Elimination of cross-reactivity by addition of an excess of cross-reactantfor radioimmunoassay of 17 alpha-hydroxypregnenolone

Polyclonal antiserum against 3 beta,17 alpha-dihydroxypregn-5-en-20-one-19- O-(carboxymethyl)-oxime bovine serum albumin (17 alpha-hydroxypregnenolone- 19-CMO:BSA), was raised in rabbits. Its main structural determinants were t he substituents on D-ring as demonstrated by its 107% cross-reaction with 1 7 alpha-hydroxyprogesterone. This unspecificity was almost completely elimi nated by addition of the excess of the cross-reactant directly to the analy tical system. The contribution of the cross-reactant from the sample in suc h a system became negligible due to saturation of the populations of polycl onal antibodies recognizing the analyte as well as the cross-reactant. The possible interference of 17 alpha-hydroxypregnenolone-3-sulfate was avoided by inserting ether extraction. The analytical system appeared to be stable to differences in cross-reactant concentrations even in samples from patie nts with pathologically elevated serum levels of 17 alpha-hydroxyprogestero ne. The radioimmunoassay was compared with the system using the unspecific antiserum alone but after separation of the cross-reactants by HPLC. As dem onstrated by parallel measurement of 125 samples of human plasma from both sexes and various ages either before and/or after adrenocorticotropin stimu lation and 17 samples with elevated basal of human plasma 17 alpha-hydroxyp rogesterone levels, an excellent correlation was achieved between both meth ods. The method, based on a simple addition of the cross-reactant, avoids t he time-consuming chromatographic separation and, in comparison with the ot her approaches for improving the specificity of polyclonal antisera, is eff icient and rapid. Mathematical analysis of the relations in equilibrium dem onstrates that such a simple approach is an efficient way for improvement o f immunoassay specificity using some polyclonal antisera. (C) 1999 Elsevier Science Inc. All rights reserved.