PubMed Health⌕ Search
Home/Archive/PubMed

PubMed · 6719116

Estrogen receptor analysis by flow cytometry.

N T Van·M Raber·G H Barrows·B Barlogie
View original source ↗Export metadata ↓

Abstract

A fluorescently labeled estradiol, N'-fluoresceino-N'-(17 beta-estradiol hemisuccinamide) thiourea (FE) was used for measuring estrogen receptor content per cell in tumor cells. The cellular content of FE was measured quantitatively by flow cytometry. Binding of FE occurs in the nanomolar concentration range, an indication of the high affinity of the labeled estradiol. Competition of FE for binding sites is observed with estrogens, but not with progestins, androgens, or glucocorticosteroids, indicating the specificity of FE binding. In contrast to other estrogen receptor assays, this new technique requires a small sample size (about 5000 cells) and permits the assessment of heterogeneity in estrogen receptor expression among tumor cells.

Explore related subjects

Binding, CompetitiveBreast NeoplasmsCell LineEvaluation Studies as TopicFemaleFlow CytometryHumansReceptors, EstrogenTemperatureTime FactorsJournal ArticleResearch Support, Non-U.S. Gov'tResearch Support, U.S. Gov't, P.H.S.

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

N T Van, M Raber, G H Barrows, B Barlogie. 1984-05-25. Estrogen receptor analysis by flow cytometry.. https://doi.org/10.1126/science.6719116

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related citations

PubMed2007

Co-immobilization of different enzyme activities to non-woven polyester surfaces.

Co-immobilization was applied to combine complementary enzyme reactions. Therefore, trypsin was co-immobilized together with both, lipase and alpha-amylase, onto the surface of non-woven polyester material. The progress of the immobilization reaction was directly monitored by investigating covalent fixation of the enzymes to the polyester flees using (1)H-MAS-NMR. Co-immobilization of the different types of enzymes to the polyester support showed retained enzymatic activity. However, a competition of binding to the support was observed. Increasing amounts of one type of enzyme reduced the degree of immobilization for the other type. In order to investigate the distribution of trypsin and alpha-amylase on the polyester support, the flees was treated with a mixture of rhodamine isothiocyanate labeled with anti-trypsin antibodies and fluorescein isothiocyanate labeled with anti-alpha-amylase antibodies. Using fluorescence microscopy, the co-immobilization was analyzed by selective excitation of both chromophores at 480 and 530 nm, respectively. In addition, fluorescence spectroscopy was applied by direct labeling of trypsin and lipase prior to co-immobilization to the polyester support. A special prism of plexiglass was constructed, which fit into a 10 x 10 mm fluorescence cuvette in that way that a diagonal plane was formed within the cuvette. The non-woven support was fixed in the cuvette and fluorescence spectra were obtained to characterize the amount of different enzymes linked to the support. Using FRET it was demonstrated that a uniform distribution of the various enzyme species was achieved, where the different enzyme activities are bound on the support in close neighborhood to one another.

Binding, Competitive↗
PubMed2007

Scintillation proximity assay as a high-throughput method to identify slowly dissociating nonpeptide ligand binding to the GnRH receptor.

Many nonpeptide antagonists of the gonadotropin-releasing hormone (GnRH) receptor, as well as other drug targets, possess a broad range of dissociation kinetic rate constants. Current methods to accurately define kinetic rate parameters such as K(on) and K(off) are time and labor intensive, prompting the development of a screening assay to identify slowly dissociating compounds for follow-up rate constant determination. The authors measured inhibition binding constants (K(i)) for GnRH receptor antagonists after 30 min and 10 h of incubation and observed several compounds with markedly decreased K(i) values over time (Ki(30 min)/Ki(10 h) > 6). They used scintillation proximity assay technology to perform these binding experiments because this homogeneous assay does not have a fixed termination end point as does filtration binding, permitting successive readings to be taken from the same assay plate over an extended period of time. They also used a quantitative method of kinetic rate analysis to confirm that a large disparity between a compound's K(i) value at 30 min and 10 h could identify compounds that dissociate slowly. Thus, the K(i) ratio can be used to screen for and select compounds to test using more quantitative, albeit lower throughput methods to accurately define kinetic rate constants.

Binding, Competitive↗
PubMed2007

The anomalous behaviour of exogenous 25-hydroxyvitamin D in competitive binding assays.

The Vitamin D International External Quality Assessment Scheme (DEQAS) was established in 1989 to monitor the performance of assays for 25-hydroxyvitamin D (25-OHD) and 1,25-dihydroxyvitamin D (I,25(OH)(2)D). This is achieved through the quarterly distribution of five samples of human serum. Results are used to calculate an All-Laboratory Trimmed Mean and a Method Mean for each of the methods used by participants. In July 2005, participants were asked to assay serum to which 50.9 nmol of either 25-OHD(3) or 25-OHD(2) had been added as ethanolic solutions. The final concentration of ethanol in the serum was 0.7%. The distribution also included a sample of the original serum (OS) containing 0.7% pure ethanol. The percentage recoveries of exogenous 25-OHD(3) (R1) and 25-OHD(2) (R2) were calculated for each method. Results (OS nM, R1 and R2) were as follows: DiaSorin RIA (n=53); 39.2, 82.1%, 83.3%, DiaSorin Liason (n=16); 36.8, 81.4%, 88.6%, IDS RIA (n=21); 36.4, 54.2%, 29.1%, IDS OCTEIA (n=16); 47.3, 78.8%, 56.4%, Nichols Advantage (n=21); 58.9, 46.4%, 43.2%, HPLC (n=9); 42.6, 112.2%, 97.1%, LC-MS (n=4); 34.0, 111.5%, 118.1%. The IDS RIA and Nichols assays gave unexpectedly low recoveries. This does not appear to be a calibration problem or the effect of ethanol.

Binding, Competitive↗