged 12 to 18 months, and the multi-epitope component is used to immunize rabbits aged 9 to 12 months. After immunization, serum antibodies containing antigens of the corresponding components are obtained, and these serum antibodies are purified by protein A column chromatography. The purified antibodies are mixed in a ratio of 1:1:1, and the obtained mixed antibodies are tested for coverage with the original antigen, and the coverage value is 85.6~92.7%.
[0023] Comparative Example 1 This comparative example provides a method for preparing a polyclonal antibody, wherein the obtained Escherichia coli cell lysis protein is fully dissolved in a buffer solution to separate the antigen protein component. The antigen protein component is used to immunize 9-12 month old rabbits, and serum antibodies are obtained after the immunization is completed. The serum antibodies are purified by protein A column chromatography, and the coverage rate is detected with the original antigen after purification, and the coverage rate value is 56.3-68.2%. Compared with Example 1, this comparative example does not perform grouping, and directly immunizes rabbits. The results show that the coverage rate of the obtained antibodies is significantly reduced.
[0024] Comparative Example 2 This comparative example provides a method for preparing a polyclonal antibody, wherein the obtained CHO cell lysate protein is fully dissolved in a buffer solution to separate the antigen protein component. The antigen component is used to immunize sheep aged 12 to 18 months, and serum antibodies containing the corresponding components containing the antigen are obtained after the immunization is completed. The serum antibodies are purified by protein A column chromatography, and the purified antibodies and the original antigens are tested for coverage, and the coverage value is 53.3 to 65.4%. Compared with Example 2, this comparative example does not perform grouping, and directly immunizes sheep. The results show that the coverage of the obtained antibodies is significantly reduced.
[0025] Comparative Example 3 For the content of implementation case 1, the obtained Escherichia coli cell lysis protein was fully dissolved in a buffer solution, and high molecular weight, medium molecular weight and low molecular weight antigen protein components were obtained through preparative chromatography. The antigen components were used to immunize three rabbits aged 9 to 12 months with the same adjuvant. After the immunization, serum antibodies containing antigens of the corresponding components were obtained. These serum antibodies were purified by protein A column chromatography, and the purified antibodies were mixed in a ratio of 1:1:2. The obtained mixed antibodies were tested for coverage with the original antigen, and the coverage value was 61.2~72.5%.
Claims
1. A method for preparing a high coverage polyclonal antibody, characterized in that: The steps include: (S1) Fully analyzing the antigenic substances, distinguishing and separating them according to one or more characteristics, and preparing antigens of different components; (S2) using the antigens of different components obtained in step (S1) to immunize young and middle-aged animals of appropriate age, respectively, to obtain serum antibodies, collect and purify them, and obtain antibodies of different components from corresponding sources; (S3) Mix the different component antibodies in a certain proportion to obtain a polyclonal antibody product with high coverage.
2. The method for preparing a high coverage polyclonal antibody according to claim 1, characterized in that: The characteristic is any one of the molecular weight, polarity, antigenic epitope, and solubility in water of the antigenic substance.
3. The method for preparing a high coverage polyclonal antibody according to claim 1, characterized in that: The animal includes any one of goats, sheep, rabbits, alpacas, chickens, geese, and calves.
4. The method for preparing a high coverage polyclonal antibody according to claim 1, characterized in that: In step (S2), the antigens of different components are used to immunize different types of animals.
5. The method for preparing a high coverage polyclonal antibody according to claim 1, characterized in that: Antibodies from different sources are reasonably matched based on antibody coverage.
6. A high coverage polyclonal antibody, characterized in that: The method is obtained by the preparation method according to any one of claims 1 to 5.
7. A high coverage polyclonal antibody according to claim 6, characterized in that: The coverage of the polyclonal antibodies is 75-93%.