A sensitive, specific, reproducible and practical radioimmunoassay for the determination of 5-methoxytryptophol (ML) in pineal glands of different species has been developed. High-affinity specific antisera were produced by immunization of sheep with ML–bovine serum albumin. Iodinated ML, used as the radiolabel, was synthesized by direct iodination of ML using 1,3,4,6,-tetrachloro-3,6-diphenylglycouril as the oxidant. Sensitivity of the assay was 0·005 pmol/tube. The validity of the assay was checked using classical techniques. Cross-reactivity with other indoles was negligible. Parallel inhibition curves were obtained for rat, hamster, sheep and tortoise pineal homogenates. Using thin-layer chromatography, tortoise pineal immunoreactivity also co-chromatographed with standard ML. Samples (n = 5) with ML concentrations of 0·013, 0·052 and 0·209 pmol/tube had intra-assay coefficients of variation of 9·8, 5·7 and 7·6% respectively. Their respective interassay coefficients of variation were 17·7 16·5 and 11·4% (n = 8). The pineal concentration of ML was found to be species dependent. Afternoon ML levels were 0·052± 0·002 (s.e.m.) pmol/gland in the rat (n = 16), 0·539 ±0·089 pmol/gland in the hamster (n = 16), 1·73±0·225 nmol/g in the sheep (n = 10) and 7·15± 0·465 pmol/gland in the tortoise (n = 4). The ratio of ML:melatonin content in the pineal gland also showed a large interspecies variation with values of 0·02 in the rat, 0·22 in the sheep, 2·7 in the hamster and 17 in the tortoise.
J. Endocr. (1986) 110, 177–184