SOSIP.v8.3 Binding Assay

Half-well 96-well plates were coated overnight at RT with Galanthus nivalis lectin (Vector laboratories) at 20 µg/mL in 0.1 M NaHCO₃ pH 8.6. The next day plates were washed and Casein blocking buffer (ThermoScientific) was added. After 30 min a 2 µg/mL concentration of untagged 16055 SOSIP.v8.3 in Casein was added for 2 h at RT. Plates were then washed and a 1:100 dilution of rabbit sera in TBS/2% skimmed milk/20% sheep serum was added for 30 min. Next, mAbs were added for 1.5 h at RT at a final concentration that gave 80% of maximal binding signal as determined by an ELISA in the absence of serum (D11A.F9: 0.1 µg/mL; D11A.F2: 0.1 µg/mL; RM19R: 2 µg/mL, in TBS/2% skimmed milk/20% sheep serum). Plates were then washed three times and a 1:3000 dilution of HRP-labeled donkey anti-human IgG (Jackson Immunoresearch) in Casein was added for 1 h at RT. Washing and colorimetric detection then proceeded as explained for Serum antibody ELISA above. Residual binding was determined as described previously^{27 (link)}.