The Quantification of Antigen-Specific T Cells by IFN-γ ELISpot

The enzyme-linked immune absorbent spot (ELISpot) assay allows for the quantification of the number of cells producing a particular secreted analyte. As T lymphocytes secrete cytokines such as interferon (IFN)-γ upon binding of the T cell receptor with its cognate antigen epitope, IFN-γ ELISpot allows for the measurement of antigen-specific T cells in an immune sample. Immune cells are isolated from the vaccinated subject and incubated with the epitope/antigen of interest on polyvinylidene difluoride (PVDF)-lined microplates precoated with a capture antibody to IFN-γ. Cytokine spots are then detected utilizing an IFN-γ-specific detection antibody and an enzyme-linked conjugate. Here, we describe the quantification of OVA-specific CD8 and CD4 T cells from mouse splenocytes to measure vaccine-induced cellular responses.