FMS-like tyrosine kinase 3 ligand (Flt3L) is a crucial hematopoietic cytokine primarily produced by bone marrow stromal cells and lymphocytes. It functions as a key regulator for the proliferation, differentiation, and survival of early hematopoietic progenitor cells and dendritic cells. Due to its vital role in stimulating the immune system, Flt3L is extensively investigated as a potential therapeutic agent in cancer immunotherapy and for enhancing immune reconstitution following bone marrow transplantation. Its mechanism of action is exclusively mediated through binding and activation of its cognate receptor, Flt3 is a type III receptor tyrosine kinase expressed on the surface of target cells. This specific ligand-receptor interaction induces Flt3 dimerization and autophosphorylation, initiating critical intracellular signaling cascades, such as the MAPK and PI3K pathways, that drive cell proliferation and differentiation.Thus a functional ELISA assay was conducted to detect the interaction of recombinant human Flt3L and recombinant human Flt3. Briefly, Flt3L was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μL were then transferred to Flt3-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST and incubated for 1h with anti-Flt3L pAb, then aspirated and washed 3 times. After incubation with HRP labeled 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. The binding activity of recombinant human Flt3L and recombinant human Flt3 was shown in Figure 1, the EC50?for this effect is 0.06262 μg/mL.