Saliva is tested at IISBR in duplicate using a commercially-available enzyme-linked immunosorbent assay kit (ELISA) from Salimetrics following manufacturer’s protocol without modification. This test employs a 96-well microtitre plate coated with a monoclonal antibody (Ab) to 17β-estradiol. 17β-Estradiol in standards, controls and unknown samples compete with 17β-estradiol-linked horseradish peroxidase (HRP) for available antibody binding sites. Bound 17β-estradiol peroxidase is measured by the reaction of the peroxidase enzyme on the substrate tetramethylbenzidine (TMB), producing a blue color. The reaction is stopped and the resultant yellow color is read at 450nm on a spectrophotometer (PowerWave HT, BioTek Instruments). The amount of 17β-estradiol peroxidase detected, as measured by the intensity of color, is inversely proportional to the amount of 17β-estradiol present in the standards, controls, and samples.
Briefly, 100 µL of standard, controls, and unknown saliva is pipetted in duplicate onto 96-well microtitre plates and incubated for 2 hours with estradiol conjugated to HRP at room temperature. Plates are washed 4 times, followed by addition of a TMB substrate. Plates are incubated at room temperature in the dark for 30 minutes. Following incubation the reaction is stopped with 2M Sulfuric Acid (NH2SO4) and optical density (OD) read at 450nm with a 630nm correction filter. Concentrations of controls and samples are calculated from a standard curve generated using a 4-parameter non-linear regression curve fit (Gen5, BioTek).