Programmed Cell Death 1 Ligand 2 (also known as PD-L2, B7-DC) is a member of the B7 family of proteins that provide signals for regulating T-cell activation and tolerance. Mature human PD-L2 consists of a 201 amino acid (aa) extracellular domain (ECD) with one V-like and one C-like Ig domain, a 21 aa transmembrane segment, and a 32 aa cytoplasmic domain. PD-L2 has also been designated as CD273 (cluster of differentiation 273). PD-L2 is an immune checkpoint receptor ligand which plays a role in negative regulation of the adaptive immune response. PD-L2 is one of two known ligands for Programmed cell death protein 1 (PD-1), thus a functional binding ELISA assay was conducted to detect the interaction of recombinant human PD-1 and recombinant human PD-L2. Briefly, biotin-linked PD-L2 were diluted serially in PBS, with 0.01% BSA (pH 7.4). Duplicate samples of 100μl were then transferred to PD-1-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST 3 times and incubation with Streptavidin-HRP for 30min, then wells were aspirated and washed 5 times. With the addition of substrate solution, wells were incubated 15-25 minutes at 37℃. Finally, add 50µl stop solution to the wells and read at 450nm immediately. The binding activity of PD-1 and PD-L2 was shown in Figure 1, and this effect was in a dose dependent manner.