Optimization of a melanotropin-receptor binding assay by reversed-phase high-performance liquid chromatography

We describe here a procedure, which should be generally applicable to a wide variety of polypeptide hormones, for analyzing with reversed-phase high-performance liquid chromatography the stability of radioiodinated ligands. Specifically, the integrity of mono [125I]beta-melanotropin during storage and under various assay conditions has been examined. The hormone was found to be quite stable during storage and when binding assays were conducted at 0-1 degree C. There was, however, a decrease in its stability when assays were conducted at higher temperatures (15, 25 and 37 degrees C), the instability increasing with temperature and being greater when cells were present. Employing heat-inactivated instead of untreated bovine serum albumin in the buffer used in the assay provided only a modest improvement in the stability of [125I]beta-melanotropin.