Monoclonal Antibodies

Milstein and Kohler, Nobel Prize 1984

Antibodies can be coupled to fluorochromes

• 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)

FACS Analysis

CD4 and CD8 can be used to subset cells of different developmental stages

CD8

83%

1.6%

4.7%

CD8

CD4

CD8

CD; clusters of differentiation• CD1-CD247; cell surface staining

Intra-cellular protein stainingSolubilize cells before staining

Intra-cellular cytokine stainingPlug up golgi with brefeldin A and let secreted proteins accumilate

Intra-cellular staining for phospho-specific forms of signal transducers

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

Transgenic mice

The deletion of specific genes

can be accomplished

by homologous

recombination.

Gene knockout in embryonic

stem cells enables mutant

mice to be produced.

Cre lox technology allows conditional knockouts

The role of recessive lethal genes in

lymphocyte function can be studied using

RAG-deficient chimeric mice