Retinol Binding Protein 4 (RBP4) is a 21-kDa secreted glycoprotein belonging to the lipocalin family, predominantly synthesized in the liver and adipose tissue. It functions as the primary carrier of retinol (vitamin A) in the bloodstream, facilitating retinol transport to target tissues and regulating systemic vitamin A homeostasis. RBP4 plays critical roles in vision, epithelial cell differentiation, immune function, and metabolic regulation, with dysregulation linked to obesity, type 2 diabetes, and cardiovascular diseases. RBP4 binds to retinoic acid-BSA conjugate, enabling targeted delivery of retinoic acid and enhancing its bioavailability in biological systems.Thus a functional ELISA assay was conducted to detect the interaction of recombinant human RBP4 and recombinant human retinoic acid-BSA conjugate. Briefly, RBP4 was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to retinoic acid-BSA conjugate-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST and incubated for 1h with anti-RBP4 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 RBP4 and recombinant human retinoic acid-BSA conjugate was shown in Figure 1, the EC50?for this effect is 13.88μg/mL.