ALPHASCREEN™ for 384- and 1536-Well Microformatted Assays Authors R. Bosse, C. Illy, L. Beaudet, CJ Wang, D. Chelsky Figure 4. Many Assays Validated for ALPHASCREEN in 1-20 µl formats Figure 2. Principles of A LPHASCREEN (cont.) HTS Applications: ABSTRACT A powerful, homogeneous, non-radioactive technology is now available for a broad range of HTS and genomics applications. Based on the proximity of two very small beads and an amplified luminescence, ALPHASCREEN results in a very high signal output with robust signal/background ratios. These characteristics allow the technology to be applied to very small volume assays, without changing assay component concentrations. A large variety of coated beads make a wide range of assays possible with off-the-shelf reagents. Assays currently validated include serine/threonine and tyrosine kinases, protease, helicase, functional cAMP, ligand/receptor, protein/protein, transcription factor/DNA, low affinity interaction (1 µM), PCR, DNA/RNA quantitation, and SNP analysis. The ALPHAQUEST-HTS microplate analyzer, with a 40-plate stacker and internal bar code reader, rapidly processes 96, 384 or 1536-well plates. Figure 1. Principles of A LPHASCREEN Figure 5. Reagents are Available in Kits or Bulk Serine/Theonine Kinase Figure 3. A LPHAQUEST HTS Microplate Analyzer cAMP Detection Kit Phosphotyrosine (P-Tyr (PY20)) Detection Kit Phosphotyrosine (P-Tyr (PT66)) Detection Kit GST Detection Kit Digoxin/Digoxigenin Detection Kit Fluorescein Detection Kit Mouse IgG Detection Kit Rabbit IgG Detection Kit Goat IgG Detection Kit Human IgG Detection Kit C-Myc-Peptide Detection Kit His-Tag-peptide Detection Kit Tyrosine Kinase Protease Helicase Functional cAMP (whole cell + membrane) Ligand/Receptor When biological interactions bring the Donor and Acceptor beads into close proximity, reactive oxygen, generated by irradiation of the Donor beads, initiates a luminescence/fluorescence cascade in the Acceptor beads. This process leads to a highly amplified signal with output in the 520-620 nm range. Protein/Protein Interaction Low Affinity Interaction (1µM) p53-HDM2 Transcription Factor/DNA interaction The ALPHAQUEST-HTS is a four-detector instrument, optimized for ALPHASCREEN chemistry, using highly efficient diode excitation at 680 nm with high performance optics detecting emission at 520-620 nm. With a 40-plate stacker and bar code reader, the ALPHAQUEST-HTS reads 96-well plates in under 1 minute, 384-well plates in 2.5 minutes and 1536-well plates in 8.5 minutes. Laser irradiation of the Donor beads at 680 nm generates a flow of short-lived singlet oxygen molecules. When the Acceptor beads are not in proximity, the reactive oxygen decays and there is no signal. HA-peptide Detection Kit FLAG-peptide Detection Kit ALPHASCREEN ™ Figure 9. TNF a / TNFaR1 Binding Assay Figure 7. Forskolin Stimulation of Adenylate Cyclase in CHO Cells: 384-Well Format 2. add 2.5 µl forskolin (300 nM-1 mM) incubate 30 minutes at 37ºC 3. add 15 µl donor bead / acceptor bead / biotin-cAMP mix incubate 2 hours at RT 4. read plate ALPHASCREEN cAMP has been designed to directly measure levels of cAMP produced upon modulation of adenylate cyclase activity by G-protein coupled receptors. ALPHASCREEN cAMP is based on the competition between endogenous cAMP and exogenously added biotincAMP. The capture of cAMP is achieved by using a specific antibody conjugated to Acceptor beads. The assay is efficient at measuring both agonist and antagonist activities on Gi and Gs coupled GPCRs. ALPHASCREEN cAMP is specific and reliable. This assay is highly competitive with existing cAMP assays in terms of ease of use, sensitivity, dynamic range and time to completion. ALPHASCREEN: Key Features Protocol: 1. add 10,000 cells resuspended in 5 µl DMEM In the absence of cells, a cAMP standard curve is generated (top right). In the presence of 10,000 cells, forskolin stimulation generates a dose-response curve in terms of raw signal on the ALPHAQUEST Analyzer (middle right). Using the standard curve, the data is converted to cAMP levels (bottom right). Assay volume is 7.5 µl for stimulation of cells and 22.5 µl after the lysis/capture step. Figure 10. TNF a Assay in 384 and 1536-Well Format Figure 8. Forskolin Stimulation of Adenylate Cyclase in CHO Cells: 1536-Well Format 40000 1. add 3,000 cells resuspended in 0.8 µl DMEM 2. add 0.8 µl forskolin (600 nM-600 µM) incubate 30 minutes at 37ºC 3. add 5 µl of donor bead / acceptor bead / biotin-cAMP mix incubate 2 hours at RT Homogeneous Non-Radioactive Protocol: ALPHASCREEN TNFa has been designed to directly measure TNFa binding to the soluble subunit of the TNF receptor type 1 (sTNFaR1). The assay is based on the capture of biotinylated-TNFa by streptavidincoated Donor beads and the capture of sTNFaR1 by anti-TNFaR1 antibody-coated Acceptor beads. ALPHAScreen signal (cps) Figure 6. Cell-Based GPCR Functional Assay for cAMP 384-well 1536-well NS 30000 ALPHAQUEST-HTS 4-Detector Microplate Analyzer 20000 10000 0 96, 384, 1536-Well Formats 14 Products Available Now in Kit and Bulk Format 10 25 50 75 3 5 7 9 Sample volume (µl per well) Wide Range of Assays Validated 4. read plate In the absence of cells, a cAMP standard curve is generated (top right). In the presence of 3,000 cells, forskolin stimulation generates a dose-response curve in terms of raw signal on the ALPHAQUEST Analyzer (middle right). Using the standard curve, the data is converted to cAMP levels (bottom right). Assay volume is 1.6 µl for stimulation of cells and 6.6 µl after the lysis/capture step. The TNFa assay has been performed in both 384 and 1536-well format in volumes ranging from 75 µl down to 3 µl. The loss of signal, however, in going from a 75 µl assay in 384 to 3 µl in 1536 is only threefold. Signal to Background ratios also remain robust as assay volume drops. These results were obtained without changing any assay component concentrations or altering the assay in any way. HTS and Genomics Applications BioSignal Packard Inc., 1744 William St., Suite 600, Montreal (Quebec), H3J 1R4 Packard Instrument Company, 800 Research Parkway, Meriden, Connecticut 06450