TITLE: anti-SARS-CoV-2 spike RBD antibody discovery from phage display library
AUTHORS: Eve Ngoh, Bei Wang, Cheng-I Wang

[STEPS]
?. [Bio-panning Round 1]
Day 1: 1st round bio-panning
?. [Bio-panning Round 1]
Add 2 µM mouse IgG1 (as mouse Fc blocker) to theHX02 human Fab phage library (Humanyx Pte Ltd) and incubate at RT, 30min.
?. [Bio-panning Round 1]
Wash 60 µl of DynaBeads M-280 Streptavidin (Invitrogen #11205D) with 1 ml PBS twice.
?. [Bio-panning Round 1]
Add 100 nM of biotinylated SARS-CoV-2 RBD-mFc protein in casein to the beads and incubate at room temperature for 1 hour.
?. [Bio-panning Round 1]
Wash beads 4 times with 1 ml PBS.
?. [Bio-panning Round 1]
Add the pre-mix of phage library and mouse IgG1 to the washed beads of biotinylated SARS-CoV-2 RBD-mFc.
?. [Bio-panning Round 1]
Mix for 1 hour at room temperature.
?. [Bio-panning Round 1]
Wash beads 7 times with 1.5 ml 0.1% PBST.
?. [Bio-panning Round 1]
Add 250 µl of 0.1M TEA (Triethylamine) to beads at room temperature for 15 min to elute bound phage.
?. [Bio-panning Round 1]
Collect eluted phage using magnet and neutralize pH with 125 µl of 1M Tris pH 8.
?. [Bio-panning Round 1]
Infect neutralized phage to TG1 cells at 37°C for 45 min and spread phage infected cells onto 150 mm AG agar plates (2xYT agar, 100µg/ml Ampicillin, 2% Glucose).
?. [Bio-panning Round 1]
Incubate agar plates at 37°C overnight.
?. [Bio-panning Round 1]
Day 2: Harvest and package phages
?. [Bio-panning Round 1]
Harvest the phage infected TG1 cells by scraping up the cells on the agar plates and let it grow to OD600nm ~0.5 at 37°C, shaking at 250rpm.
?. [Bio-panning Round 1]
Infect the bacteria culture with M13K07 helper phage (NEB #N0315S) at 37°C for 45 min.
?. [Bio-panning Round 1]
Culture the M13K07 helper phage infected cells at 30°C for 7 hrs with shaking at 250 rpm in AKG media (2x YT media, 100 µg/ml Ampicillin, 25 µg/ml Kanamycin).
?. [Bio-panning Round 1]
Centrifuge down the bacterial cell culture and precipitate phage (supernatant) by adding 1/5 volume of PEG (20% PEG 6000, 2.5M NaCl) to the supernatant.
?. [Bio-panning Round 1]
Incubate at 4°C overnight.
?. [Bio-panning Round 1]
Day 3: Phage preparation
?. [Bio-panning Round 1]
Centrifuge down the PEG-precipitated phage and discard supernatant.
?. [Bio-panning Round 1]
Resuspend the phage pellet with 500 µl of PBS so that it is ready for use for Bio-panning Round 2.
?. [Bio-panning Round 2]
Day 3:  2nd round bio-panning
?. [Bio-panning Round 2]
Wash 50 µl DynaBeads M-280 Streptavidin (Invitrogen # 11205D) with 1 ml PBS twice.
?. [Bio-panning Round 2]
During the 2nd wash, split the beads into 2 tubes (tube 1 and tube 2).
?. [Bio-panning Round 2]
Add 10nM of biotinylated SARS-CoV-2 RBD-mFc protein in casein to tube 1 beads and incubate at room temperature for 1 hr.
?. [Bio-panning Round 2]
Wash beads 4 times with 1 ml PBS.
?. [Bio-panning Round 2]
Add 2 µM mouse IgG1 (as mFc blocker) to the Round 1 amplified phage and incubate at RT, 30 min.
?. [Bio-panning Round 2]
Add the pre-mix of phage and mouse IgG1 to the tube 2 of washed beads from Step 4.2.
?. [Bio-panning Round 2]
Rotate at room temperature for 1 hr.
?. [Bio-panning Round 2]
Collect supernatant (contains phage where streptavidin-binding phage have been removed by bead binding) using magnet and add supernatant to the washed biotinylated SARS-CoV-2 RBD-mFc coated beads from Step 4.4 above.
?. [Bio-panning Round 2]
Rotate at room temperature, 1hr.
