Protein Tyrosine Phosphatase Receptor Type C (CD45) is one of the most abundant leukocyte cell surface glycoproteins and is expressed exclusively upon cells of the hematopoietic system. CD45 functions positively to regulate lymphocyte activation by servesing to dephosphorylate and activate members of the Src-tyrosine kinase family. it has been reported that SEMA4D can associate with the phosphatase CD45 at the surface of T cells and that triggering of CD45 on T cells, using monoclonal antibodies (mAb), induces shedding of SEMA4D. Thus a functional binding ELISA assay was conducted to detect the interaction of recombinant human CD45 and recombinant human SEMA4D. Briefly, biotin-linked CD45 were diluted serially in PBS, with 0.01% BSA (pH 7.4). Duplicate samples of 100 ul were then transferred to SEMA4D-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 CD45 and SEMA4D was shown in Figure 1, the EC50 for this effect is 6.47 ug/mL.