Characterization of an in situ IFN-gamma ELISA assay which is able to detect specific peptide responses from freshly isolated splenocytes induced by DNA minigene immunization.

An in situ IFN-gamma ELISA assay has been developed and optimized for both freshly isolated and peptide-restimulated splenocytes. This assay is based on the ELISPOT assay, but utilizes a soluble chromagen, making it readily adaptable to high-throughput analysis. We show that in both the primary and restimulation assays this technique is more sensitive than either a traditional supernatant ELISA or the 51Cr-release assay, in that responses are observed in the in situ ELISA that are not detectable in these other assays. On a per-cell basis, the sensitivity of the in situ ELISA is approximately one IFN-gamma secreting cell/10(4) plated cells. The in situ IFN-gamma ELISA was utilized to describe the kinetics of the IFN-gamma response to DNA vaccination with pMin.1. For freshly isolated splenocytes, the peak response for all the peptides tested was observed from 10 to 12 days after immunization, with responses seen to some peptides as early as 7 days. When a 6-day in vitro peptide restimulation step was added, responses were seen for all the peptides tested after 7 days of in vivo immunization. This data demonstrates that a single intramuscular administration of a DNA vaccine can induce T-cell responses that can be detected in freshly isolated splenocytes.