?. [Bio-panning Round 2]
Wash beads 15 times with 1.5ml 0.1% PBST.
?. [Bio-panning Round 2]
Add 120 µl of 0.1 M TEA (Triethylamine) to beads at room temperature for 15 min to elute bound phage.
?. [Bio-panning Round 2]
Collect eluted phage using magnet and neutralize pH with 60 µl of 1M Tris pH 8.
?. [Bio-panning Round 2]
Infect neutralized phage into HB2151 cells at 37°C for 45min and spread phage infected cells onto 90 mm AG agar plates (2xYT agar, 100 µg/ml Ampicillin, 2% Glucose).
?. [Bio-panning Round 2]
Incubate agar plates at 37°C overnight.
?. [Bio-panning Round 2]
Day 3: Collect the agar plates and proceed with the Fab screening by ELISA.
?. [Binding avidity ELISA of the Fab supernatant to SARS-CoV-2 spike RBD protein]
Day 3: Pick colonies for Fab production
?. [Binding avidity ELISA of the Fab supernatant to SARS-CoV-2 spike RBD protein]
Day 3: Coat ELISA plates.
?. [Binding avidity ELISA of the Fab supernatant to SARS-CoV-2 spike RBD protein]
Day 4: Continue with binding avidity ELISA using Fab supernatant
?. [Binding avidity ELISA of the Fab supernatant to SARS-CoV-2 spike RBD protein]
Pick colonies from [Biopanning Round 2] Step 5 into 96-well plate 1 containing 100 µl of AG media (2xYT media, 100 µg/ml Ampicillin, 2% Glucose) per well.
?. [Binding avidity ELISA of the Fab supernatant to SARS-CoV-2 spike RBD protein]
Shake the plate 1 at 37°C, 300 rpm for 4 hrs.
?. [Binding avidity ELISA of the Fab supernatant to SARS-CoV-2 spike RBD protein]
Prepare a new 96-well plate 2 with 270 µl/well of 2xYT media + 100 µg/ml Ampicillin.
?. [Binding avidity ELISA of the Fab supernatant to SARS-CoV-2 spike RBD protein]
Transfer 10 µl of each bacterial culture from 96-well plate 1 (Step 6.2) to respective wells on 96-well plate 2.
?. [Binding avidity ELISA of the Fab supernatant to SARS-CoV-2 spike RBD protein]
Culture the 96-well plate 2 at 37°C, 300 rpm, 1.5 hrs.
?. [Binding avidity ELISA of the Fab supernatant to SARS-CoV-2 spike RBD protein]
When the cultures from the 96-well plate 2 is slightly turbid, add 40 µl of 8 mM IPTG (prepared in 2xYT + 100 µg/ml Ampicillin) to each well. This will give a final 1 mM IPTG in each well.
?. [Binding avidity ELISA of the Fab supernatant to SARS-CoV-2 spike RBD protein]
Culture the 96-well plate 2 at 30°C overnight, 300 rpm.
?. [Binding avidity ELISA of the Fab supernatant to SARS-CoV-2 spike RBD protein]
Coat 96-well ELISA plate with 70 µl per well of 5 µg/ml NeutrAvidin Protein (ThermoFisher scientific, #31000) in carbonate coating buffer (8.4 g/L NaHCO3, 3.56 g/L Na2CO3, pH 9.5).
?. [Binding avidity ELISA of the Fab supernatant to SARS-CoV-2 spike RBD protein]
Incubate ELISA plate at 4°C overnight.
?. [Binding avidity ELISA of the Fab supernatant to SARS-CoV-2 spike RBD protein]
Wash each well of the ELISA plate from Step 7 with 0.05% PBST (Tween-20), 4 times.
?. [Binding avidity ELISA of the Fab supernatant to SARS-CoV-2 spike RBD protein]
Block each well with 200 µl casein (Thermo Fisher Scientific, #A37528) at room temperature for 2 hrs.
?. [Binding avidity ELISA of the Fab supernatant to SARS-CoV-2 spike RBD protein]
Wash each well of the ELISA plate with 0.05% PBST, 4 times.
?. [Binding avidity ELISA of the Fab supernatant to SARS-CoV-2 spike RBD protein]
Add 70 µl of 0.2 µg/ml biotinylated recombinant SARS-CoV-2 spike protein RBD-mFc (Sino Biological, 40592-V05H) in casein per well of the ELISA casein-blocked plate. Incubate at room temperature, 1 hr.
