The PathHunter PD1 Signaling Bioassay consists of human cells engineered to stably express an ED-tagged immune checkpoint receptor, PD-1, while EA is fused to either the full length or the phosphotyrosine-binding SH2 domain of an intracellular signaling protein, 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. The bioassay kit contains all reagents needed for the assay including pre-qualified cryopreserved cells, detection reagents, cell plating reagent, positive control agonist, and assay plates.