17-Beta-Hydroxysteroid Dehydrogenase Type 14 (HSD17b14) is an enzyme mainly expressed in the liver and other tissues. It belongs to the hydroxysteroid dehydrogenase family and participates in the metabolism of steroid hormones. HSD17b14 can catalyze the interconversion of specific steroid substrates, influencing the balance of steroid hormone levels in the body.Brsides,HSD17b14 can regulate cellular pH and metabolic processes by binding to Carbonic Anhydrase VIII (CA8), affecting activities related to steroid metabolism and ion balance to maintain cellular stability.Thus a functional binding ELISA assay was conducted to detect the interaction of recombinant human HSD17b14 and recombinant human CA8 . Briefly, biotin-linked HSD17b14 were diluted serially in PBS, with 0.01% BSA (pH 7.4). Duplicate samples of 100μl were then transferred to CA8-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST 3 times and incubation with Streptavidin-HRP for 30min, then wells were aspirated and washed 5 times. With the addition of substrate solution, wells were incubated 15-25 minutes at 37℃. Finally, add 50μl stop solution to the wells and read at 450nm immediately. The binding activity of HSD17b14 and CA8 was shown in Figure 1, the EC50?for this effect is 0.23ug/mL.