High binding 96 well plate

SmCB-specific serum IgG was assessed by ELISA as described elsewhere (10 (link)). Briefly, high binding 96-well plates (Greiner Bio-One, Frickenhausen Germany) were coated with rSmCB (0.5 μg/ml) in 100 mM bicarbonate/carbonate buffer (pH 9.6) overnight at 4°C. After blocking plates with 2% bovine serum albumin (BSA; Sigma Aldrich) in PBS-Tween 20 (PBS-T: 0.05%; Fisher Scientific, Ottawa, ON, Canada) (blocking buffer), serum samples were added to the plates in duplicate. Plates were incubated for 1 h at 37°C then washed with PBS (pH 7.4) and horseradish peroxidase (HRP)-conjugated anti-mouse IgG (Sigma Aldrich) was diluted 1:20,000 in blocking buffer and applied. Again, plates were washed with PBS, and 3,3’,5,5’-tetramethyl benzidine (TMB) substrate (Millipore, Billerica, MA) was used for detection followed by the addition of H₂SO₄ (0.5M; Fisher Scientific). Optical density (OD) was measured at 450 nm with an EL800 microplate reader (BioTek Instruments Inc., Winooski, VT), and concentration of SmCB specific IgG was calculated by extrapolation from the IgG standard curve.

Total anti-RBD antibodies were determined in plasma samples by a homemade ELISA. Briefly, high-binding 96-well plates (Greiner Bio-One) were coated with 3 μg/mL of recombinant wild-type SARS-CoV-2 receptor binding domain (RBD) (ACROBiosystems) diluted in 0.5 mM of carbonate-bicarbonate buffer pH 9.6 (Sigma-Aldrich) and incubated overnight at 4°C. Plates were washed with PBS-0.05%Tween-20 and blocked for 1 hour with PBS-2%BSA at 37°C. Plasma samples were serially diluted in PBS-1%BSA in triplicates (1:40, 1:240 and 1:1440), added to plates, and incubated for 2 hours at 37°C. A mix of biotinylated goat anti-human k and λ light chain were used at 1:2500 (Bethyl Laboratories, Inc., A80-115B and A80-116B) for detection, followed by avidin-HRP diluted at 1:2000 (ThermoFisher), for 30 minutes at room temperature in the dark and mild agitation. The detection was carried out with 3,3’,5,5’-tetramethylbenzidine (TMB) (Invitrogen) and quenched with 1 M H₂SO₄. Two plasma samples collected before the SARS-CoV-2 pandemic were included as negative controls, whereas an RBD-specific monoclonal antibody (Human Anti-SARS-CoV-2 Spike RBD Monoclonal Antibody, clone BIB116, Creative Diagnostics) was included as positive control. The optical density (OD) was measured by using TECAN Sunrise™ at 450 nm and 620 nm, and the area under the curve (AUC) was determined with GraphPad Prism 9.4.

A 2 μg/mL concentration of GPC-I53-dn5B diluted in Casein blocking buffer (Thermo Scientific) was added for 2 h on high-binding 96-well plates (Greiner). Four-fold serial dilutions of mAbs diluted to 2.5 μg/mL in Casein were then added for 2 h. A 1:3000 dilution of HRP-labeled goat anti-rabbit IgG (Jackson Immunoresearch) in Casein was added for 1 h. Up to now, between each step, plates were washed three times with TBS. Next, plates were washed five times with TBS, 0.05% Tween-20. Colorimetric detection was performed as described above in Serum antibody ELISA. All procedures were performed at RT. The midpoint binding concentration (IC₅₀) was determined by calculating the concentration of mAb that gave 50% of the maximal response from the sigmoidal binding curve.