Experiment Table CD4+ T lymphocyte counts and viral RNA Results Tetramer Staining Results Neutralizing Antibody Assays Results Viral Sequencing Results Peptide-binding Assays Results ELISPOT assays Results Conclusion   Navigation Home Methods Results Conclusion Quiz Links

ELISPOT Assay   Synopsis: The role of the ELISPOT in this experment is to determine the ability of the immune system to recognize the wild type p11C epitope as compared to the mutant p11C and respond with production of interferon-gamma (IFN-g). The production of IFN-g indicates a positive response by the immune system to the epitope. Little or no IFN-g production indicates an escape from immune recognition by the epitope.  Does the ELISPOT measuring the surface expression of components or intracelluar components of cells?   Answer ELISPOT measures surface expression. The more cells that express a specific surface molecule. The more are bound Procedure: 1. Plates of 96 wells were coated with 100ul each of 10ug anti-human IFN-g antibodies. 2. 2 x 10^5 peripheral blood mononuclear cells (PBMC) are added to the wells.   3. Varying concentrations of peptide (p11C or mutant p11C) are placed in the wells and then incubated for 18 hours. Would you expect to see the response measured by the ELISPOT grow or shrink with lower concentrations of peptides? Answer The response would most likely grow as the greater amount of peptide would bind more cells. 4. The wells were washed several times and then incubated with biotinylated rabbit anti-human IFN-g and streptavidin.     4. The NBT/BCIP chromogen is added next. 5. Data was collected as the number of Spot Forming Cells (SFC) per 106 PBMCs.   6. The spots will form around sites where IFN-g is present.     What are these spots indicative of?  Answer A region of localized IFN-g production, each spot is probably the site where a single IFN-g producing cell was located What type of response would a high number of spots indicate? Answer A large amount of IFN-g being produced by many cells.     NEXT