Comparison of 3 AT1 Receptor Binding Assays: Filtration Assay, ScreenReady™ Target, and WGA Flashplate®

Hee, R. M. van der; Deurholt, Tanja; Gerhardt, C.C.; Groene, Els M. de

doi:10.1177/1087057104271330

Cited by 8 publications

(2 citation statements)

References 20 publications

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“…Previous reports show that rat brain and rat liver homogenates yield similar binding affinities for σ 1 ligands [13–15] and rat liver has already been established as the preferred tissue for σ 2 binding studies [2]. Receptor expression levels of 2 pmol/mg or greater are required for detection with tritiated ligands and the typical sample sizes of 2–100 μg total protein per well used in 96-well filtration assays [16–18]. Rat liver P 2 contains densities of both subtypes of σ receptors that exceed this requirement [13, 19, 20], making it a suitable receptor source for the proposed assay platform.…”

Section: Introductionmentioning

confidence: 99%

A 96-well filtration method for radioligand binding analysis of σ receptor ligands

Fishback

Rosen

Bhat

et al. 2012

Journal of Pharmaceutical and Biomedical Analysis

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σ Receptors represent a potential drug target for numerous therapeutic indications including cancer, depression, psychostimulant abuse, and stroke. Most published radioligand binding studies for σ receptors utilize a low throughput method employing a “cell harvester.” Higher throughput methods are required to facilitate efficient screening of large numbers of novel compounds. In this study, a series of reference compounds was analyzed with a new medium-throughput 96-well filtration method and the results were compared to those obtained using the conventional cell harvester-based method. The 96-well assay utilized rat liver membranes for the determination of both known σ receptor subtypes (σ1 and σ2) because this tissue contains high densities of both subtypes and fulfills criteria required for reliable use with the 96-well format. The new method gave comparable Ki values for reference ligands analyzed in parallel with samples prepared in rat brain membranes and processed on the traditional cell harvester. For σ1 receptors, equivalent affinity values were observed for both methods/tissues. For σ2 receptors, approximately 2-fold higher affinities were observed for most compounds in liver, as compared to brain membranes, but excellent correlation with brain-derived values was maintained. To further demonstrate the utility of the new method it was used to screen a novel series of 2(3H)-benzothiazolone compounds, resulting in the identification of several analogues with nanomolar affinity and greater than 50-fold specificity for σ1 versus σ2 receptors.

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Section: Introductionmentioning

confidence: 99%

A 96-well filtration method for radioligand binding analysis of σ receptor ligands

Fishback

Rosen

Bhat

et al. 2012

Journal of Pharmaceutical and Biomedical Analysis

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“…WGA, by binding to the N-acetylglucosamine residues on membrane glycoproteins, allows for immobilization of cell membranes or whole cells on the bead surface for receptor binding assays or guanosine 5Ј-O-(3-[ 35 S]thio)triphosphate (GTP␥S) functional assays (discussed later). 74,93 Streptavidin-functionalized beads allow for any biotinylated substance such as peptides or proteins to be captured. [35][36][37][38][39]41,46,55,58 Protein A-or antibody-coupled beads allow for the capture and detection of any substance that can be specifically recognized by an antibody and thus becomes analogous to the radioimmunoassay (RIA).…”

Section: Scintillation Materialsmentioning

confidence: 99%

Scintillation Proximity Assays in High-Throughput Screening

Glickman

Schmid

Ferrand

2008

ASSAY and Drug Development Technologies

102

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Scintillation proximity assays (SPAs) have become a powerful tool for high-throughput screening (HTS) because they can measure the activity and binding of very diverse classes of drug targets. By applying the basic principles of ligand-receptor binding and enzyme kinetics, it is possible to build a large variety of miniaturized, high-throughput assays and screen millions of compounds. SPAs are enabled by the diversity of radiolabeled molecules and affinity tags that are commercially available. These synthetic radiotracers allow for minimal disturbance of the natural binding interactions. This article will present a comprehensive review of the technique and provide detailed information on its applications related to HTS, highlighting the major uses and giving some suggestions for future research.

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