Monoclonal Antibodies

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Monoclonal Antibodies Milstein and Kohler, Nobel
Prize 1984
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Antibodies can be coupled to fluorochromes
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• Immunoprecipitation
• Immunoblot (western blot)
• Reverse genetics ( got clone, can make ab and
  study genetics)
• Got antibody, can clone gene (Brian Seed)
• Immunofluorescence Microscopy
• Fluorescence Activated Cell Sorter (Herzenberg,
  70s)

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FACS Analysis
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(No Transcript)
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CD4 and CD8 can be used to subset cells of
different developmental stages
CD4
83
1.6
CD8
4.7
CD8
CD8
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CD clusters of differentiation

• CD1-CD247 cell surface staining

Intra-cellular protein staining Solubilize cells
before staining Intra-cellular cytokine
staining Plug up golgi with brefeldin A and
let secreted proteins accumilate
Intra-cellular staining for phospho -specific
forms of signal transducers
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Purification of lymphocyte subpopulations (from
most to least expensive and most efficient)

• Sort by FACs
• Purify using antibody coated beads
• Purify by antibody and complement
• Nylon wool for T cells
• Adherence to plastic for Macrophages

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Transgenic mice
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The deletion of specific genes can be
accomplished by homologous recombination.
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Gene knockout in embryonic stem cells enables
mutant mice to be produced.
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Cre lox technology allows conditional knockouts
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The role of recessive lethal genes in lymphocyte
function can be studied using RAG-deficient
chimeric mice