?. [Binding avidity ELISA of the Fab supernatant to SARS-CoV-2 spike RBD protein]
Wash each well of the ELISA plate with 0.05% PBST, 4 times.
?. [Binding avidity ELISA of the Fab supernatant to SARS-CoV-2 spike RBD protein]
Add 20 µl of 7% milk (in PBS) to each well. This will give a final [milk] to 2% after addition of 50 µl Fab supernatant culture.
?. [Binding avidity ELISA of the Fab supernatant to SARS-CoV-2 spike RBD protein]
Centrifuge the overnight 96-well plate 2 culture from Step 6.7 at 4000 rpm, 10min to pellet the IPTG-induced HB2151 cells. Without disturbing the cell pellet, gently transfer 50µl of each culture supernatant into respective wells in binding ELISA plate.
?. [Binding avidity ELISA of the Fab supernatant to SARS-CoV-2 spike RBD protein]
Incubate the ELISA plate at room temperature, 2 hrs.
?. [Binding avidity ELISA of the Fab supernatant to SARS-CoV-2 spike RBD protein]
Wash each well of the ELISA plate with 0.05% PBST, 4 times.
?. [Binding avidity ELISA of the Fab supernatant to SARS-CoV-2 spike RBD protein]
Add 70 µl of (1:3000 in casein) Peroxidase-AffiniPure F(ab')2 Fragment Goat Anti-Human IgG, F(ab')2 Fragment Specific (JACKSON ImmunoResearch, #109-036-097) in each well. Incubate the ELISA plate at room temperature, 1 hr.
?. [Binding avidity ELISA of the Fab supernatant to SARS-CoV-2 spike RBD protein]
Wash each well of the ELISA plate with 0.05% PBST, 5 times.
?. [Binding avidity ELISA of the Fab supernatant to SARS-CoV-2 spike RBD protein]
Add 70 µl of TMB One Component HRP (SurModics, #TMBW-1000-01) per well. Stop each reaction with 70 µl 1M HCl.
?. [Binding avidity ELISA of the Fab supernatant to SARS-CoV-2 spike RBD protein]
Measure OD450nm and OD570nm (baseline).
?. [Binding avidity ELISA of the IgG antibodies to SARS-CoV-2 and SARS-CoV spike RBD proteins]
Coat ELISA plates.
?. [Binding avidity ELISA of the IgG antibodies to SARS-CoV-2 and SARS-CoV spike RBD proteins]
Coat ELISA plates with 70 µl per well of 5 µg/ml NeutrAvidin Protein (ThermoFisher scientific, #31000) in carbonate coating buffer (8.4 g/L NaHCO3, 3.56 g/L Na2CO3, pH 9.5).
?. [Binding avidity ELISA of the IgG antibodies to SARS-CoV-2 and SARS-CoV spike RBD proteins]
Incubate ELISA plates at 4°C overnight.
?. [Binding avidity ELISA of the IgG antibodies to SARS-CoV-2 and SARS-CoV spike RBD proteins]
On next day, continue with binding avidity ELISA using IgG antibodies
?. [Binding avidity ELISA of the IgG antibodies to SARS-CoV-2 and SARS-CoV spike RBD proteins]
Wash each well of the ELISA plates from Step 9 with 0.05% PBST (Tween-20), 4 times.
?. [Binding avidity ELISA of the IgG antibodies to SARS-CoV-2 and SARS-CoV spike RBD proteins]
Block each well with 200 µl casein (Thermo Fisher Scientific, #A37528) at room temperature for 2 hrs.
?. [Binding avidity ELISA of the IgG antibodies to SARS-CoV-2 and SARS-CoV spike RBD proteins]
Wash each well of the ELISA plates with 0.05% PBST, 4 times.
?. [Binding avidity ELISA of the IgG antibodies to SARS-CoV-2 and SARS-CoV spike RBD proteins]
Add 70 µl of 0.2 µg/ml biotinylated recombinant SARS-CoV-2 spike protein RBD-mFc (Sino Biological, 40592-V05H) or biotinylated recombinant SARS-CoV spike protein RBD-His (Sino Biological, 40150-V08B2) in casein into respective wells. Incubate at room temperature, 1 hr.
?. [Binding avidity ELISA of the IgG antibodies to SARS-CoV-2 and SARS-CoV spike RBD proteins]
Wash each well of the ELISA plate with 0.05% PBST, 4 times.
