Figure 1. KILR ADCC Assay Principle. Target cells expressing the relevant antigen are engineered to stably express a housekeeping protein tagged with a reporter β-gal ED fragment called enhanced ProLabel® (ePL). The β-gal enzyme is inactive when the reporter fragment (also called the KILR reporter protein) is not paired with its larger β-gal EA fragment. On incubation of KILR target cells with a test antibody and appropriate effector cells (e.g. KILR CD16 Effector Cells), effector cell-mediated killing releases the tagged protein into the media. The presence of both EA and ED fragments leads to the formation of the active β-gal enzyme that hydrolyzes the substrate to give a chemiluminescent output detected on any benchtop luminometer.

Figure 2. ADCP assays measures antibody-dependent phagocytosis of target cells that is typically mediated by primary human macrophages. Target cells expressing the relevant antigen are engineered to stably express a housekeeping protein that is tagged with the small enhanced β-gal enzyme donor fragment ePL. Monocytes from primary human PBMCs from a single healthy donor are differentiated with M-CSF for 7 days to produce M0 macrophages or further stimulated with IFNγ for 24 hours to produce M1 macrophages. Both M0 and M1 macrophages can mediate ADCP. On the day of the assay, the KILR target cells are opsonized with the antibody of interest, then co-cultured with the isolated M0 or M1 macrophages for 24 hours at 37°C. Since macrophages digest the target cells, all cells are lysed and the detection reagent containing the complementing β-gal EA fragment is added to the lysed cells. Complementation of ePL and EA facilitates quantitation of the remaining amount of KILR protein present. A high chemiluminescent signal indicates minimum killing, while a low signal correlates with increased killing.