Current Pharmaceutical Biotechnology, 2000, 1, 265-281 265

A Multi-Modality Assay Platform for Ultra-High Throughput Screening

A. Fowler*, I. Davies and C. Norey

Amersham Pharmacia Biotech UK Limited, Amersham Place, Little Chalfont, Buckinghamshire,

England, HP7 9NA.

Abstract: The demand for increased throughput during primary screening using less
reagents is changing the way of drug discovery. Searching for hits using high throughput
screening in 96-well format plates is being replaced by the use of higher density plates,
such as 384 and 1536-well formats. The analysis of radiometric assays by scintillation
counters is becoming limiting since only 12 wells can be counted at a time. Charged
coupled device (CCD) camera based instruments, that image the whole plate in one
exposure, speed up detection and are compatible with any microplate footprint.
Researchers are also demanding a choice of detection methods, including fluorescence, luminescence and
radioactivity, and require imagers suitable for all applications. LEADseeker Homogenous Imaging System
is a multi-modality platform offering imaging technology and assay toolboxes for radiometric, fluorescent and
luminescent based assays. LEADseeker allows the very rapid analysis of high density formats enabling ultra-
high throughput screening of a range of biological assays. Research areas that can be studied using this
system include enzyme assays, receptor binding and molecular interactions.

INTRODUCTION imaging times are up to 30 seconds dependent on

The drive for assay miniaturization and the
move towards a choice of detection methods for
high throughput screening (HTS) is changing the
way researchers are detecting assays. Throughput
of miniaturized radioactive assays is currently
limited by scintillation counters since they can
only count up to 12 wells at a time, resulting in
count times of approximately 40 minutes per 384-
well plate, causing bottlenecks in HTS. Even with
the short read times (<1 second/well) used for
luminescent assays, the total time to read a whole
plate is just under 5 minutes. For shorter-lived
substrates such as horse radish peroxidase (HRP)-
luminol, the luminescence signal may change
substantially in five minutes, adding to intra-well
variation and normalisation problems. These
problems may be overcome by the use of imagers
that have the potential to image the entire plate at
once with the resolution required for any number
of wells. The exposure time on LEADseeker for
luminescence applications is usually less than 1
minute and more commonly less than 20 seconds
per plate. Radiometric assays are commonly
imaged for 5 minutes, for fluorescent assays,
*Address correspondence to this author at the Amersham Pharmacia
Biotech UK Limited, Amersham Place, Little Chalfont, Buckinghamshire,
England, HP7 9NA; Tel: +44 29 20526417; Fax: +44 29 20526474; Email:
leadseeker@eu.apbiotech.com
the particular application. Another potential source
of inaccuracies is the variation in sensitivity
between the different photomultiplier tubes
(PMTs) in the same instrument, resulting in
variable data being observed in wells emitting the
same amount of light. This is usually overcome by
normalisation against reference plates. Despite
this, statistical errors between readings given by
individual PMTs will inevitably occur,
contributing to instrument noise. Imager systems
are not subject to these problems because light
from all the wells in the plate is received
simultaneously.
LEADseeker, from Amersham Pharmacia
Biotech, is a multi-modality assay platform which
is capable of imaging radiometric, fluorescent and
luminescent based assays.
MULTI-MODALITY IMAGING PLATFORM
LEADseeker homogeneous imaging system
consists of a cooled charged coupled device
(CCD) camera, cooling unit, telecentric lens,
automated excitation and emission filter changers,
tungsten halogen excitation light source, X, Y and
Z plate mechanism and tip/tilt/rotate camera
movements for automated focusing. The
instrument can be used in three modes: as a

1389-2010/00 $25.00+.00 © 2000 Bentham Science Publishers Ltd.

