Saliva is tested at IISBR 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 estriol. Estriol in standards, controls and unknown samples compete with estriol-linked horseradish peroxidase (HRP) for available antibody binding sites. Bound estriol 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 estriol peroxidase detected, as measured by the intensity of color, is inversely proportional to the amount of estriol present.
Briefly, 100 µL of standard, controls, and unknown saliva (*Note* Samples from pregnant participants are diluted 1:2 prior to plating by adding 150ul saliva in to 150ul estriol assay diluent) are pipetted onto 96-well microtitre plates and incubated for 24 hours shaking with prepared estriol conjugated to HRP at 2-8ºC. Plates are washed 4 times followed by addition of a TMB substrate and incubation shaking at room temperature in the dark for 45 minutes. Following incubation, the reaction is stopped with 2M Sulfuric Acid (NH2SO4)and optical density (OD) read at 450nm with a 490nm 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).