anti-SARS-CoV-2 spike RBD antibody discovery from phage display library v1

• DOI: 10.17504/protocols.io.bitykepw
• Publication Date: 2020-08-01T01:45:52Z
• Authors: Eve Ngoh; Bei Wang; Cheng-I Wang
• Keywords: Panning (audio); Streptavidin

Day 1: 1st round bio-panning 1.1Add 2 µM mouse IgG 1 (as Fc blocker) to theHX02 human Fab phage library (Humanyx Pte Ltd) and incubate at RT, 30min. 1.2Wash 60 µl of DynaBeads M-280 Streptavidin (Invitrogen #11205D) with ml PBS twice. 1.3Add 100 nM biotinylated SARS-CoV-2 RBD-mFc protein in casein the beads room temperature for hour. 1.4Wash 4 times PBS. 1.5Add pre-mix washed RBD-mFc. 1.6Mix hour temperature. 1.7Wash 7 1.5 0.1% PBST. Bio-panning Round 1protocols.io| https://dx.doi.org/10.17504/protocols.io.bitykepw 1.8Add 250 0.1M TEA (Triethylamine) 15 min elute bound phage. 1.9Collect eluted using magnet neutralize pH 125 1M Tris 8. 1.10Infect neutralized TG1 cells 37°C 45 spread infected onto 150 mm AG agar plates (2xYT agar, 100µg/ml Ampicillin, 2% Glucose). 1.11Incubate overnight. 2Day 2: Harvest package phages 2.1Harvest by scraping up on let it grow OD 600nm ~0.5 37°C, shaking 250rpm. 2.2Infect bacteria culture M13K07 helper (NEB #N0315S) min. 2.3Culture 30°C hrs rpm AKG media (2x YT media, µg/ml 25 Kanamycin).protocols.