A Modified Competitive Protein-Binding Determination for Cortisol

A modified competitive protein-binding assay for cortisol using ammonium sulfate precipitation of the rabbit alpha-globulin binding protein to separate free and protein-bound steroid was developed. 90% of the label was precipitated in the absence of added cortisol, and standard curves sensitive in the range of 0–15 ng cortisol were obtained. Steroid specificity was similar to that reported for rabbit plasma. Extraction solvent blanks had no effect on the assay, and cortisol recovery from serum was 99 ± 9%. Corresponding plasma and serum samples had similar cortisol levels (plasma/serum = 1.05), indicating that either can be used to determine circulating cortisol. Analysis of 20 human sera (mean = 15.2 μg/100 ml cortisol) yielded a standard deviation of 0.75 μg/100 ml and a coefficient of variation of 5.0%, indicating better duplicate reproducibility than reported for similar assays using an adsorbent. It was concluded that ammonium sulfate rather than adsorbent separation of free and protein-bound cortisol was easier, faster and more reproducible.