Biodrugs 2005; 19 (6): 383-392

TECHNOLOGY 1173-8804/05/0006-0383/$34.95/0

© 2005 Adis Data Information BV. All rights reserved.

Application of Scintillation Proximity Assay in

Drug Discovery
Shaogui Wu1,2 and Bailing Liu1
1 Chengdu Institute of Organic Chemistry, Chinese Academy of Sciences, Chengdu, China
2 Graduate School of the Chinese Academy of Sciences, Beijing, China

Contents
Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 383
1. The Principle of Scintillation Proximity Assay (SPA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 384
2. The Choice of Radioisotope . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 384
3. The Support Materials Used in SPA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 384
4. Scintillant Quenching . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 385
5. Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 386
5.1 Receptor-Ligand Binding Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 386
5.2 Application in Drug Screening . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 386
5.3 Application in Radioimmunoassays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 387
5.4 Application in Enzymatic Activity Measurement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 388
5.5 SPA Based on Other Radioisotopes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 389
5.6 Latest Advances . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 390
6. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 390

Abstract Scintillation proximity assay (SPA), characterized by its speed, sensitivity, reliability, and the fact that no
separation step is required, has become an important technique in high-throughput screening (HTS) for new
drugs, and for investigating their biological interactions. The SPA technique now plays a key role in HTS, in that
it can be used in many assay formats including radioimmunoassays (RIAs), ligand-receptor binding assays, and
enzyme assays. The SPA-based enzyme assay is usually designed in three formats corresponding to different
enzymes: signal removal format for hydrolytic enzymes, signal addition format for polymerase and transferase
enzymes, and product capture format for antibodies, DNA probes, receptors or other specific binding proteins.
The use of SPA in RIAs has been facilitated by new carriers, such as membranes that can be configured in
various shapes and sizes, allowing the assay to be performed on samples from many sources including tissue,
serum, plasma or cells. This review presents the principles of SPA, discusses supporting materials and quenching
effects, as well as detailed examples of the latest advances.

