17-Beta-Hydroxysteroid Dehydrogenase Type 3 (HSD17b3) belongs to the HSD17B family with NAD(P)H/NAD(P) -dependent oxidoreductase activity that catalyzes the interconversion between 17-ketosteroids and 17-hydroxysteroids to maintain the balance between less potent (17-keto) and more potent (17β-hydroxy) forms of estrogens and androgens. This isoform of 17 beta-hydroxysteroid dehydrogenase is expressed predominantly in the testis and catalyzes the conversion of androstenedione to testosterone. 3-oxo-5-alpha-steroid 4-dehydrogenase 2 (SRD5a2 ) is a enzyme which plays a central role in sexual differentiation and androgen physiology. A functional ELISA assay was conducted to detect the interaction of recombinant human HSD17b3 and recombinant human SRD5a2. Briefly, HSD17b3 was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to SRD5a2-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST and incubated for 1h with anti-HSD17b3 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 HSD17b3 and recombinant human SRD5a2 was shown in Figure 1, the EC50?for this effect is 27.6 ng/mL.