Steroid 5 Alpha Reductase 2 (SRD5a2) is a microsomal enzyme critical in steroid metabolism, primarily converting testosterone to dihydrotestosterone (DHT), a potent androgen. Expressed in androgen-sensitive tissues (e.g., prostate, skin), it regulates male sexual development, hair growth, and prostate function. Additionally, the SRD5a2-CYP17A1 complex optimizes androgen synthesis: CYP17A1 generates testosterone precursors, while SRD5a2 amplifies androgen potency via DHT production. This collaboration ensures efficient testosterone utilization and targeted DHT formation, critical for physiological androgen effects and pathological conditions.Thus a functional ELISA assay was conducted to detect the interaction of recombinant human SRD5a2 and recombinant human CYP17A1. Briefly, CYP17A1 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-CYP17A1 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. Measured by its binding ability in a functional ELISA. When recombinant SRD5a2 is lmmobilized at 2 ug/mL(100 uLwell), the concentration of CYP17A1 that produces 50% optimal bindingresponse is found to be approximately 0.307 ug/mL.