Modern high-throughput screening (HTS) technology for drug
discovery and many other applications is a multidisciplinary field
involving biology, biochemistry, molecular biology, analytical
chemistry, synthetic chemistry, automation engineering, and com-
puter science.[1] Scintillation proximity assay (SPA) is an emerg-
ing technology that enables radioactive assays in high-throughput
format. This assay technique was initially developed by Hart and
Greenwald in 1979[2] and has become a mature and well estab-
lished detection approach in HTS for new drug discovery. Com-
mon therapeutic targets for HTS are enzymes, cell-surface recep-
tors, nuclear receptors, ion channels, and signal transduction pro-
teins. Compounds that interact with these targets are usually
384
identified using in vitro biochemical assays.[3] Significant im-
provements in combinatorial chemistry over the last several years
have enabled a large number of chemical compounds to be synthe-
sized.[4-6] Meanwhile, there has also been a tremendous increase in
the number of possible targets after completion of the Human
Genome Project (http://www.ornl.gov/sci/techresources/
Human_Genome/home.shtml), providing both opportunities and
requirements for high-throughput assays.[7-10] These two factors
have promoted the application of SPA in HTS,[11] and the SPA
technique has been widely applied to radioimmunoassays,[12,13]
receptor-ligand binding assays,[14,15] enzymatic activity as-
says,[15-18] RNA transcript detection,[19] protein-peptide interac-
tions, among others. Beyond all of these, SPA has also highlighted
many possibilities for the design of new detection techniques in
the future. Without doubt, new advances in SPA would further its
applications in HTS and related fields.
SPA is a solid phase homogeneous technique. A major advan-
tage of SPA over the conventional assay techniques is the conve-
nient operation, because no separation step is needed in the whole
process of the assay and the basic assay relies on a ‘mix and read’
format. Consequently, the homogeneous format makes this assay
more simple, robust, and amenable for automation than a
filter-based assay. SPA also offers high detection sensitivity and
can be operated in a 96- or 384-well format with minute sample
volumes (5–150μL). Additionally, automated operation (using
automated pipeting) increases the assay throughput. Therefore,
SPA permits the design of high-flux assay formats for screening a
large number of compounds in chemical collections. In this article,
the basic principles of SPA are reviewed and examples of its
applications in HTS for drug discovery and other purposes are
described.
1. The Principle of Scintillation Proximity Assay (SPA)
When a radioisotope such as tritium decays, it releases high-
energy β particles, which have a limited transferring path-length in
aqueous solutions (1.5μm for 3H and ~17.5μm for 125I Auger
electrons) [figure 1]. If the radioligand is not ‘captured’ onto the
SPA bead (figure 1a), energy from radioactive decay is dissipated
into the aqueous environment and gives no light emission, leaving
the disintegration undetected. Conversely, if the radioligand is
captured on the surface of the SPA bead (figure 1b), the radioiso-
tope is brought into close proximity to the scintillant and the β
particles effectively transfer their energy to the scintillant, result-
ing in light emission that can be detected by liquid scintillation
counters.[3] Hence, the bound and free radiolabeled ligand can be
© 2005 Adis Data Information BV. All rights reserved.
Wu & Liu
detected and no physical separation, such as centrifugation, wash-
ing, and filtration, is necessary, making the operation more conve-
nient and easily amenable to automation than other binding assay
techniques.[20-22]
2. The Choice of Radioisotope
For more than 50 years, biologists have successfully used
radioactive isotopes (3H, 14C, 32P, 35S, and 125I) to study in vivo
metabolism, distribution, and binding of small amounts of com-
pounds.[23] Because their radiation energy is relatively strong, a
tiny amount will meet the requirement for an assay. However,
disadvantages include harmful radioactive rays, impracticality of
long-term storage, and experiments involving compounds with
very short half-lives.
The path length of the β particle is determined by its energy,
and varies for different isotopes (see table I). For example, β
particles from 3H have an average path-length of approximately
1.5μm,[24] which easily meets the distance requirement for SPA.
The Auger electrons emitted by 125I, which have energies in the
same range as β particles and travel between 1 and 17.5μm in
aqueous media, also satisfy the distance requirement. Therefore
3H and 125I are ideal isotopes for labeling ligands in SPA.
Other isotopes of interest, such as 14C, 32P, and 35S, have
longer path-lengths with mean ranges of approximately 58, 126,
and 66μm, respectively, and are less suited to application of the
proximity principle. Although SPA has recently been adapted to
utilize 35S and 32P,[25-28] the use of 14C is limited because the
specific activities of 14C-labeled compounds are generally too low
(approximately 60 ci/mmoL),[20] even though the path-length of
14C is less than that of 35S. Nonetheless, some scientists have
successfully applied 14C in SPA (see section 5.5).
3. The Support Materials Used in SPA
The core of this technique is the support material: the SPA
bead. The most commonly used SPA beads are polyvinyltoluene
a b
β emission
β emission
SPA bead SPA bead
Fig. 1. The principle of scintillation proximity assay (SPA). (a) Free: no
signal generated; (b) bound: signal generated.
Biodrugs 2005; 19 (6)
Application of Scintillation Proximity Assay in Drug Discovery
Table I. Radioactive isotopes in common use
Isotope characteristic 3H 14C
Half-life 12.32 (y) 5730 (y)
Radioactive rays β β
Average energy (keV) 5.7 49
Transmission distance in air (cm) 0.6 22
Path-length in aqueous solution (μm) 1.5 58
(PVT) beads (diameter ~5μm; density ~1.05 g/cm3) and yttrium
silicate (YSi) beads, which are commercially available from
Amersham Biosciences (GE Healthcare).[29-31] High-efficiency
scintillant has been incorporated inside the PVT matrix, which can
be excited by the high-energy β emission generated from the decay
of radioactive isotopes when the SPA bead is within the effective
path length for energy transference.[32] The surface of this SPA
bead is coated with hydrophilic polyhydroxy film that reduces
hydrophobicity of the bead to reduce nonspecific interactions. This
film has been chemically derived to covalently couple generic
capture molecules, which can selectively bind molecules of inter-
est including receptors, proteins, or other disease targets. SPA
beads with the following capture molecules are available commer-
cially: protein A, avidin, streptavidin, wheatgerm agglutinin
(WGA), glutathione, and various polyclonal secondary antibo-
dies.[33,34] All of these capture molecules are routinely used as one
member of a detection-pair system. These beads are easily pipetted
using automated liquid handling devices into 96-well plates and
therefore are easily accommodated in HTS operation. Because
PVT beads have a lower density than YSi beads, they can remain
in suspension longer, which is beneficial for automated HTS.
However, some ligands bind nonspecifically to the surface of PVT
beads and may increase the background noise. The heavier density
of YSi beads allows the beads to settle for bottom counting, which
can reduce nonspecific interference.
Other support materials include scintillating microplates, in
which scintillant is directly incorporated into the polymeric matrix
or coated on the inner surface of the wells.[32,35,36] Two kinds of
these plates, Flash-plate® 1 and Scintistrip-plate® are commercial-
ly available from NEN Life Science Products (Boston, MA, USA)
and Wallac-Oy (Turku, Finland), respectively. When SPA is car-
ried out in these microplates, it is necessary to remove all liquid
from the wells. With an appropriate washing/air-drying procedure
before measuring (but not a ‘mix and measure’ technique) these
plates offer the advantage of minimizing nonproximity effects,
1 The use of trade names is for product identification purposes only and does not imply endorsement.
© 2005 Adis Data Information BV. All rights reserved.
385
32P 35S 125I
14.3 (d) 87.4 (d) 60 (d)
β β Auger electron
690 49 32
7.2 24 2.4
126 66 1–17.5
which contribute to background noise. Therefore, these plates are
also accommodated to SPA based on other long path-length iso-
topes such as 33P, 35S, etc.
4. Scintillant Quenching
Although SPA is an accepted and versatile technique, the
quenching effect is a serious shortcoming. Some chemical or
colored samples absorb a proportion of the light emission from the
scintillator, which causes a severe reduction (quenching) of signal
and subsequently interferes with the efficiency of the scintillation
counter. Two types of quenching occur with SPA: chemical
quenching and color quenching.
Chemical quenching takes place when the energy is transfer-
ring between solvent and scintillator. Some chemical quenching
agents adsorb a part of the energy and give off heat that is then
dissipated in the medium, which reduces the transformation effi-
ciency of energy to light emission. However, the effect of chemi-
cal quenching is slight and can be ignored in many conditions
(such as in YSi-based SPA).
Color quenching takes place when light emitted by the stimulat-
ed scintillator is absorbed or dissipated by the colored solution.
Most colored or fluorescent compounds, especially dyes, have
absorption spectra that overlap the emission spectra of the scintil-
lant, which results in quenching of the light emission.
The degree of quenching can be calculated by analyzing the
quenched and unquenched samples. The standard curve can be
made from a series of standards of the same activity but with
different quenching levels. Therefore, it is possible to compensate
for and correct the quenching effect. A color quench correction
program can be applied to automatically overcome this issue by
the appropriate scintillation counter. If some compounds with
false positive activity are picked up by SPA, further assays, such
as absorbance or filtration-based radioactive assays, will be neces-
sary to eliminate them and identify the genuine ‘hits’.
Biodrugs 2005; 19 (6)
386 Wu & Liu