266 Current Pharmaceutical Biotechnology, 2000, Vol. 1, No. 3 Fowler et al.

manual system for assay development, as a

workstation (Fig. 1) containing a microplate
carousel and stacking system capable of
containing one hundred and twenty 384-well
plates or one hundred and thirty nine 1536-well
plates, or as a fully automated system, for
integration into automatic systems by an
anthropomorphic robotic arm. In this format,
LEADseeker is compatible with all major robotic
systems, including Aurora, CRS, Beckman,
Zymark, Robocon and Scitech. For use with either
of the automated systems, plate identification is
via a bar code reader.
The camera, from Spectral Instruments,
contains a grade 1, thinned, back-illuminated CCD
chip mounted in a light tight case. The chip is
thinned to increase sensitivity to relatively low
light output assays such as radiometric proximity
assays that require the use of low energy isotopes. Fig. (2). Variation of dark noise (integrated optical density
The chip has an imaging area of 24.6mm x (IOD) units) with temperature. Noise levels are taken from 1
minute exposures at a binning factor of 3x3, using an area
24.6mm, at 1024 x 1024 pixels to enable high equivalent to one well of a 384-well plate.
resolution imaging of microplates. As well as
being sensitive to light, CCD chips are also
sensitive to heat. To reduce this thermal noise, the elements and collects parallel light from samples
operating temperature of the chip is cooled to - in all wells of a microplate. This removes parallax
100°C under vacuum by a proprietary refrigerant errors by collecting light equally from all wells
gas. Dark noise is therefore substantially reduced with no shading (Fig. 3 and Fig. 4).
(Fig. 2) and any remaining noise can mostly be
attributed to electronic read-out noise. In LEADseeker, as with any other CCD based
system, it is necessary to account for the variation
Standard lens types suffer from parallax error in response across the chip. In order to facilitate
where light is distorted and shadowing can occur multi-modality operation, two correction methods
in wells that are not at the centre of the imaging are available. The “Flat Field Correction” (FFC)
field. These problems can be avoided by using a method is an image correction method. A plate
telecentric lens. In LEADseeker, the telecentric containing a constant level of signal is imaged
Borealis™ lens is made up of multiple lens (Fig. 5a) and the differences in intensity observed

Fig.(1). Automated multi-modality LEADseeker imager workstation.

A Multi-Modality Assay Platform Current Pharmaceutical Biotechnology, 2000, Vol. 1, No. 3 267

Fig. (3). Schematic representation of standard and telecentric lenses.

Fig. (4). Images showing the amount of parallax error obtained with standard and telecentric lenses. The image on the left
shows a 96-well microplate viewed through a standard lens. The white crescent shapes around the edges of each well are the
well walls. Only in the centre of the plate can the bottoms of the wells be completely seen. With a telecentric lens (image on
right), the well bottoms are completely visible in all areas of the plate.

