Indirect ELISA was performed by coating BSA-Estradiol ~30 ng/well (by Estradiol amount), followed by blocking with 1% BSA. Serial diluted Estradiol antibody 68600-1-Ig was added to the plates and incubated at 37℃. HRP-Goat anti-mouse was used for detection (top X-right Y, blue curve). Competitive ELISA was performed similarly except that different concentration of Estradiol was mixed in 30ng/mL primary antibody (bottom X-left Y, red curve).

BSA-Estradiol was coated in 96 well plate at ~30 ng/well (by Estradiol amount), followed by blocking with 1% BSA. Different concentrations of Estradiol standard as well as diluted urine from different donor were mixed with 30 ng/mL Estradiol antibody 68600-1-Ig respectively. Urine Estradiol was calculated based on standard curve. Please notice that the level from different donor might be different between individuals.