Competitive ELISA analysis of Estriol / Estradiol / Estrone / 16-Epiestriol was performed by coating wells of a 96-well plate with 50 µl per well of Estriol-BSA antigen diluted in carbonate/bicarbonate buffer, at a concentration of 5 µg/mL overnight at 4℃. The wells of the plate were washed, blocked with StartingBlock blocking buffer, and then incubated with 50 µL per well of an Estriol antibody at a concentration of 4 µg/mL along with Estriol / Estradiol / Estrone / 16-Epiestriol / PBS buffer only for 2 hours at room temperature. The plate was washed and incubated with 50 µl per well of an HRP-conjugated goat anti-rabbit IgG secondary antibody at a dilution of 1/10,000 for one hour at room temperature. Detection was performed using an Ultra TMB Substrate for 5 minutes at room temperature in the dark. The reaction was stopped with sulfuric acid and absorbances were read on a spectrophotometer at 450 nm.