Impairment of G protein—coupled seven-transmembrane (7 TM) receptor function has been implicated in a variety of different pathologic conditions, suggesting that the discovery of specific antagonists may lead to the development of successful therapeutic agents. The effect of different agents on receptor-ligand interaction is often measured directly in a receptor binding assay; however, this assay format can be time consuming and does not detect agents that interact at sites distal to the native ligand binding site. Cyclic adenosine monophospate (cAMP) represents a ubiquitous second messenger generated in response to ligand binding to many 7 TM receptors. The present study describes the practical adaptation of scintillation proximity methodology, using FlashPlate™ (NEN Life Sciences, Boston, MA) technology to evaluate cAMP production. The bioassay is based on competition between endogenously produced cAMP and exogenously added radiolabeled [125I]-cAMP. Cyclic AMP capture is mediated through an anti-cAMP antibody onto a microplate well surface. Removal of unbound radioligand is not necessary because only ligand within ≤20 μm of the plate surface is detected due to the proximity effect. The data indicate that the use of scintillation proximity technology allows accurate and specific evaluation of G protein—coupled receptor function as measured by cAMP production and is suitable for high throughput screening.
This report compares several types of liquid handling equipment presently used in HTS. The devices include 96-well automated pipettors such as the Carl Creative PlateTrac™ (Harbor City, CA), Matrix PlateMate™ (Hudson, NH), Tomtec Quadra-96T? (Hamden, CT) and a Zymark RapidPlate-96™ (Hopkinton, MA) integrated into a full robotic system. A general set of considerations including ease of programming, assay-completion time, accuracy and precision of liquid dispensing, and low-volume pipetting were evaluated. Both a protease screen and a cell-based reporter gene assay were used as model systems for comparison. The data indicate that the Carl Creative PlateTrac has an advantage in several areas. These include the ease in programming, reduction in assay run time, and increased accuracy and precision in liquid dispensing, especially for volumes of 1 μl or less. However, both the Matrix Platemate and Zymark robotic systems may be used to perform complicated multi-step tasks involving multidirectional plate transfer, which is not possible on the current PlateTrac. Advantages and limitations of each piece of equipment are discussed further in this report.
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