Red fluorescent neutravidin beads

As described recently (32 (link)), biotinylated antigen gp120 MN was coupled to red fluorescent neutravidin beads (Thermo Fisher Scientific). Immune complexes were formed with plasma samples for 30 minutes at 37°C at 3 dilutions (1:100, 1:500, 1:2500) in order to calculate the AUC. Lyophilized guinea pig complement was resuspended according to manufacturer’s instructions (Cedarlane), and 2 μL per well was added in veronal buffer with 0.1% gelatin (Boston BioProducts). After 20 minutes of incubation at 37°C, immune complexes were washed and C3 was detected with a fluorescein-conjugated goat IgG fraction to guinea pig complement C3 (catalog 855385, MpBio). Complement-coated beads were acquired on a BD LSR II with a high-throughput sampler, and C3 deposition was reported by gating on single beads and C3⁺ events of 2 independent runs. AUC was calculated in Prism.

ADCD assays were performed as previously described^{26 (link)}. Briefly, SARS-CoV-2 S and RBD were biotinylated (Thermo Fisher Scientific) and coupled to 1 μm red fluorescent neutravidin beads (Thermo Fisher Scientific) for 2 h at 37 °C, and excess antigen was washed away afterwards. For the formation of immune complexes, 1.82 × 10⁸ antigen-coated beads were added to each well of a 96-well round bottom plate and incubated with 1:10 diluted samples at 37 °C for 2 h. Lyophilized guinea pig complement was reconstituted according to the manufacturer’s instructions (Cedarlane) with water, and 4 μl per well was added in gelatin veronal buffer containing Mg²⁺ and Ca²⁺ (GVB⁺⁺, Boston BioProducts) to the immune complexes for 20 min at 37 °C. Immune complexes were washed with 15 mM ethylenediaminetetraacetic acid in PBS, and fluorescein-conjugated goat IgG fraction to guinea pig complement C3 (MP Biomedicals) was added. After staining, samples were fixed with 4% paraformaldehyde, and sample acquisition was performed via flow cytometry (IntelliCyt, iQue Screener Plus) using a robot arm (PAA). All events were gated on single cells and bead-positive events; the median of C3-positive events is reported. All samples were run in duplicate on separate days.

Complement deposition was measured as described previously with a bead-based assay [51 (link)]. Briefly, biotinylated antigen gp140 ConS was coupled to red fluorescent neutravidin beads (Thermo fisher). Plasma samples were diluted 1:10 in PBS and allowed to form immune complexes with antigen coupled beads for 2 h at 37°C. Lyophilized guinea pig complement was resuspended according to manufacturer’s instructions (Cedarlane) and 4 μl per well was added in gelatin veronal buffer containing Mg²⁺ and Ca²⁺ (GVB⁺⁺, Boston BioProducts) and incubated with the washed immune complexes for 20 min at 37°C. C3 deposition was detected with a fluorescein-conjugated goat IgG fraction to guinea pig complement C3 (MpBio). Complement coated beads were acquired via flow cytometry (IntelliCyt, iQue Screener plus) and C3 deposition was reported by gating on single beads and C3 positive events of two independent runs.