1. Antibody Production BIT 120 Chapter 12 (Part of Immunology lecture)

2. Where do antibodies come from? Get antibodies from blood (animal) by centrifugation- increases gravitational force on a sample; more rapid settling of sample- force red/white blood cells to bottom- plasma on top- antibodies in plasma of blood

3. Isolating Antibodies Subject samples to affinity chromatography – column with solid support has antigen- plasma passed over column, only specific antibodies will stick to column; –Get rid of what you don’t want, then remove antibodies from column- raise salt solution- becomes unbound- collect purified version of what you want at the bottom

4. Example Insulin- put on column, run plasma over it- get a collection of antibodies that all bind to insulin- called polyclonal antibodies Each clone recognizes a different epitope- portion of insulin molecule – p. 270- different portion of molecule- Polyclonal can bind less well to other things so greater cross reaction

5. Monoclonal Antibodies need sometimes monoclonal antibodies- a pool that recognizes and binds a single epitope of a protein hard to isolate monoclonals from polyclonals how to produce monoclonals: from B cells- can’t culture a long time in vitro and how to select the right B cell

6. Producing mAB hybridomas - fusion of 2 different cells to produce a single cell that expresses properties of both Use myeloma cells- cancerous form of B cells. No longer produce antibodies, but grow in culture indefinitely; have your insulin B cell – don’t grow in culture long Fuse insulin antibody B cell with myeloma- so long lasting in vitro while secreting insulin antibodies. HAT- hypoxanthine, aminopterin, and thymine- myeloma cannot detoxify these cells –B cells die naturally –Myeloma cells die –Only hybridoma cells live

9. How to use these Antibodies ELISA- enzyme linked immunoabsorbant assay (antibody sandwich) magic bullets Western blot analysis See overheads