Comparison of separation techniques in radioimmunoassays for 17-hydroxyprogesterone

Separation techniques have been studied in the development of a direct radioimmunoassay to determine levels of 17-hydroxyprogesterone in serum. The same highly specific sheep antiserum was used throughout, together with the same amount of 125I-labelled 17-hydroxyprogesterone to which was added sodium salicylate to eliminate interference by endogenous binding proteins in serum samples. In one approach, dextran-coated charcoal was employed to adsorb the free fraction and, in another, the antibodies were covalently coupled to magnetisable particles. The antiserum was also adsorbed to assay tubes either directly or indirectly through second (double) antibodies. Analytical recovery and specificity were similar irrespective of the separation technique as was the correlation with results obtained by a reference assay. Levels of 17-hydroxyprogesterone in sera from normal adults and from treated and untreated patients with congenital adrenal hyperplasia due to 21-hydroxylase deficiency were also similar. However, the assay employing dextran-coated charcoal for separation showed the best precision and resulted in the greatest sensitivity, while the use of antibodies adsorbed indirectly to assay tubes was superior in terms of practicability.