The preparation of monoclonal antibodies, how are monoclonal antibodies prepared?

Monoclonal antibodies are highly homogeneous produced from single B cell clones.

1. Antibodies that only target a specific epitope. Hybridoma antibody technology is based on cell fusion technology, which fuses sensitized B cells with the ability to secrete specific antibodies and myeloma cells with infinite reproduction ability to form a B-cell hybridoma.

By culturing a population of single hybridoma cells with this property, specific antibodies against one epitope can be produced, known as monoclonal antibodies.

Monoclonal antibodies are produced from artificially prepared hybridoma cells, which are formed by the fusion of an antigen-activated B cell with a myeloma cell. Advantages of monoclonal antibodies: high purity, high sensitivity, strong specificity, less cross-reactivity, and low cost of preparation.

Disadvantages: There are certain technical requirements, and it is easy to lose epitopes through chemical processing of antigens.

1. Immunized animals.

Immunized animals are the process of immunizing mice with the target antigen to produce sensitized B lymphocytes before lifting and bending.

2. Cell fusion.

The mice were sacrificed by eyeball enucleation and bloodletting, and the spleen was removed by aseptic operation, and the spleen was squeezed and ground in a flat dish to prepare a splenocyte suspension. Prepared syngeneic myeloma cells are mixed with mouse splenocytes in a certain proportion and the pro-fusion agent polyethylene glycol is added. Under the action of polyethylene glycol, various lymphocytes can fuse with myeloma cells to form hybridoma cells;

3. Selective culture.

The purpose of selective culture is to screen confluent hybridoma cells, generally using HAT selective medium;

4. Screening and cloning of hybridoma positive clones.

The sensitive, rapid and specific immunological method was used to screen out the positive hybridoma cells that could produce the required monoclonal antibodies, and the clonal expansion was carried out.

5. Mass preparation of monoclonal antibodies.

The mass preparation of monoclonal antibodies generally adopts animal induction method and in vitro culture method.

1. To prepare monoclonal antibodies, it is necessary to obtain monoclonal B lymphocytes that can synthesize specific antibodies, but such B lymphocytes cannot grow in vitro;

2. Experiments have found that myeloma cells can grow and multiply in vitro, and cell hybridization technology is used to combine myeloma cells and immune lymphocytes into one to obtain hybrid myeloma cells;

3. Hybrid myeloma cells inherit the characteristics of two parental cells, which not only have the characteristics of B lymphocytes to synthesize specific antibodies, but also have the characteristics that myeloma cells can be cultured and proliferated in vitro.