阅读说明：本技术 一种制备抗人源TNF-α单克隆抗体的方法及其用途 (Method for preparing anti-human TNF-alpha monoclonal antibody and application thereof ) 是由 李治寰 林立龙 卢佳豪 于 2019-10-21 设计创作，主要内容包括：本发明属生命科学技术领域,具体为一种单克隆抗体的制备,该抗体可用于ELISA和免疫印迹等生物学研究实验。本发明还公开了上述单克隆抗体制备的过程以及在ELISA实验与Westerp blot实验中的检测效果。表明该抗体可用于ELISA和免疫印迹等生物学研究实验。(The invention belongs to the technical field of life science, and particularly relates to preparation of a monoclonal antibody, which can be used for biological research experiments such as ELISA (enzyme-linked immunosorbent assay) and immunoblotting. The invention also discloses the preparation process of the monoclonal antibody and the detection effect in an ELISA experiment and a Westernblot experiment. The antibody can be used for biological research experiments such as ELISA, immunoblotting and the like.)

1. A protein antigen (patent of invention CN201210572674.X) for producing high titer anti-TNF-alpha antigen antibody.

2. An immunized female mouse, strain BALB/c, for injection of the protein antigen of claim 1 as a host for antibody production.

3. A method for producing a monoclonal antibody against TNF- α according to claim 2, by hybridoma technology, which comprises fusing antigen-immunized mouse spleen cells and mouse myeloma cells to produce the immortalized monoclonal hybridoma cell line 2C 11. The antibody is obtained by sequential amplification culture, injecting hybridoma cell 2C11 in logarithmic growth phase into abdominal cavity of BALB/C mouse, and subsequently killing mouse to obtain ascites and further purifying.

4. A hybridoma cell line 2C11 producing the anti-TNF- α protein according to claim 3.

5. A monoclonal antibody that produces the anti-TNF- α protein of claim 4.

6. The monoclonal antibody of claim 5 can be used in biological research experiments such as ELISA and immunoblotting.

Technical Field

The invention belongs to the technical field of life science, and particularly relates to preparation of a monoclonal antibody, which can be used for biological research experiments such as ELISA (enzyme-linked immunosorbent assay) and immunoblotting. The invention also discloses a preparation process of the monoclonal antibody and detection effects in an ELISA experiment and a Western blot experiment. The antibody can be used for biological research experiments such as ELISA, immunoblotting and the like.

Background

Tumor Necrosis Factor (TNF) is a type of cytokine that induces the death of certain tumor cells by stimulating the secretion of interleukin-1 by the cells. TNF is both a pro-inflammatory cytokine and an anti-inflammatory cytokine and is of great interest for relevant research and treatment of autoimmune diseases, cancer and infectious pathogens.

As a member of the TNF family, TNF- α is a cytokine that is secreted primarily by macrophages and binds primarily to TNFRSF1A/TNFR1 and TNFRSF 1B/TNFR. The gene encoding the TNF-alpha protein is located at position 6p21.3 of the chromosome. Initially TNF-alpha protein was identified as a tumor-death causing mediator produced in serum after lipopolysaccharide induction in animals. The research finds that the abnormal expression of TNF-alpha is related to the development of rheumatoid arthritis, psoriatic arthritis and inflammatory bowel disease, and is closely related to the occurrence of breast cancer, liver cancer and skin cancer. Since inhibition of TNF- α activity has a certain effect on the treatment of these diseases, studies on the structure and biological function of TNF- α and the development of inhibitors thereof have become important subjects for the study of inflammation and tumors.

In biological research, antibodies are widely used for qualitative and quantitative analysis of antigens. The monoclonal antibody can be used for analyzing the fine structure of the antigen and testing the unknown structural relationship of the antigen and the antibody. The new anti-TNF-alpha monoclonal antibody with high cost performance can provide an effective tool for TNF-alpha related research. The hybridoma technology for preparing the murine monoclonal antibody is a very mature antibody preparation technology, but has the problems of long preparation period, high cost, unstable antibody effect caused by low antibody titer and the like. Based on the limitations, the invention uses a novel TNF-alpha antigen and adopts a unique hybridoma technology to prepare a high-titer anti-TNF-alpha monoclonal antibody.

Disclosure of Invention

The invention aims to provide a monoclonal antibody. The antibody is a monoclonal antibody of human TNF-alpha protein.

Another objective of the invention is to provide a preparation process and a method for preparing high-titer antibodies against human TNF-alpha protein.

Another purpose of the invention is to show that the anti-TNF-alpha antibody prepared by the method can be used for biological research experiments such as ELISA, immunoblotting and the like. The antibody has good effect in ELISA and immunoblotting experiments.

