The measurement of testosterone and oestradiol-17β using iodinated tracers and incorporating an affinity chromatography extraction procedure

The development of sensitive radioimmunoassays (RIA) for testosterone and oestradiol-17β, utilising 125I-radioligands, is described. Use of an homologous bridge at the same site of attachment, for both the radioligand and the steroid-carrier protein conjugate employed in raising antibodies, normally results in a loss of assay sensitivity and precision. This was overcome in the oestradiol assay by utilising an heterologous configuration at the site of attachment (11α vs 11β). In contrast, for testosterone, even though an homologous bridge and site of attachment was used for the radioligand and the steroid-carrier protein conjugate, a very sensitive assay with extremely high antibody titres (dilution of 1:2 × 106) was achieved. This finding was repeated with a different antiserum suggesting that the “bridge binding” phenomenon may be related to the position of attachment to the steroid molecule. In addition, an antibody-Sepharose 4B affinity chromatography extraction procedure has been developed for both oestradiol and testosterone. This approach allows the measurement of very low concentrations of steroids from large volumes of a variety of biological fluids. As antibody-linked Sepharose 4B uses high concentrations of antibody, steroids of similar structure are extracted from biological fluids. However, the cross-reactivity of these related steroids are very low in the RIA's, ensuring good specificity.