PMID: 6539551Jun 1, 1984Paper

Development and validation of a melatonin radioimmunoassay using radioiodinated melatonin as tracer

Acta Endocrinologica
O VakkuriO Vuolteenaho

Abstract

A new radioimmunoassay (RIA) using [125I]-melatonin as tracer for determination of melatonin in biological fluids has been developed. Melatonin antisera were raised in rabbits by immunization with bovine thyroglobulin conjugate of N-acetyl-5- methoxytryptophan . Two high-affinity and specific antisera were obtained. Unlike in previous studies melatonin was radioiodinated directly. Iodo-Gen was used as the oxidant. Radioiodinated melatonin was purified by TLC for use in RIA. Melatonin was extracted from plasma, serum and urine samples (1 ml) with chloroform. Using the extraction the sensitivity of the RIA method was 18 fmol/ml of original sample. Plasma, serum and urine extracts diluted parallelly with synthetic melatonin in RIA. HPLC analysis of plasma and serum extracts showed only one immunoreactive peak co-eluting with synthetic melatonin. Majority of urine immunoreactivity co-eluted with synthetic melatonin, but 7-23% contaminating immunoreactivity was also observed. Daytime values for rat plasma, human serum and urine melatonin were 30-60, 20-40 and 50-130 fmol/ml and the respective night values were 160-300, 180-370 and 230-470 fmol/ml. Thus a characteristic diurnal rhythm of melatonin was observed in all cases. The urina...Continue Reading

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