across the whole field are then normalised by the
software in subsequently imaged test plates. This
has been shown to be appropriate for both
radiometric and luminescence work. The “Well By
Well Correction” (WBWC) method is a data
correction procedure and is recommended for
fluorescence applications because of the added
complication of both excitation light delivery and
cross chip detection variation. Briefly, successive
calibration plates, containing uniform and different
known levels of signal in each plate, are imaged
and the data obtained for each signal level is used
to construct a calibration curve for each separate
well (Fig. 5b). Determinations from the wells of
268 Current Pharmaceutical Biotechnology, 2000, Vol. 1, No. 3
Fig. (5). Calibration methods. a) Whole field image derived from a LEADseeker 384-well radiometric reference plate,
containing equivalent signal in each well. The different colours represent the variation in CCD response across the field. The
FFC accounts for this variation in subsequent test images. b) An example of a WBWC calibration curve derived for a single
well of a 384-well plate, constructed using a range of three Cy3 fluor concentrations and a buffer blank.
subsequent test plates are then related to the
respective well calibration curve to determine a
normalised read value.
REAGENT TOOLBOXES
As part of a multi-modality platform, assay
reagents are necessary for radiometric, fluorescent
and luminescent technologies. Radiometric assays
suitable for high throughput imaging are based on
scintillation proximity assays (SPA) [1]. In
proximity assays, scintillant-containing beads are
chemically treated on the surface to enable the
coupling of molecules such as enzyme substrates
and receptor proteins. Low to medium energy
isotopes, such as 3H, 125I, 33P and 35S, are used in
such assays, so that when a radiolabelled molecule
binds to a bead via interaction with a biochemical
or cellular target molecule, the energy emitted
stimulates the scintillant within the bead to emit
light. This emitted light can be imaged using a
CCD camera. Unbound radioactive molecules are
not close enough to stimulate the beads to emit
light. Since such molecules are not detected in the
assay, there is no need to remove them from the
assay, thus eliminating time-consuming separation
steps. Since CCD chips are most sensitive to light
emitted in the red region of the spectrum,
europium-containing beads have been developed
for use with LEADseeker [2]. Polystyrene (PS)
and yttrium oxide (YOx) beads are available with
several bead coatings: streptavidin, wheat germ
agglutinin (WGA), protein A and nickel-chelate
for use with his tags.
Fowler et al.
An inherent problem of homogeneous assays is
colour quenching. The majority of compounds that
cause colour quenching are yellow or brown in
colour, and therefore, absorb light in the blue
region of the spectrum. Since PMT-based
scintillation counters are most sensitive to blue
light, protocols have to be incorporated to correct
for quenching. This quenching effect is less of a
problem with red-emitting LEADseeker beads
(Fig. 6).
To miniaturise low signal 384-well plate assays
and develop assays in 1536-well plates with
reduced quantities of reagents, the detection limit
of the imager is important. Using tritium-labelled
biotin bound to LEADseeker imaging beads, the
minimum detectable limit (MDL, defined as 3 x
background standard deviation) can be calculated
using PS and YOx beads in 384 and 1536-well
plates (Fig. 7). In 384-well plates for yttrium oxide
beads, the MDL is 215dpm, which is equivalent to
2.5fmol biotin at the specific activity of
40Ci/mmol. In 1536-well plates, the MDL is
54dpm, equivalent to 625amol biotin at the
specific activity of 40Ci/mmol. For PS beads, the
MDL’s are higher (430dpm, 5fmol and 215dpm,
2.5fmol for 384 and 1536-well plates,
respectively), due in part to the inherently lower
light output of these beads, but also to the small
amount of background phosphorescence that is
apparent in the images. Detection limits of these
orders should make it possible to image assays
with very low signal levels in 384-well plates and
to miniaturise many assays into 1536-well plate
formats with reduced quantities of reagents.
A Multi-Modality Assay Platform Current Pharmaceutical Biotechnology, 2000, Vol. 1, No. 3 269

Fig. (6). Quench effect of coloured dyes with SPA (Yttrium silicate (YSi) /Polyvinyltoluene (PVT)) and LEADseeker
(YOx/PS) beads. Beads were labelled with [3H] biotin in a 384-well microplate and imaged for 10 minutes. Dye concentrations
range from 0 to 3mg/ml in a tripling dilution series from bottom to top. The false colour images show that YOx and PS beads
do not alter in intensity in the presence of tartrazine, but that YSi and PVT SPA beads are both quenched. All four bead types
are quenched by the blue dye.

Fig. (7). Images of 384 and 1536-well plate dilution series. Each row contains [3H] biotin, as indicated, coupled to 0.1mg
streptavidin coated PS or YOx bead in 10mM Hepes, pH 7.5. The final volume in 384-wells was 20µl and the plate was imaged
by coincidence averaging, 2 x 10 minute exposures, using a binning factor of 3x3. For 1536-well plates, the total volume was
4µl and a binning factor of 2 x 2 was used.

An attractive alternative to the use of available from several suppliers (such as

radioactivity in high-throughput screening has
been the use of fluorescence technologies [3].
These approaches necessitate the labelling of one
or more of the assay components with a
fluorescent species. This in itself generally
requires that the fluor contain an active group for
conjugation purposes and also that it has other
chemical/physical properties that are compatible
with the biological assay. Such reagents are
Molecular Probes), other examples include the
family of CyDye fluors and quenchers available
from Amersham Pharmacia Biotech (Table 1).
Cyanine dyes were first synthesised in the mid
1800’s for the photographic industry as spectral
sensitisers or desensitisers for silver halide
emulsion films [6]. However, in recent years,
interest in these dyes for high technology
270 Current Pharmaceutical Biotechnology, 2000, Vol. 1, No. 3 Fowler et al.

