Radiolabeling of melatonin using different oxidizing agents for immunoassay purpose

Abstract

An efficient method was developed for radioiodination of melatonin with ¹²⁵I. Chloramine-T and iodogen were used as oxidizing agents. The radioiodination was carried out via electrophilic substitution of the H atom with the I⁺ cation. Indirect iodination using Bolton–Hunter reagent was also performed. The influence exerted on the radiochemical yield by the melatonin concentration, pH of the reaction mixture, type of oxidizing agent, and reaction time was studied. The shelf life and specific activity of the product were determined. The radiochemical yield and product purity were determined by HPLC and paper electrophoresis. The immunoreactivity of the tracer was checked using its specific antibody. Specific and sensitive polyclonal antimelatonin was prepared using two New Zealand rabbits, which were injected with 5-methoxy-N-acetyltryptamine–bovine serum albumin conjugate as immunogen. The tracer prepared by the indirect method with iodogen had high specific activity, high yield, and long shelf life; it was also more selective in the melatonin assessment using radioimmunoassay technique.