ELISA Assay for Protein Binding

For ELISA assay^{14 (link)}, Peptides (1.0 μg per well) dissolved in H₂O were coated onto ELISA plates and incubated at 4 °C overnight. Then, 2% BSA was used to block plates at 4 °C overnight. For HA and S binding, 150 ng HA1 or S in solution I buffer (Sino Biological Inc., Cat^# 11055-V08H4) was incubated with peptides at 37 °C for 1 h. The binding abilities of peptides to HA1 or S proteins were determined by incubation with rabbit anti-His-HRP (Invitrogen, Cat^# R93125, 1: 2,000) at room temperature for 30 min. The reaction was developed by adding 50 μl of TMB single solution (Life Technologies, Cat^# 002023) for 15 min at 37 °C and stopped with 50 μl of 1 M H₂SO₄. Readings were obtained in an ELISA plate reader (Victor 1420 Multilabel Counter; PerkinElmer) at 450 nm.