CD40, also known as Tumor Necrosis Factor Receptor Superfamily, Member 5 (TNFRSF5), is a 45-50 kDa type I transmembrane glycoprotein member of the TNF receptor superfamily. Mature human CD40 consists of a 173 amino acid (aa) extracellular domain, a transmembrane domain, and a 62 aa cytoplasmic domain. CD40 is expressed on the surface of B cells, dendritic cells, macrophages, monocytes and platelets, as well as endothelial and epithelial cells. Interaction of CD40 with its ligand, CD40 Ligand, leads to the aggregation of CD40 molecules resulting in the initiation of bidirectional intracellular signaling in both CD40 and CD40 Ligand expressing cells. A binding ELISA assay was conducted to detect the interaction of recombinant human TNFRSF5 and recombinant human TNFSF5. Briefly, TNFRSF5 was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100μl were then transferred to TNFSF5-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST and incubated for 1h with anti-TNFRSF5 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 50µL stop solution to the wells and read at 450/630nm immediately. The binding activity of TNFRSF5 and TNFSF5 was shown in Figure 1, the ED50 for this effect is 120 ng/mL.