An ADCC Assay

Human eosinophils (5 × 10⁴/well) were seeded in 96-well U-bottom tissue culture plates (Nunc, 163320). Purified autologous NK cells were washed with a culture medium and added at 2.5 × 10⁵ cells/well (at a target cell–to–effector cell ratio of 1:5). One of the Abs was added along with 100 pM rhIL-5 as described in the text, and the plates were incubated for 20 h at 37°C and 5% CO₂ for induction of ADCC. The addition of 100 pM rhIL-5 was due to maintain the basal survival of eosinophils during the assay period. After that, the plates were centrifuged at 300 g for 5 min, and 50 μl of the supernatant was removed from each well for an assay of lactate dehydrogenase to determine any potential ADCC activity of the added Ab. The lactate dehydrogenase assays were performed as follows: to each supernatant, 50 μl of CytoTox96 Assay reagents (Promega, G1780) was added, the plate was covered by an opaque box to protect it from light and was incubated for 30 min at room temperature. At the end of color development, 50 μl of Stop Solution (Promega, G183A) was added, and absorbance was measured at 490 nm. The absorbance value of the culture medium was subtracted as a background. A spontaneous release was defined as absorbance of wells containing only the target cells, and a maximum release was defined as absorbance of wells containing target cells lysed with 10× lysis solution (Promega, G182A). The percentage of ADCC for each Ab was calculated according to the following formula: ADCC (%) = 100 × (A – B)/(C – B), where A is an experimental release, B is a target cell spontaneous release, and C is a target cell maximal release with 10× lysis solution (24).