Hrp conjugated goat anti rabbit igg h l

For Western blot analysis, 10⁶ Vero-hSLAM cells (8 (link)) were either infected with MeV-eGFP or MeV-RNP^tracker at a multiplicity of infection (MOI) of 0.1 or left uninfected and then harvested at 24 hpi and lysed in 300 μl of denaturing protein lysis buffer (62.5 mM Tris [pH 6.8], 2% [wt/vol] sodium dodecyl sulfate, 10% [vol/vol] glycerol, 6 M urea, 5% [vol/vol] β-mercaptoethanol, 0.01% [wt/vol] bromphenol blue). Samples were heated at 95°C for 10 min and 10 μl/lane were loaded on 10% SDS-PAGE gels. Western blots were performed as previously described (44 (link)). The antibodies used for detection were polyclonal rabbit anti-N₅₀₅ (1:5,000 [70 (link)]), polyclonal rabbit anti-P₂₅₄ (1:5,000 [70 (link)]), monoclonal mouse anti-GFP (clone GF28R, 1:1,000, catalog no. 14-6674-82 [Thermo Fisher Scientific]), horseradish peroxidase (HRP)-conjugated mouse monoclonal anti-β-actin (1:25,000, catalog no. A3854-200UL [Sigma-Aldrich]), HRP-conjugated goat anti-mouse IgG(H+L) (1:10,000, catalog no. 401215 [Millipore Sigma]), and HRP-conjugated goat anti-rabbit IgG(H+L) (1:25,000, catalog no. 111-035-144 [Jackson ImmunoResearch]).

The SARS-CoV-2 variants, including WHU01, Beta (B.1.351), and Omicron subvariants (BA.1.1, BA.2.3, BA.5, XBB.1, and BQ.1), were preserved in the Guangzhou Customs District Technology Center BSL3 laboratory. The SARS-CoV-2 variants BA.5, XBB.1 and BQ.1 were provided by the Guangdong Provincial Center for Disease Control and Prevention, China. Experiments related to authentic SARS-CoV-2 neutralization were conducted in the BSL3 laboratory of the Guangzhou Customs District Technology Center. Briefly, prediluted antibody or ACE2-Ig proteins were mixed with 100 FFU of SARS-CoV-2 and coincubated at 37°C for 1 h. Then, 50 μL of mixture was transferred to a 96-well plate preseeded with Vero E6 cells. After being maintained at 37°C for 1 h, the cells were covered with MEM containing 1.6% CMC and cultured for 24 h. Cells were then fixed with 4% paraformaldehyde solution, permeabilized with 0.2% Triton X-100, and incubated with anti SARS-CoV/SARS-CoV-2 nucleocapsid rabbit polyclonal antibody (PAb) (Sino Biological; no 40143-T62) and HRP-conjugated goat anti-rabbit IgG (H+L) (Jackson ImmunoResearch; no. 109-035-088). Virus infection foci were stained by KPL TrueBlue peroxidase substrate and read by a CTL ImmunoSpot S6 Ultra reader (Cellular Technology, Ltd.).

FRNT assay was used for the evaluation of the Abs (peptide) neutralization effect. Vero E6 cells were seeded into a 96-well plate 1 day before infection. The next day, two-serially diluted Abs (peptide) and SARS-CoV-2 (80–120 FFU) were combined in DMEM (2% FBS) and incubated at 37 °C for 1 hour, then 50 μl mixtures were added into 96-well plate seeded with Vero E6 cells and incubated in 37 °C for 1 hour with rocking every 15 min. Then mixtures were removed and 100 μl MEM containing 1.2% Carboxymethylcellulose (1.2% CMC) was added. The medium was discarded after 24-hour post-infection, and the cell monolayer was fixed with 4% paraformaldehyde buffer at RT for 2 hours and permeabilized with 0.2% Triton X-100 for 20 min. Then the plates were sequentially stained with rabbit anti-SARS-CoV-2 N IgG (Cat. No. 40143-T62, Sino Biological Inc) and HRP-conjugated goat anti-rabbit IgG(H + L) (No.109–035-088, Jackson ImmunoResearch) at 37 °C for 1 hour respectively. The reactions were developed with KPL TrueBlue Peroxidase substrates and CTL ImmunoSpot S6 Ultra reader (Cellular Technology Ltd) was used to calculate the numbers of SARS-CoV-2 foci. The half-maximal inhibitory concentration (IC₅₀) was determined by 50% focus reduction neutralization test titers (FRNT50) used to evaluate the potency of Abs in inhibiting SARS-CoV-2 replication.