Affinity chromatography using competitive elution separates polyclonal glucocorticoid antisera into fractions of varying cross-reactivity.
Affinity chromatography of glucocorticoid antisera using cross-reacting steroid-Sepharose columns and competitive elution with the immunising steroid has allowed the separation of polyclonal antibodies into fractions of varying cross-reactivity. Elution was at neutral pH in the presence of 20% acetonitrile followed by dissociation of the eluted immunoglobulin-steroid complex, by dialysis. A polyclonal cortisol antibody with an initial 70% cross-reactivity to 11-deoxycortisol yielded a fraction with 10% cross reactivity and improved affinity. This fraction was suitable for determining plasma cortisol on patients undergoing the metyrapone test whereas another fraction of similar affinity but higher cross reactivity to 11-deoxycortisol, as well as the intact antiserum, grossly over-estimated plasma cortisol on these patients. This technique should permit the use of antibody fractions for immunoassay when the intact antiserum may be unsatisfactory due to lack of specificity.