A Radioimmunoassay for Adrenocorticotropic Hormone (ACTH) in Unextracted Plasma of Various Animals

Abstract

A radioimmunoassay of plasma ACTH, useful for various mammalian species was devel-oped. The RIA for ACTH involves use of commercially available antiserum and 125I labelled hormone, and permits measurement of immunoreactive ACTH in unextracted plasma of various mammals. Physiologi-cal validation of the method was obtained from the following studies. Intact male rats had elevations of immunoreactive plasma concentrations of ACTH after the onset of immobilization stress. A dose related increase in plasma concentrations of ACTH was obtained in adult male rats by the injection of ACTH releasing hormone (CRH).The specific steps in the procedure of assay were following. All steps were performed under cool condi-tion (4C). Basal buffer was 0.063 M Na2HPO4 containing 0.013M ethylenediaminetetraacetic acid 2Na salt (EDTA) and 0.02% NaN3 adjusted to pH 7.4. RIA buffer (basal buffer containing 0.1% Triton X-100 and 250 KIU/ml aprotinin) was made into BSA•RIA buffer (for dilution of standards or samples: containing 1% protease free bovine serum albumin), NRS•RIA buffer (for dilution of antiserum to ACTH: contain-ing 0.4% normal rabbit serum) and PEG•RIA buffer (for dilution of antiserum to rabbit gamma globulin: containing 4% polyethylene glycol). BSA•EDTA buffer (separation buffer) was also prepared by adding 1% BSA to basal buffer.Plasma or serum (1-40 μl) were diluted to 100μl with BSA•RIA buffer. Standards or samples (100μl) were put in the polystylene tubes and 100 μl of antiserum were added to each tubes. After incubation at 4C for 24 h, 100μl of labelled ligand were added and incubated for another 24 h. Then, antiserum to rabbit gamma globulin was added to each tube. After further incubation for 24 h, 1 ml of separation buffer was added to each tube and antibody bound hormone was separated from the free hormone by centrifugation at 4C, 1700 g for 30 min. The supernatant was carefully decanted and the precipitate was counted in auto-matic gamma counter. Plasma or serum concentrations of ACTH were calculated using a standard curve.