Rabbit monoclonal antibody anti sars cov 2 s1

After the highest immunoreactivity was found by ELISA, the corresponding IgY-R were subjected to a blocking assay to assess binding interactions of RBD to ACE2 protein on the Vero E6 cells surface. For this, Vero E6 cells were harvested and washed with FACS buffer. 1x10⁶ cells were blocked with FACS buffer with 5% of normal mouse serum for 30 min at 37°C. Then, the cells were incubated with a mix containing 8 mg/mL of purified IgY-R and 8 µg/mL of RBD (Sino Biological), for 2 hours at 37°C. To remove the IgY and RBD residual not attached to Vero E6, the cells were washed with FACS buffer twice. After, the mix was marked with rabbit monoclonal antibody anti-SARS-CoV-2 S1 (1:200) (Sino Biological) as the primary antibody for 1 hour at 37°C, followed by goat anti-rabbit IgG Alexa Fluor 488 (1:200) as the secondary (Abcam). Cells were acquired by the flow cytometer FACS Canto II (BD Biosciences), and data analyzed using the software FlowJo v.10.6 (BD Biosciences). Graphics were constructed using the software GraphPad Prism 8.0.1.

Vero-E6 cells (Cod. CRL-1586^™, ATCC®, USA), which were previously cultured in DMEM/F12 (HyClone, USA) + 10% fetal bovine serum (FBS) (HyClone, USA), were harvested and washed with DPBS with 5% FBS (FACS buffer). Approximately 10⁶ cells were blocked with FACS buffer and 5% of normal mouse serum (Abcam, USA) for 30 min at 37°C. Then, the cells were incubated with the purified RBD (8 μg/mL) for 2 h at 37°C. To remove the excess of RBD not attached to Vero E6, the cells were washed with FACS buffer twice. After that, the mix was marked with rabbit monoclonal antibody anti-SARS-CoV-2 S1 (1:200) (Sino Biological, China) as the primary antibody for 1 h at 37°C, followed by the addition of the secondary goat anti-rabbit IgG antibody conjugated with Alexa Fluor 488 (1:200) (Abcam, USA). Finally, cells were acquired by the BD FACSCanto^™ II flow cytometer (BD Biosciences, USA). The data was analyzed using the software FlowJo v.10.6 (BD Biosciences, USA), and the graphics were generated with GraphPad Prism 8.0.1. For the interpretation of results, the percentage of positive cells indicates the binding of RBD to Vero E6 cells.