A one-tube micromethod for radioimmunoassay of plasma cortisol.

A micro-scale method is presented for measurement of cortisol. Serum or plasma, 2 microliter, is diluted with buffer and the solution heated at 60 degrees C in the presence of high-affinity antibody and [3H] cortisol. Corticosteroid-binding globulin in the serum is denatured, releasing cortisol and making it available to the antibody, which is stable during the incubation. After cooling, ammonium sulfate solution is added, followed by a toluene-based scintillant that does not dissolve in the aqueous phase. The vial is shaken to extract the free cortisol into the scintillant. Antibody bound cortisol remains in the aqueous phase and does not produce any scintillation. The amount of [3H] cortisol that is free at the end of the incubation, and which therefore gives rise to the measured counts, is related to the concentration of cortisol in the original serum, and may be calculated from a standard curve. The assay is sensitive and precise. Ranges are presented for normal and pathologic subjects.