Table 1. LEADseeker Fluorescent Reagents: CyDye Probe Series and Properties. Values for Extinction Coefficients
and Quantum yield in Phosphate Buffered Saline (PBS) are reproduced from References [4,5]

Fluor Formula weight Excitation maxima Emission maxima Lifetime Extinction coefficient Quantum yield
(M-1cm-1)

Cy3 766 548nm 562nm <0.3ns 150,000 [4] 0.04 [4]

Cy3.5 1102 581nm 596nm 0.5ns 120,000 [5] 0.15 [5]

Cy5 792 646nm 664nm 1.0ns 250,000 [4] 0.28 [4]

Cy5.5 1128 673nm 692nm 1.0ns 190,000 [5] 0.23 [5]

Cy3B 658 558nm 572nm 2.8ns - 0.67

Cy5Q 907 644nm - - - -

Cy7Q 933 739nm - - - -

applications such as optical data storage [7] and
fluorescent labelling probes for biological studies
[8] has increased. They are characterised by a
resonance structure comprising a polymethine
chain bearing two terminal nitrogen atoms and an
overall positive charge. Modification of the
heterocycle and/or methine chain allows for dyes
to be synthesised which have a range of excitation
and emission wavelengths [9], with a Stokes’ shift
of approximately 15nm. Presently, CyDye probes
have excitation and emission wavelengths ranging
between 560nm-700nm (Fig. 8).
Cyanine dyes have large extinction coefficients,
fluorescent lifetimes in the region of 1-3ns and are
generally detected away from the natural
autofluorescence of biomolecules. They have also
been found to be insensitive to many relevant
chemical environments such as pH and DMSO.
However, in some circumstances, significant
excitation energy can be lost through the dyes
interconverting into a number of stereoisomeric
conformations and also by other non-fluorescent
pathways, such as rotational and translational
modes of vibration [10]. If the relaxation pathways
of the molecule could be restricted, the preferred

Fig. (8). Emission maxima of different CyDye fluors.

