Radioimmunoassays for melatonin using an antiserum raised against a novel antigen.

The highly specific antiserum to melatonin (MT) was produced, and the sensitivity and specificity of the radioimmunoassays (RIAs) using this antiserum was investigated. An immunogen was synthesized by binding 6-hydroxy-melatonin (6-OH-MT) to bovine serum albumin with a spacer of 6-bromohexanoic acid ethyl ester. Rabbits were immunized subcutaneously once a month with 1.0 mg of the immunogen for 7 months. The antiserum was characterized by a crossreactivity test by RIA using 125I-labelled MT. The antiserum specifically recognized MT but hardly did other analogues except for 6-OH-MT with a crossreactivity of only 0.4%. Two RIAs were established using [o-methyl-3H]MT and 2-[125I]iodo MT. In both RIAs, inhibition curves for MT were obtained in the range of 10 pg to 10 ng and 1-2 ng of MT inhibited the value of the assay at "0" by half. The similarity of the RIAs in sensitivity and inhibition by unlabelled MT is discussed on the basis of the structures of radioactive MTs and that of the immunogen.