V 5 tween 20

Uncoated 2HB 96-well plates (Thermo Fisher Scientific) were coated with 0.5 μg/mL SIVmac239 gp120 (Immune Technology, New York, NY) in coating buffer (2.25mg/mL Na₂CO₃ and 4.395mg/mL NaHCO₃ in distilled H₂O). Plates were incubated overnight at 4°C. The next day, plates were washed with wash buffer (1X PBS with 0.05% V/V TWEEN-20 [Thermo Fisher Scientific]) and blocked in a buffer containing 1X PBS supplemented with 10% FBS for one hour at room temperature. After blocking, plates were washed and serial dilutions of plasma were added for 90 minutes at room temperature. Plasma samples were heat-inactivated at 56°C for 30 minutes, prior to use. Serial dilutions of anti-SIV gp120 monoclonal antibody (B404, NIH HIV Reagent Program, Division of AIDS, NIAID, NIH) were plated as a positive control. Next, plates were washed with wash buffer and anti-monkey IgG HRP (SouthernBiotech, Birmingham AL) was diluted 1:10,000 in blocking buffer and added to each well. Plates were incubated for one hour at room temperature. Plates were then washed with wash buffer and TMB substrate (3,3’5,5’ – tetramethylbenzidine, Thermo Fisher Scientific) was added to each well. After a 15 minute incubation, HCl (1N) was added to each well to stop the reaction. ELISA plates were immediately read using a GloMax^®-Multi Detection System microplate reader (Promega, Madison WI) at 450 nm absorbance.