The cluster of differentiation (CD) antigen CD28 is a member of the immunoglobulin subfamily. 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. CD80 and CD86 are expressed on antigen presenting cells and CD86 is primary ligand for CD28. Thus a functional binding ELISA assay was conducted to detect the interaction of recombinant rat CD28 and recombinant human CD86. Briefly, CD28 was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to CD86-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 rat CD28 and recombinant human CD86 was shown in Figure 1, the EC50 for this effect is 0.07 ug/mL.