Drawings

FIG. 1 Signal network of TNF- α protein interaction with other proteins.

FIG. 2 is a preparation scheme of a TNF- α monoclonal antibody.

FIG. 3 TNF-. alpha.monoclonal antibody was used in Western blot assay application (Serum was diluted 10 ten thousand times the above 3 times post-immunization Serum, Isotype was Isotype control antibody to 2C 11).

FIG. 4 TNF-. alpha.monoclonal antibodies can be used in ELISA experiments (Serum was diluted 10 ten thousand times the above 3 times post-immunization Serum, Isotype was Isotype control antibody to 2C 11).

Detailed Description

Preparation of 1 TNF-alpha monoclonal antibody

TNF-alpha protein antigen was produced (patent of invention CN201210572674.X), and female BALB/c mice (6 weeks old) were immunized. The first immunization was performed by antigen emulsification using Freund's complete adjuvant, and subcutaneous 6-point injection was performed, and the amount of antigen injected per mouse was 100. mu.g. After 14 days, a second immunization was performed, using Freund's incomplete adjuvant to emulsify antigen, and 6 injections were performed subcutaneously, and the amount of antigen injected per mouse was 100. mu.g. After 14 days, the 3 rd immunization was performed in the same manner as the second immunization. After 14 days, a small amount of blood was collected from the tail of the mice and subjected to serum titer ELISA, and the mice with the highest antibody titer (1: 500000) were selected for booster immunization without emulsification, and 100. mu.g of antigen protein was intraperitoneally injected into each mouse.

3-5 days after 4 th immunization, splenocytes of the killed mice are taken to be fused with SP2/0 cells, and the stable hybridoma cells are obtained by HAT culture medium (HAT Supplement (50X), Gibco). Screening hybridoma cells capable of secreting TNF-alpha antibody by an ELISA method, carrying out subcloning by a limiting dilution method, screening by the ELISA method to obtain a monoclonal hybridoma cell strain 2C11 capable of secreting TNF-alpha antibody, carrying out amplification culture step by step, and freezing and preserving seeds by liquid nitrogen.

Preparation and purification of ascites antibody: female BALB/C mice (8 weeks old) were intraperitoneally injected with Freund's incomplete adjuvant, 0.5ml per mouse, 3-5 days later, i.p.injection of hybridoma cells 2C11 in logarithmic growth phase, and 5X 105 cells (0.5ml) per mouse. The mice were sacrificed 10 days later to obtain ascites. Centrifuging at 5000rpm and 4 deg.C for 10min, removing precipitate, diluting ascites with 10 times volume of 1 XPB solution, mixing, and filtering with 0.45 μm filter membrane. The ascites fluid was affinity-purified by Protein G (Protein G Sepharose 4 Fast Flow, GE Healthcare) to obtain a purified TNF-. alpha.antibody.

Western blot detection application of 2 TNF-alpha monoclonal antibody

After TNF-alpha protein is mixed with 5 × loading buffer, boiling denaturation is carried out for 6min, SDS-PAGE is carried out, 20 μ l of each well is loaded, 10ng of protein is loaded per well, 400mA is transferred to a membrane for 1h, 5% skimmed milk powder is sealed for 1h at room temperature, 2C11 antibody (1 μ g/ml) is added for incubation for 1h at 37 ℃, a goat anti-mouse IgG secondary antibody (A0413, Shanghai Bitian Biotechnology limited) diluted by 1: 5000 is added after PBST is washed for 3 times, incubation is carried out for 30min at room temperature, and Western HRP substrate (WBLUR0500, Millipore) is added for development after PBST is washed for 3 times.

ELISA application of 3 TNF-alpha monoclonal antibody

TNF-alpha protein is added into a 96-well enzyme-labeled plate according to 100ng of each well, the plate is incubated at 37 ℃ for 3 hours, PBST is washed for 3 times, 5% skimmed milk powder is added for sealing at room temperature for 1 hour, 2C11 antibody (1 mu g/ml) is added for incubation at 37 ℃ for 1 hour, a goat anti-mouse IgG secondary antibody (A0413, Shanghai Binyan biotechnology limited) diluted at a ratio of 1: 5000 is added after PBST is washed for 3 times, the plate is incubated at room temperature for 30 minutes, TMB color developing solution is added for developing for 5 minutes after PBST is washed for 3 times, 2M sulfuric acid is added for stopping the reaction, and an OD value at 450nm is analyzed by a full-wavelength spectrophotometer (Thermo).