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SPA Bead Technology

Overview


Scintillation proximity assay (SPA) is a homogeneous and versatile technology for the rapid and sensitive assay of a wide range of biological processes, including applications using enzyme and receptor targets, radioimmunoassays, and molecular interactions. When 3H, 14C, and 125I radioisotopes decay, they release β-particles (or Auger electrons, in the case of 125I). 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. However, if the radioactive molecule does not associate with the SPA bead, 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 physical separation of bound and free radiochemical is required.

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SPA technology

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SPA bead types


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Table 1. SPA bead types

There are four different SPA beads: two spherical plastic beads (polyvinyl toluene, PVT and polystyrene, PS) and two crystalline beads (yttrium silicate, YSi and yttrium oxide, YOx). The plastic beads are larger in size (5-8 µm) and stay in suspension longer than the crystalline beads (average, 2.5 µm) making them more amenable to automation.

The scintillator in the PVT beads is diphenylanthracine (DPA), which is co-polymerized in the polyvinyl toluene matrix. In YSi SPA beads, naturally occurring cerium ions act as scintillators and are stably trapped within the yttrium silicate crystal lattice. Both scintillators emit a blue light (400 – 450 nm) upon activation. This blue light emission is best captured on a PMT-based scintillation counter (Tri-Carb® for single tube detection; MicroBeta2® or TopCount® for microplate detection).

Europium is the scintillator co-polymerized in the polystyrene matrix in the PS imaging beads and stably trapped within the yttrium oxide crystal lattice of the YOx imaging beads. This scintillator emits a red light (615 nm) upon activation. This red light emission is best captured on a CCD-camera based detector (ViewLux™) which provides μHTS capability by imaging an entire 384- or 1536-well microplate at one time.

Each bead type can be suitably derivatized for use in several types of SPA applications.


SPA scintillation beads

CoatingApplicationCore bead typeSizeCat. number
Streptavidin       Capture of biotinylated proteins or peptides for use in enzyme or molecular interaction assays       PVT     50 mgRPNQ0006
150 mgRPNQ0009
500 mgRPNQ0007
25 x 500 mgSPQ0032
2 gRPNQ0066
25 x 2 gRPNQ0067
Ysi 75 mgRPNQ0015
250 mgRPNQ0012
Wheat Germ Agglutinin (WGA)      Binds cell membranes and internal components for receptor binding studies      PVT    100 mgRPNQ0252
500 mgRPNQ0001
25 x 500 mgSPQ0031
2 gRPNQ0060
25 x 2 gRPNQ0063
YSi 250 mgRPNQ0011
1 gRPNQ0023
WGA PEI Type A  Extra coating for reduced non-specific binding  PVT  500 mgRPNQ0003
2 gRPNQ0061
25 x 2 gRPNQ0064
WGA PEI Type B  Extra coating for reduced non-specific binding  PVT  500 mgRPNQ0004
2 gRPNQ0062
25 x 2 gRPNQ0065
Polyethyleneimine (PEI)Extra coating for reduced non-specific bindingPVT500 mgRPNQ0097
Poly-L-lysineEnhances binding of negatively-charged membranesYSi1 gRPNQ0010
PDE  Measure of phosphodiesterase activity  YSi  500 mgRPNQ0150
2 gRPNQ0024
25 x 2 gRPNQ0029
RNA binding beadsUncoated Ysi beads that interact with primary phosphate groups in nucleotides (oligos, DNA and RNA); membranes can also bindYSi500 mgRPNQ0013
Arginine binding beadsCapture of arginine residuesYSi500 mgRPNQ0101
Copper chelate Capture and assay of His-tag fusion proteins or their binding partners PVT250 mgRPNQ0095
YSi125 mgRPNQ0096
Glutathione   Capture and assay of GST-fusion proteins   PVT 750 mgRPNQ0030
25 x 2 gRPNQ0036
YSi 50 mgRPNQ0033
500 mgRPNQ0034
Protein A    Binding of antibodies via the Fc portion of the antibody for RIA    PVT  500 mgRPNQ0019
25 x 500 mgRPNQ0031
2 gRPNQ0069
YSi 500 mgRPN143
2 gRPNQ0068
Anti-mouse antibody Capture of mouse antibody PVT500 mgRPNQ0017
YSi500 mgRPN141
Anti-rabbit antibody  Capture of rabbit antibody  PVT 500 mgRPNQ0016
25 x 500 mgRPQ0638
YSi500 mgRPN140
Anti-sheep antibody Capture of sheep antibodies PVT500 mgRPNQ0018
YSi500 mgRPN142
SPA scintillation Select-a-Bead kitReceptor binding studies (contains 100 mg of WGA PVT, WGA Ysi, WGA PEI type A, WGA PEI type B, and Poly-L-lysine beads)PVT and Ysi100 mg of 5 bead typesRPNQ0250

