New and Highly Sensitive AlphaScreen camp Assay to Measure Femtomol camp Variations in Cell and Membrane Based Assays

Transcription

1 New and Highly Sensitive AlphaScreen camp Assay to Measure Femtomol camp Variations in Cell and Membrane Based Assays 1 Illy, C., 1 Boissonneault, M., 1 Bouchard, N., 2 Dimeo, J., 1 Hamann, G., 1 Legault, M., 1 Lian, J., 1 Pinard, G., 1 Wong, S.H., 1 Banks, P. and 1 Bossé, R. 1 PerkinElmer BioSignal Inc., 1744 William, Montreal, Quebec, Canada, H3J 1R4 2, 549 Albany Street, Boston, MA02118, USA chantal.illy@perkinelmer.com

2 1 Abstract Using a new, higher purity biotinylated camp derivative, we have developed a highly sensitive camp assay to measure the functional activity of G protein coupled receptors using the andenylate cyclase pathway. This new assay provides good sensitivity to camp allowing one to use as little as 1,000 cells per well. In addition, the response to camp provides large S/B ratios that enables the screening for both Gα i and Gα s coupled receptor antagonists with Z values above 0.5. In this contribution, we present and discuss the results obtained from screening validation experiments performed with 5-HT1a receptors expressed in CHO cells and MC4 receptors expressed in 293 cells. 2 AlphaScreen camp Assay The AlphaScreen Assay Kit (cat. # ) contains biotinylated camp probe, anti-camp-acceptor beads, streptavidin-dor beads, camp standard, 10X control buffer and 3% Tween-20 solution. 2

3 3 Features of New AlphaScreen camp Assay Kit (Cat. # ) 1. New biotinylated camp probe has labeling site matched to camp immugen labeling site 2. Higher purity of biotin-camp probe (>95%) 3. Improved kit stability 4. Detailed instructions (User Manual) to facilitate assay development and assay troubleshooting Three sizes of kit available: 1,000 assay points (cat. # D); 10,000 assay points (cat. # M) & 50,000 assay points (cat. # R) Also available: AlphaScreen camp-biotin probe supplement: 10,000 Units (cat. # M) & 50,000 Units (cat. # R) 4 Assay Protocols All assays were performed in 384-well OptiPlate microplates (PerkinElmer cat. # ) in a final volume of 25 µl and the plates read on an AlphaQuest-HTS microplate analyzer. Stimulation buffer for cells: HBSS containing 5 mm HEPES ph 7.4, 0.1% BSA and 0.5 mm IBMX Stimulation buffer for membranes: 50 mm Tris-HCl ph 7.4, 150 mm NaCl, 10 mm MgCl 2, 1.5 mm CaCl 2, 100 µm ATP, 1 µm GDP and 1 nm GTP Detection buffer: 5 mm HEPES, ph 7.4, 0.1% BSA, 0.3% Tween-20 Standard curves Add 5 µl Anti-cAMP Acceptor beads Add 5 µl camp dilutions Incubate in the dark at 23ºC for 30 min Add 15 µl biotinylated camp/streptavidin-dor beads detection mix Incubate in the dark at 23ºC for appropriate time (1 hour minimum) Cell- and membrane-based assays Add 5 µl cells/anti-camp Acceptor beads or 5 µl membranes/anti-camp Acceptor beads Add 2.5 µl antagonist or buffer (incubate for 30 min at 23ºC for membrane assays) Add 2.5 µl forskolin and/or agonist Incubate in the dark at 23ºC for 30 min Add 15 µl biotinylated camp/streptavidin-dor beads detection mix Incubate in the dark at 23ºC for appropriate time (1 hour minimum) 3

4 5 camp Standard Curves For 1 hour and O/N camp Assays Greater assay windows can be achieved using O/N incubation although with some loss in sensitivity Reduce probe concentration to Unit to achieve loss in sensitivity in O/N incubation camp LOD: 2.5 fmol 6 Forskolin Dose-Response Curves / Cell Titration The high sensitivity of the kit allows use of as little as 1,000 cells/well 4

5 7 Z Determination for Gα i -coupled Receptors Gα i -coupled serotoninergic 5-HT1a receptors expressed in CHO-K1 cells Assay conditions: biotinylated camp probe, Dor-SA and Anti-cAMP-Acceptor beads; 4 hour incubation after last addition step Z = 0.6 for antagonist screen 8 Z Determination for Gα s -coupled Receptors Gα s -coupled MC4 receptor expressed in HEK-293 cells Assay conditions: 3,000 cells/well, biotinylated camp probe, Dor-SA and Anti-cAMP-Acceptor beads; 4 hour incubation after last addition step Z = 0.8 for antagonist screen 5

6 9 Membrane-based Assays for Gαs-coupled receptors HEK-293-hMC4 membranes: SignalScreen product (cat. # ) definition for radioactive assay is 2.8 µg membranes Can use as little as 0.1 µg membranes in AlphaScreen camp assay Attests to high sensitivity of AlphaScreen camp assay 10 Membrane-based Assay Pharmacology HEK293-hMC4 membranes Appropriate pharmacology demonstrated for agonists NDP-a-MSH, MTII and SHU9119 antagonist 6

7 11 Cost Reduction Possibilities: Bead Amount/Well Reduction 1 hour incubation after last addition step camp-biotin Beads Dilution* Max Signal (Cps) IC (nm) /well 2 Units/well 5 Units/well New camp kit (cat. # ) hour incubation after last addition step camp-biotin Beads Dilution* Max Signal (Cps) IC (nm) /well 2 Units/well 5 Units/well * Dilution factor relative to standard bead concentrations ( per well for each bead) Former camp kit (cat. # ) 1 hour incubation after last addition step camp-biotin Beads Dilution* Max Signal (Cps) IC (nm) 10 nm/well 0.35 µg Acceptor; 0.5 µg Dor 0.35 µg Acceptor; 0.5 µg Dor hour incubation after last addition step camp-biotin Beads Dilution* Max Signal (Cps) IC (nm) 10 nm/well 0.35 µg Acceptor; 0.5 µg Dor 0.35 µg Acceptor; 0.5 µg Dor * Dilution factor relative to standard bead concentrations (20 ug/ml Dor & 15 ug/ml Acceptor) 2X reduction in beads (0.5U/well) with - 5U probe and 1 hr incubation: SAME ASSAY WINDOW AS WITH 1U BEADS - 1U probe and 4 hr incubation: SOME LOSS OF SENSITIVITY - USE MORE CELLS Advantage over former AlphaScreen camp kit 12 Conclusions High sensitivity fmol detection limit for camp 1,000 cells/well capability 0.1 µg membranes/well capability Excellent performance Z = 0.6 for Gα i cell assay Z = 0.8 for Gα s cell assay Appropriate pharmacology for GPCRs exp. both in cells and membranes Flexibility Wide range of incubation time possible (1-17 hrs) Work with whole cells or membranes Cost-reduction possibility without need for further miniaturization 7

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