A Pregnenolone Radioimmunoassay Utilizing a New Fractionation Technique for Sheep Antiserum

Abstract

Antibodies against pregnenolone were generated in a ewe immunized with a pregnenolone 3-bovine serum albumin conjugate. Upon fractionation of the resultant antiserum using QAE-Sephadex chromatography, a 7-8S highly negatively-charged antibody fraction was isolated possessing substantially increased sensitivity towards pregnenolone relative to the crude antiserum. On the basis of this novel fractionation procedure it was possible to devise a radioimmunoassay sensitive to 20 pg of pregnenolone. Normal serum concentrations in ng/ml obtained with this assay were: men (1.05 ± 0.19 se); women, follicular phase, (0.96 ± 0.16 se); women, luteal phase, (1.23 ± .25 se). These mean values were not significantly different from each other. It is speculated that our purified antibody preparation may represent a previously unidentified subclass of IgG in the sheep, and that the chromatographic method described may be generally useful for isolating highly sensitive antibodies against steroids or other hapten-BSA antigens in this animal.