A simplified radioimmunoassay for plasma 11-deoxycortisol.
A simple, specific, accurate, precise and sensitive radioimmunoassay procedure developed for plasma 11-deoxycortisol is described. 1. The assay employs an anti-11-deoxycortisol serum generated against 11-deoxycortisol-3-(0-carboxymethyl) oxime coupled to bovine serum albumin, crystalline 11-deoxycortisol as standard, and [3H] 11-deoxycortisol as the radioactive ligand. 2. Cross-reactivity studies performed with structurally related steroids indicated cross reactivities with 17 alpha-hydroxyprogesterone, deoxycorticosterone and progesterone of 2.0%, 1.3% and 0.4% respectively; cortisone, corticosterone, cortisol, testosterone, less than 0.1%; and estrone, 17-beta-estradiol, estriol, and metyrapone less than 0.001%. Due to the high specificity of the anti-11-deoxycortisol serum, the method is simplified by the lack of need for chromatographic purification of the organic solvent extract of the plasma prior to the radioimmunoassay. The procedure was validated by comparing values for plasma 11-deoxycortisol with and without preliminary purification by chromatography on Sephadex LH-20 columns (y = 0.99 R/-x + 4.0, r = .98). Pretreatment of the plasma with n-hexane was found to eliminate interferences from high concentrations of 17 alpha-hydroxyprogesterone or progesterone. 3. Parallel dose-response curves were demonstrated between dilutions of plasma with elevated 11-deoxycortisol concentrations and the standard reference preparation. A non-specific binding less than 4% of the total [3H] 11-deoxycortisol was routinely observed. The detection limit of the assay was approximately 10 pg of 11-deoxycortisol which corresponds to a plasma concentration of approximately 0.7 micrograms/L 4. The analytical recovery of 11-deoxycortisol added to human plasma varied from 88 to 108%, with a mean recovery of 100%. The inter-assay variation was determined by assaying (n = 30) three different quality control pools. The following data were obtained: x 1 = 3.8 +/- 0.6 micrograms/L (CV = 15.8%); x 2 = 18.5 +/- 2.0 micrograms/L (CV = 10.8%); x 3 = 43.0 +/- 3.7 micrograms/dl (CV = 8.6%).
D015065:17-Hydroxycorticosteroids / Q000097:blood*; D000918:Antibody Specificity; D002852:Chromatography, Ion Exchange; D003350:Cortodoxone / Q000097:blood* / Q000276:immunology; D006801:Humans; D011863:Radioimmunoassay / Q000379:methods* / Q000592:standards; D012016:Reference Values
D015065:17-Hydroxycorticosteroids; D003350:Cortodoxone