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Scintillation Proximity Assay

Steven D. Kahl1,  Christian C. Felder1

1Lilly Research Laboratories, Indianapolis, Indiana

Publication Name: 
Current Protocols in Neuroscience
Unit Number: 
UNIT 7.15
DOI: 
10.1002/0471142301.ns0715s30
Print Publication Date: 
January, 2005
Online Posting Date: 
February, 2005
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Abstract

Scintillation proximity assay technologies provide a rapid non-separation method to measure common biological interactions using radioactively tagged molecules. This unit identifies potential uses of the technology for the measurement of receptor-ligand binding, cAMP accumulation, GTP binding to heterotrimeric G proteins, protease activity and cellular uptake.

     
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Table of Contents

  • Unit Introduction
  • Basic Protocol 1: Saturation Analysis of [3H]5-HT Binding to the 5-HT1E Receptor
  • Support Protocol 1: Optimization of Membrane Protein Concentration
  • Support Protocol 2: Optimization of Amount of WGA SPA Beads
  • Support Protocol 3: Optimization of Incubation Time
  • Basic Protocol 2: Pharmacological Profile for the 5-HT1E Receptor Using SPA
  • Basic Protocol 3: Rapid Measurement of cAMP Accumulation in CHO Cells Stably Expressing the Muscarinic M1 Receptor
  • Basic Protocol 4: Time-Course Analysis of Rhinovirus 3C Protease Using SPA
  • Basic Protocol 5: Uptake of [14C]p-Hydroxy-Loracarbef Using Cytostar-T Plates
  • Basic Protocol 6: Muscarinic M1 Receptor–Mediated [-35S]GTP Binding: An SPA Approach Using a Specific Anti–G Protein Antibody
  • Basic Protocol 7: Miniaturization of Receptor-Radioligand Interactions Using 384-Well FlashPlates
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
  • Other Versions
     
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Materials

Basic Protocol 1: Saturation Analysis of [3H]5-HT Binding to the 5-HT1E Receptor

 Materials
  • LM(tk-) cells expressing 5-HT1E receptor (Kahl et al., 1997)
  • 50 mM Tris×Cl, pH 7.4 (appendix 2A), ice-cold
  • Bovine serum albumin (BSA)
  • Saturation assay buffer (see recipe), room temperature
  • 20 mg/ml WGA SPA beads (see recipe)
  • 1 mM 5-hydroxytryptamine (unlabeled 5-HT; see recipe)
  • 1 µM [3H]5-hydroxytryptamine ([3H]5-HT; see recipe)
  • Teflon pestle/glass tissue homogenizer (e.g., Thomas)
  • 250-ml conical centrifuge tubes (e.g., Corning)
  • Beckman refrigerated tabletop centrifuge, or equivalent
  • Beckman J2-21M centrifuge and JA-14 rotor with 250-ml bottles, or equivalent
  • 13 × 100–mm disposable polypropylene tubes
  • 96-well white polystyrene microtiter plates (e.g., clear-bottom from Corning or opaque bottom from Perkin Elmer Life and Analytical Sciences)
  • Single and multichannel pipettors
  • Microtiter plate shaker (e.g., Labline Instruments)
  • Self-adhesive microtiter plate sealers
  • Liquid scintillation cocktail (e.g., Ready Protein+, Beckman)
  • Liquid scintillation counter (e.g., Packard Tricarb) and glass scintillation vials
  • Microtiter plate scintillation counter (e.g., Perkin Elmer Life and Analytical Sciences Trilux or TopCount)
  • Additional reagents and equipment for protein assays (see cpmb unit 10.1A) and nonlinear regression analysis (unit 7.5)

Basic Protocol 2: Pharmacological Profile for the 5-HT1E Receptor Using SPA

 Materials
  • Serotonergic compounds (also see Table 7.15.4), e.g.:
    • 10 mM -methyl-5-hydroxytryptamine (see recipe)
    • 10 mM 2-methyl-5-hydroxytryptamine (see recipe)
    • 10 mM 5-carboxyamidotryptamine (see recipe)
  • 1 ml volume/well 96-well polypropylene microtiter plate
  • 50-ml conical disposable polypropylene tubes
  • Additional reagents and equipment for saturation analysis of [3H]5-HT binding to the 5-HT1E receptor (see Basic Protocol 1)
 
