Radioimmunoassay of Corticosterone using <SUP>125</SUP>I-Labeled Radio-ligand

Abstract

A radioimmunoassay (RIA) of corticosterone was developed and characterized using 125I labeled radioligand (ICN Biomedicals, U.S.A.) and a single ether extraction procedure. The intra- and interassay coefficients of variation was 7.2% and 14.6% and assay sensitivity was 16.4±3.5 pg per tube. Utilizing this assay, the antiserum for corticosterone (GDN377) made small samples (0.1-1.0μl) use possible. The ease of this technique allows handling of large numbers of samples as compared with RIA using 3H-corticosterone.The specific steps in the procedure of assay were following: 125I-corticosterone was diluted with 0.05M phosphate buffered saline (PBS) containing 1 % bovine serum albumin (BSA). Antiserum (GDN377) was diluted with 0.05M PBS containing 0.05mM ethylenediamine tetraacetic acid, 2Na salt, dihydrate (EDTA) and 0.4% normal rabbit serum (NRS) to 1 : 15000-1 : 20000 dilution. Goat anti-rabbit gamma globulin serum (ARGG) was diluted with 0.05M PBS containing 3.5% polyethylene glycol 6000 (PEG) to 1:150 dilution. Plasma or serum (0.1-1μl) and standard were diluted to 400μl with distilled water in 13 × 100mm culture tubes and extracted once with 2ml of diethyl ether. The samples were allowed to settle for 5 min to ensure separation of ether and aqueous phases. The tubes were then placed in dry ice-ethanol bath to freeze the aqueous phase. The ether phase was then decanted into 12 × 75mm culture tube and dried under reduced pressure with gentle mixing at 50C. One hundred μl of PBS containing 1% BSA, antiserum and labelled ligand (50-100Bq/100μ) were added to each tube and the extract was dissolved by agitation and incubated at 4C for 24 h. Then, 100μl of ARGG was added to each tube. After further incubation for 24 h, the antibody bound hormone was separated from the free hormone by centrifugation at 4C 1700g for 30 min. The supernatant was carefully decanted and the precipitate was counted in automatic gammacounter. Plasma or serum concentrations of corticosterone were calculated using a standard curve.