So I've decided to make a brief tutorial on how to make a hybridoma. Here we go:Hybridomas are products of cell fusion between spleen B cells (from an immunized animal) and myeloma cells (cancerous B cells). They proliferate continuously AND produce antibody. The process was developed by Georges Kohler and Cesar Milstein (1976).
(Figure 1 - Attached)To make a hybridoma:- Immunize mouse with antigen of interest
- Mouse responds to antigen: B cells are stimulated to produce antibody
- Spleen contains many antibody-producing B cells, so remove spleen and fuse B cells with myeloma cells. Polyethyelene glycol is used to fuse the cell membranes.
- Successfully fused B cells + myeloma cells = “hybridoma cells”
Culture and test for antibody production. You need to screen for hybridomas producing antibody – and make sure it is specific for the antigen of interest!
(Figure 2 - Attached)Now to produce monoclonal antibodies:- Specialized media and myeloma cells are used: HGPRT-deficient myeloma cells. The lack of HGPRT causes inability to survive in selective HAT medium. “HAT”: Hypoxanthine, aminopterin, thymidine; Aminopterin blocks “de novo” nucleotide synthesis.
- Mouse B cells DO have HGPRTso can take up hypoxanthine and thymidine from medium – so would survive for a while!
Selection process: - Myeloma cells die due to lack of HGPRT. Spleen cells die (can only undergo limited # of divisions in vitro)
Sole Survivors? Only the successfully fused “hybridoma cells” survive
(Figure 2 - Attached)Mouse spleen B cells DO have HGPRT (and make antibody). Myeloma cells do NOT have HGPRT (and are not making antibody) But myeloma cells are “immortal”
Selection process: Myeloma cells die – cannot make nucleotides
Spleen cells die (limited divisions in vitro)
Successfully fused “hybridoma cells” have properties of both:
- possess HGPRT
- Are “immortal”
And ideally – are making the antibody you want!