Avidin hrp

Proximity labeling using TurboID was performed as previously described³⁴ . Briefly, biotin was added to the culture medium at 48 h after co-transfection of FUT9-TurboID and Flag-tagged EPO with or without the L29 sequence. After incubation for 18 h, the Flag-tagged recombinant proteins were purified from the medium on anti-Flag M2 Affinity Gels, subjected to SDS-PAGE, and electroblotted to protein-blotting membrane. The proteins were detected using avidin-HRP (Thermo Scientific) and anti-Flag M2.

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.

ELISA experiments were conducted as previously described^{36 (link)}. Briefly, mouse peripheral blood was collected via cardiac puncture immediately after euthanasia into BD SST microcontainer tubes (02-675-185) and sera were harvested after centrifugation. Flat-bottom 96-well plates were coated with capturing antibodies in 50 μL 0.1 M NaHCO₃ solution at pH 9.5 O/N at 4°C. The plates were then emptied, blocked with 200 μL 1% bovine serum albumin (VWR, 97061-422) in PBS, and washed 3 times with PBS containing 0.05% Tween-20 (Sigma-Aldrich, P1379). 50 μL of sera at appropriate dilutions was added and incubated O/N at 4°C. The plate was then incubated in sequential orders with 50 μL of biotinylated detection antibodies for 2–3 hours, 50 μL of avidin-HRP (ThermoFisher, 18-4100-51) for 30 minutes, and 100 μL of TMB solution (ThermoFisher, 00-4201-56) at 25°C, with 3–4 washes with PBS-Tween in between each incubation steps. The colorimetric reaction was stopped with 100 μL of 1M H₃PO₄ (Sigma-Aldrich, P5811) after 5–10 minutes of adding TMB and absorbance at 450 nm was measured with a Synergy HTX plate reader (BioTek). Concentrations of antigens were determined using standard curves constructed with purified recombinant proteins and calculated with Gen5 3.02.2 (BioTek).

The S1/S2-specific IgG were quantified with LIAISON SARS-CoV-2 S1/S2 IgG (DiaSorin) according to the manufacturer’s instructions, and expressed as IU/ml. The RBD-specific antibodies (i.e. IgM, IgA, IgG1 and IgG3) were determined by an in-house ELISA and expressed as area under the curve (AUC). Briefly, high-binding 96-well plates (Greiner Bio-One) were coated with 3 µg/ml of recombinant SARS-CoV-2-RBD (Creative Diagnostics) and incubated overnight at 4 °C. After 1 h blocking with PBS-2% BSA at 37 °C, plasma was serially diluted in duplicates, and incubated for 2 h at 37 °C. The following biotinylated antibodies were used: goat anti-human kappa and lambda light chain for total antibodies (Bethyl Laboratories, Inc.), rabbit monoclonal anti-human IgM and IgA (Abnova), mouse anti-human IgG1 (BD Biosciences) and IgG3 (Southern Biotech); followed by avidin-HRP (ThermoFischer Scientific) for 30 min at RT. The detection was carried out with 1 × 3,3’,5,5’-Tetramethylbenzidine and quenched with 1 M H₂SO₄. In each run, a plasma sample collected before the SARS-CoV-2 era was included. Additionally, the RBD-specific monoclonal antibody (Human Anti-SARS-CoV-2 Spike RBD Monoclonal antibody, Creative Diagnostics) was included as positive control for total RBD antibodies detection. Optical density (OD) was measured with Tecan SunriseTM at 450 and 620 nm.