Cluster of Differentiation 90 (CD90), a glycosylphosphatidylinositol-anchored glycoprotein, is widely expressed on stem cells, T cells, and stromal cells. It regulates cell proliferation, adhesion, and migration, playing key roles in development, immunity, and tumor progression. CD90 interacts with EPHA4, a receptor tyrosine kinase, mediating signaling pathways that modulate cell fate and tissue homeostasis. Briefly, EPHA4 was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to CD90-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST and incubated for 1h with anti-EPHA4 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 450/630nm immediately.Measured by its binding ability in a functional ELISA. When Recombinant CD90 is lmmobilized at 2 µg/mL(100 uLwell), the concentration of EPHA4 that produces 50% optimal bindingresponse is found to be approximately 0.178 µg/mL.