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 androstenedione. Androstendione in standards, controls and unknown samples compete with androstenedione-linked horseradish peroxidase (HRP) for available antibody binding sites. Bound androstenedione 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 androstenedione peroxidase detected, as measured by the intensity of color, is inversely proportional to the amount of androstenedione present.
Briefly, 50 µL of standard, controls, and unknown saliva are pipetted onto 96-well microtitre plates and incubated for 2 hours shaking with transferrin conjugated to HRP at room temperature. Plates are washed 4 times followed by addition of a TMB substrate and incubation 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. Concentrations of controls and samples are calculated from a standard curve generated using a 4-parameter non-linear regression curve fit (Gen5, BioTek).