Direct radioimmunoassay of plasma cortisol.
The simplification of the measurement of circulating cortisol by direct radioimmunoassay of plasma samples sets the problem of inhibiting the carrier proteins competing with antibodies. This was accomplished by exploiting the much higher effectiveness of pH and temperature variations on steroid binding to carrier proteins than to antibody sites. A solid-phase system was set up, using antisera to cortisol-21-BSA conjucates coupled to CNBr-activated cellulose. The standardized procedure consisted of an incubation at pH 3.5 and room temperature, directly assaying 10 mul of plasma. A methodological and clinical validation of the measurement was carried out through a series of tests aimed at assessing the reliability of results (assay of steroid-deprived plasma, recovery test and serial dilution of samples, comparison between different antisera and with different methods including extraction, responsiveness to well-established physiological situations). The results obtained are reported and the validity of the method discussed in terms of more general applicability to steroid assay.
Direct radioimmunoassay of plasma cortisol. The simplification of the measurement of circulating cortisol by direct radioimmunoassay of plasma samples sets the problem of inhibiting the carrier proteins competing with antibodies. This was accomplished by exploiting the much higher effectiveness of pH and temperature variations on steroid binding to carrier proteins than to antibody sites. A solid-phase system was set up, using antisera to cortisol-21-BSA conjucates coupled to CNBr-activated cellulose. The standardized procedure consisted of an incubation at pH 3.5 and room temperature, directly assaying 10 mul of plasma. A methodological and clinical validation of the measurement was carried out through a series of tests aimed at assessing the reliability of results (assay of steroid-deprived plasma, recovery test and serial dilution of samples, comparison between different antisera and with different methods including extraction, responsiveness to well-established physiological situations). The results obtained are reported and the validity of the method discussed in terms of more general applicability to steroid assay.