To determine Antibody-dependent NK cell activation, MaxiSporp ELISA plates (Thermo Fisher) were coated with respective antigen for 2h at RT and then blocked with 5% BSA (Sigma-Aldrich). 50 μl 1:50 diluted plasma sample or monoclonal Abs was added to the wells and incubated overnight at 4°C. NK cells were isolated from buffy coats from healthy donors using the RosetteSep NK cell enrichment kit (STEMCELL Technologies, MA, USA) and stimulated with rhIL-15 (1ng/ml, STEMCELL Technologies) at 37°C overnight. NK cells were added to the washed ELISA plate and incubated together with anti-human CD107a (BD, 1:40, clone: H4A3), brefeldin A (Sigma-Aldrich, MO, USA), and monensin (BD) for 5 hours at 37°C. Next, cells were surface stained for CD56 (BD, 1:200, clone: B159), CD16 (BD, 1:200, clone: 3G8), and CD3 (BD, 1:800, UCHT1). After fixation and permeabilization with FIX & PERM Cell Permeabilization Kit (Thermo Fisher), cells were stained for intracellular markers MIP1β (BD, 1:50, clone: D21–1351) and IFNγ (BD, 1:17, clone: B27). NK cells were defined as CD3-CD16+CD56+ and frequencies of degranulated (CD107a+), INFγ+ and MIP1β+ NK cells determined on an iQue analyzer (Intellicyt)(49 (link)).