Avidin hrp

Pru p 3-specific serum antibody (IgE and IgG1) was measured at 0 and 42 days in mice sera by ELISA. Briefly, 96-well microtiter polystyrene ELISA plates (Corning, NY, USA) were coated with 5 μg/ml of Pru p 3 in coating buffer, pH 9.6, overnight at 4 °C. Fifty μl of serum was added in duplicates (diluted 1:8 for IgE and 1:50 for IgG1) and incubated overnight at 4 °C. After washing, the plates were incubated with biotinylated labelled goat anti-mouse IgE at 1:1000 (BD Pharmingen, San Diego, CA, USA) and goat anti-mouse IgG1 at a 1:3000 dilution (BD Pharmingen) for 1 hour at room temperature. After washing, avidin-HRP (BD Pharmingen) solution at 1:5000 for IgE and 1:1000 for IgG1 was added and samples were incubated for 30 minutes at room temperature. Then, 50 μl of ready to use TMB substrate (BD Pharmingen) was added and incubated for 10 minutes in dark condition. The enzymatic reaction was stopped with 50 μl of H₂SO₄(2 N) and absorbance was read at 450 nm. The ELISA results were expressed in optical density.

Plate-bound PVDF membranes (Millipore Corporation, Billerica, MA, USA) were pre-treated with 70% ethanol. Ethanol was removed and the membranes were washed with PBS. Anti-mouse IFN-γ coating antibody (BD Biosciences) was applied to the membranes and incubated overnight at 4 °C. The coating antibody was decanted, the plates were washed with PBST, and then blocked with PBS containing 10% CS for 1 h. Duplicates of 10⁶ splenocytes treated with 2 µM env or gag peptides pools (NIH Reagents) in 200 µL RPMI-1640 complete media were added to each well, and incubated at 37 °C overnight in a humidified 5% CO₂ incubator. The cell suspensions were discarded and the plate was washed with distilled water and PBS to remove all splenocytes. Biotinylated anti-mouse IFN-γ detection antibody was added into each well and incubated for 2 h at RT. Plates were washed with PBST and incubated with avidin-HRP (BD Bioscience) for 15 min followed by five additional washes with PBST. Soluble HRP substrate, 3-amino-9-ethylcarbazole (AEC), in citrate buffer (Sigma) was added to each well and incubated at RT for 1 h. Spots were counted by ZellNet Consulting Inc (Fort Lee, NJ, USA).

Single cell suspensions (2×10⁵/well) were cultivated in Millipore HTS HA plates coated with anti-mouse IFNγ. Cells were stimulated with 1 µg/ml CSP_245–253 peptide and in some experiments with 0.1 µg OVA 257–264. Supernatants were removed after 18 hours and the number of IFNγ-producing cells was determined using ELISPOT, as described elsewhere [18] . Antibodies and Avidin-HRP were purchased from BD Pharmingen (Heidelberg, Germany). The synthetic peptides SYIPSAEKI and SIINFEKL, corresponding to CD8+ T-cell epitopes CSP 245–253 and OVA 257–264 respectively, were obtained from MWG (Ebersberg, Germany).