 


SPA imaging beads

CoatingApplicationCore bead typeSizeCat. number
Streptavidin       Capture of biotinylated proteins or peptides for use in enzyme assays or molecular interaction assays       PS    50 mgRPNQ0263
500 mgRPNQ0261
25 x 500 mgRPNQ0285
2 gRPNQ0306
25 x 2 gRPNQ0307
YOx  50 mgRPNQ0273
500 mgRPNQ0271
25 x 500 mgRPNQ0283
Wheat Germ Agglutinin (WGA)       Binds cell membranes and internal components for receptor binding studies with partially purified cell membrane preps or fractionated, solubilized receptor preps by immobilized receptors attached via glycosylation sites       PS    50 mgRPNQ0262
500 mgRPNQ0260
25 x 500 mgRPNQ0281
2 gRPNQ0308
25 x 2 gRPNQ0290
YOx  50 mgRPNQ0272
500 mgRPNQ0270
25 x 500 mgRPNQ0282
WGA PEI Type A PEI treatment decreases non-specific binding to beads PS 50 mgRPNQ0286
500 mgRPNQ0287
WGA PEI Type B PEI treatment decreases non-specific binding to beads PS 50 mgRPNQ0288
500 mgRPNQ0289
Polyethyleneimine (PEI) PEI treatment decreases non-specific binding to beads PS 50 mgRPNQ0297
500 mgRPNQ0098
Poly-L-lysine    Enhances binding of negatively-charged membranes    PS   50 mgRPNQ0295
500 mgRPNQ0294
2 gRPQ0786
25 x 2 gRPQ0787
YOx2 gRPQ0328
Membrane binding beads  Receptor binding studies  YOx  500 mgRPNQ0280
25 x 500 mgRPNQ0296
2 gRPQ0785
Nickel chelate   Capture and assay of His-tag fusion proteins   PS 50 mgRPNQ0267
500 mgRPNQ0266
YOx 50 mgRPNQ0277
500 mgRPNQ0276
Glutathione Binding and assaying GST-fusion proteins YOx 50 mgRPNQ0279
500 mgRPNQ0278
Protein A   Capture of antibodies via the Fc portion of the antibody for RIA; best for rabbit, mouse, or guinea pig   PS 50 mgRPNQ0265
500 mgRPNQ0264
YOx 50 mgRPNQ0275
500 mgRPNQ0274
Anti-mouse antibody Affinity purified IgG designed for capture of mouse antibody PS500 mgRPNQ0298
YOx500 mgRPNQ0300
Anti-rabbit antibody Affinity purified IgG designed for capture of rabbit antibody PS500 mgRPNQ0299
YOx500 mgRPNQ0301
SPA Imaging Select-a-Bead kitReceptor binding studies (contains 500 mg of WGA PS, WGA YOx, WGA PEI type A, WGA PEI type B, PEI PS and poly-L-lysine beads)PS and YOx500 mg of 6 typesRPNQ0321

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Choosing between SPA Scintillation Beads and SPA Imaging Beads


The choice of SPA bead depends on several parameters and these are summarized in Table 2.

Table 2. SPA scintillation beads vs. SPA imaging beads

 

SPA Scintillation Beads

SPA Imaging Beads

Feature

Benefit

Instrument

PMT readers, standard beta counters

CCD Imagers

SPA Scintillation Beads emit in the blue region and SPA Imaging Beads emit light in the red region of spectra

Match bead to instrument type (e.g. CCD imagers are more sensitive in the red region)

Throughput

Medium to high

Medium to ultra high

Choice of throughput

Match instrument and bead choice with throughput needs

Format

tubes; 96- or 384-well format

96-, 384-, 1536-well plates and above

Choice of format

Increase throughput and decrease assay component costs

Emission

Blue region (400 nm)

Red region (615 nm)

Choice of emission properties

Reduce color quench caused by orange/red colored compounds through use of SPA Imaging Beads

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Suitable radioisotopes for SPA beads


The most important characteristic when selecting a radioisotope for SPA is the pathlength of the decay particle (Table 3). In general, the shorter the pathlength of the decay particle, the better-suited is it for SPA.

  • Tritium and Iodine-125 are ideally suited to SPA
  • Carbon-14, Sulfur-35, and Phosphorus-33 have been used successfully with SPA
  • Other gamma emitters (Calcium-45, Rubidium-86, Selenium-75, and Cobalt-65 have all been used successfully in SPA assays).