Table 7.15.4 Stock Solutions of Serotonergic Compoundsa, b
CompoundMix conc.Final assay conc.10 mM stock (µl)Saturation assay buffer (µl)
5-HT30 µM10 µM
      3
997
-Me-5-HT3 mM1 mM300700
2-Me-5-HT300 µM100 µM
   30
970
5-CT300 µM100 µM
   30
970
 aDifferent concentration ranges are used to cover a complete competitive response for each compound.
 bAbbreviations:5-HT, 5-hydroxytryptamine; -Me-5-HT, -methyl-5-hydroxytryptamine; 2-Me-5-HT, 2-methyl-5-hydroxytryptamine; 5-CT, 5-carboxyamidotryptamine.

Basic Protocol 3: Rapid Measurement of cAMP Accumulation in CHO Cells Stably Expressing the Muscarinic M1 Receptor

 Materials
  • CHO cells (ATCC CCL-61) stably expressing the human M1 muscarinic receptor (Felder et al., 1989)
  • DMEM/ F-12/10% FBS medium (see recipe) with HEPES
  • Phosphate-buffered saline (PBS; appendix 2A)
  • Trypsin/EDTA solution: 0.05% trypsin/0.53 mM tetrasodium EDTA (e.g., Life Technologies)
  • Enzyme (trypsin)-free cell dissociation solution (Specialty Media)
  • Dilution buffer (see recipe)
  • 1 mM 3-isobutyl-1-methylxanthine (IBMX; Sigma) or 10 µM RO20-1724 (Calbiochem) in dilution buffer
  • Carbachol (Research Biochemicals)
  • Lysis buffer: 1% (w/v) dodecyltrimethylammonium bromide in 50 mM sodium acetate, pH 5.8
  • 500 µM adenosine 3¢:5¢-cyclic monophosphate (cAMP), sodium salt (Sigma)
  • 50 mM sodium acetate buffer, pH 5.8
  • Beads/antibody/[125I]cAMP mixture (see recipe)
  • 75-cm2 and 225-cm2 tissue culture flasks (Costar)
  • 96-well microtiter plates (white plate; clear bottom; Fisher or Costar)
  • Microtiter plate sealing tape (Wallac)
  • Microtiter plate scintillation counter (e.g., Wallac Trilux or Packard TopCount)
  • Additional reagents and equipment for cell culture and counting cells (appendix 3B)

NOTE: All culture incubations are performed in a 37°C, 5% CO2 incubator unless otherwise specified.

NOTE: All solutions and equipment coming into contact with living cells must be sterile, and aseptic technique should be used accordingly.


Basic Protocol 4: Time-Course Analysis of Rhinovirus 3C Protease Using SPA

 Materials
  • Protease assay buffer (see recipe)
  • Stop solution: 10% (w/v) orthophosphoric acid
  • 50 nM biotinylated 125I-labeled rhinovirus 3C protease substrate stock solution (see recipe)
  • 50 µM purified rhinovirus 3C protease stock (store at –80°C; Birch et al., 1995)
  • 20 mg/ml streptavidin SPA beads (see recipe), 4°C
  • 13 × 100–mm disposable polypropylene tubes
  • 96-well white polystyrene microtiter plates (e.g., clear bottom from Corning or opaque bottom from Perkin Elmer Life and Analytical Sciences)
  • Single and multichannel pipettors
  • Microtiter plate shaker (e.g., Labline Instruments)
  • Self-adhesive microplate sealers
  • Microtiter plate scintillation counter (e.g., Perkin Elmer Life and Analytical Sciences Trilux or TopCount)

