ABSTRACT
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.
Subject(s)
Enzyme-Linked Immunosorbent Assay/methods , Interferon-gamma/analysis , Interferon-gamma/biosynthesis , Peptides/immunology , Spleen/immunology , Vaccines, DNA/administration & dosage , Amino Acid Sequence , Animals , Antigen-Presenting Cells/immunology , Chromium Radioisotopes , Enzyme-Linked Immunosorbent Assay/statistics & numerical data , Evaluation Studies as Topic , HLA-A2 Antigen/genetics , Humans , Injections, Intramuscular , Jurkat Cells , Kinetics , Mice , Mice, Transgenic , Peptides/genetics , Sensitivity and Specificity , Vaccines, DNA/geneticsABSTRACT
The electrophysiological and pharmacological properties of two mammalian cell lines stably transfected with cDNAs encoding recombinant human N-methyl-D-aspartate (NMDA) receptor subtypes NMDAR1A/2A and NMDAR1A/2B are described. In whole-cell electrophysiological recordings, application of NMDA/glycine elicited inward currents at negative holding potentials in human NMDAR1A/2A (hNMDAR1A/2A)- and hNMDAR1A/2B-expressing cells. The current-voltage relationships determined in both cell lines in the presence and absence of external Mg++ were similar to those observed with recombinant rat NMDA receptors. Power spectra calculated from NMDA/glycine-induced currents for both NMDA receptor-expressing cell lines suggested a kinetically homogeneous population of channels. Immunoprecipitation with an anti-NMDAR1A antibody coprecipitated the corresponding NMDAR2 subunit with the NMDAR1A, suggesting that heteromeric complexes are formed in these stable cell lines. Stimulation of NMDA receptors evoked an increase in intracellular Ca++, which was used to characterize their pharmacological properties. NMDA displayed less intrinsic activity than did glutamate in both NMDA receptor-expressing cell lines and was a 4-fold more potent agonist at hNMDAR1A/2B than hNMDAR1A/2A. NMDA/glycine-evoked increases in Ca++ levels were inhibited by CGS 19755, (+/-)-3-(2-carboxypiperazin-4-yl)propyl-1-phosphonate, MK-801, ketamine and ifenprodil. (+/-)-3-(2-Carboxypiperazin-4-yl)propyl-1-phosphonate was a 3-fold more potent antagonist at hNMDAR1A/2A than hNMDAR1A/2B, whereas ifenprodil was markedly more selective toward hNMDAR1A/2B, being 250-fold more potent than against hNMDAR1A/2A. These data suggest that cells stably expressing recombinant heteromeric hNMDAR1A/2A and hNMDAR1A/2B represent pharmacologically valid experimental systems to study human NMDA receptors.
