Nunc maxisorp flat bottom clear plates

For each phage clone, 4 different conditions were tested—Direct: Spike-RBD-Fc, Competition: Spike-RBD-Fc with an equal concentration of Spike-RBD-Fc in solution, Negative selection: ACE2-Fc/Spike-RBD-Fc complex, and Control: Fc. 384-well Nunc Maxisorp flat-bottom clear plates (Thermo Fisher Scientific) were coated with 0.5 μg/mL of NeutrAvidin in PBS overnight at 4°C and subsequently blocked with PBS + 0.02% Tween-20 + 2% BSA for 1 hr at room temperature. Plates were washed 3X with PBS containing 0.05% Tween-20 (PBST) and were washed similarly between each of the steps. 20 nM of biotinylated Spike-RBD-Fc, ACE2-Fc/Spike-RBD-Fc complex, or Fc diluted in PBSTB was captured on the NeutrAvidin-coated wells for 30 min, then blocked with PBSTB + 10 μM biotin for 30 min. Phage supernatant diluted 1:5 in PBSTB were added for 20 min. For the competition samples, the phage supernatant was diluted into PBSTB with 20 nM Spike-RBD-Fc. Bound phage were detected by incubation with anti-M13-HRP conjugate (Sino Biological)(1:5000) for 30 min, followed by the addition of TMB substrate (VWR International). The reaction was quenched with the addition of 1 M phosphoric acid and the absorbance at 450 nm was measured using a Tecan M200 Pro spectrophotometer.