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 melatonin. Melatonin in standards, controls and unknown samples compete with melatonin-linked horseradish peroxidase (HRP) for available antibody binding sites. Bound melatonin 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 melatonin peroxidase detected, as measured by the intensity of color, is inversely proportional to the amount of melatonin present.
Briefly, 100 µL of standard, controls, and unknown saliva is pipetted in duplicate onto 96-well microtitre plates and incubated for 3 hours shaking with melatonin conjugated to HRP at 2-8°C. Plates are washed 4 times followed by addition of a TMB substrate and incubation while shaking 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 were calculated from a standard curve generated using a 4-parameter non-linear regression curve fit (Gen5, BioTek).