Vitamin D Binding Protein (DBP, encoded by the GC gene, also termed Gc-globulin, product B810Hu) is a ~58 kDa multifunctional serum glycoprotein belonging to the albumin gene family. Synthesized primarily in the liver, it is encoded on human chromosome 4q11–q13 and comprises 458 amino acids with three structural domains. DBP is the principal carrier of vitamin D3 (VD3) and its metabolites (e.g., calcidiol, calcitriol), solubilizing these lipophilic molecules and mediating their systemic transport to target tissues. Beyond vitamin D transport, it sequesters extracellular G-actin and modulates macrophage activation and immune responses. DBP specifically binds the VD3 moiety of VD3-BSA conjugate with high affinity via its N-terminal sterol-binding pocket. Thus a functional ELISA assay was conducted to detect the interaction of recombinant human DBP and VD3-BSA. Briefly, DBP was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to VD3-BSA-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST and incubated for 1h with anti-DBP 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 DBP and VD3-BSA was shown in Figure 1, the EC50 for this effect is 0.163 µg/mL.