How to make a Hybridoma!

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!