Antibody-Mediated Phagocytic Activity Assay

Characterization of the phagocytic activity of serum antibodies was performed as described previously^{77 (link)}. Briefly, 1 uM yellow-green fluorescent beads (Thermo Fisher, F8813) were covalently conjugated to spike or RBD antigens. Beads were then incubated with serum samples for 4 hr with THP-1 cells (ATCC TIB-202). Cells were fixed and analyzed by flow cytometry using a MACSQuant Analyzer (Miltenyi Biotec). Scores were calculated as the percentage of cells that phagocytosed one or more fluorescent beads multiplied by the MFI of this population. S309 and VRC01 antibodies were included as positive and negative controls, respectively. Additional control wells with no added antibody were used to determine the level of antibody-independent phagocytosis. Serum samples were assayed at three different dilutions, which were determined by an initial pilot experiment to determine the optimal dilution series for measuring signal compared to background. All samples were run in three biological replicates.

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Hederman A.P., Natarajan H., Wiener J.A., Wright P.F., Bloch E.M., Tobian A.A., Redd A.D., Blankson J.N., Rottenstreich A., Zarbiv G., Wolf D., Goetghebuer T., Marchant A, & Ackerman M.E. (2022). SARS-CoV-2 mRNA vaccination elicits broad and potent Fc effector functions to VOCs in vulnerable populations. medRxiv.

Publication Preprint 2022

yellow-green fluorescent beads MACSQuant Analyzer

Antibodies Antibody Antigens Biological Cells Dilution Flow cytometry Phagocytic Phagocytosis Serum Thp 1 cells

Corresponding Organization : Dartmouth College

Other organizations : Dartmouth–Hitchcock Medical Center, Johns Hopkins Medicine, Johns Hopkins University, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hadassah Medical Center, University Medical Center, Centre Hospitalier Universitaire de Saint-Pierre, Université Libre de Bruxelles

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Variable analysis

independent variables

Concentration of yellow-green fluorescent beads covalently conjugated to spike or RBD antigens

dependent variables

Percentage of THP-1 cells that phagocytosed one or more fluorescent beads
Mean fluorescence intensity (MFI) of the population of cells that phagocytosed one or more fluorescent beads

control variables

Incubation time of beads with serum samples and THP-1 cells (4 hours)
Cell line used (THP-1 cells, ATCC TIB-202)
Flow cytometry instrument used (MACSQuant Analyzer, Miltenyi Biotec)

controls

Positive control: S309 antibody
Negative control: VRC01 antibody
Additional control: No added antibody to determine the level of antibody-independent phagocytosis

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