Maxisorp 384 well plates

The ability of complement activation by the Fc variants was measured by ELISA. MaxiSorp 384-well plates (Thermo Fisher Scientific) were directly coated with the antibodies for 1 h at room temperature. After blocking with TBS-T containing 0.5% BSA and 1× Block Ace (DS Pharma Biomedical) for 2 h at room temperature, pooled human complement serum (Innovative Research) or AG129 mouse serum was added onto the plates and incubated for 1 h at 25 °C. The plates were incubated with anti-C3b antibody clone 6c9 (Thermo Fisher Scientific) for 1 h at 25 °C, followed by incubation with a peroxidase-conjugated antibody to mouse IgG₁ (Yamasa Corporation, Chiba, Japan) for 30 min at room temperature. Washes in PBS-T (pH 7.4) were performed after each subsequent step. TMB substrate (Thermo Fisher Scientific) was subsequently added, and the signal was measured by a plate reader at a wavelength of 450 nm. The complement activation was confirmed to be suppressed in this assay when adding EDTA into the serum.

MaxiSorp 384-well plates (Thermo Fisher Scientific) were incubated for 24h with 1 μg ml⁻¹ recombinant SARS-CoV-2 RBD (Acro, SPD-C52H3), SARS-CoV-2 S2 (Acro, S2N-C52H5), HCoV-OC43 S (Sino Biological, 40607-V08B), or HCoV-HKU1 S (Sino Biological, 40606-V08B), in carbonate-bicarbonate buffer. Plates were washed with PBST (PBS + 0.1 % Tween20) six times between each step. Plates were blocked with PBS + 1 % BSA for 1h at RT, then mAbs were added at concentrations of 10, 1, 0.1, and 0.01 μg ml⁻¹ in PBS + 1% BSA and plates were incubated for 24h at 4°C. Plates were incubated for 1h at RT with secondary HRP-conjugated goat anti-human IgG antibodies (Bethyl Laboratories) and developed with TMB substrate (Thermo Fisher Scientific). Development was stopped with 2 N sulfuric acid. Four dilutions of positive-control plasma and secondary antibody only, as well as BSA only served as positive and negative controls. Plates were read on a GloMax Explorer Microplate Reader (Promega). ELISA assays were performed at least twice, in duplicate or triplicate.

The interaction of Fc variants with human C1q was measured by ELISA. MaxiSorp 384-well plates (Thermo Fisher Scientific) were directly coated with the anti-CD154 antibodies overnight at 4 °C. After blocking with TBS-T containing 0.5% BSA and 1× Block Ace (DS Pharma Biomedical, Osaka, Japan) for 2 h at room temperature, 3 μg/mL of human C1q (Sigma-Aldrich, St. Louis, MO, USA) was added onto the plates and incubated for 1 h at room temperature. At room temperature, the plates were incubated with sheep anti-human C1q antibody-HRP conjugate (Bio-Rad, Hercules, CA, USA) for 1 h. Washes in PBS-T (pH 7.4) were performed after each subsequent step. TMB substrate (Thermo Fisher Scientific) was subsequently added, and the signal was measured by a plate reader at a wavelength of 450 nm (test wavelength) and 570 nm (reference wavelength).