a
The ligand binding and protein coating are achieved by mixing
ligand, protein, and SPA beads in sequence. No additional steps
β emission
such as pre-coating and washing procedures are required. For the
+
purpose of measuring the quantitative relation in interactions,
equation 1 was derived to accurately determine the binding affini-
ty for non-tight and tight binding inhibitors. For non-tight binding
Receptor Radioligand
inhibitors, the binding affinity Ki can be calculated from its
relation to IC50 (the concentration of inhibitor at which 50% of the
radioligand binding is reduced).
+ IC50
Ki =
1 + L/KL
Inhibitor (Eq. 1)
For tight binding inhibitors, Ki can be obtained by fitting a plot
b of RL/RL0 versus [I0] in light of equation 2:
R+L RL RL − (Kiapp + I0 − R0) + (Kiapp + I0 − R0)2 + 4 × R0 × Kiapp
+ KL =
I RL0 2 × R0
Ki (Eq. 2)
RI where = Ki × (1 + L0/KL).
Kiapp
Fig. 2. Schematic diagram of a competitive binding assay (reproduced This quantitative assay provided an important theoretical basis
from Sun et al.,[39] with permission from Elsevier). I = inhibitor; Ki = equilibri-
for high-throughput assays to screen chemical structures in a
um dissociation constant for I; KL = equilibrium dissociation constant for L;
L = radioligand; R = receptor; RI = inhibitor-bound receptor; RL = radioli- chemical library.
gand-bound receptor.
5.2 Application in Drug Screening
5. Applications
The SPA technique has been used successfully in HTS for drug
discovery. Many novel chemical structures have been identified as
5.1 Receptor-Ligand Binding Assay ‘hits’ or active molecules, which can be developed into potential

