Hello all,
I'm using ELISPOT to measure the response of lymphocytes from mice immunized with full ovalbumin protein. Would I need to pulse my splenocyte feeder cells with full ova protein or can I just use the immunodominant peptides? My collaborator believes that we should use full protein, as just using peptides would cause us to lose some of the ova-specific lymphocytes recognizing regions outside of the immunodominant epitopes. I argue that the majority of the response will be directed towards the immunodominant epitopes anyways, so we should just use the peptides. This would save us time, as we wouldn't need to wait for antigen processing to occur in the feeder cells before we irradiate them.
Hi.
Both should work, although you will miss the response directed to non-immunodominant peptide response when you use the immunodominant peptide. Irradiated splenocytes are pretty good APCs and should process and present peptide in the period of time that it takes for a robust T cell response. We generally do ELISA and proliferation instead of ELISPOT but can get good cytokine secretion between 24 and 48 hrs and T cell proliferation is maximal at 48-72 hrs.
Mireia
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