Culture in HAT media

Culture in HAT media

• Unfused myeloma cells (HGPRT-) die out because they cannot make purines.
• Unfused spleen cells (containing B-cells) die out because they have a limited life span
• The only cells that will survive are the fused B-cell: myeloma cells = hybridomas

2. Test fused cells for antibodies by ELIZA
3. Subculture and clone positives.
4. Test supernatants for antibodies
5. "Expand' or scale up the best clone (all derived from a single cell = monoclonal)

6. Hybridoma cultures producing monoclonal antibodies can be maintained indefinitely in vitro or in vivo

7. MAbs can be developed into drugs, and injected into a person to seek out, bind, and target for destruction the antigen against which the antibody was raised.

The combination of antibody specificity (B cell) + unlimited proliferation (myeloma):

Because the human body eventually develops an immune response against the monoclonal antibody (made from a mouse B-cell), most monoclonal antibody-based drugs today are so-called humanized monoclonal antibodies: the mouse antibody is carefully digested to release just the antigen-binding variable region, which is then swapped into a human antibody missing its variable region. This prevents an immune response to the antibody itself.

A fully humanized monoclonal antibody has the antigen binding murine complementarity-determining regions interspersed within the variable regions of the light (light gray) and heavy (dark gray) chains of the Fab portion of the engineered antibody. A chimeric antibody has the entire antigen-binding murine component of the variable region of the Fab section is maintained integrally (handout).

See figures from Access Excellence and for humanized monoclonal antibodies.