Anti his hrp

Peptides (0.5–8.0 μg per well), including FBP, P9R, P9RS (13) or ACE2 (100 ng) 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 virus or spike protein binding to peptides, viruses or spike protein were diluted in PBS and then were added to ELISA plates for binding to the coated peptides at room temperature for 1 h. After washing the unbound viruses or spike protein, the bound viruses were lysed by RLT buffer of RNeasy Mini Kit (Qiagen, Cat^# 74106) for viral RNA extraction. Viral RNA copies of binding viruses were measured by RT-qPCR. The bound spike protein was detected by anti-His-HRP (Invitrogen, Cat^# R93125, 1: 2,000) or rabbit anti-spike (Sino, Cat^# 40590-T62, 1:8000) with secondary goat-anti-rabbit HRP (Invitrogen, Cat^# 656120, 1:4000) by reading OD₄₅₀.

For ELISA assay^{38 (link)}, peptides (1.0 μg per well) dissolved in H₂O, ACE2 (100 ng), and chondroitin sulfate (CS, Sigma, Cat^# 230699, 300 ng) or heparan sulfate (HS, Sigma, Cat^# H7640, 300 ng) dissolved in PBS were coated onto ELISA plates and incubated at 4 °C overnight and was blocked at 4 °C overnight. Spike or ACE2 was added to 4H30 wells for binding and then determined by incubation with rabbit anti-spike (Sino, Cat^# 40590-T62, 1:8000) or rabbit anti-ACE2 (Takara, Cat^# A4612, 1:6000), or anti-His-HRP (Invitrogen, Cat^# R93125, 1:2000) at 37 °C for 30 min. 4H30 was added to CS or HS wells for binding and then determined by incubation with rabbit anti-HBD2 (Life Technologies, Cat^# PA5-103126). For determining spike and ACE2 binding, 4H30 or PBST was added to ACE2 for binding and then the spike was added to ACE2 for further incubation at 37 °C for 30 min. The binding ability of spike to ACE2 was determined by incubation with rabbit anti-spike (Sino, Cat^# 40590-T62, 1:8000) at 37 °C for 30 min and then incubation with goat-anti-rabbit HRP (Life Technologies, Cat^#656120, 1:4000) at 37 °C for 30 min. The reaction was developed by adding 100 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.