A Sensitive Radioimmunoassay for Corticotropin Using a Fully Biologically Active125I-Labeled Ligand*

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Abstract
The human corticotropin (ACTH) analog, Phe2,Nle4-ACTH-(l–38) was iodinated by the chloramine-T procedure and the product was purified by reverse phase high performance liquid chromatography. The specific radioactivity of [125I]Tyr23,Phe2,Nle4-ACTH-(l–38) was determined by comparing the antiserum binding curves of the iodinated peptide and [3H]ACTH of known specific activity. This method gave a value of 1800 ± 75 Ci/mmol, which is close to the theoretical radioactivity expected for the introduction of a single 125I atom into the peptide. [125I]Tyr23,Phe2,Nle4-ACTH-(l–38) was as potent as ACTH in stimulating corticosterone production in isolated rat adrenocortical cells. The concentrations for half-maximal steroidogenesis were 36.5 ±6.1 pM for the 125I derivative and 37.6 ± 6.7 pM for ACTH. By the use of this 125I-labeled ligand, a highly sensitive RIA capable of detecting 1 pg ACTH was developed. The antiserum employed in this study appeared to be directed against residues 11–13 of ACTH.