Conventional receptor-ligand binding assays are often complex
and time consuming,[23,37,38] but the application of SPA can greatly
facilitate this type of assay. Receptors are directly immobilized
onto the surface of SPA beads via a number of coupling approach-
es. When a suitable radioligand binds to a receptor, it will be in
proximity to stimulate the bead to emit light. Unbound ligands are
too distant from the beads to transfer their energy and thus are not
detected. Sun and colleagues[39] described an optimization of a
generic binding assay to determine the ligand-receptor interactions
based on SPA. They immobilized peroxisome proliferator-activat-
ed receptors (PPARs) on the surface of SPA beads, and light
emission was generated by excitation of the scintillant inside the
bead as a result of binding of a radiolabeled ligand to immobilized
PPARs. The intrinsic binding affinity of unlabeled ligands is
determined by competitive displacement of the radioligand (figure
2a). The binding equilibrium of receptor, radioligand, and inhibi-
tor can be established as shown in figure 2b.
[3H]-dihydronovobiocin
hv
GyrB43
β emission
SPA bead
*
Av-Bio
Fig. 3. Schematic diagram of the gyrase B (GyrB) subunit scintillation
proximity assay (SPA). An ATP-binding site is located in GyrB43 and both
coumarin and cyclothialidine antibacterials act through binding to it. When
radiolabeled inhibitors such as [3H] dihydronovobiocin bind to this site, the
radioisotope is in proximity to the SPA bead, and the scintillant inside the
bead is excited by the β particles from the radioactive decay to emit light. A
scintillation counter can be used to record the emission signal (reproduced
from Gevi and Domenici,[50] with permission from Elsevier). Av-Bio = avi-
din-biotin; hv = photon emission.

