The cluster of differentiation (CD) antigen CD28 is a central co-stimulatory molecule for TCR-mediated activation such as cytokine production and T-cell proliferation upon ligand binding and TCR stimulation. It is a member of the immunoglobulin subfamily, other members of the subfamily include ICOS, CTLA4, PD1, PD1H, TIGIT and BTLA. Cytotoxic T-Lymphocyte Associated Antigen 4 (CTLA4) and CD28 are highly homologous and compete for the same ligands, CTLA4 can also bind with CD28 to form a dimer. Thus a functional binding ELISA assay was conducted to detect the interaction of recombinant mouse CD28 and recombinant rat CTLA4. Briefly, rmCD28 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-CD28 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 mouse CD28 and recombinant rat CTLA4 was shown in Figure 1, the EC50?for this effect is 2.82 ug/mL.