Basic Protocol 5: Uptake of [14C]p-Hydroxy-Loracarbef Using Cytostar-T Plates

 Materials
  • Human adenocarcinoma cell line Caco-2 (obtained from Dr. J. Fogh at the Research Unit of Memorial Sloan-Kettering Cancer Center, Rye, NY)
  • DMEM/F12/10% FBS medium without HEPES (see recipe)
  • Trypsin/EDTA solution: 0.05% trypsin/0.53 mM tetrasodium EDTA (e.g., Life Technologies; store at 4°C)
  • 4 mg/ml rat tail collagen, type II (see recipe)
  • 0.5% (v/v) acetic acid
  • Ammonium hydroxide
  • 20 mM [14C]p-hydroxy-loracarbef (see recipe)
  • Flux buffer (see recipe)
  • Phosphate-buffered saline (PBS; appendix 2A)
  • 75-cm2 sterile tissue culture flasks (sterile, tissue culture treated; Costar)
  • Cytostar-T microtiter plate (sterile, tissue culture treated; Amersham Biosciences)
  • Multichannel pipettor
  • 8-channel aspirator
  • Self-adhesive microplate seals
  • Microtiter plate scintillation counter (e.g., Perkin Elmer Life and Analytical Sciences Trilux or TopCount)
  • Additional reagents and equipment for cell culture and counting cells (appendix 3B)

NOTE: All culture incubations are performed in a 37°C, 5% CO2 incubator unless otherwise specified.

NOTE: All solutions and equipment coming into contact with living cells must be sterile, and aseptic technique should be used accordingly.


Basic Protocol 6: Muscarinic M1 Receptor–Mediated [-35S]GTP Binding: An SPA Approach Using a Specific Anti–G Protein Antibody

 Materials
  • Membrane preparation from CHO cells expressing recombinant human M1 muscarinic receptor (Perkin-Elmer Life and Analytical Sciences)
  • Oxotremorine-M (Sigma)
  • Dimethyl sulfoxide (DMSO)
  • Compound dilution buffer: muscarinic assay buffer (see recipe) containing 4% (v/v) DMSO (add 2 ml DMSO to 48 ml assay buffer; prepare fresh each day)
  • Muscarinic assay buffer (see recipe)
  • [-35S]GTP in stable aqueous solution (Perkin-Elmer Life and Analytical Sciences), store up to 60 days at –20°C (see recipe for preparing [-35S]GTP ligand solution)
  • 10% (v/v) Nonidet P-40 (NP-40; Roche), 4°C
  • Rabbit anti-Gq/11 antibody (Santa Cruz Biotechnology; see recipe for preparing diluted antibody)
  • SPA PVT antibody-binding beads, anti-rabbit reagent (Amersham Biosciences; store according to manufacturer's recommendations)
  • 50-ml disposable polystyrene tubes
  • 10-ml disposable glass tubes
  • 96-well deep-well polypropylene microtiter plate (1 ml/well capacity)
  • Tissue homogenizer (e.g., PowerGen 700, Fisher)
  • 50-ml polystyrene reagent reservoirs
  • Single and multichannel pipettors
  • 96-well polystyrene microtiter plate (white, with clear bottom; Corning Costar)
  • Self-adhesive microtiter plate seals
  • Microtiter plate shaker (e.g., Vortex Genie, Scientific Industries)
  • Tabletop centrifuge (e.g., AccuSpin 1R, Fisher) with microtiter plate carrier
  • Microtiter plate scintillation counter (e.g., Perkin-Elmer Life and Analytical Sciences Trilux)

Basic Protocol 7: Miniaturization of Receptor-Radioligand Interactions Using 384-Well FlashPlates

 Materials
  • Miniature SPA assay buffer (see recipe)
  • 1.5 mM S(–)-propranolol (see recipe)
  • 500 nM [125I]iodocyanopindolol (see recipe)
  • Membrane preparation (2.0 µg/ml) from HEK293 cells expressing 2 adrenergic receptor (Perkin Elmer Life and Analytical Sciences)
  • 13 × 100–mm disposable polypropylene tubes
  • FlashPlates (Perkin Elmer Life and Analytical Sciences, store at 4°C):
    • 96-well Basic (uncoated) FlashPlate
    • 96-well WGA FlashPlate
    • 384-well Basic (uncoated) FlashPlate
    • 384-well WGA FlashPlate
  • Multichannel pipettors and corresponding reagents reservoirs
  • Gamma counter (e.g., ICN Micromedic)
  • Microtiter plate shaker (e.g., Vortex Genie, Scientific Instruments)
  • Self-adhesive microtiter plate seals
  • Microplate scintillation counter (e.g., Perkin Elmer Life and Analytical Sciences Trilux or TopCount)
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Figures

