ANTIBODY LABELING AS A TOOL FOR DEVELOPMENTAL RESEARCH

I.What are antibodies? II.How are specific antibodies produced A.Polyclonal - antibodies come from a variety of lymphocyte clones, each of which produces an antibody against the antigen of interest. Typically produced by immunizing an animal such as a rabbit, goat or donkey and then isolating a fraction of the blood serum that contains the antibodies you want. B.Monoclonal - a single antibody against the antigen of interest is produced by a clone of activated hybridoma cells (hybridoma = an activated lymphocyte fused with a myeloma (cancer) cell). Hybridoma cells are artificially produced and comprise a cell line that is effectively immortal. Thus, once you’ve made your hybridoma, you can produuce as much antibody as you want. III. How are they used (You somehow have to be able to “see” where the antibody is binding.) A. Direct (visible probe is directly attached to the antibody that binds to your antigen) and indirect methods (the visible probe is linked to a secondary antibody that binds to the type of antibody that attaches to your antigen of interest) of labeling B. Types of “visible” labels 1. Fluorescent - e.g. FITC, RITC 2. Opaque - e.g. gold particles 3. Radioisotopes 4. Enzmyatic reactions - DAB

The scanning electron micrograph above, shows a human macrophage (gray) approaching a chain of Streptococcus pyogenes (yellow). Riding atop the macrophage is a spherical lymphocyte. Both macrophages and lymphocytes can be found near an infection, and the interaction between these cells is important in eliminating infection.

1.Macrophage digests bacteria. 2.Resulting peptides are attached to MHC II (major histocompatibility complex). Some complexes remain internal (in the macrophage) and others are released. 3.MHC II - peptide complex binds to surface of both macrophage and B-lymphocyte. 4.Helper T-cell “recognizes” peptide on macrophage. When it contacts B-lymphocyte with same peptide it activates the B-lymphocyte. 5.The B-lymphocyte begins dividing to form a clone of many B-lymphocytes that can produce antibodies against this particular peptide. 6.The clone cells differentiate into plasma cells that produce large quantities of antibody that can bind to the peptide that is characteristic of the bacteria. 7.Antibodies bind to the bacteria and act as markers that will be either recognized by other white blood cells that digest the bacteria or bound to complement that will kill the bacterium. How an immune response works

Antibody structure IgG

Kohler & Milstein, 1974

More detail

An embryonic day 14.5 mouse forelimb in which all the limb musculature has been indentified by whole mount immunohistochemistry using an anti-muscle myosin antibody. Using Optical Projection Tomography (OPT) techniques a 3-D rendering of the muscles has been produced.

Vinculin enables cells to move within developing tissues and organs of the embryo and spark the healing of wounds.

Dopamine neurons in the rat substantia nigra are seen here as red triangular and oval shapes with dendrites (arrowheads) extending from the cell bodies (arrows). This brain section was stained immunohistochemically using an antibody that recognizes the dopamine synthesizing enzyme, tyrosine hydroxylase, and a secondary antibody labeled with rhodamine Red-X, a fluorescent red molecule.

Programmed Cell Death in Cultured Cells. The same field of view is seen in A, B, and C. The same two cells are identified by arrows. Exposure to ultraviolet light causes some cells to undergo apoptosis. A. Differential interference contrast (DIC) microscopy reveals all cells in this field of view. B. Immunocytochemical labeling demonstrates that some of the cells contain active caspase-3, an enzyme that triggers the cell death program, indicating that the red cells are dying. C. Fragmented DNA, a hallmark of programmed cell death, is labeled green in two cells (also in A and B, arrows) using the TUNEL technique, which attaches fluorescent molecules to termini of fragmented DNA. The presence of fragmented DNA suggests that these two green cells are relatively far along in the cell death process.

A protein specific to Drosophila trachea is marked by an antibody that binds to it and is observed by confocal laser microscopy. The wild-type embryo (upper pane) shows an intricate network of tubular structure. The network is disrupted in mutants of the Drosophila oncogene pnt (lower panel). wild-type embryo mutant embryo

Monoclonal antibody that binds to a part of the connective tissue sheath surrounding a nerve. Visualized using a secondary antibody with fluorescene isotheocyanate (FITC, bright yellow green) linked to it.

Monoclonal antibody against small cardioactive peptide (SCP, a neurotransmitter and possibly neurohormone) used to visualize the neurons in the brain of a slug that contain this peptide (bright yellow green, one large and one small in each buccal ganglion). This is another prep using secondary antibodies).