Serum Estradiol Quantification using ELISA

Circulating estrogen levels were quantified according to the manufacturer’s protocol using the 17 beta Estradiol ELISA Kit (cat no. ab108667, Abcam). This kit was validated by Marino FE et al.^{80 (link)}. Briefly, 25 µl of blood serum samples, prepared standards, or controls were added in duplicate to a 96 well plate. 200 µl of the 17 beta Estradiol-HRP Conjugate were added to each well followed by incubation at 37 °C for two hours. Samples, standards, and controls were aspirated, and wells were washed three times with 300 µl of diluted washing solution (soak time >5 seconds) using an automated plate washer (BioTek 50TS). After washing, 100 µl tetramethylbenzidine (TMB) Substrate Solution was added to each well, and the plate was incubated for 30 minutes in the dark at room temperature. After incubation, 100 µl of Stop Solution was added into all wells in the same order and at the same rate as the substrate solution. Absorbance was measured at 450 nm with Spectramax M3 plate reader (Molecular Devices) within 30 minutes of adding the Stop Solution. Construction of standard curve and subsequent analyses were performed in Microsoft Excel.