Rabbit anti sars cov spike polyclonal antibody

To evaluate the infectivity of the constructed recombinant adenoviruses, A549 (human lung adenocarcinoma epithelial cell line) cells were transduced with a MOI of 10 of Ad5.SARS‐CoV‐2‐S1. At 6 h after infection, cells were washed three times with PBS, and then incubated with serum‐free media for 48 h. The supernatants of A549 cells transduced with Ad5.SARS‐CoV‐2‐S1 were subjected to SDS‐PAGE and Western blot. Briefly, after the supernatants were boiled in Laemmli sample buffer containing 2% SDS with beta‐mercaptoethanol (β‐ME), the proteins were separated by Tris‐Glycine SDS‐PAGE gels and transferred to nitrocellulose membrane. After blocking for 1 h at room temperature (RT) with 5% nonfat milk in PBS‐T, rabbit anti‐SARS‐CoV spike polyclonal antibody (1:3000) (Sino Biological) was added and incubated overnight at 4°C as primary antibody, and HRP‐conjugated goat anti‐rabbit IgG (1:10 000) (Jackson immunoresearch) was added and incubated at RT for 2 h as secondary antibody. After washing three times with PBS, the signals were visualized using ECL Western blot substrate reagents and Amersham Hyperfilm (GE Healthcare). Mock (AdΨ5)‐infected A549 cells and 100 ng of recombinant SARS‐CoV‐2‐S1 (Sino biological, 1–685 amino acids with ten histidine tag) were used as negative and positive controls, respectively.