Sphingosine Kinase 1 (SPHK1) is a lipid kinase that catalyzes the phosphorylation of sphingosine to generate sphingosine-1-phosphate (S1P), a potent bioactive lipid mediator regulating cell proliferation, survival, and immune responses. SPHK1 is localized in the cytosol and translocates to the plasma membrane upon activation, playing critical roles in inflammation, cancer progression, and vascular biology. Its overexpression is associated with various cancers and inflammatory diseases.Besides,N-Acylsphingosine Amidohydrolase 2 (ASAH2) has been identified as an interactor of SPHK1, thus a functional binding ELISA assay was conducted to detect the interaction of recombinant human SPHK1 and recombinant rat ASAH2. Briefly, SPHK1 was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to ASAH2-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST and incubated for 1h with anti-SPHK1 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. The binding activity of recombinant human SPHK1 and recombinant rat ASAH2 was shown in Figure 1, the EC50 for this effect is 0.06 μg/mL.