LANCE™ cAMP –Increased Sensitivity, Stability and Overall Assay Performance with Gαs and Gαi Coupled Receptors in a Miniaturized 1536-Well Format. Patricia Kasila, Harry Harney PerkinElmer Life and Analytical Sciences, Boston, MA 02118 0 -14 -13 -12 -11 -10 -9 -8 -7 0 0 -6 -1 1 2 Victor 2V 3691 16364 -9.965 -1.923 1.0850e-010 0.50 0.90 ViewLux 271.1 883.4 -10.31 -1.382 4.8670e-011 EnVision 1 nM 2430 7942 0.2902 2.314 1.951 BOTTOM TOP LOGEC50 HILLSLOPE EC50 0.78 Z' = Victor2V 1 nM 5097 13133 0.1330 3.025 1.358 0.85 Signal Victor2V ViewLux 500 7500 400 5000 -10 -9 -8 -7 -6 0 -4 -5 BOTTOM TOP LOGEC50 HILLSLOPE EC50 0.88 Z' = Results 5HT1A 11 EnVision 3445 9269 -7.654 1.144 2.2170e-008 0.82 Victor 2V 5726 11878 -7.922 1.399 1.1970e-008 0.56 EnVision Victor2V ViewLux 300 200 -3 -2 -1 0 1 2 3 4 Log [Spiperone (nM)] Log [8OH-DPAT (M)] ViewLux 351.9 764.7 0.2662 2.297 1.846 0.94 250 600 EnVision 2500 0 -11 3 Log [Propranolol (nM)] EnVision 1858 8796 -9.981 -2.201 1.0440e-010 Z' = 0 500 10000 5000 0 -2 Log [Epinephrine (M)] BOTTOM TOP LOGEC50 HILLSLOPE EC50 Victor2V ViewLux 250 10000 750 15000 Signal Signal 500 10000 Forskolin Dose Response Cell Titration EnVision Eu Standard Curve SA 5K 2.5K 1K 0.5K EnVision Signal Free cAMP 1000 20000 500 10000 Biotin-cAMP Victor V ViewLux 1500 20000 15000 1500 30000 Standard Curve ViewLux 341.8 680.7 -7.724 1.135 1.8880e-008 BOTTOM TOP LOGEC50 HILLSLOPE EC50 0.96 EnVision 2661 5915 0.03118 -0.7193 1.074 Z' = 0.55 Victor 2V 4638 9867 0.1014 -0.5918 1.263 ViewLux 317.2 512.0 0.1123 -0.8150 1.295 0.50 0.47 Detection Instruments 10000 1000 10000 500 5000 Forskolin Dose Response Cell Titration EnVision EnVision Victor 2V ViewLux 12500 5K 2.5K 1K 0.5K 10000 Signal Ex@340nm 615nm 615nm 750 250 8 2 SA Victor2V ViewLux Signal (ADU) Eu 500 EnVision EnVision In the Presence of Excess cAMP Signal (ADU) EuropiumStreptavidin 10000 1000 20000 1000 750 Signal En erg yT ran sfe r Ex@340nm 20000 Antagonist Dose Response Agonist Dose Response Signal (ADU) In the Absence of Free cAMP Agonist Dose Response Results 2 Adrenergic Receptor 5 Antagonist Dose Response 1 nM Epinephrine Results 5HT1A Signal (ADU) 2 Ø LANCE™ cAMP Assay • 2 µL of cAMP standards, forskolin, agonist, antagonist in PBS • 2 µL cells diluted in Stimulation Buffer containing cAMP antibody or Stimulation Buffer containing cAMP antibody without cells added to the wells containing standards • Incubate for 30 –60 minutes at room temperature • 4 µL of Detection Buffer containing Biotin-cAMP and SA-Eu • Incubate for 60 minutes at room temperature • Read on an appropriate reader 10 Signal (ADU) Schematic Representation of LANCE cAMP Assay 7 Signal (ADU) Time resolved fluorescence (TRF) assays exhibit low background and high signalto noise ratios, two attributes critical for robust HTS assays. LANCE™ refers to homogeneous TRF applications using TR-FRET. In the previous version of the LANCE cAMP assay, energy was transferred from europium labeled antibody to an acceptor, allophycocyanin (APC) labeled to cAMP. The assay has now been improved by using an antibody labeled with the acceptor, Alexa Fluor® 647 which significantly improved assay sensitivity, stability and overall performance. The principle of the assay involves the loss of energy transfer as the quantity of cell derived cAMP increases, thereby competing and therefore decreasing the amount of biotinylated-cAMP/streptavidin europium available to bind to the Alexa dye labeled anti-cAMP antibody. Due to the format change, the assay has now been proven to provide superior performance with Gαs and sometime difficult Gαi coupled receptors in a 1536-well format. Data presented here will demonstrate the sensitivity and precise results obtained