A Multi-Modality Assay Platform
route would more likely be via fluorescence. Cy3B
(Fig. 9) contains a tricyclic system across the
polymethine chain which fixes the chromophore,
preventing flexibility within the molecule.
Consequently, it is found to be approximately 3
times as intense on labelling of a biological
conjugate when compared with Cy3. In addition,
the molecule has an increased fluorescence
lifetime of 2.8ns compared with Cy3 (which has a
lifetime of <0.3ns). This increase in lifetime
suggests the potential for use in fluorescence
polarization assays as a replacement for
fluorescein.
HO3S
O biological entity such as streptavidin. This can be
C O
N N
O
Fig. (9). Cyanine probes: Cy3B (N-hydroxysuccinimide
(NHS) ester).
Another common assay approach, particularly
in the field of protease cleavage, is the use of a
dual labelled peptide substrate, incorporating both
a fluorescent “Donor” and non-fluorescent energy
acceptor, or “Quencher” [3]. In order to optimise
such an approach with the CyDye fluors (as the
donor), a family of CyDye quenchers (or “dark”
dyes) have been developed (Fig. 10).
The presence of a dinitrobenzyl moiety may
allow for orientation of the benzyl ring in relation
to the chromophore which could allow for either
HO3S
N+ N N+
O2N
NO2
O O
N O
A. CY5Q (NHS ester)
Fig. (10). Cyanine probes: Quenchers.
Current Pharmaceutical Biotechnology, 2000, Vol. 1, No. 3 271
collision quenching or the generation of radical
anion pairs [11]. From a range of hybridization
experiments (data not shown) it has been found
that use of these CyDye quenchers with a CyDye
donor can be compared to and considered more
efficient than some other donor dyes with Dabcyl
quencher systems.
A further alternative to radiometric assays is the
use of chemiluminescence. Chemiluminescent
reactions generally involve the formation of a
metastable intermediate which upon
decomposition, gives out visible light. A
chemiluminescent molecule serves as the substrate
for an enzyme, typically horseradish peroxidase or
O alkaline phosphatase, that is usually linked to a
N used to report on the binding of streptavidin to a
biotinylated moiety, such as a peptide or
O oligonucleotide.
Light output profiles of chemiluminescent
substrates vary considerably with the enzyme-
substrate combination in use. The light output
profile of horseradish peroxidase reaches
maximum level quickly but also deteriorates
quickly. Alkaline phosphatase substrates tend to
reach a maximum level after 20-40 minutes but
may then persist at or near this level for several
hours. This is illustrated in the data shown in (Fig.
11).
Short-lived substrates such as HRP-luminol are
ideally suited for imaging since the whole plate is
imaged in 20-60 seconds. This results in less error
due to signal decay compared to counting on a
PMT based system where counting a plate may
take as long as five minutes. Wavelength shifters
may also be incorporated in reaction mixtures that
SO3H HO3S SO3H
N
NO2
NO2
O
O
O N O
B. CY7Q (NHS ester)
272 Current Pharmaceutical Biotechnology, 2000, Vol. 1, No. 3
Fig. (11). Comparison of signal duration from chemiluminescent alkaline phosphatase (AP) and horse radish peroxidase (HRP)
substrates. Data was collected by making repeat 30 second exposures on LEADseeker over 2 hours incubation at ambient
temperature.
alter the wavelength of emitted light from
(typically) 420nm to 500-600nm. These
particularly suit the sensitivity range of CCD-
based detection systems, which have better
sensitivity towards the red region of the spectrum
[12]. Substrates that naturally emit longer
wavelength light have been reported [13].
ASSAY APPLICATIONS SUITABLE FOR
IMAGING
Imaging systems can be used to study a range
of applications including enzyme assays, receptor
binding studies and molecular interactions.
PROTEASE ASSAYS
An example of a protease assay suitable for
imaging utilises the metallo-endoproteinase, Asp-
N. Asp-N is often used for peptide mapping and
protein sequencing. The enzyme cleavages an 8-
mer peptide on the N-terminal side of aspartic acid
(D) [14]. The assay has been configured in a
quench format in which the N-terminus of the
peptide has been labelled with Cy3B and the C-
terminus with Cy5Q. Therefore, prior to protease
cleavage the intact peptide cannot generate a
fluorescent signal at Cy3B emission wavelengths
(following excitation with light at Cy3B
wavelengths) due to quenching by Cy5Q.
Proteolytic digestion of the peptide releases the
quench effect, so excitation at Cy3B wavelengths
results in the concomitant emission at Cy3B
Fowler et al.
wavelengths (Fig. 12). This assay format has also
been adapted for use with other proteases.
Assays were carried out in black opaque 384-
well or 1536-well microplates by adding substrate
peptide, Cy3B-YVADAPVK-Cy5Q to a final
assay concentration of 100nM in a volume of 50µl
(384-well assay) or 5µl (1536-well assay) 50mM
Tris buffer, pH8, containing 0.005% (v/v)
Tween™ 20. To 384-well assays, 5µl Asp-N
enzyme at 1µg/ml was added, resulting in a final
assay volume of 55µl. In 1536-well assays, 0.5µl
of enzyme at 1µg/ml was added, resulting in a
final volume of 5.5µl. In no enzyme control wells,
the volume was made up using assay buffer.
Following incubation at room temperature for
between 0 and 60 minutes, imaging was performed
on LEADseeker at 540nm excitation and 570nm
emission wavelengths; exposure times of 60
seconds (384-well assay) and 90 seconds (1536-
well assay) were used (Fig. 13). A WBWC method
was employed with four standard concentrations
of between 0 and 100nM Cy3B.
KINASE ASSAYS
Protein kinases play an important role in the
control of many of the key steps in cellular
processes, such as signal transduction and cell
cycle control [15]. Due to the nature of the
enzymes, kinases are inherently suitable for
radiometric proximity based screening assays.
Incubation with [γ-33P] adenosine 5’-triphosphate
(ATP) and a kinase, under the appropriate assay
A Multi-Modality Assay Platform Current Pharmaceutical Biotechnology, 2000, Vol. 1, No. 3 273

Fig. (12). Schematic representation of the Asp-N peptide cleavage assay.

