SPA Product Guide

18-1171-48 AA SPAproduct guide 1 SPA product guide Contents Page Scintillation proximity assay—versatile technology for rapid and sensitive assays 2 The principle of SPA 3 Advantages of SPA bead-based assays 3 Choosing between SPA Scintillation Beads and SPA Imaging Beads 4 SPA bead types 5 SPA bead coatings 6 Suitable radioisotopes for SPA 6 Application of SPA technology to enzyme assays 7 Assay format 7 SPA bead types and coatings used for enzyme assays 8 Application of SPA technology to kinase assays 9 SPA bead types and coatings used for kinase assays 9 Application of SPA technology to receptor-binding assays 11 SPA bead types and coatings for receptor-binding assays 12 Typical applications for receptor-binding SPAs 12 Application of SPA technology to molecular interaction research 14 SPA bead types and coatings for studying molecular interactions 14 Application of SPA technology to radioimmunoassays 16 Application of SPA technology to cell-based assays 17 Principle of Cytostar-T Scintillating Microplates 18 Application of SPA technology to drug metabolism and pharmacokinetics 19 Receptor Membrane Preparations 19 Scintillation counter settings 20 Multiwell plates suitable for counting SPA Scintillation Beads 20 Multiwell plates suitable for measuring SPA Imaging Beads 21 Ordering information 22 Related products 23 2 Scintillation proximity assay—versatile technology for rapid and sensitive assays Scintillation proximity assay (SPA) is a homogeneous and versatile technology for the rapid and sensitive assay of a wide range of biological processes. The assay format requires no separation steps, is amenable to automation, and is supported by more than 10 years of application data generated independently and by Amersham Biosciences scientists. SPAs using either SPA Scintillation Beads or SPA Imaging Beads can be read on scintillation counters and CCD imagers respectively. Versatile A range of bead types and coatings z For use with enzyme and receptor targets, radioimmunoassays, and molecular interactions z Various pack sizes available from stock for assay development and research z Bulk packs available for screening applications z Custom bead coatings on request SPA beads are suitable for use with a range of radioisotopes z 3H, 125I, 33P, and 14C can be used as radiolabels z No requirement for protein modification Compatible with any scintillation counter or CCD imager z SPA Scintillation Beads can be used in tubes, 96- or 384-well microplates z SPA Imaging Beads can be used in any format microplate No separation step Reduction in handling steps z Increased assay precision and assay reproducibility z Improved safety z No need for filters and scintillation cocktails Reduction in radioactive liquid waste z Decreased disposal costs Amenable to automation z Increased throughput Proven track record z Leading radiometric screening technology z Supported by more than 10 years of application data generated by Amersham Biosciences and leading pharmaceutical companies z Over 600 literature references are available on our Web sites at www.amershambiosciences.com/spa and www.amershambiosciences.com/leadseeker 3 Advantages of SPA bead-based assays SPA bead-based assays have many advantages over plate-based methods z The higher surface area of SPA beads allows the concentration of key assay components (e.g. receptor membrane) to be increased or decreased, thus giving greater flexibility in assay design. z The higher binding capacity of SPA beads gives higher signal allowing a potential reduction in the amount of radioactive label required. z The amount of SPA bead can be altered to optimize the sensitivity of an assay. z SPA beads are format independent making it easier to transfer assays to higher density plates. z A choice of four different core bead matrices allows optimization of signal and background over a wide range of applications (Table 2). z SPA beads are not dependent on one plate type or supplier (Table 11). The principle of SPA When certain radioactive atoms decay, they release β-particles. The distance these particles travel through an aqueous solution is dependent on the energy of the particle. If a radioactive molecule is held in close enough proximity to a SPA Scintillation Bead or a SPA Imaging Bead, the decay particles stimulate the scintillant within the bead to emit light, which is then detected in a PMT- based scintillation counter or on a CCD-based imager respectively (Table 1). However, if a radioactive molecule is free in a solution containing SPA beads, the decay particles will not have sufficient energy to reach the bead and no light will be emitted. This discrimination of binding by proximity means that no separation of bound and free ligand is required. Fig 1. Basic SPA concept. Different colored dyes can affect the signal produced in a SPA using either SPA Scintillation Beads or SPA Imaging Beads (Fig 3). The dyes used