LANCE cAMP Cell-based Assay is Ideal for Automating HTS of GPCRs
Use of ESC’s and Primary Cells Increasing in GPCR and Kinase HTS
LANCE® cAMP Cell-based Assay is Ideal for Automating High Throughput Screens of G-Protein Coupled Receptors for Greater Precision and Productivity
- A homogenous, easy-to-use assay that uses TR-FRET technology for cAMP detection
- High sensitivity, low background, and high signal-to-noise ratios
- Flexible sample formats—work with whole cells or membranes
- Ultra HTS-compatible—simple conversion between 96-, 384-, or 1536-well plate formats results in dramatic savings
- High precision—Z' = 0.8 for G?i cell assay, 0.8 for G?s cell assay
- Easy to automate for greater productivity—simple one-step protocol (following cell stimulation)
- Read on any TRF-compatible instrument, such as PerkinElmer’s ViewLux®, EnVision® and VICTOR™ readers
G-protein coupled receptors (GPCRs) represent the largest family of integral membrane-bound receptors in the human genome. These receptors are the most important class of pharmaceutical targets, mainly due to their sheer diversity as signaling proteins and involvement in many disease states. Stimulation of GPCRs by agonists triggers a cascade of events resulting in the activation of adenylate cyclase, which in turn catalyzes the synthesis of cyclic adenosine monophosphate (cAMP) in the presence of ATP. Levels of intracellular cAMP are often measured to assess the effect of test compounds on GPCR-mediated adenylate cyclase activation or inhibition.
A myriad of assays that measure levels of cAMP are commercially available. Most of these assays are based on the competition between a labeled-derivative of cAMP and endogenous cAMP for binding to specific anti-cAMP antibodies.
Principle of LANCE cAMP Assay
PerkinElmer’s LANCE cAMP assay is a homogeneous time-resolved fluorescence resonance energy transfer (TR-FRET) immunoassay, which exhibits low background and high signal-to-noise ratios, two attributes critical for robust high throughput screening (HTS) assays. The assay is designed to measure cAMP produced upon modulation of adenylyl cyclase activity by GPCR and allows measurement of both agonist and antagonist activity on G?s- and G?i-coupled receptors in whole cells and G?s-coupled receptors in membrane preparations. LANCE cAMP assay is based on the competition between cAMP produced by cells and exogenously added, europium (Eu)-labeled cAMP tracer complex for binding sites on cAMP-specific antibodies labeled with Alexa Fluor® 647 dye. The Eu-labeled cAMP tracer complex is formed by the tight interaction between biotinylated cAMP (b-cAMP) and streptavidin-labeled europium chelate (Eu-SA). As shown on Figure 1, the principle of the LANCE cAMP assay involves the loss of energy transfer as the quantity of cell-derived cAMP increases, thereby decreasing the amount of biotinylated cAMP available to bind to the anti-cAMP antibody.
Fig. 1. Principle of the LANCE cAMP Assay.
Automation of the LANCE cAMP Assay for HTS
The LANCE cAMP assay is simple to use and requires only 90 minutes for completion. The assay is ideal for high throughput screens and is easily adaptable for use in 96-, 384- and 1536-well OptiPlate™ microplates, with comparable results in all formats.
Fig. 2. Comparison of the LANCE cAMP assay workflow (A) with that of Competitor Y that uses luminescence for detection (B). Results were measured on a ViewLux.
Sensitivity of the LANCE cAMP Assay
The LANCE cAMP assay is sensitive and highly reproducible for detection of cAMP produced upon modulation of adenylyl cyclase activity by GPCRs. Sensitivity of the assay was determined by incubating stimulation buffer containing Alexa 647-labeled anti-cAMP antibody with a series of cAMP dilutions for 30 minutes at room temperature. Detection buffer containing biotinylated cAMP (b-cAMP) and streptavidin-labeled with europium chelate was then added and incubated for 60 minutes at room temperature. cAMP standard curves were run in 384- and 1536-well microplates. Assay results were read using the EnVision multilabel plate reader and ViewLux CCD imager uHTS plate reader.
LANCE cAMP assay showed sensitivity with IC50 values in the low nM range, demonstrating lower background and higher sensitivity in both 384- and 1536-well formats (Figures 3A and 3B).
Fig. 3. LANCE cAMP standard curve generated in 384-well (A) and 1536-well (B) microplates on a ViewLux.
LANCE cAMP Assays for Measuring G?i-Coupled Receptor Activation in Whole Cells
G?i-coupled receptor activation is commonly assayed by measuring the reduction of cAMP levels resulting from the inhibition of forskolin -induced stimulation of adenylate cyclase. This approach is very indirect and often suffers from a very narrow signal window, making it unsuitable for high throughput screening. The performance of the LANCE cAMP assays for G?i-coupled receptor analysis was evaluated by running stimulation assays in 384-and 1536-well microplates. The sensitivity of each assay platform was determined using a human CHO 5-HT1A serotonin receptor cell line that over-expresses G?i-coupled receptors. Cells were stimulated with either increasing concentrations of forskolin or a combination of forskolin and increasing concentrations of the agonist 8-OH DPAT. Cells were lysed after stimulation and the extracts were analyzed.
In both 384- and 1536-well formats, the agonist 8-OH DPAT reversed the forskolin -induced cAMP production. The assay showed a similar sensitivity in both multi-well formats (Figure 4A and 4B).
Fig. 4. CHO-5HT1A cells were stimulated for 30 minutes with either increasing concentrations of forskolin (blue curve) or a combination of 1 µM forskolin and increasing concentrations of agonist 8-OH DPAT (red curve) in 384-well (A) and 1596-well (B) formats. Results were measured on a ViewLux.
Precision and reproducibility the LANCE cAMP assay was assessed by performing Z' analysis on cells treated with various combinations of forskolin and 8-OH DPAT in 24 replicate assays. The assay was capable of delivering robust and reproducible performance, as demonstrated by maximum Z’ values in all 24 replicates performed in 384- and 1596-well formats (Figures 5A and 5B).
Fig. 5. The LANCE cAMP assay delivers robust and reproducible performance in 24 separate 384-well (A) and 1596-well (B) plates as automated on a JANUS Automated Work Station with detection on the ViewLux Imager.
Summary
LANCE cAMP assay is designed for direct measurement of receptor-mediated adenylate cyclase activation/inhibition in G-protein coupled receptors. The assay yields rapid and reproducible data with whole cells as well as membrane preparations (data not shown). The robust performance of the LANCE cAMP assay is ideal for high throughput screening of cAMP, and increases productivity and efficiency during characterization of both agonist and antagonist drug leads. This assay has high sensitivity and high precision in both 384- and 1536-well formats with comparable results in all formats and is easy to fully automate using the JANUS Automated Workstation and ViewLux Screening Platform.
Look for the next LANCE cAMP article in the Fall GPCR News!