conditions, results in the incorporation of the
radiolabelled phosphate in the peptide or protein
substrate. Use of a biotinylated, glutathione-S-
transferase (GST) or his-tagged substrate allows
capture onto streptavidin, glutathione or nickel
chelate beads, respectively. The close proximity of
bound [33P] to the scintillant containing beads
causes the emission of light that is detected by the
imaging system. A schematic example of a
proximity kinase assay using streptavidin coated
beads is shown in (Fig. 14).
Assays of the mitogen-activated protein (MAP)
kinase enzyme, extracellular signal-regulated
kinase 1 (Erk-1) have been imaged in 384 [16] and
1536-well format. A MAP kinase time course was
performed in 1536-well format using 12.5pmoles
biotinylated myelin basic protein (bMBP), 20nCi
[γ-33P]ATP, 5pmoles unlabelled ATP, 0.05µg Erk-
1 in 50mM MOPS, pH 7.2, containing 5mM
MgCl2. The reaction volume was 5µl. Assays were
incubated for increasing times, up to 60 minutes,
at 37°C, prior to the addition of a ‘stop’ solution
containing 500µM unlabelled ATP, 5mM EDTA,

Fig. (13). Time based cleavage of Asp-N peptide substrate.

Cleavage of the peptide substrate over time was followed both in the presence (n) or absence (s) of enzyme in both (a) 384-
well and (b) 1536-well assays. Values are means ± standard error of the mean (SEM) (n=3 for 384-well assay and n=4 for
1536-well assay) and are expressed as nominal signal units. For reference, maximum signal:background levels achieved were
in the order 12:1 (384-well assay) and 14:1 (1536-well assay), expressed as the total Cy3B signal obtained after extensive
cleavage (approximately 3 hours) above that of the uncleaved (no enzyme) peptide control.
274 Current Pharmaceutical Biotechnology, 2000, Vol. 1, No. 3
Fig. (14). Schematic concept of a kinase proximity assay using streptavidin coated beads.
0.1% (v/v) Triton X-100 and 50µg streptavidin
coated PS LEADseeker beads in PBS. The final
volume was 8µl. Plates were centrifuged for 10
minutes at 1000 x g to reduce non-proximity
effects. Medium energy isotopes are incompatible
with the use of PS beads in suspension due to their
relatively long path length producing higher
backgrounds. These limitations can be overcome
by settling or centrifuging beads, or floating them
by the addition of caesium chloride. Plates were
then imaged for 5 minutes. The effect of time on
Erk-1 kinase activity was shown by a signal
increase. Images using PS beads were obtained
(Fig. 15) and used to construct a typical time
course graph (Fig. 16). Linear phosphorylation of
bMBP was observed up to approximately 30
minutes.
Fig. (15). Image of an Erk-1 kinase time course (n=3). A 5 minute image was taken. Non specific binding (NSB) was
determined in the absence of enzyme.
Fowler et al.
Using this assay protocol, the inhibitory effects
of staurosporine, a non-specific inhibitor of protein
kinases [17], on Erk-1 has been studied.
Staurosporine, dissolved in DMSO, was added to
assays covering concentrations of 0.01µM to
50µM. The effect of staurosporine on Erk-1
activity was shown by a signal decrease with
increasing staurosporine concentrations. From a
typical inhibition curve (Fig. 17) an IC50 value of
4.1µM was obtained.
Other kinases suitable for radiometric imaging
include p56lck, which is critical for T-cell
activation [18]. Assays for this enzyme have been
performed in both 384 and 1536-well format to
look at the effect of a range of inhibitors [19].
A Multi-Modality Assay Platform
Fig. (16). Erk-1 time course. An increase in enzyme activity
over time was seen as an increase in integrated optical
density units. Values are means ± standard deviation (SD)
(n=3).
An alternative method to study kinase activity,
which removes the need for radioactivity, is the
use of chemiluminescence. Mouse anti-
phosphotyrosine antibody immobilised onto
microplates can be used to capture biotinylated
tyrosine phosphopeptide. These captured
phosphopeptides can be detected by reaction with
streptavidin conjugated to alkaline phosphatase. A
schematic diagram illustrating the assay format is
shown in (Fig. 18).
This assay format has been used with two
phosphopeptides, tyrosine kinase biotinylated
phosphopeptide 2 (Biotin-EGPWLEEEEEA[pY]
GWMDF-amide) and tyrosine kinase biotinylated
phosphopeptide 3 (Biotin-RRLIEDAE[pY]AARG
-amide, both from Pierce). Black 384-well
Fig. (18). Schematic representation of chemiluminescent immunoassay format.
