Radioimmunoassay of melatonin in rat serum

1. 1. Melatonin was coupled to albumin by means of a N_a-p-carboxybenzyl, a N_a-propionic acid, methylene, or a diazotized p-aminobenzoic acid bridge and injected into rabbits or sheep. 2. 2. All coupling procedures stimulated antisera which bound melatonin with the greatest affinity; N-acetylserotonin and 6-hydroxymelatonin cross-reacted with the antisera at 3-5%. Other naturally occurring indolealkylamine derivatives showed less cross-reactivity. 3. 3. Coupling the hapten melatonin to protein through a bridge at or near the indole N can stimulate antisera which are relatively specific to melatonin and suggest that antisera which bind specifically to any other individual indolealkylamine derivative could be produced using these coupling procedures. 4. 4. Using the methylene bridged antisera, a radioimmunoassay for melatonin was developed and validated for rat serum. In addition to tests for reliability and parallelism, serum estimates of melatonin by radioimmunoassay correlated .98 with gas chromatography-mass spectrometry. 5. 5. Serum melatonin levels have a characteristic 24-hour rhythm with a crest late in the dark period (L:D 12:12). Following the injection of 50 ug of melatonin, serum levels reach 5-20 fold higher concentrations than physiological levels.