The PathHunter Checkpoint Signaling Assay consists of human cells engineered to stably express an ED-tagged immune checkpoint receptor, while EA is fused to either the full length or the phosphotyrosine-binding SH2 domain of an intracellular signaling protein such as Grb2 or SHP1. Engagement of the receptor by its ligand, or agonist antibodies results in phosphorylation of the receptor’s cytosolic tail. The SH2-domain fused to EA binds the phosphorylated receptor, forcing complementation of ED and EA, resulting in formation of an active β-gal enzyme, which hydrolyzes the substrate to generate a chemiluminescent signal.