Quantifying Cytokine and Antibody Responses

Millititer HA 96-well microtiter plates with nitrocellulose bases (Millipore, Bedford, MA) were coated with either purified rat α-mouse IFN-γ (clone R46A2; BD Pharmingen, San Diego, CA) or recombinant trans-sialidase (TS) as described (25 (link)). For IFN-γ ELISPOT analyses, spleen cells (2.5×10⁵ cells/well) plus antigen presenting cells [1×10⁵ A20J cells alone (Control A20; ATCC), A20J cells stably transfected with the TS gene (A20-TS), or A20J cells pulsed with 2.5 μg/mL of the immunogenic CD8⁺ H2K^d restricted TS peptide IYNVGQVSI; A20-TSKd1)] were cultured overnight at 37°C. TS-specific antibody ELISPOT analyses were done as previously described (24 (link)). Briefly, 1-5×10⁵ spleen cells were added to TS-coated ELISPOT plates. Following overnight incubation at 37°C, anti-mouse IgG or IgA biotin was added to detect T. cruzi-specific IgG and IgA antibody secreting cells, respectively (SouthernBiotech). Results are represented as the number of spot-forming cells (SFC) or antibody-secreting cells (ASC) per million spleen cells or absolute number per spleen. Background responses to A20J cells were subtracted from experimental antigen responses in results shown from IFN-γ ELISPOT assays.