© 2005 Adis Data Information BV. All rights reserved. Biodrugs 2005; 19 (6)
Application of Scintillation Proximity Assay in Drug Discovery
Membrane containing
secondary antibody
[125I]cAMP
cAMP
2° antibody
1° antibody
Fig. 4. Principle of scintillation proximity radioimmunoassay with micro-
porous membranes. A secondary antibody is immobilized in the pore sur-
face of the membranes, onto which a primary antibody was bound. When a
radioligand such as [125I] cyclic adenosine monophosphate (cAMP) binds
to the primary antibody, the scintillant inside the polymeric membrane is
stimulated by the radioactive rays to emit light that can be measured easily
by a liquid scintillation counter (reproduced from Mansfield et al.,[13] with
permission from Elsevier).
drugs with a desired therapeutic activity, such as agonist, antago-
nist, antibacterial, etc.[40-45] Recently, much attention has been
focused on the development of novel coumarin and noncoumarin
antibacterials.[46-48] Bacterial DNA gyrase is the site of action of
naturally occurring antibacterials, including the broad-spectrum
coumarin antibiotics and the cyclic peptide inhibitors. They both
act through binding to a site located on the gyrase B (GyrB)
subunit and consequent inhibition of the adenosine triphosphatase
(ATPase) reaction. The principle of the GyrB SPA is illustrated in
figure 3. A radiolabeled ligand such as [3H] dihydronovobiocin
binds to GyrB, which is in turn captured by the SPA bead via the
streptavidin-biotin (Av-Bio) system. The radioactive decay is in
proximity of the bead, which results in the excitation of the
scintillant to emit photons that can be measured by a scintillation
counter. From this assay, IC50s of 42, 64, and 11nM were obtained
for novobiocin, dihydronovobiocin, and the cyclothialidine analog
GR 122222X, respectively, which is consistent with the results
from other methods.[49] However, the SPA technique is rapid,
robust, and reliable compared with conventional HTS techniques.
This assay can be conducted in 96-well (384-well or more) micro-
titer plates and no additional separation steps are required, which
greatly facilitates drug discovery efforts and is amenable for
automated HTS of large chemical collections for novel drugs.
© 2005 Adis Data Information BV. All rights reserved.
387
5.3 Application in Radioimmunoassays
The application of the SPA technique to radioimmunoassays is
referred to as the scintillation proximity radioimmunoassay
(SPRIA). Characterized by its speed, simple operation, accepted
precision, and preferable reproducibility, SPRIA has inherited
many advantages of SPA. It is apparent that this high-throughput
homogeneous assay is superior to the conventional radioimmu-
noassay that uses ammonium sulfate precipitation as the separa-
tion method. SPRIAs are amenable to automation and processing
of a large number of samples. Such assays may be applied in place
of traditional separation-based radioimmunoassays. Tadepalli and
Quinn[51] applied this technique to quantitate acyclovir in samples
from pre-clinical and clinical studies after the administration of
valaciclovir (a prodrug of acyclovir).[51] Serres et al.[52] used this
technique in determination of phosphrothioate antisense
oligonucleotide plasma concentrations.
SPA beads are also the main supporting materials currently
used in SPRIAs. Recently, Mansfield et al.[13] reported a new
SPRIA based on the use of microporous membranes (figure 4).
Organic or inorganic fluors were incorporated into the matrix of
microporous polymeric membranes. A secondary antibody was
immobilized onto their surfaces prior to use. These membranes
were employed in competitive binding assays in which a separa-
∗
Cleavage site
SPA bead
+ Protease + Protease
inhibitor
SPA bead
Av-Bio
+
∗
Fig. 5. Schematic diagram of the principle of scintillation proximity assay
(SPA) used in detecting human cytomegalovirus (HCMV) protease inhibi-
tor. The peptide substrate (the thick horizontal black line) is attached to the
SPA bead through a streptavidin-biotin (Av-Bio) system, which contains a
cleavage site (black rectangle). The peptide is phosphorylated with radio-
labeled adenosine triphosphate (ATP) at the serine residue near its car-
boxyl terminus. The scintillant inside the SPA bead is stimulated by the
radioactive decay and a high radioactive signal can be detected. The
cleavage of the peptide by HCMV separates the phosphate label from the
part related to the SPA bead and the counts per minute (CPM) will de-
crease. However, the presence of protease inhibitors can prevent this
cleavage. The radioactive signal changes can be used to detect protease
inhibitors (reproduced from Baum et al.,[25] with permission from Elsevier).
Biodrugs 2005; 19 (6)
388 Wu & Liu

tion procedure was not necessary for bound and unbound radioli-
gands.[13] The results were nearly identical to those obtained using
the standard radioimmunoassay, although less time was used and
the assay procedure was simplified greatly. This membrane-based
SPRIA is applicable in the detection of various radioanalytes and
can be configured in a number of sizes and shapes.
5.4 Application in Enzymatic Activity Measurement
Conventional approaches for quantitating enzymatic activity
are often time consuming, labor intensive, or not sensitive enough,
especially for high-throughput studies. These limitations prompted
the development of a new, versatile, simple, solid-phase technique
based on SPA. SPA has been a routine tool used in enzymatic
activity measurement and is well documented in the litera-
ture.[20,53-55] For example, Zhang et al.[56] reported a 96-well homo-
geneous SPA for the study of DnaB-stimulated Escherichia coli
primase activity and the identification of E. coli primase inhibi-
tors.[56] Macarron et al.[57] used SPA in the measurement of the
aminoacyl-tRNA synthetase aminoacylation activity.[57] They
found it was faster and more amenable to HTS than traditional
approaches.
In SPA enzyme assays, the conversion of the substrate to
product is monitored by either removal or addition of radioisotope
to the component, which is captured on the SPA bead. Three basic
formats of SPA enzyme assays have been developed: signal re-
moval, signal addition, and product capture. In the first format
(signal removal), the radiolabeled substrate is biotinylated to the
SPA bead, which results in the scintillation of the bead. When the
catalysis of a specific enzyme separates the radioactive moiety
from the biotinylated portion of the substrate molecule, the light
signal will decrease. This assay is suitable for hydrolase activities
such as those of proteases, nucleases, phospholipases, and ester-
ases.
In the signal addition format, which is suitable for molecular
transfer activities (e.g. transferases, polymerases, and kinases), the
acceptor substrate is biotinylated and the donor substrate is radio-
labeled. The radiolabel is transferred from the donor substrate to
the receptor substrate, which is in turn captured by the SPA bead
and results in the generation of a signal.
The last assay format is product capture, in which the product
generated from the action of the enzyme is captured via bio-
specific recognition by antibodies, DNA probes, receptors, or
other specific binding proteins on the beads. The antibody or probe
must be able to discriminate adequately between the generated
product and the substrate. The choice of the format and detailed
design for the enzymatic activity assay are in accordance with
actual conditions such as the properties of the enzyme and corre-
sponding substrates.

