Softmax pro 5 microplate reader

Assays were performed essentially as described previously (Malherbe et al., 2020 (link)). To assay plasma antibody responses, half-well ELISA plates (Costar) were coated with recombinant SIVmac251 Gag (NIH HIV Reagent Program) or recombinant monomeric SIVmac239 gp120 at a concentration of 1 μg/ml (Gag) or 2 μg/ml (Env) in 0.2M H₂CO₃ buffer pH 9.4 at 4°C overnight. Plates were washed in binding buffer (PBS pH 7.4 + 0.1% Triton X-100) and blocked with 150 μl PBS containing 5% dried milk and 1% goat serum for 1 hr at room temperature. Blocking buffer was discarded and eight 3-fold serial dilutions of plasma or control antibodies were added to unwashed cells in 50 μl binding buffer. Purified polyclonal anti-SIV immunoglobulin was used as a positive control and naïve macaque immunoglobulin was used as the negative control. After 1 hr at RT, plates were washed 3x and then probed for 1 hr with 50 μl 1:5000 dilution of a horseradish peroxidase-conjugated goat anti-human IgG Fc fragment-specific polyclonal antibody (Jackson ImmunoResearch). Plates were then washed 5x, and bound Ab was visualized by the addition of 50 μl tetramethylbenzidine (Southern Biotech) for 10 min before stopping the reaction with 50 μl 1N H₂SO₄. Optical density was immediately quantified on a SoftMax Pro 5 microplate reader (Molecular Devices) at 450 nm.