Radioimmunoassay of human transcortin

A specific, sensitive and simple radioimmunoassay (RIA) system for human transcortin was developed. A highly purified transcortin was prepared from pooled human serum by the following four successive steps; ammonium sulfate fractionation, cortisol-Sepharose column chromatography, Ultragel AcAA column chromatography and hydroxylapatite column chromatography. Anti-transcortin antibody was raised by immunizing rabbits. The RIA employing 125I-labeled transcortin preparation and polyethylene glycol solution for separation of free and bound-form was sensitive to transcortin in concentrations as low as 10 ng/ml. This RIA was reliable in the tests of dilution, reproducibility and recovery. The presence of cortisol does not interfere with the assay. Also a test of cross reactivity revealed that the system was not influenced by human serum albumin in a concentration 10,000 times that of transcortin. The transcortin concentrations determined by the RIA (Y) and those by the conventional steroid-binding assay (X) revealed a good correlation (Y = 1.01 X + 6.25) in normal serum, and the immunoreactivity and steroid binding activity revealed a good correlation in heat and acid-inactivated transcortin. With some total cortisol concentrations given, the transcortin concentrations were inversely correlated with protein-unbound cortisol concentrations. The present assay is useful not only for biochemical research but also for clinical studies, in which the determination of transcortin makes it possible to evaluate the concentration of protein-unbound cortisol which is the physiologically active fraction in serum.