Antibody Production
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Antibody Production

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Antibody Production Antibody Production Presentation Transcript

  • Antibody Production BIT 120 Chapter 12 (Part of Immunology lecture)
  • 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
  • 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
  • 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
  • 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
  • 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
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  • How to use these Antibodies
    • ELISA- enzyme linked immunoabsorbant assay (antibody sandwich)
    • magic bullets
    • Western blot analysis
    • See overheads