Current Pharmaceutical Biotechnology, 2000, Vol. 1, No. 3 275
Fig. (17). Inhibition of Erk-1 kinase activity by
staurosporine. Values are means ± S.D. (n=4).
microplate wells were coated with 50µl/well
mouse anti-phosphotyrosine antibody at 4.6 µg/ml
in 0.05M Na2CO3/NaHCO3 buffer, pH 9.6 for 3
hours at ambient temperature and washed three
times with 75µl/well phosphate buffered saline
(PBS)/0.05% (v/v) Tween 20 wash buffer. Wells
were treated with 75µl/well 0.2% (w/v) I-Block™
(Tropix Inc.) in PBS/Tween 20 blocking buffer for
1 hour at ambient temperature, drained over
absorbent paper, then sealed with adhesive film
and stored at 4°C until required.
Dilutions (50µl/well) of tyrosine kinase
biotinylated phosphopeptides 2 or 3 in 0.2%
I-Block in PBS/Tween 20 were added to plate
wells and incubated for 30 minutes at ambient
temperature, then washed with 3 x 50µl wash
276 Current Pharmaceutical Biotechnology, 2000, Vol. 1, No. 3
12000
10000
IOD units
8000
6000
4000
Phosphopeptide 2
2000 Phosphopeptide 3
0
0 0.5 1 1.5
pmoles phosphopeptide / well
Fig. (19). Detection of biotinylated phosphopeptides by
chemiluminescent immunoassay using LumiPhos 530
alkaline phosphatase substrate in 384-well plates. Less than
19.5 femtomoles phosphopeptide 2 or phosphopeptide 3
were detected with LumiPhos 530 after 2 hours incubation.
buffer. Streptavidin-enzyme conjugate in blocking
buffer (50µl/well) was added to plate wells,
incubated (30 minutes, ambient), then washed with
3 x 75µl/well wash buffer, followed by 2 x 75µl
20mM Tris, 1mM MgCl2, pH 9.5 assay buffer).
LumiPhos 530 streptavidin-alkaline phosphatase
conjugate was used for chemiluminescent
detection at 50µl/well.
For photomultiplier instrument-based detection
(i.e. Wallac 1450 MicroBeta™), the instrument
was used in chemiluminescence mode with a count
time of 0.2 seconds per well, in order to reduce the
total plate acquisition time. For CCD imaging,
LEADseeker was used to record chemil-
uminescence from plates with exposures of 30
seconds or less with 2 x 2 binning. Data from both
PMT- and CCD-based systems was recorded 2
hours after the time of addition of chemil-
uminescent substrate. Similar detection sensitivity
was demonstrated in 96 (data not shown), 384
(Fig. 19) and 1536-well formats (data not shown).
The same plate read on different instruments
gave information on the relative performance of
LEADseeker and a PMT-based plate reader
(Wallac MicroBeta), as shown in (Fig. 20). This
data is also summarised in (Table 2).
Using LEADseeker, the time required to image
a complete microplate is considerably less than
Fowler et al.
300
250
Signal:Noise
200
150
100 LEADseeker
50 Microbeta
0
0 10 20 30
nM phosphopeptide 2 added
Fig. (20). A comparison of normalised signal to noise
data taken from the same 384-well microplate read on
LEADseeker (u) and on a Wallac MicroBeta plate reader
(o). Signal to noise was calculated as mean signal / 3 x
standard deviation of the signal from control wells. The
error bars shown represent relative standard deviation
from triplicate wells. No more than 6 minutes elapsed
between reading the microplate on the two different
instruments.
that taken for PMT-based luminometers even
though in the latter systems, the time required per
well is shorter. Furthermore, the statistical
variation observed across the wells of a microplate
is significantly reduced. This is partly due to the
constantly changing luminescence output and
partly due to variation between photomultipliers
reading different areas of the plate. This problem
is worse for the shorter-lived HRP-based
substrates because of the greater variability of
output.
RECEPTOR BINDING ASSAYS
Receptor binding assays have traditionally been
screened using radiometric filter binding assays or
SPA. Miniaturization of these assays is possible
using imaging proximity assays. Receptors can be
immobilised directly onto imaging beads via a
number of coupling methods, including the use of
WGA, which binds to N-acetyl-β-D-glucosamine
residues in membranes and selects for membrane
glycolipids and glycoproteins. Once immobilised,
the receptor is close enough to the bead so that,
should a suitably radiolabelled ligand bind to the
receptor, it will be in close enough proximity to
stimulate the bead to emit light. This is shown
schematically in (Fig. 21).
A Multi-Modality Assay Platform Current Pharmaceutical Biotechnology, 2000, Vol. 1, No. 3 277

