Preparation steps of monoclonal antibody

Immunizing animals is the process of immunizing mice with target antigen to produce sensitized B lymphocytes. Antigen enters peripheral immune organs through blood circulation or lymphatic circulation, which stimulates the corresponding B lymphocytes to clone, activate, proliferate and differentiate into sensitized B lymphocytes.

2, cell fusion:

Kill mice with carbon dioxide gas, take out the spleen by aseptic operation, squeeze and grind it in a Petri dish to prepare spleen cell suspension. The prepared homologous myeloma cells were mixed with mouse spleen cells in a certain proportion, and the fusion accelerator polyethylene glycol was 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 fused hybridoma cells, and HAT selective medium is generally used.

4. Screening and cloning of hybridoma positive clones:

Only a few hybridoma cells growing in HAT medium secrete monoclonal antibodies with predetermined specificity, so they must be screened and cloned. Limited dilution method is usually used to clone hybridoma cells.

5. Mass preparation of monoclonal antibodies:

A large number of monoclonal antibodies are mainly prepared by in vivo induction and in vitro culture of animals.

Extended data:

Advantages of monoclonal antibodies:

1. Hybridoma can "permanently" survive in vitro and be passaged. As long as the cell line does not undergo gene mutation, it can continuously produce antibodies with high specificity and uniformity.

2. A large number of highly specific and homogeneous antibodies can be obtained from relatively impure antigens.

3. Because we can obtain "infinite" homogeneous antibodies, it is suitable for immunological analysis methods characterized by labeled antibodies.

4. Because of its high specificity and single biological function, monoclonal antibodies can be used for in vivo radioimmunoimaging and immunotherapy.

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