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A direct urinary ELISA for estrone-3-glucuronide has been produced following cloning and characterisation of a monoclonal antibody to the above estrogen metabolite. The ELISA follows our established pattern of absorbing a thyroglobulin conjugate, to which estrone-3-glucuronide has been coupled, to the wells of a microtitre plate using guanidine hydrochloride. A competition reaction between either standards/samples and the adsorbed hormone compete for antibody combining sites. The assay is completed by addition of an anti-mouse Ig-peroxidase complex and read at 492 nm following additions of O-phenylenediamine substrate in under 4 h. The correlation between urinary "total estradiol" and "total estrone and estradiol" is very good and, in conjunction with our ELISA for pregnanediol glucuronide, has allowed for the improved clinical management of infertile and subfertile women.

ELISA technology using immobilized steroid conjugates has allowed the rapid screening and characterization of hybridoma supernatants. Although cortisol-3CMO-BSA was the immunogen, a monoclonal antibody with exceptionally high cross-reactivity (greater than 1000%) to prednisone was obtained. Characterization of the antigenic determinants shows a requirement for overall glucocorticoid 21-hydroxyl and 17 alpha-hydroxyl groups with additional high specificity for the 1,2-double bond and 11-position. There is potential use for this antibody in the assay of prednisone.

A direct ELISA for plasma cortisol is described which is carried out in a standard 96 well microtitre plate. In this ELISA cortisol-thyroglobulin conjugate is immobilised to the microtitre plate and competes with cortisol in the standard or plasma sample for antibody binding sites. Following washing the rabbit cortisol antibody bound to immobilised cortisol is incubated with peroxidase labelled goat antirabbit IgG. Following further washing o-phenylenediamine is added, colour developed, and the plate read at 492 nm on a standard ELISA plate reader. This ELISA shows good agreement with RIA and its sensitivity, specificity and precision allow its use in the routine steroid laboratory.

A mouse was immunised with an antigen(s) purified by oestradiol-Sepharose affinity chromatography of pooled oestrogen-receptor positive cytosols from human breast cancer tissue. One antibody secreting clone was identified which precipitated labelled antigen and which also stained MCF-7 cells. Culture supernatant and ascites fluid were used for immunofluorescence, SDS-PAGE-Western blotting, photoaffinity labelling and binding studies. The antibody staining of MCF-7 cells was inhibited by preincubation in oestrogen-receptor positive cytosol but was unaffected by oestrogen-receptor negative cytosol. MCF-7 cells stained whether cultured in the presence or absence of oestradiol. The oestrogen-receptor negative cell lines MDA-MB-231 and MDA-MB-330 did not stain. Binding studies with 16-alpha-iodooestradiol using breast cancer tissue cytosols followed by immunoprecipitation showed activity only with oestrogen-receptor positive cytosols with optimal binding activity at 4 degrees C, unaffected by molybdate, but reduced at 25 degrees C or in the presence of 0.4 M KCl. Binding studies with MCF-7, MDA-MB-231 and MDA-MB-330 cytosols and nuclear fractions only showed activity with the MCF-7 cytosol and MCF-7 particulate fractions. The antibody recognised a 48 K species in both MCF-7 cytosol and nuclear fractions but not in the cytosol and nuclear extracts of oestrogen-receptor negative cell lines. Photoaffinity labelling using 16 alpha-iodooestradiol suggests the 48 K antigen does not bind oestradiol directly. The relationship of this antigen to the classical oestrogen-receptor and receptor complex awaits further clarification.

The use of affinity chromatography in the presence of 20% acetonitrile combined with a decreasing pH gradient has allowed the fractionation of two cortisol antisera into components of varying affinity. The high affinity fractions generate considerably improved ELISA standard curves compared to the intact sera but do not grossly alter the specificity of the antisera. Accordingly, the high affinity fraction of the least cross-reactive antiserum was used for a plasma cortisol ELISA. The cortisol ELISA, although returning slightly higher values than RIA, was comparable in the ability to distinguish dexamethasone suppression and cortisol response to synacthen and should thus prove of value in the assay of plasma cortisol.

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.

A rapid, single extraction ELISA for testosterone in plasma is described, using a standard 96 well microtitre plate. Testosterone is covalently bonded to bovine thyroglobulin and passively adsorbed in guanidine hydrochloride to the ELISA plate, giving an immobilised antigen approach which simplifies subsequent assay standardisation for steroid hormone assays. The addition of standard, sample and first antibody (rabbit anti-testosterone), which is unique for each different assay, is followed by a general procedure which includes washing, addition of peroxidase labelled goat antirabbit IgG, further washing and finally, addition of o-phenylenediamine substrate with colour development and reading of the plate at 492 nm on an automatic ELISA processor. The ELISA assay is compared to a testosterone RIA with 125I-label and has similar specificity and precision to the latter with a quicker processing time, and is more cost effective. The added advantages that ELISA assays confer over RIAs in terms of isotope purchase and disposal make this an ideal procedure for use in a routine steroid laboratory.