Improved Enzyme Immunoassay Method for Melatonin: Application to the Determination of Serum Melatonin in Rats, Sheep, and Humans

Melatonin influences physiological processes such as reproduction (1), immune regulation (2), and aging (3). Recent findings have also indicated its potent antioxidative properties (4)(5). This hormone is known to exhibit a circadian rhythm, with high concentrations during the dark phase. Pineal function can be evaluated by the measurement of melatonin in body fluids (6)(7) by the use of specific and sensitive immunoassays.Although RIA is widely used, Ferrua and Masseyeff (8) in 1985 first reported an immunoassay for melatonin with enzyme-labeled antibodies. They used anti-melatonin IgG coupled with horseradish peroxidase as a nonisotopic label. Although their assay procedure was simple and rapid, the detection limit of the assay was 5 pg/well, which may not be adequate to measure daytime melatonin concentrations. In 1993, Yie et al. (9) reported a competitive solid-phase enzyme immunoassay (EIA) for melatonin with melatonin-sodium- p -carboxybenzyl-alkaline phosphatase (MT-pcb-AP) as a nonisotopic label. They validated the assay with a detection limit of 1.0 fmol/well (0.2 pg/well), but the assay required overnight incubation steps over several days.Our aim was to modify the method of Yie et al. (9) so that it could be completed within 1 or 2 days. We describe here the method and its validation in rat, sheep, and human serum samples after extraction with dichloromethane.Polystyrene multiwell plates (modified flat bottom; Corning 25805-96), serum samples [rat (cat. no. S7648), sheep (cat. no. S2263), and human (cat. no. H1388)], bovine albumin (cat. no. A9418), thimerosal (cat. no. T8784), diethanolamine (cat. no. D8885), and melatonin (cat. no. M5250) were purchased from Sigma Chemical. MT-pcb-AP (cat. no. CIA101a) and purified anti-melatonin serum (cat. no. CIA101, lot no. S380, 24-4-89) were obtained from CIDtech Inc. The specificity of the purified anti-melatonin serum has been reported (9). p -Nitrophenyl phosphate, disodium salt (cat. no. 1.06850) …