Blocker casein in tbs

Cells were washed with ice-cold PBS and harvested in cold PBS. For the preparation of whole cell extracts, pellets were resuspended in lysis buffer containing 20 mM Tris-Cl (pH 8.0), 137 mM NaCl, 10% glycerol, 1% Nonidet P-40 and a mixture of protease inhibitors. Samples were sonicated by using a Bioruptor (Diagenode) for 16 min at medium power, with an interval of 30 s between pulses. Following sonication, samples were centrifuged for 2 × 5 min at 21,000g. After preclearing, IP was performed overnight at 4 °C by using the indicated antibodies and protein G-Sepharose. IP was followed by five washes in 1 ml lysis buffer performed at 4 °C, and protein complexes were denatured in Laemmli sample buffer (2% SDS, 10% glycerol, 60 mM Tris-Cl (pH 6.8), 0.01% bromophenol blue, 100 mM dithiothreitol (DTT)) for 5 min at 95 °C and resolved by NuPAGE Novex 4–12% Bis-Tris Protein Gels (Invitrogen catalog no. NP0336PK2). After electronic transfer, the PVDF membrane was blocked by incubation at room temperature for 1 h in Blocker Casein in TBS (Thermo Scientific, catalog no. 37532). Complexes were revealed by Clarity Western ECL substrate (Bio-Rad catalog no. 170-5061), as recommended by the manufacturer.

384-well Maxisorp plates (Thermo Fisher) were coated overnight at room temperature with 3 μg/mL in 20mM Tris pH 8 and 150mM NaCl of SARS-CoV-2 S2P (Pallenson et al 2017) or SARS-CoV-2 A222V-D614G S2P, produced as previously described in Walls et al. (2020) (link). Briefly, Expi293F cells were transiently transcribed with a plasmid containing the spike protein and supernatant was clarified six days later prior to Ni Sepharose resin purification and flash freezing. Plates were slapped dry and blocked with Blocker Casein in TBS (Thermo Fisher) for one hour at 37°C. Plates were slapped dry and S2E12 (Tortorici et al., 2020 ) or S309 (Pinto et al., 2020 (link)) antibodies were serially diluted 1:3 with a starting concentration of 1000nM in TBST or NIBSC human plasma (20/130 https://www.nibsc.org/documents/ifu/20-130.pdf) was serially diluted 1:3 starting at 1:4 of original concentration in TBST and added to the plate for one hour at 37°C. Plates were washed 4x with TBST using a 405 TS Microplate Washer (BioTek) followed by addition of 1:5,000 goat anti-human Fc IgGHRP (Thermo Fisher) for one hour at 37°C. Plates were washed 4x and TMB Microwell Peroxidase (Seracare) was added. The reaction was quenched after 1–2 minutes with 1 N HCl and the A450 of each well was read using a Varioskan Lux plate reader (Thermo Fisher).

For ELISA experiments with NTD-targeted mAbs, 384-well Maxisorp plates (Thermo Fisher Scientific) were coated overnight at 4 °C with 2 μg/mL of S glycoprotein in 20 mM HEPES pH 8 and 150 mM NaCl. For ELISA experiments with RBD-targeted mAbs, 384-well Maxisorp plates (Thermo Fisher Scientific) were coated overnight at 4 °C with 4 µg/mL of hACE2-His in 20 mM Sodium Phosphate pH 8 and 100 mM NaCl. The antibodies that were used were purified previously^{13 (link),36 (link)}. Plates were slapped dry and blocked with Blocker Casein in TBS (Thermo Fisher Scientific 37532) for one hour at 37 °C. Plates were slapped dry and mAbs were serially diluted in TBST with an initial concentration of 50 µg/ml. Plates were left for one hour at 37 °C and washed 4X with TBST, then 1:5000 Goat anti-Human (Thermo Fisher Scientific A18817) was added. Plates were left for 1 h at 37 °C and washed 4x with TBST, and then TMB Microwell Peroxidase (Seracare 5120-0083) was added. The reaction was quenched after 4 min with 1 N HCl and the A450 of each well was read using a BioTek plate reader.