Cluster Of Differentiation 4 (CD4) is a glycoprotein expressed on the surface of immune cells, primarily T-helper cells, macrophages, and dendritic cells. It serves as a co-receptor for the T-cell receptor (TCR) during antigen presentation, binding to major histocompatibility complex class II (MHC-II) molecules on antigen-presenting cells (APCs). This interaction stabilizes TCR-MHC-II binding and initiates immune signaling. CD4 also acts as a primary receptor for HIV-1, enabling viral entry into host cells. Besides,Chemokine C-X-C-Motif Receptor 4 (CXCR4) has been identified as an interactor of CD4, thus a functional binding ELISA assay was conducted to detect the interaction of recombinant mouse CD4 and recombinant mouse CXCR4. Briefly, biotin-linked CD4 were diluted serially in PBS, with 0.01% BSA (pH 7.4). Duplicate samples of 100μl were then transferred to CXCR4-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 CD4 and CXCR4 was shown in Figure 1, the EC50 for this effect is 2.57µg/mL.