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 sim IFN-gamma ELISA assay has been developed and optimized for both f reshly isolated and peptide-restimulated splenocytes. This assay is based o n the ELISPOT assay, but utilizes a soluble chromagen, making it readily ad aptable to high-throughput analysis. We show that in both the primary and r estimulation assays this technique is more sensitive than either a traditio nal supenatant ELISA or the Cr-51-release assay, in that responses are obse rved 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 on e IFN-gamma secreting cell/10(4) plated cells. The in situ IFN-gamma ELISA was utilized to describe the kinetics of the IFN-gamma I response to DNA va ccination with pMin.1. For freshly isolated splenocytes, the peak response for all the peptides tested was observed from 10 to 12 days after immunizat ion, 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 demonst rates that a single intramuscular administration of a DNA vaccine can induc e T-cell responses that can be detected in freshly isolated splenocytes. (C ) 2000 Elsevier Science B.V. All rights reserved.