Cluster of Differentiation 4 (CD4) is a transmembrane glycoprotein expressed primarily on helper T cells, regulatory T cells, and dendritic cells. As a key immune checkpoint molecule, it interacts with MHC class II molecules to modulate T cell activation, proliferation, and cytokine secretion, playing pivotal roles in adaptive immunity, inflammation, and immune homeostasis. CD4 binds to CXCR4, a G protein-coupled chemokine receptor, facilitating cell migration and signaling crosstalk in immune responses and tissue homeostasis.Briefly, CXCR4 was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to CD4-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST and incubated for 1h with anti-CXCR4 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 450/630nm immediately.Measured by its binding ability in a functional ELISA. When Recombinant CD4 is lmmobilized at 2 μg/mL(100 uLwell), the concentration of CXCR4 that produces 50% optimal bindingresponse is found to be approximately 3.97 μg/mL.