Melatonin ELISA

The Melatonin ELISA kit is a competitive immunoassay using a capture antibody technique. Melatonin present in pretreated saliva and controls as well as in the calibrators compete with biotinylated melatonin for the binding sites of the highly specific polyclonal Kennaway G280 anti-melatonin antibody (5, 6) during an overnight incubation. During this incubation the formed antibody-melatonin-biotin complexes are captured onto the pre-coated wells of the microplate. After washing away unbound melatonin-biotin conjugate the enzyme label (streptavidin conjugated to horseradish peroxidase) is added, which binds to the free biotin sites captured on the coated wells during a 60-minute incubation step. Unbound enzyme label is then removed by a second washing step and TMB (tetramethylbenzidine) substrate is added to the wells. In a further 30-minute incubation step, a chromophore is formed (turns from colorless to blue) in inverse proportion to the amount of melatonin originally present in the samples, controls and calibrators. The color turns from blue to yellow after the addition of an acidic stop solution and can be measured at 450 nm. See instructions for use for references.