Endothelial Nitric Oxide Synthase (eNOS), also known as Nitric Oxide Synthase 3 (NOS3) or Constitutive NOS (cNOS), is an enzyme that plays a crucial role in maintaining cardiovascular health. It belongs to the nitric oxide synthase (NOS) family of enzymes, which are responsible for catalyzing the production of nitric oxide (NO) from L-arginine. eNOS is primarily expressed in endothelial cells, which line the interior of blood vessels.NOSIP can directly interact with eNOS and induce conformational change or spatial position change of eNOS, thus inhibiting enzyme activity of eNOS.Thus a functional ELISA assay was conducted to detect the interaction of recombinant human eNOS and recombinant human NOSIP. Briefly, eNOS was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to NOSIP-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST and incubated for 1h with anti-eNOS 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 rat eNOS and recombinant human NOSIP was shown in Figure 1, the EC50?for this effect is 0.05ug/mL.