Antibody-Dependent NK Cell Activation Assay

An assay for measuring antibody-dependent NK cell activation (ADNKA) has been described previously.⁷¹ (link)^,72 (link) Flat-bottom 96-well ELISA plates (Thermo Fisher, #439454) were coated with biotinylated PfRH5 antigen, then blocked with PBSA. Plasma samples from test subjects were diluted in PBSA, added to the plates, and incubated for 2 h at 37°C. Primary human NK cells were purified from buffy coats from healthy donors using the RosetteSep human NK cell enrichment cocktail (StemCell, #15065), then resuspended in R-10 media containing 10 μg/mL brefeldin A (Sigma, #B7651), GolgiStop (BD Biosciences, #554724), and fluorescent anti-CD107a. The ELISA plates were washed three times with PBS, then isolated NK cells (25,000/well) were added and incubated at 37°C for 5 h. The cells were then stained for surface CD56 and CD3, permeabilized, stained with fluorescent antibodies to IFN-γ and MIP-1β, fixed, and acquired on an Intellicyt iQue Screener PLUS flow cytometer. Gates were drawn on singlet, CD56⁺/CD3^- cells, and results were reported as the percentages of these cells that expressed surface CD107a, intracellular MIP-1β, or intracellular IFN-γ.