Production

Mouse monoclonal antibodies are traditionally produced by the hybridoma methods:

1. The antigen is injected into mice (or rats). The antigen is often injected in combination with an adjuvant, to enhance the immune response, even though the use of adjuvant generally leads to severe side-effects.

2. After an appropriate interval (5-–21 days), the immunised animals are killed and lymphoid cells (including progenitor antibody-producing cells) are isolated from the spleen.

3. The lymphoid cells are fused with myeloma cells which have been grown in vitro.

4. The two original cell types and the newly formed hybrids are cultured in a selective medium, such as HAT (hypoxanthine/aminopterin/thymine) medium, which only allows the hybridoma cells to grow.

5. The supernatant media from the numerous in vitro microcultures exhibiting a recognisable growth of hybridomas are screened for secretion of the desired antibody, by using various immunoassay procedures.

6. The selected cells are subcultured in vitro, using special cloning procedures to ensure that each in vitro culture consists of hybridomas with a single antibody specificity only.

7. The propagation of cloned hybridoma cells, can be accomplished either by continuing to grow the cells in vitro, or by propagating them in vivo in the form of ascites tumours.