Table 3. 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

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SPA bead applications


Ligand receptor binding

GTPγS assays

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General SPA FAQs


Q. What liquid scintillation counter window settings do I need to use for counting an SPA assay?
A. For counting SPA assays in a liquid scintillation counter or instruments other than the TopCount counter or MicroBeta counter, set the windows to wide open or count in a ³²P channel. Make certain that you use a blank such as beads plus radioactive tracer, as well as any other assay-specific controls to assess origins of any detectable background.


Q. What is the difference in SPA counting efficiency as compared to liquid scintillation counting?
A. SPA counting is not as efficient as liquid scintillation counting. Typically, counting efficiency of PVT SPA beads will be 40% and YSi SPA beads will be 60% of the expected liquid scintillation counts.


Q. What is the average size of the YSi and PVT SPA beads?
A. The yttrium silicate (YSi) and polyvinyl toluene (PVT) SPA beads range in size from 2-8 µm. The PVT SPA beads average approximately 5 µm in diameter, while the YSi beads are irregular-shaped crystals averaging 2.5 µm in diameter.


Q. How does SPA work with gamma emitters?
A. [125I] and other gamma emitters decay by a process termed "electron capture". This type of decay gives rise to particles named Auger (pronounced 'oh-zhay') electrons and these electrons are detectable by SPA beads. The most common gamma-emitters that have been used in SPA include Iodine-125, Calicium-45, Rubidium-86, Selenium-75, and Cobalt-65.


Q. What is the maximum centrifuge speed I can use to spin down the SPA beads?
A. As a rule of thumb treat the SPA beads as you would cells, spinning at no more than 2,000 rpm in a standard centrifuge without braking. Though higher speed or centrifugal g-forces will not affect the integrity of the SPA bead itself, is not recommended since the molecular interactions of interest may not be stable to such conditions.


Q: How are proteins and other molecules linked or coated onto SPA beads?
A. Proteins or other macromolecules are covalently linked to SPA beads (PVT, PS, YSi, or YOx) in one of two ways:

  1. By direct coupling of proteins to the chemically activated surface
  2. By pre-coating the naked bead with polylysine or polyethyleneimine (PEI), followed by chemical cross-linking of the macromolecule.


Q. What kind of plates do I use with my SPA assays?
A. Most any type of microplate can be used for SPA. However, plates that have high reflective optics and low associated phosphorescence are preferable-these plates are typically opaque white. If you are using a bottom reading instrument, the use of white-walled plates would be best. Additionally, there are plates available that may be used to reduce the non-specific binding of radiolabeled ligand to plate walls which could cause an increase in background signal. They may not be useful for all applications but should be considered as a tool that may reduce the non-specific binding of your labeled compound to the microplate well.


Q. What fluor or scintillator is present in the PVT and YSi SPA beads?
A. The scintillator in the PVT beads is diphenylanthracine (DPA), which is co-polymerized in the polyvinyl toluene matrix. In YSi SPA beads, naturally occuring cerium ions act as scintillators and are stably trapped within the yttrium silicate crystal lattice.


Q. What is meant by the term 'non-proximity effect' and how can it be minimized?
A. A non-proximity effect, or "NPE", is low-level background signal caused by stimulation of SPA beads by unbound radioactivity in solution. NPE generally can occur with higher energy isotopes such as 33P, 35S, and 14C. The potential for NPE can be minimized by maximizing the ratio of solution to beads- mainly by centrifuging or allowing the beads to settle prior to counting, and by increasing the volume of the assay.


Q. What is the mechanism of cell membrane binding to Wheat Germ Agglutinin and poly-lysine SPA beads?
A. Cell membranes couple to the wheat germ agglutinin (WGA) coated SPA beads through interactions between WGA and N-acetylglucosamine residues that are present in glycosylated cell surface proteins. In the case of poly-lysine coated SPA beads, salt-bridges form between the positively charged poly-lysine sites on the bead and the negatively charged lipids that comprise the cellular membrane.


Q. Why are some SPA beads coated with PEI along with WGA?
A. SPA (Scintillation Proximity Assay) beads may be coated with WGA (wheat germ agglutin) alone, or in concert with the agent PEI (polyethyleneimine). The PEI helps minimize non-specific background.

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Custom services


PerkinElmer offers custom radiochemicals, custom SPA beads, custom plate barcoding, and other services. If you are interested in custom services, please contact our custom teams:

ON>POINT® Custom Services

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