SARS-CoV-2 Variant Antibody Phagocytosis Analysis

An assessment of antibody-dependent monocyte-mediated phagocytosis (ADMP) was performed on the THP-1 cells. For this, the recombinant S-proteins of D614G, B.1.617.2, and BA.1 SARS-CoV-2 variants (Miltenyi Biotec) were biotinylated using a protein-biotinylating kit (Lumiprobe), followed by incubation for 12 h with fluorescent 1 µm neutravidin particles (Invitrogen, F8775) at 4 °C. Next, the resultant particles were incubated with heat-inactivated serum samples at 37 °C for 2 h. The THP-1 cells were maintained in RPMI medium (PanEco) supplemented with 10 mM L-Glutamine and 10% FBS (PanEco). A total of 25 × 10⁴ of the THP-1 cells were seeded in a 96-well plate in serum-free AIM-V medium (Gibco), and then the S-protein-covered fluorescent particles were added and incubated overnight at 37 °C, 5% CO₂. Next, the cells were washed with PBS, fixated with 4% PFA, and analyzed using flow cytometry using the CytoFlex LX (Beckman Coulter). The antibody-dependent phagocytic activity index was calculated as follows: (% of cells containing particles × MFI (mean fluorescent intensity) of cells containing particles)/10⁴ [24 (link),25 (link)].