Cluster Of Differentiation 8a (CD8a) is an approximately 32-34 kDa cell surface glycoprotein found on cytotoxic T lymphocytes. It belongs to the immunoglobulin family and is a key component of the immune system, particularly in the context of T-cell biology. Its interaction with MHC class I molecules is crucial for T-cell development, activation, and the mediation of the immune response against infected or cancerous cells.CD4 and CD8 molecules play a key role in the interaction between T cells and antigen presenting cells (APCs), thus a functional binding ELISA assay was conducted to detect the interaction of recombinant human CD8a and recombinant human CD4. Briefly, biotin-linked CD8a were diluted serially in PBS, with 0.01% BSA (pH 7.4). Duplicate samples of 100 ul were then transferred to CD4-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 50 μl stop solution to the wells and read at 450 nm immediately. The binding activity of recombinant human CD8a and recombinant human CD4 was shown in Figure 1, the EC50?for this effect is 0.59 ug/mL.