Peptide stimulation typically works very well in ELISpot. This is what most vaccine papers using this method employ. Given that the peptide induces a response it is very reliable. In every quality control at Mabtech we include the CEF peptide pool. As long as the donor is a responding one, it always works. One should make sure that the cells are of good quality. Viability should be over 84% and this includes both dead and apoptotic cells.
By contrast, some researchers are investigating whole proteins. These need to go through much more extensive processing and presentation and here there can be limitations in getting the T-cells to respond in certain scenarios. One might need to go up very high in cell numbers/well and one might need to supplement with anti-CD28 antibody stimulation to see a clear response. Some go to even greater extent and load autologoous dendritic cells with antigen and add those in great numbers to isolated T-cells. Or in the case you are refering to: mix in cells infected with virus expressing the antigen.
All of this is ofcourse possible but one should always start with the easy approach: Take your peptide and run an ELISpot at 250,000 cells/well and 400,000 cells/well. Titrate the peptide so you know you get optimum stimulatino. Usually stimulation is effective at 2ug/ml but go up to 4-5 just to be sure. Make sure your cells are in good condition. Get a precoated Mabtech IFNg ELISpot kit. Select an ALP kit like this one:
https://www.mabtech.com/products/human-ifn-gamma-elispot-pro-kit-alp_3420-2apt-2
For your initial testing don't coat yourself. Most often when ELISpot goes wrong it is when you make mistake in coating. Precoated kits from Mabtech have been done under optimal conditions which include the etoh activation. Performance is much much better compared to coating yourself but not doing the etoh.
