IFN-γ ELISPOT assay.

PBMCs were isolated from whole blood by Ficoll gradient centrifugation and tested for IFN-γ ELISPOT responses to pools of synthetic peptides representing RSV proteins F, G, and N (15-mers overlapping by 11 amino acids). The F peptides were divided into two pools, F1 (amino acids [aa] 1 to 295) and F2 (aa 285 to 574). Ninety-six-well plates with polyvinylidene difluoride (PVDF) membranes (Millipore) were coated overnight at 4°C with anti-IFN-γ monoclonal antibody (U-Cytech). PBMCs were added to the blocked plates at 4 × 10⁵/well. Peptide pools were added to the cells at approximately 2-μg/ml final concentration per peptide. After overnight incubation in a 37°C, 5% CO₂ incubator, bound IFN-γ was detected with biotinylated anti-IFN-γ antibody (U-Cytech), followed by alkaline phosphatase-conjugated streptavidin (BD PharMingen) and NBT-BCIP (nitroblue tetrazolium–5-bromo-4-chloro-3-indolylphosphate) substrate (Pierce). Spots were enumerated using a digital imager and an automated counting system (AutoImmun Diagnostika [AID]), and responses were normalized to spot-forming cells (SFC) per 1 × 10⁶ PBMCs. A positive response was defined as an antigen-specific response that was at least 55 spot-forming cells per 1 × 10⁶ PBMCs and at least 3-fold over the corresponding unstimulated (mock) control.