?. [Binding avidity ELISA of the IgG antibodies to SARS-CoV-2 and SARS-CoV spike RBD proteins]
Add 70 µl per well of each concentration of different clones of anti-SARS-CoV-2 spike RBD IgGs (3-fold serial dilution) into respective wells. Incubate at room temperature, 1 hr.
?. [Binding avidity ELISA of the IgG antibodies to SARS-CoV-2 and SARS-CoV spike RBD proteins]
Wash each well of the ELISA plate with 0.05% PBST, 4 times.
?. [Binding avidity ELISA of the IgG antibodies to SARS-CoV-2 and SARS-CoV spike RBD proteins]
Add 70 µl of (1:3000 in casein) Peroxidase-conjugated AffiniPure F(ab')2 Fragment Goat Anti-Human IgG, Fcγ Fragment Specific (JACKSON ImmunoResearch, #109-036-098) to each well. Incubate at room temperature, 1 hr.
?. [Binding avidity ELISA of the IgG antibodies to SARS-CoV-2 and SARS-CoV spike RBD proteins]
Wash each well of the ELISA plates with 0.05% PBST, 5 times.
?. [Binding avidity ELISA of the IgG antibodies to SARS-CoV-2 and SARS-CoV spike RBD proteins]
Add 70µl TMB One Component HRP (SurModics, #TMBW-1000-01) per well. Stop each reaction with 70 µl 1M HCl.
?. [Binding avidity ELISA of the IgG antibodies to SARS-CoV-2 and SARS-CoV spike RBD proteins]
Measure OD450nmand OD570nm (baseline).
?. [Competition ELISA of the anti-SARS-CoV2 spike RBD IgG antibodies]
Coat ELISA plates.
?. [Competition ELISA of the anti-SARS-CoV2 spike RBD IgG antibodies]
Coat ELISA plates with 70 µl per well of 1 µg/ml ACE2_hFc protein in carbonate coating buffer (8.4 g/L NaHCO3, 3.56 g/L Na2CO3, pH 9.5).
?. [Competition ELISA of the anti-SARS-CoV2 spike RBD IgG antibodies]
Incubate ELISA plates at 4°C overnight.
?. [Competition ELISA of the anti-SARS-CoV2 spike RBD IgG antibodies]
On next day, continue with the competition ELISA using IgG antibodies.
?. [Competition ELISA of the anti-SARS-CoV2 spike RBD IgG antibodies]
Wash each well of the ELISA plates from Step 11 with 0.05% PBST (Tween-20), 4 times.
?. [Competition ELISA of the anti-SARS-CoV2 spike RBD IgG antibodies]
Block each well with 200 µl casein (Thermo Fisher Scientific, #A37528) at room temperature for 2 hrs.
?. [Competition ELISA of the anti-SARS-CoV2 spike RBD IgG antibodies]
During the 2 hours incubation time of blocking, in a separate 96-well plate, add final concentration of 0.5 nM biotinylated SARS-CoV-2 spike protein RBD-mFc with different concentrations of different anti-SARS-CoV-2 spike RBD IgG antibodies (3-fold serial dilution) in a total mixture of 100 µl per well. Incubate the plate at room temperature, 1 hr.
?. [Competition ELISA of the anti-SARS-CoV2 spike RBD IgG antibodies]
Wash the casein blocked ELISA plates from Step 12.2 with 0.05% PBST, 4 times.
?. [Competition ELISA of the anti-SARS-CoV2 spike RBD IgG antibodies]
Add the pre-incubated mixture from Step 12.3 at 100 µl/well into respective wells of the ELISA plates. Incubate at room temperature, 1 hr.
?. [Competition ELISA of the anti-SARS-CoV2 spike RBD IgG antibodies]
Wash each well of the ELISA plates with 0.05% PBST, 4 times.
?. [Competition ELISA of the anti-SARS-CoV2 spike RBD IgG antibodies]
Add 70 µl (1:3000 in casein) per well of streptavidin-HRP (Biolegend, #405210) to each well of the ELISA plates. Incubate at room temperature, 1 hr.
?. [Competition ELISA of the anti-SARS-CoV2 spike RBD IgG antibodies]
Wash each well of the ELISA plates with 0.05% PBST, 4 times.
?. [Competition ELISA of the anti-SARS-CoV2 spike RBD IgG antibodies]
Add 70µl TMB One Component HRP (SurModics, #TMBW-1000-01) per well. Stop each reaction with 70 µl 1M HCl.
?. [Competition ELISA of the anti-SARS-CoV2 spike RBD IgG antibodies]
Measure OD450nmand OD570nm (baseline).