  • Figure 7.15.1
    Scintillation proximity assay. (A) With the free radioligand, the energy of the radiation is absorbed and dissipated into the buffer due to the low energy of the emitted particles. (B) In SPA, antibodies, agglutinins, and other molecules covalently attached to the scintillation proximity bead surface or attached to the bottom of a scintillation plate (not represented in this diagram) serve as acceptor molecules to bring the radioligand in close proximity to the bead or plate, which contains the chemical scintillant. The energy absorbed by the scintillant is reemitted as light, which is then detected by a scintillation spectrophotometer.

    View Image
  • Figure 7.15.2
    Schematic representation of a typical SPA for radioligand binding to receptor-enriched membranes. Cell membranes containing the receptor are incubated with radioligand, followed by an addition of WGA-coated SPA beads, which capture the membranes to the bead via interactions with the glycoprotein residues. The energy released by the bound radioligand excites the scintillant incorporated in the SPA bead, resulting in the emission of detectable light. Energy released from unbound radioligand is too far away from the SPA bead and is dissipated in the assay medium. No separation of bound and unbound radioligand is required for the SPA technique. Competitive compounds that bind at the same site as the radioligand result in a dose-dependent loss of SPA signal.

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  • Figure 7.15.3
    Plate setup for saturation binding analysis.

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  • Figure 7.15.4
    Saturation analysis of [3H]5-HT binding to membranes enriched in 5-HT1E receptors using the SPA procedure. Nonspecific binding (triangles) is determined in the presence of excess unlabeled 5-HT (1 µM). Specific binding (circles) is the difference between total binding (squares) and nonspecific binding. Nonspecific binding increases linearly and mimics the signal for the nonproximity effects (NPE) of [3H]5-HT and WGA SPA beads in the absence of added membrane protein. Nonlinear regression analysis yields a dissociation constant (Kd) of 5.6 nM and the maximum number of binding sites (Bmax) as 22.2 pmol/mg.

    View Image
  • Figure 7.15.5
    Amount of membrane protein used for [3H]5-HT binding to the 5-HT1E receptor with SPA in the absence (total; solid circles) or presence (NSB; open circles) of excess unlabeled 5-HT using techniques described in Basic Protocol 1 and Basic Protocol 2. Higher amounts of membrane protein (>15 µg/well) will saturate the limiting amount of WGA SPA beads (0.5 mg) and result in a reduced signal due to non-SPA bead bound receptor.

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  • Figure 7.15.6
    Determination of optimal WGA SPA bead concentration in a receptor/radioligand binding assay. In this example, [3H]5-HT was incubated with receptor-enriched membranes prior to the addition of various concentrations of WGA SPA beads. Total binding is represented by solid circles and specific binding is represented by squares. Nonspecific binding (NSB; open circles) was determined in the presence of 1 µM unlabeled 5-HT. Since the amount of SPA beads used is also driven by economics, this data demonstrates that 0.5 mg of WGA SPA beads is adequate for capture of nearly all of the specific binding. Higher amounts of WGA SPA beads/well result in increased costs without increased specific signal.

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  • Figure 7.15.7
    The incubation time following the addition of WGA SPA beads is a critical parameter in the SPA techniques for receptor/radioligand binding. Here [3H]5-HT is incubated with 5 µg of receptor-enriched membranes followed by an incubation with WGA SPA beads for the indicated amount of time. No appreciable signal from total binding (triangles) or nonspecific binding (NSB; squares) occurs after a 2-hr incubation. The primary incubation of radioligand and membranes was also investigated and determined to be optimal at 30 min (not shown).

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  • Figure 7.15.8
    Plate setup for competitive binding analysis (four compounds).

