Immunology Laboratory

Microbiology 542

University of Massachusetts, Amherst

Taught by Eric Martz with the Microbiology and Molecular Biology Laboratory Services Staff Dr. Elizabeth Stuart, Tom Carpenter, Pat Haviland, Pat Madden, and Gail Suprenant and the Teaching Assistants

See the Class Home Page.

This course includes exercises in:

Protein Immunochemistry and Immunoassays

• Purification of IgG by sodium sulfate precipitation, dialysis, and ion exchange chromatography. All protein recoveries are quantitated with the Bradford assay, for which each student prepares a standard curve.
• Fragmentation of purified IgG into F(ab')2 and Fab divalent and monovalent fragments with pepsin and papain. Purification with molecular sieve gel filtration. Crystallization of the Fc fragment.
• Purification of IgG by affinity chromatography on staphylococcal protein-A:sepharose 4B columns.
• Polyacrylamide gel electrophoresis with silver staining and immunoblotting with horseradish peroxidase-conjugated antibody staining (PAGE and western blots).
• Enzyme-linked immunoassay (ELISA).

Cellular Immunology

• Microscope alignment, cell counting with microscopic chamber (hemocytomerer), observation of human blood smears and lymph node histology.
• Mouse dissection with identification of lymphoid organs, and preparation of spleen and/or thymus cells. [Optional.]
• Microscopic immunofluorescence.
• Antibody plus complement-mediated cytolysis.
• Flow cytometric analysis. Mouse spleen and thymus lymphocyte populations are stained and analyzed with over a dozen antibodies which distinguish functional lymphocyte subpopulations by surface receptor markers. Two-color (fluorescein and phycoerythrin) data are acquired on a Becton-Dickinson FACScan. Students analyze listmode data with free PC/Windows software (WinMDI) and interpret their data in the context of other data in the class matrix. See examples of data from past classes.
• Flow cytometric sorting (demonstration of the FACStar Plus).