Quantitative immunological detection of estrogen receptors in nuclear pellets from human breast cancer biopsies.

A rapid, convenient method for the quantitative determination of estrogen receptors (ER) under high salt (0.6 M KCl) conditions (such as in extracts of nuclear pellets from human breast cancer biopsies) using a commercially available kit [estrogen receptor enzyme immunoassay (ER-EIA) Monoclonal; Abbott] is described. This assay has been validated using breast tumor cytosol ER preparations. It determines total (both occupied and unoccupied) ER, it is insensitive to KCl at concentrations up to 0.8 M, and it can be used with ER preparations having very low protein concentrations. Results obtained using the ER-EIA method for breast tumor nuclear extracts have been compared to those obtained using the hydroxylapatite method, and higher values have been found using the ER-EIA method. The reasons for this discrepancy may be due to: the sensitivity of ER binding to hydroxylapatite in high concentrations of KCl; the temperature dependent degradation of the receptor complex at 30 degrees C, the temperature commonly used to achieve exchange between radioactive estradiol and the endogenously bound estradiol; and possible detection of immunoreactive, but non-ligand-binding forms of ER. The possibility that occurrence of "free" receptors in high salt extracts from nuclear pellets may be an artifact is discussed. The availability of this ER-EIA suitable for nuclear ER determinations opens the possibility of extending correlations between the clinical course of breast cancer and the levels of the ER form (nuclear) that are thought to be of greatest physiological significance.

Abbott monoclonal enzyme immunoassay measurement of estrogen receptors in human breast cancer: a European multicenter study.

A new enzyme immunoassay (Abbott ER-EIA Monoclonal) for the determination of estrogen receptor in cytosols from breast tumor specimens has been developed by Abbott Laboratories. To establish the correlation of the results from this new technique with currently existing steroid binding methods, a multicenter study was conducted in eight European laboratories. All participants followed the same protocol consisting of a familiarization phase, a proficiency evaluation, and a comparison of existing steroid binding methods with the new immunoassay using panel samples and clinical specimens. ER-EIA was compared with the multipoint dextran coated charcoal assay in six laboratories, four of which followed the EORTC protocol; of the remaining two laboratories, one used a single saturating dose assay, the other an isoelectric focusing assay. The results show no significant difference between reducing agents when used in the ER-EIA. Reproducibility for the immunoassay (interassay coefficient of variation, 6%, interlaboratory coefficient of variation, 11-19%) was somewhat better than that for the steroid binding methods (interlaboratory coefficient of variation, 12-32%). The correlation between the methods was dependent on the origin of the lyophilized specimens. In breast tumor samples, an excellent correlation, (not statistically different from 1) was found between the ER-EIA and the steroid binding method in six laboratories. One laboratory showed a slope of 1.1 for the correlation line; the laboratory using isoelectric focusing showed a slope of 1.9. The mean value determined by the enzyme immunoassay in premenopausal women was 74 fmol/mg cytosol protein, and in postmenopausal women it was 187 fmol/mg cytosol protein with no significant difference in the slope of the correlation line. Results suggest the usefulness of the new standardized enzyme immunoassay for routine use in the clinical laboratory.

[Epidemiology of FeLV and FIV infection in the Federal Republic of Germany]. / Epidemiologie der FeLV- und FIV-Infektion in der Bundesrepublik Deutschland.

In a nationwide study 6101 cats were tested for presence of feline leukemia virus (FeLV) antigen and for feline immunodeficiency virus (FIV) antibodies utilizing enzyme linked immunosorbent assay (ELISA). 21.8% of the German cat population may be FeLV- and/or FIV-positive. Of the animals tested, 13.4% were found to be FeLV carriers, while 8.4% showed evidence of FIV infection. Infection with both viruses was identified in 2.1% of the cats tested. Of the animals showing clinical symptoms, nearly one cat in three was found to be carrying either one or both of the viruses. Male cats were more likely to be infected then were females, similarly as were free-roaming cats, compared with confined cats; domestic cats, compared with purebred cats; and cats > 6 years old, compared with younger cats.

Use of high-performance liquid chromatography in the evaluation of the synthesis and binding of fluorescein linked steroids to estrogen receptors.

A fluorescein-linked estrogen was synthesized as a non-invasive, non-radiochemical means of detecting the levels and distribution of estrogen receptors in histological preparations of breast and endometrium. 17 alpha-Ethynylestradiol-21-carboxylic acid was coupled via octane-1,8-diamine to fluorescein-isothiocyanate yielding a promising ligand, N-fluoresceinyl-5,N"-[8-(3,17 beta-dihydroxy-19-nor-17 alpha-pregna-1,3,5 (10)-triene-20-yne-21-carboxylic acid amide)]octylthiourea (F8DE) for an estrogen receptor. High-performance liquid chromatography on preparative reversed-phase C18 columns was used to purify the final product. Using cytosolic receptor preparations from bovine uterus and human uterus and breast cancer, the binding of F8DE was determined by competition analyses to have a Kd value of 10(-8) M. High- and low-molecular-weight forms of estrogen receptors were separated on TSK 3000SW and 4000SW columns by high-performance size-exclusion chromatography. Specific binding of radio labeled estradiol-17 beta to these forms was inhibited in the presence of F8DE, indicating association with the fluorescein-linked steroid.