to generate the data in figure 3 were at concentrations of up to 3 mg/ml, which is in excess of that liable to be found in typical compound libraries, while all wells have identical amounts of radiolabel and SPA bead. The data show no significant attenuation of signal from the SPA Imaging Beads at any concentration of tartrazine, whereas the SPA Scintillation Beads report a lower level of signal as dye concentration increases. The presence of Chicago Sky Blue (CSB) causes quenching of the SPA Imaging Bead signal from a concentration of 0.03 mg/ml; the YSi and PVT beads only begin to show quenching at 0.1 mg/ml of CSB. Given that there is a higher proportion of yellow/orange/red compounds than blue compounds in most compound libraries, the issue of color quenching is reduced by conversion to SPA Imaging Beads. Choosing between SPA Scintillation Beads and SPA Imaging Beads The choice of SPA bead depends on several parameters and these are summarized in Table 1. Table 1. SPA bead selection guide 4 SPA Scintillation SPA Imaging Feature Benefit Beads Beads Instrument PMT readers, CCD Imagers SPA Scintillation Beads Match bead to instrument standard beta emit in the blue region type (e.g. CCD imagers are counters and SPA Imaging Beads more sensitive in the emit light in the red red region). region of spectra (fig 2). Throughput Medium to high High to ultra high Choice of throughput Match instrument and bead choice with throughput needs Format 96- or 384- 96-, 384-,1536-well Choice of format Increase throughput and well plates plates and above decrease assay component costs Emission Blue region Red region Choice of emission Reduce color quench (400 nm) (615 nm) properties caused by orange/red colored compounds through use of SPA Imaging Beads SPA Scintillation Beads SPA Imaging Beads Benefit Polyvinyltoluene (PVT) Polystyrene (PS) Easy to dispense and suited to automation and high- throughput screening. Used for the majority of SPA scintillation and SPA imaging applications Yttrium silicate (YSi) Yttrium oxide (YOx) Possess greater scintillation counting efficiency but less suited to automation because their higher density makes them settle more rapidly than PVT or PS beads SPA bead types SPA Scintillation Beads and SPA Imaging Beads are available in different core materials and their benefits are described in Table 2. Table 2. SPA bead type selection guide. 5 Fig 2. Wavelength emissions from SPA Scintillation Beads (PVT and YSi) and SPA Imaging Beads (PS and YOx ). The overlaid spectrum indicates the compound colors that have the potential to quench bead signal. Fig 3. Plate map showing effect of color quenching on an imaged assay, on SPA Scintillation Beads (PVT and YSi), and SPA Imaging Beads (PS and YOx). SPA bead coatings SPA beads are coated with coupling molecules such as wheat germ agglutinin (WGA), streptavidin, antibodies, glutathione, and copper or nickel chelate. These coupling molecules allow a variety of assay reagents such as cell membranes, biotinylated peptides, and fusion proteins to be captured onto the bead. The choice of bead coating will be determined by the application under investigation. Table 3 gives guidance on which bead coating is most suited to a range of typical applications. Table 3. SPA bead coatings and their recommended applications. Typical bead coating Application Wheat germ agglutinin (WGA) Receptor-ligand binding Antibody binding beads Radioimmunoassay Glutathione Signal transduction/molecular interactions Copper chelate Nickel chelate Antibody binding beads Streptavidin Streptavidin Lipid modifying enzyme Streptavidin DNA/RNA modifying enzyme Streptavidin Protease/nuclease Streptavidin Kinase Streptavidin Transferase Suitable radioisotopes for SPA The most important characteristic when selecting a radioisotope for SPA is the pathlength of the decay particle (Table 4). In general, the shorter the pathlength of the decay particle, the more suited it is to SPA. z Tritium and iodine-125 are ideally suited to SPA z Carbon-14, sulfur-35, and phosphorus-33 have been used successfully with SPA Table 4. Radioisotopes suitable for SPA and their decay particle average pathlengths. Isotope Average pathlength of decay particle in aqueous solution Tritium 1.5 µm Iodine-125 2e-: 1.0 µm, 17 µm Carbon-14 50 µm Sulfur-35 65 µm Phosphorus-33 125 µm 6 Application of SPA technology to enzyme assays Enzyme SPA applications can be used to identify potential new enzyme inhibitors, measure enzyme activity, and perform kinetic analysis. Activity of the following enzymes can be determined by enzyme SPA . z Hydrolases such as proteases and nucleases z DNA modifying enzymes such as helicase and integrase z Transferases z Phosphodiesterases z Lipid modifying enzymes Examples of enzyme SPA applications can be found as Proximity News articles on our SPA Web site at www.amershambiosciences.com/spa and as Leads articles on our LEADseeker™ Web site www.amershambiosciences.com/leadseeker. These Web sites also include bibliographies containing further publications citing enzyme SPA applications. Assay format The format of an enzyme SPA depends on the action of the enzyme being studied. 