Horseradish peroxidase hrp labeled streptavidin

The following reagents and kits were used in the present study: Rabbit polyclonal anti-tyrosine hydroxylase (TH; cat. no. 2792; Cell Signaling Technology, Inc., Boston, MA, USA), rabbit polyclonal anti-OX-42 (cat. no. orb11009; Biorbyt, Cambridge, UK), hydroethidine (Molecular Probes, Eugene, OR, USA), LPS (Sigma-Aldrich, St. Louis, MO, USA), heparin (Qianhong Bio-pharma Co., Ltd., Changzhou, China), SST (ProSpec, East Brunswick, NJ, USA), biotinylated goat anti-rabbit secondary antibody (cat. no. A0277) and horseradish peroxidase (HRP)-labeled streptavidin (Beyotime, Shanghai, China), bicinchoninic acid (BCA) kit (Beyotime), Rat TNF-α ELISA kit, Rat IL-1β ELISA kit and Prostaglandin E2 ELISA kit (PGE2; USCN, Wuhan, Hubei, China).

In a binding assay, 0.1 μg/mL (50 μL) 5-Helix protein was coated on a 96-well half-area plate (Costar, New York, NY, USA) at 4 °C overnight. The coated plates were then blocked with 3 % non-fat milk at 37 °C for 2 h. Then, serially diluted biotinylated SARS-CoV-2 HR2P peptide or HIV-1 HR2 peptide was pipetted into each well for 1 h. Bound HR2 peptide was detected by horseradish peroxidase (HRP)-labeled Streptavidin (Beyotime Biotechnology, Beijing, China). The absorbance at 450 nm was detected by an Infinite M200 reader (Tecan, Männedorf, Sweden). In a separate binding assay, the wells of a plate were coated with 2 μg/mL Streptavidin (Sangon Biotechnology, Shanghai, China). After blocking, biotinylated SARS-CoV-2 or HIV-1 HR2P peptides were added. After washes, serially diluted 5-Helix was added. Finally, the bound 5-Helix was detected by using HRP-conjugated anti-His₆ antibody (Proteintech, Wuhan, China), and the absorbance at 450 nm was detected as described above [15 (link)].