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  • Figure 7.15.9
    Competitive inhibition of [3H]5-HT binding to 5-HT1E membranes using the SPA technique. In this example, various concentrations of unlabeled serotonergic compounds were added to the wells of a microplate along with [3H]5-HT and receptor-enriched membranes. The resulting SPA signal was plotted versus the concentration of the added compound. Since the unlabeled compounds were capable of binding at the same site as the radioligand, dose-dependent reductions in signal were observed. IC50 concentrations and dissociation constants (Ki) were calculated using nonlinear regression analysis as described in unit 7.5.

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  • Figure 7.15.10
    Plate setup for cAMP assay. A standard curve is run on each plate and can be run with either duplicate or triplicate samples. The wells labeled blank in positions G1 and H1 contain all the assay ingredients but not cAMP or antibody. The wells labeled B0 in positions G2 and H2 contain all the assay ingredients but not a cAMP standard. Sodium acetate assay buffer should be used to substitute for these components.

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  • Figure 7.15.11
    (A) Standard curve and (B) final dose-response curve for carbachol-stimulated cAMP accumulation in CHO cells stably expressing the M1 muscarinic receptor.

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  • Figure 7.15.12
    Schematic representation for a protease assay using SPA. A biotinylated peptide substrate labeled on the C-terminus with 125I is incubated with a specific protease enzyme followed by capture of the substrate with streptavidin-coated SPA beads. If appropriate cleavage of the substrate occurs, the energy release by the 125I molecule is too far away from the SPA bead and a low signal results.

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  • Figure 7.15.13
    Plate setup for enzymatic time course.

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  • Figure 7.15.14
    Peptide substrate cleavage time course. Biotinylated [125I]labeled peptide substrate was incubated in the presence and absence of enzyme for the indicated times, followed by the addition of streptavidin SPA beads in stop buffer. Reduction of signal indicates that the peptide substrate is cleaved.

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  • Figure 7.15.15
    Schematic for principle of Cytostar-T plate. Radiolabel incorporated into adherent cells (through active transport or cell receptor binding) excites the scintillant contained in the base plate, resulting in the release of detectable light. Nonincorporated label in the assay medium is too far away from the base plate and does not result in a signal.

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  • Figure 7.15.16
    Cobblestone effect for 14-day post-confluent Caco-2 cells. Viewed at 20× 7 magnification under a light microscope.

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  • Figure 7.15.17
    Plate setup for dipeptide transport in Cytostar-T microplate.

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  • Figure 7.15.18
    Transport in Caco-2 cells was measured in a Cytostar-T plate with various concentrations of [14C]p-hydroxy loracarbef at three different temperatures.

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  • Figure 7.15.19
    Antibody capture assay for [-35S]GTP binding.

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  • Figure 7.15.20
    Plate setup for [-35S]GTP assay.

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  • Figure 7.15.21
    Percent maximal stimulation of the M1 muscarinic receptor induced by increasing concentrations of oxotremorine. Radioligand was [-35S]GTP. n = 3 replicates of a typical experiment. Bars represent SEM. Data was fit using nonlinear regression for a sigmoidal concentration-response curve (GraphPad Prism; see unit 7.5). EC50 value = 138 nM.

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  • Figure 7.15.22
    Plate setup for competitive inhibition of [125I]iodocyanopindolol binding to 2 adrenergic receptor membranes using (A) 96-well microplate, and (B) 384-well microplate.

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  • Figure 7.15.23
    Competitive inhibition of [125I]iodocyanopindolol binding to membranes containing 2 adrenergic receptors using 96- and 384-well FlashPlates. In this example, various concentrations of unlabeled S(–)-propranolol were added to the wells of either a 96-well or 384-well FlashPlate along with [125I]-iodocyanopindolol and receptor-enriched membranes. Two types of FlashPlates were tested: uncoated (Std) and coated with wheat germ agglutinin (WGA). Percent specific binding was determined from the resulting signal and plotted against the concentration of added S(–)-propranolol. Since the unlabeled S(–)-propranolol was capable of binding at the same site as the radioligand, concentration-response reductions in signal were observed. The IC50 values were calculated using non-linear regression analysis as described in unit 7.5. Tables 7.15.16 and 7.15.17 list the calculated results. These results indicate that there is no change in sensitivity for either WGA or non-coated plates for either plate density. n = 4, error bars represent the standard deviation.

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