1. Signal increase assays are used to measure the activity of kinases, polymerases, and transferases because these enzymes add radiolabelled molecules to the substrate immobilized on the bead (Fig 4a). 2. Signal decrease assays are used to measure the activity of enzymes such as proteases and nucleases because they cleave radiolabelled substrate from the bead (Fig 4b). 7 Fig 4. Enzyme SPA concept. (a) Signal increase format (b) Signal decrease format. SPA bead types and coatings used for enzyme assays 1. The majority of enzyme SPAs use biotinylated substrates that may be either immobilized on, or subsequently captured by, streptavidin-coated SPA Scintillation Beads or SPA Imaging Beads. The biotin-streptavidin system provides a reliable and reproducible high-affinity capture mechanism. Further details on biotinylation techniques can be found in Proximity News 39, a copy of which is available on our SPA Web site at www.amershambiosciences.com/spa. 2. Arginine Binding YSi SPA Beads are used in the Nitric Oxide Synthase (NOS) Screening Kit (TRKQ7160). Figure 5 shows timecourse data whereby [3H]arginine is converted to citrulline by the action of NOS. The product, [3H]citrulline, does not bind to the beads and the action of NOS is therefore observed as a reduction in SPA counts. 8 3. GST-tagged fusion protein enzyme substrates can be captured onto SPA beads coated with glutathione. 4. His-tagged fusion proteins can be captured onto copper chelate coated SPA Scintillation Beads or Nickel chelate coated SPA imaging Beads Table 5. SPA bead types and coatings commonly used in enzyme SPA applications. Instrument Core bead type Coating Product code PMT readers PVT Streptavidin RPNQ0009, RPNQ0007, RPNQ0006 Copper chelate RPNQ0095 YSi Streptavidin RPNQ0015, RPNQ0012 Copper chelate RPNQ0096 Glutathione RPNQ0033, RPNQ0034 PDE RPNQ0150 Arginine binding SPA beads RPNQ0101 CCD imagers PS Streptavidin RPNQ0261, RPNQ0263 Nickel chelate RPNQ0266, RPNQ0267 YOx Streptavidin RPNQ0271, RPNQ0273 Nickel chelate RPNQ0276, RPNQ0277 Bulk order quantities are available for larger scale research experiments and high-throughput screens. For a range of fully optimized enzyme SPA kits, please visit our Web site at www.amershambiosciences.com/spa. A full listing of SPA beads is shown on page 22. Fig 5. Timecourse data for the Nitric Oxide Synthase (NOS) Screening Kit (TRKQ7160). 9 SPA bead types and coatings used for kinase assays Typically, kinase SPAs are configured by using streptavidin-coated SPA beads and biotinylating the peptide or protein substrate. Alternatively, fusion protein binding SPA beads can also be used. A list of kinase applications can be found in the bibliography section on our Web sites at www.amershambiosciences.com/spa and www.amershambiosciences.com/leadseeker Fig 6. Kinase SPA concept. Fig 7. Inhibition of p56lck kinase activity by PP2 and nocodazole in 1536-well format using Polystyrene SPA Imaging Beads. Each data point is the mean ± SEM of six replicates. Application of SPA to kinase assays Kinases play pivotal roles in many signal transduction cascades and consequently kinase activities remain a key focus of academic and pharmaceutical research. Amersham Biosciences has developed kinase SPA applications for tyrosine and serine/threonine kinases using both peptide and protein substrates. z Cyclin dependent kinases z MAP kinases z Protein kinase A z Protein kinase C isoforms The basic principle of kinase SPA involves the transfer of [33P]phosphate from [33P]ATP to the tyrosine, serine, or threonine in the peptide or protein substrate. The phosphorylated peptide is then captured by a streptavidin-coated SPA bead (fig 6). Table 6. SPA bead types and coatings commonly used in kinase SPA applications. Instrument Core bead type Coating Product code PMT readers PVT Streptavidin RPNQ0009, RPNQ0007, RPNQ0006 Copper chelate RPNQ0095 YSi Streptavidin RPNQ0015, RPNQ0012 Copper chelate RPNQ0096 CCD imagers PS Streptavidin RPNQ0261, RPNQ0263 Nickel chelate RPNQ0266, RPNQ0267 YOx Streptavidin RPNQ0271, RPNQ0273 Nickel chelate RPNQ0276, RPNQ0277 Bulk order quantities are available for larger scale research experiments and high-throughput screens. For a range of fully optimized kinase SPA kits, please visit our Web site at www.amershambiosciences.com/spa. A full listing of SPA beads is shown on page 22. 