Development and validation of a radioimmunoassay for serum melatonin

A radioimmunoassay using N-[3-(4-hydroxy-3-[4-hydroxy-3- [125I]iodophenylpropionyl)]-5-methoxytryptamine as tracer for determination of melatonin in the serum of different species is described. Melatonin antisera were raised in rabbits by immunization with a bovine serum albumin conjugate of N-[3-(2-aminoethyl)-5-methoxy indole] hemisuccinamide. A single high affinity, specific antiserum was obtained. In contrast to previous studies, the tracer was synthesised in one step in the absence of water, giving an excellent yield of highly pure product. No chromatographic purification step was needed. Polyethylene glycol in combination with goat antirabbit immunoglobulins was used to separate bound and unbound tracer. Sera were delipidized with Lipoclean prior the extraction of melatonin with diethyl ether. This sample preparation allows the determination of melatonin in the presence of widely varying amounts of lipids in human, rat and hamster serum. Using this extraction procedure, the sensitivity of the radioimmunoassay was approximately 1 ng/l of serum. Dilutions of sera and of synthetic melatonin gave the same parallel response in the radioimmunoassay. High performance liquid chromatography analysis of a serum extract showed only one immunoreactive peak co-eluting with synthetic melatonin. Characteristic diurnal rhythms of melatonin were observed in all species. All assay components including standards and serum controls are stable for at least 1 year at 4 degrees C, thus facilitating the determination of melatonin in a routine laboratory.