with Gαs and Gαi whole cell cAMP assays in 1536-well formats. Results 2 Adrenergic Receptor Signal 4 Abstract Signal 1 Methods 7500 5000 2500 0 0 0 -3 -2 -1 0 1 2 3 0 -4 4 -3 -2 Log [cAMP(nM)] Alexa cAMPspecific Antibody BOTTOM TOP LOGEC50 HILLSLOPE EC50 Em@665nm TR-FRET Signal No Emission No TR-FRET Signal EnVision 1326 14198 0.6214 -0.8746 4.182 Z' = 0.53 -1 0 1 2 -3 3 -2 -1 Victor V 2783 18721 0.6102 -0.9600 4.075 ViewLux 212.1 992.3 0.7237 -0.9498 5.292 0.63 BOTTOM TOP LOGEC50 HILLSLOPE EC50 0.90 Z' = 5K 1478 9649 -0.4881 -0.9244 0.3250 2.5K 1535 10097 -0.3106 -0.8678 0.4891 0.84 0.81 0 1 2 3 0 4 -4 Log [cAMP(nM)] [Forskolin (µM)] 2 1K 1867 11082 -0.03100 -0.7710 0.9311 0.5K 2375 11518 0.2961 -0.6799 1.978 0.73 EnVision™ 0 BOTTOM TOP LOGEC50 HILLSLOPE EC50 0.53 EnVision 1286 10797 0.4975 -1.092 3.144 Z' = -2 -1 0 1 2 Victor3V™ 3 [Forskolin (µM)] Victor 2V 2403 18480 0.6389 -1.063 4.354 0.70 -3 ViewLux 232.5 1007 0.5438 -1.073 3.498 0.66 5K 1356 7138 -0.9658 -0.9898 0.1082 BOTTOM TOP LOGEC50 HILLSLOPE EC50 Z' = 0.68 0.54 2.5K 1429 8309 -0.8610 -0.8388 0.1377 1K 1983 9484 -0.2644 -0.7964 0.5440 0.49 0.5K 2986 10150 0.3777 -0.5996 2.386 0.82 HTS ViewLux™ uHTS Multilabel plate readers provides the speed and flexibility needed to perform many types of assays and plate densities. 0.88 HTS Forskolin Dose Response Cell Titration Victor2 V Forskolin Dose Response Cell Titration ViewLux 5K 2.5K 1K 0.5K 15000 10000 5000 1000 5K 2.5K 1K 0.5K 750 500 1000 20000 5K 2.5K 1K 0.5K 10000 250 0 -4 -3 -2 -1 0 1 2 -4 -3 [Forskolin (µM)] BOTTOM TOP LOGEC50 HILLSLOPE EC50 Z' = 5K 2874 12798 -0.4764 -1.060 0.3339 2.5K 3133 14079 -0.3122 -0.9864 0.4873 0.96 0.90 -2 -1 0 1 2 3 0.89 0.5K 4361 15492 0.2994 -0.8802 1.993 0.92 750 500 BOTTOM TOP LOGEC50 HILLSLOPE EC50 Z' = 5K 231.0 713.3 -0.3844 -1.039 0.4127 2.5K 247.2 778.0 -0.2049 -1.012 0.6238 0.81 0.96 0 0 -4 [Forskolin (µM)] 1K 3302 15395 0.01354 -0.8324 1.032 5K 2.5K 1K 0.5K 250 0 3 ØExcellent performance in a 1536-well format ØExcellent performance of both Gαs and G αi coupled receptors Forskolin Dose Response Cell Titration ViewLux Forskolin Dose Response Cell Titration Victor2V Signal 20000 Signal (ADU) ØLANCE cAMP Assay (PerkinElmer) •The kit includes the following components: •Biotinylated- cAMP •Streptavidin Europium •Alexa Labeled Antibody •cAMP Standard •Detection Buffer ØOptiPlate (1536-well white plate) (PerkinElmer Catalog # 6005228) ØCell line - 2 adrenergic receptor cell line (PerkinElmer MCL501) and 5HT1A receptor cell line (PerkinElmer MCL-508) ØChemicals-Forskolin (Sigma# 6886) Agonists (Epinephrine Sigma# E4250), (8OH-DPAT Sigma# H140) Antagonists(Propanolol Sigma# P-0884) (Spiperone Sigma# S-7395) 9 Results 2 Adrenergic Receptor Signal (ADU) 6 Materials Signal 3 12 Results 5HT1A 1K 277.6 858.4 0.08303 -0.9162 1.211 0.5K 331.8 878.6 0.3973 -0.9344 2.496 0.93 0.72 -3 -2 -1 0 1 2 -4 3 -3 BOTTOM TOP LOGEC50 HILLSLOPE EC50 Z' = 2.5K 2884 15003 -0.6973 -0.8192 0.2008 0.98 0.81 -1 0 1 2 3 [Forskolin (µM)] [Forskolin (µM)] 5K 2663 13195 -0.8630 -0.9198 0.1371 -2 1K 3949 17090 -0.06978 -0.7822 0.8516 0.98 0.5K 5376 17532 0.6670 -0.6225 4.645 0.76 BOTTOM TOP LOGEC50 HILLSLOPE EC50 Z' = 5K 244.2 729.9 -0.8753 -1.005 0.1333 0.85 2.5K 255.3 824.3 -0.7462 -0.9362 0.1794 0.67 1K 299.0 908.3 -0.1737 -0.8112 0.6704 0.97 Conclusion 0.5K 370.8 912.4 0.5286 -0.6911 3.377 ØCorrect pharmacology as compared to literature values ØDramatic cost savings §Requires less reagents/well §Requires less cells/well ØUltra High Throughput Sreening capability Ø Simple protocol Ø Easily adapted to robotic systems 0.64 PerkinElmer Life and Analytical Sciences, 710 Bridgeport Avenue, Shelton, CT USA (800) 762-4000 (+1) 203 925-4602 www.perkinelmer.com