Goat anti human igg conjugated to horseradish peroxidase

MaxiSorp 96-well microtiter plates (Nunc) were coated overnight at 4°C with 20 ng of recombinant WNV NS1 protein (Native Antigen) in 50 μl of sodium bicarbonate buffer, pH 9.3. Subsequently, plates were washed four times with PBS and blocked with ELISA buffer (PBS containing 2% BSA and 0.05% Tween 20) for 1 h at 37°C. Plates then were incubated with anti-WNV NS1 murine MAbs at 10 μg/ml for 1 h at room temperature. Without washing, anti-WNV NS1 human MAbs were added to the plates at preoptimized concentrations and incubated for 10 min at room temperature. The plates then were washed four times in PBS containing 0.05% Tween 20 and incubated with goat anti-human IgG conjugated to horseradish peroxidase (1:2,000 dilution; Jackson ImmunoResearch 109-035-088) for 30 min at room temperature. After washing, plates were developed using 3,3',5,5'-tetramethylbenzidine substrate (Agilent) for 5 to 10 min. The reaction was stopped using 2 N H₂SO₄, and absorbance (450 nm) was read using a TriStar microplate reader (Berthold Technologies).

MaxiSorp 96-well plates were coated overnight at 4°C with 35 ng of recombinant human Fcγ receptor I (CD64) protein (R&D Systems) in 50 μl of sodium bicarbonate buffer, pH 9.3. Plates then were washed and blocked as described above. Serial dilutions of the IgG1-WT and -LALA variants of WNV-96 were added to the plates and incubated for 2 h at room temperature. Subsequently, the plates were washed and incubated with goat anti-human IgG conjugated to horseradish peroxidase (1:2,000 dilution; Jackson ImmunoResearch 109-035-088) for 45 min. The plates then were washed and developed as described above for the NS1 ELISAs.