Serum concentration of antigen-specific IgG can substantially bias interpretation of antibody-dependent phagocytosis assay readout

Because virus neutralization cannot solely explain vaccine-induced, antibody-mediated protection, antibody effector functions are being considered as a potential correlate of protection (CoP). However, measuring effector functions at a fixed serum dilution for high throughput purposes makes it difficult to distinguish between the effect of serum antibody concentration and antibody properties such as epitopes, subclass, and glycosylation. To address this issue, we evaluated antibody-dependent cellular phagocytosis (ADCP) assay against SARS-CoV-2 spike. Adjustment of serum samples to the same concentration of antigen-specific IgG prior to the ADCP assay revealed concentration-independent differences in ADCP after mRNA vaccination in subjects with and without prior SARS-CoV-2 infection not detectable in assay performed with fixed serum dilution. Phagocytosis measured at different concentrations of spike-specific IgG strongly correlated with the area under the curve (AUC) indicating that ADCP assay can be performed at a standardized antibody concentration for the high throughput necessary for vaccine trial analyses.