PD-1 (Programmed Death-1 receptor), also known as CD279, is a receptor expressed on T cells responsible for modulating T cell activation. Like CTLA4, PD-1 is classified as an immune checkpoint inhibitory receptor. When PD-1 protein binds to PD-L1, it initiates a negative signaling cascade inhibiting activation of T cells. The cytoplasmic tail contains two tyrosine residues that form the immuno receptor tyrosine-based inhibitory motif (ITIM) and immuno receptor tyrosine-based switch motif (ITSM) that are important for mediating PD-1 signaling. Normally, PD-1 helps keep T cells from attacking other cells in the body. However, many cancers take advantage of this by expressing high amounts of PD-L1 allowing cancer cells to evade the body's own immune response. Blocking the PD-1:PD-L1 interaction has proven successful in treating many different cancer types. A functional binding ELISA assay was conducted to detect the interaction of recombinant rat PD1 and recombinant human CTLA4. Briefly, PD1 was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to CTLA4-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST and incubated for 1h with anti-PD1 pAb, then aspirated and washed 3 times. After incubation with HRP labelled secondary antibody for 1h at 37℃, wells were aspirated and washed 5 times. With the addition of substrate solution, wells were incubated 15-25 minutes at 37℃. Finally, add 50 μL stop solution to the wells and read at 450/630 nm immediately. The binding activity of recombinant rat PD1 and recombinant human CTLA4 was shown in Figure 1, the EC50?for this effect is 0.04 ug/mL.