Cortisol directly determined in serum by fluoroimmunoassay with magnetizable solid phase

We developed a direct fluoroimmunoassay for cortisol in serum. This method involves cortisol labeled at the 3 position with fluorescein, and antibodies to cortisol coupled to magnetizable cellulose/iron oxide particles. Sodium salicylate is used as a blocking agent to prevent interference from endogenous binding proteins in serum. Serum sample and labeled cortisol are incubated with the antibody-coupled solid phase for 30 min. The solid phase is then separated and washed to remove free labeled cortisol and endogenous fluorophores of the sample. Finally we elute the antibody-bound fraction of the labeled cortisol into an alkaline methanolic medium and measure its fluorescence. The separation, wash, and elution steps are facilitated by magnetic sedimentation. The assay is sufficiently sensitive, specific, and reliable for routine use and correlates acceptably (r = 0.92) with an established radioimmunoassay. Precision (CV) ranged from 4 to 10% in experiments on three pooled sera; analytical recovery for sera supplemented with as much as 360 microgram of cortisol per liter was 91 to 109%.