10 Application of SPA technology to receptor-binding assays Receptor-binding SPAs can be configured to determine receptor kinetics, saturation binding, or to detect inhibitors of radioligand binding. SPA has been successfully applied to receptor-binding assays by immobilizing receptors directly to SPA beads via a number of coupling methods. SPA receptor-binding assays have been developed for a wide range of receptor types, including: z Serotonin z Acetylcholine z Chemokine z Cytokine z Steroid z Inositol triphosphate SPA technology also enables the binding of GTP to G-protein coupled receptors (GPCRs) following agonist binding to the GPCR to be measured. SPA is compatible with receptors prepared from a number of different sources: z Cell membrane preparations from tissue z Cell membrane preparations from cultured cells z Soluble purified receptors z Solubilized receptors from tissues and cultured cells The principle of receptor-binding SPAs involves radiolabelled ligand binding to a receptor immobilized on the surface of a SPA bead. The bound ligand is held in close enough proximity to the bead to stimulate scintillant within the bead to emit light. Unbound radioligand is too distant from the bead to transfer energy and therefore goes undetected (Fig 8). 11 Fig 8. Receptor-binding SPA concept. SPA bead types and coatings used for receptor-binding assays 1. Receptor-binding SPAs are usually formatted using wheat germ agglutinin (WGA)-coated SPA beads. WGA binds N-acetyl-β-D-glucosaminyl residues, N-acetyl-β-D-glucosamine oligomers, and glycoproteins present in cell membranes to capture cell membranes expressing the receptor of interest. 2. The use of polyethyleneimine to either treat WGA or as a separate bead coating can further reduce non-specific binding, 3. Poly-L-lysine is used for binding negatively charged cellular membranes. 4. Soluble and solubilized receptors can be captured onto antibody binding SPA beads via an antibody specific to the receptor. 5. The receptor protein can be biotinylated and captured onto streptavidin-coated beads. Typical applications for receptor-binding SPAs Proximity News, 27, describes the steps required to develop a receptor-binding SPA and can be found on our SPA Web sites at www.amershambiosciences.com/spa and www.amershambio- sciences.com/leadseeker. Further examples of receptor-binding SPA applications can be found as Proximity News articles on the SPA Web site, which also includes a bibliography containing an additional 172 publications citing receptor-binding SPA applications. The LEADseeker Web site contains application sets detailing receptor-binding applications using SPA Imaging Beads. Data from a typical receptor-binding assay is shown in Figure 9 whereby competition analysis was performed on membranes from HEK cells expressing the human melanocortin 4 receptor. The radiolabelled ligand, [125I]NDP-α-MSH - [Nle4-D-Phe] melanocyte stimulating hormone and the competitor (unlabelled NDP-α-MSH) were added together with WGA-coated Polystyrene SPA Imaging Beads at T = 0 into the wells of 384-well plates. The assays were incubated overnight at room temperature without shaking and the plates were then read for 5 min on the LEADseeker Multimodality Imaging System. 12 Fig 9. Melanocortin 4 receptor imaging SPA competition analysis. Values shown as Means +/- SD (n =3). Table 7. SPA bead types and coatings that may be used in receptor-binding assays. Instrument Core bead type Coating Product code PMT readers PVT WGA RPNQ0252, RPNQ0001 WGA PEI Type A RPNQ0003 WGA PEI Type B RPNQ0004 Polyethyleneimine RPNQ0097 YSi WGA RPNQ0011 Poly-L-lysine RPNQ0010 PVT and YSi SPA Scintillation Select-a-Bead Kit* RPNQ0250 CCD imagers PS WGA RPNQ0260, RPNQ0262 WGA PEI Type A RPNQ0286, RPNQ0287 WGA PEI Type B RPNQ0288, RPNQ0289 YOx Poly-L-lysine RPNQ0295, RPNQ0294 PS and YOx SPA Imaging Select-a-Bead Kit† RPNQ0291 * Contains 100 mg each of WGA PVT; WGA YSi; WGA PEI Type A, WGA PEI Type B, and poly-L-lysine to allow quick and convenient receptor assay development using SPA Scintillation Beads. † Contains 50 mg each of WGA PS; WGA YOx; WGA PEI Type A, WGA PEI Type B, and poly-L-lysine to allow quick and convenient receptor assay development using SPA Imaging Beads. Bulk order quantities are available for larger scale research experiments and high-throughput screens. For a range of fully optimized receptor-binding SPA kits, please visit our Web sites at www.amershambiosciences.com/spa and www.amershambiosciences.com/leadseeker A full listing of SPA beads is shown on page 22. 13 Application of SPA technology to molecular interaction research SPA technology has been successfully applied to the study of a variety of molecular interactions. z Protein:protein interactions z Protein:peptide interactions z Protein:DNA interactions z Cell adhesion molecule interactions Many low-affinity binding events, such as occur between cell adhesion molecules, can be disrupted by the separation and wash steps required in heterogeneous techniques such as filtration. However, with SPA, these low-affinity binding events can be studied because there is no separation step. SPA bead types and coatings for studying molecular interactions Various SPA bead types and coatings have been used for molecular interaction a