As indicated in the schematic overview, biotinylated HEK-derived recombinant proteins (ACE2, nucleoprotein, spike trimer, spike monomer, spike RBD, spike S1, spike S2) were coupled to streptavidin-coated PMMA beads (PolyAn), each at a concentration of 10 nM. After incubation with plasmapheresis samples of COVID-19⁺ patients or healthy donors, captured antibodies were detected with anti-Igk-PE (130-123-246). Flow cytometry was performed using a MACSQuant® Analyzer. Comparison to the negative controls (ACE2 and healthy donor plasma) and the different plasma dilutions show the high specificity of the antigens and the sensitivity of the assay. 

Quantification of the above presented analysis of patient-derived SARS-CoV-2 antibodies using our biotinylated antigens in a bead-based immunoassay. Results for plasma from a COVID-19⁺ patient (circle) or a healthy donor (square) are shown.

As indicated in the schematic overview, ACE2-expressing cells (Vero E6 cells) were incubated with biotinylated recombinant spike RBD, monomer, or trimer (all produced in HEK cells). Different protein concentrations were used, and ACE2-bound proteins were subsequently detected by fluorescent staining with streptavidin-PE. Flow cytometry was performed using a MACSQuant Analyzer. Cell debris and dead cells were excluded from the analysis based on scatter signals and propidium iodide fluorescence. The results show the titratable, high-affinity binding of all three recombinant spike proteins to cell surface ACE2, with the trimer showing the highest affinity.