Diazepam Binding Inhibitor (DBI), also known as acyl-CoA-binding protein (ACBP), is a 10 kDa multifunctional protein widely expressed in the brain, liver, and steroidogenic tissues. It's implicated in anxiety regulation, energy homeostasis, and neurodegenerative processes. DBI overexpression has been observed in Alzheimer's disease (AD) brains, suggesting its involvement in neuropathology.?DBI interacts with Apolipoprotein E (APOE)?by modulating lipid metabolism in astrocytes, potentially influencing APOE-mediated cholesterol transport in AD progression.DBI-APOE interaction alters astrocytic lipid metabolism, impacting neurodegenerative processes.Thus a functional ELISA assay was conducted to detect the interaction of recombinant human DBI and recombinant dog APOE. Briefly, DBI was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to APOE-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST and incubated for 1h with anti-DBI 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 DBI and recombinant dog APOE was shown in Figure 1, the EC50?for this effect is 9.47 μg/mL.