A simple method for separating unbound and bound cortisol in a radioimmunoassay.
A cortisol radioimmunoassay in which unbound cortisol is partitioned into the organic phase of a toluene: water scintillation fluid mix at 0 to 5 degrees C is described. Antibody-bound cortisol remained in the aqueous phase. Since liquid scintillation spectrometers detect photons generated from the [3H]cortisol only in the organic phase, the system effectively separates antibody bound from unbound [3H]cortisol. Regression coefficients including linear, quadratic, and cubic components of standard curves were between 0.980 and 0.999. Cross-reactivity was 3% or less with 11 other steroids and cholesterol except for cortisone (16%) and prednisone (12%). Intra- and interassay coefficients of variation were 8 and 13%, respectively. The lower limit of sensitivity of the assay was 1.4 ng/ml. Recoveries of added mass averaged 97.5%. The correlation between concentrations of glucocorticoids assayed by competitive binding to dog plasma and the current procedure was 0.90. The assay procedure described simplifies separation of unbound from antibody-bound cortisol.
D000818:Animals; D000915:Antibody Affinity; D001667:Binding, Competitive; D006854:Hydrocortisone / Q000302:isolation & purification*; D011817:Rabbits; D011863:Radioimmunoassay; D012997:Solvents; D013696:Temperature
D012997:Solvents; D006854:Hydrocortisone