Ph O Ph

R
OH
ST1 R= O ST10 R= O

ST11 R= OH
ST2 R= O O
Et
OH
ST3 R= O ST12 R=
N Et Br
OH Et
ST4 R= O
Et
ST13 R= O
N H Br
ST5 R= O
Et
OH O N
ST14 R= O
O
ST6 R= O O
O
O

ST7 R= O

O OH O
ST8 R= O
O ST15 R=
O O
O
ST9 R= O NH 2
O ST16 R= N
H
Fig. 6. Chemical structures of membrane-soluble scintillators (reproduced from Culliford et al.,[65] with permission from Elsevier). ST = structure.

© 2005 Adis Data Information BV. All rights reserved. Biodrugs 2005; 19 (6)
Application of Scintillation Proximity Assay in Drug Discovery 389

Liposomes
Scintillant molecules

Incorporation

Incubation with
Fusion
HeLa cells
HeLa cell membrane

Influx/incorporation Efflux assay Radioligand binding

assay

Scintillant molecule
Light emitted from scintillant
Movement of substrate
Radiolabeled substrate/ligand
Monolayer of cultured cells

Fig. 7. Schematic diagram of the principle of cell-based scintillation proximity assay following application of membrane soluble scintillators (MSS) to
cultured cells (reproduced from Culliford et al.,[65] with permission from Elsevier). The scintillation of MSS cannot occur in an aqueous environment but can
proceed in a non-aqueous environment (such as lipid bilayer) in the presence of a radioactive decay. MSS can be incorporated into liposomes and then
fused into the membrane of HeLa cells. Therefore, the radiolabeled ligand or substrate in close proximity to the cell membrane (e.g. when the [14C]-
methionine is taken up by the HeLa cells) will cause the light emission of scintillator in the cell membrane, which can be monitored by a scintillation counter.
This system can be used in radiochemical and radioligand binding assays either in vivo or on microsomal preparations obtained from tissues.

5.5 SPA Based on Other Radioisotopes
The path-lengths of the β emissions for 33P and 35S are 126 and
66μm, respectively, which seem less suitable for the application in
SPA than 3H (1.5μm) and 125I (~17.5μm), because longer path-
lengths will often bring about the ‘no-proximity effect’ and se-
verely reduce the signal-to-noise ratio. However, Baum et al.[25]
successfully applied the SPA technique in detecting human
cytomegalovirus (HCMV) protease UL80 inhibitor using 33P.[25]
This is the first report that 33P is indeed compatible with the SPA
system. The peptide substrate used in the assay contains an HCMV
protease cleavage site and is biotinylated to the SPA bead at its
amino terminus (figure 5). The peptide was also phosphorylated
with radioactive ATP at its carboxyl terminus. The peptide is
incubated with protease prior to binding to SPA beads. Cleavage
of the peptide by the HCMV separates the radiolabel from the SPA
bead-related moiety and results in a concomitant decrease in the
radioactive signal, which is prevented by the presence of a prote-
ase inhibitor. This new SPA is amenable to other proteases with a
known cleavage site.
The path-length of 35S emission is 66μm, which is more
applicable for SPA than the 126μm path length of 33P. 35S-based
SPA seems to be more common than that of other radioactive
isotopes except 3H and 125I.[58-60] Delapp and colleagues[61,62]
reported an antibody-capture [35S] GTPγS SPA and used it in the
analysis of G-protein-coupled receptors (GPCRs) pharmacolo-
gy.[61,62]
Although the specific activities of 14C-labeled compounds are
generally too low, reports about 14C-based SPA have appeared
frequently in the literature in recent years. For example, Williams
and coworkers[63] reported the application of 14C-based SPA for

