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

Abstract

The development of sensitive radioimmunoassays (RIA) for testosterone and oestradiol-17 beta, 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 (11 alpha vs 11 beta). 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 X 10(6] 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.

Effects of IGF-I bioavailability on bovine preantral follicular development in vitro

“…Nunc-Immuno Maxisorp 96-well plates were coated with donkey anti-sheep serum by incubating overnight at 4 8C in the presence of donkey anti-sheep serum (250 mg/ml) made up in carbonate buffer pH 9.6 (100 ml/well). The primary antibody was raised in sheep against 17b-estradiol 6-(O-carboxymethy)-oxime: BSA (Webb et al 1985).…”

Direct in vivo effects of leptin on ovarian steroidogenesis in sheep

“…Antibody titres were determined monthly, using 125 I-labelled human leptin as a tracer incubated with serial dilutions of plasma as previously described (Webb et al 1985), with the animals slaughtered after the last immunisation. The serum was collected and titrated and the serum that gave the highest antibody titre (sheep 6E-546: 54% binding at 1:10 000 dilution) was used for the passive immunisation study.…”

Expression of mRNA encoding IGF-I, IGF-II and type 1 IGF receptor in bovine ovarian follicles

“…Oestradiol-17 was also measured by RIA as described by Webb et al (1985). IGF-I was measured by RIA (Guttierrez et al 1997b), after acid gel filtration to remove IGFBPs, using a rabbit polyclonal antibody raised against human recombinant IGF-I (Armstrong et al 1990).…”