A miniature radioimmunoassay for melatonin for use with small samples from invertebrates

Melatonin has been identified in a number of invertebrates, but little is known of its functions or its circadian control. Studies in this area are hindered by the lack of an assay for melatonin that is appropriate for use with the small sample volumes obtainable from most invertebrates and with the large numbers of sequential samples generated by circadian studies. We describe and validate a novel, miniature radioimmunoassay (RIA) protocol for these purposes. Sample volumes of c.50 microl were extracted with methanol and the extracts dried in 6 x 50 mm glass tubes. The entire assay was conducted in these tubes. The incubation volume was 200 microl. Bound ligand was precipitated and washed in these tubes, then 400 microl scintillation fluid was added and the tubes were mounted inside conventional scintillation vials for counting. Losses of material due to transfer between containers were thus eliminated. Previous RIAs for melatonin employed multiple transfers between containers and their use with small sample volumes can result in loss of substantial proportions of the sample. Two hundred samples could be processed together. The assay sensitivity was 2 pg melatonin and recovery of internal standards was c.100%. Two commercially available antisera gave closely similar results. The assay was used to quantify melatonin titers in the haemolymph of the insect Rhodnius prolixus (Hemiptera); scotophase haemolymph contained 270 pg/ml melatonin, compared with minimal values (c.40 pg/ml) during the photophase, revealing a daily rhythm. This assay opens the door for analyses of the functions and circadian regulation of melatonin in invertebrates.