© 2005 Adis Data Information BV. All rights reserved. Biodrugs 2005; 19 (6)
390
analysis of Na+/Cl–-dependent neurotransmitter transporter activi-
ty. Mattingly et al.[64] developed a new membrane-based SPA for
detecting and quantifying 14CO2.[64] They used a polymeric mem-
brane, which could simultaneously capture and quantify 14CO2
successfully, as the support material. Compared with traditional
detection approaches for 14CO2, it exhibited obvious advantages
such as easy operation, low cost, and minimization of radioactive
waste.
5.6 Latest Advances
Further advances in SPA are continuously occurring. A novel
cell-based SPA for studying protein function and activity in vi-
tro was reported by Culliford et al.,[65] Chapman et al.,[66] and
McCairn et al.[67] They used membrane-soluble scintillants
(MMS), which have a scintillant ‘head’ group (2,5-diphenylox-
azole) and a lipophilic ‘tail’ (figure 6). In the presence of a
radioactive source, these MSS have no scintillant activity in an
aqueous environment, but do in a non-aqueous environment, such
as a lipid bilayer (e.g. liposome or cell membrane). MSS li-
posomes are incorporated and fused with the plasma membranes
of cells in culture and can be excited by radiolabeled molecules in
close proximity to the cell membrane to elicit a scintillation signal.
This system has been used to successfully monitor [14C] methio-
nine uptake in HeLa cells, and may be used in radiochemical and
radioligand binding assays either in vivo or on microsomal prepa-
rations obtained from tissues (figure 7). This is obviously a great
technical advance in SPA, since even the live cell can be used as
the support material and no protein purification is needed. In other
words, this new improvement not only further simplifies the SPA
procedure, but also effectively avoids the inactivation that often
results from purification and immobilization to the disease target/
receptor. It can be used in situations where the assays should be
performed in a cellular environment to ensure the activity of
proteins. This methodology has been successfully employed in
radioligand-binding studies and in a variety of flux and uptake
assays and incorporation assays. This technique is clearly a prom-
ising development in the utilization of SPA.
6. Conclusion
SPA plays an important role in HTS, radioimmunoassays,
ligand-receptor binding assays, enzyme assays, and so on. Howev-
er, SPA has many shortcomings. For example, SPA is a costly
assay (including the expensive SPA reagents) and scintillation
cocktails maybe radioactive and toxic. Disposal of experimental
waste is also costly and environmentally unfriendly. Issues that are
© 2005 Adis Data Information BV. All rights reserved.
Wu & Liu
yet to be resolved include how to reduce the nonspecific binding
and increase the signal-to-noise ratio so as to enhance the reliabili-
ty of the detection result, how to avoid the use of radioisotopes,
and how to protect the protein/enzyme (acceptor or donor) from
inactivation due to immobilization or labeling during the assay.
Currently, development of non-isotopic detection systems, cell-
based assays, and low-cost assays are concentrated on resolving
these disadvantages. It is anticipated that the rapid progress of
SPA will combine with the latest advances in scientific technology
to continuously improve this technique and expand its applica-
tions.
Acknowledgments
This project was supported by the National Natural Science Foundation of
China (20574071).
The authors have no conflicts of interest relevant to the contents of this
article.
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Correspondence and offprints: Professor Bailing Liu, Chengdu Institute of
Organic Chemistry, Chinese Academy of Sciences, Chengdu 610041, China.
E-mail: Blliuchem@hotmail.com
Biodrugs 2005; 19 (6)