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 testosterone. Testosterone in standards, controls and unknown samples compete with cotinine linked horseradish peroxidase (HRP) for available antibody binding sites. Bound testosterone 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 testosterone peroxidase detected, as measured by the intensity of color, is inversely proportional to the amount of Testosterone present.
Briefly, 25 µL of standard, controls, and unknown saliva are pipetted in duplicate onto 96-well microtitre plates and incubated for 1 hour shaking with testosterone 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 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).