Table 2. A Comparison of Values Obtained from LEADseeker and Wallac MicroBeta PMT Plate Reader

MicroBeta LEADseeker

Instrument noise
38.9 0.96
(values in empty wells, % relative standard deviation (RSD))

Phosphopeptide Phosphopeptide

2 3 2 3

Z' factor * 0.78 0.75 0.91 0.81

Percent relative standard deviation: average values for signal wells 5.0 6.4 1.8 4.5

Signal:Noise
163 140 344 301
mean(signal) / 3 x S.D.(blank).

*
Z'-factor calculations were based on the work of Zhang and co-workers [20].

Several receptor binding assays have been
imaged using radiolabelled ligands such as the
chemokine [125I]macrophage inflammatory
protein-1α (MIP-1α), which has a high affinity for
the CCR1 chemokine receptor and [3H]imipramine
which binds to the serotonin transporter protein
[21]. To study the 5-HT7 receptor, which plays a
key role in cognitive and behavioural processes,
the ligand [3H]5-carboxamidotryptamine ([3H]5-
CT) has been used [22]. Assays have been
miniaturized to 384-well format using
LEADseeker. Assays contained 2.5µg cloned rat
5-HT7 receptor, 14nCi [3H]5-CT and 750µg WGA
coated YOx beads. Non-specific binding was
determined by the addition of 40µM 5-CT. The
final assay volume was made up to 50µl by the
addition of 50mM Tris, pH 7.4, 0.5mM EDTA,
10mM MgCl2 and 0.1% (w/v) sodium ascorbate.
After incubation at room temperature for 2 hours,
assays were imaged for 10 minutes. This resulted
in a signal to background ratio of greater than 5:1
(Fig. 22).
A displacement curve was obtained using
increasing amounts of unlabelled 5-CT and an
EC50 value of 2.6nM was determined (Fig. 23).
Another receptor type suitable for assaying
using imaging are G-protein coupled receptors.
The quantification of agonist-induced activity can
be measured via increased [35S]GTPγS binding
[23].

Fig. (21). Schematic concept of a proximity receptor binding assay.

278 Current Pharmaceutical Biotechnology, 2000, Vol. 1, No. 3
Fig. (22). 5-HT7 receptor binding assay using [3H]5-CT.
Values are means ± SEM (n=3)
MOLECULAR INTERACTIONS
The study of molecular interactions are other
examples of assays which are highly suited to
fluorescent detection. An example of this is the
study of the transcription factor, NF-κB. NF-κB
has received extensive interest over the past
decade because of its pivotal role in cellular
processes, such as inflammation and apoptosis
[24-25]. The interaction between the p65 subunit
of NF-κB and a 19 base pair double stranded (ds)
DNA sequence containing the 10 base pair
recognition site for NF-κB can be examined using
a fluorescence resonance energy transfer (FRET)
assay. The assay comprises of a Cy3 labelled anti-
Fig. (24). Schematic representation of the NF-κB competition binding assay.
Fowler et al.
Fig. (23). [3H]5-CT displacement curve. Values are means
± SEM (n=2).
GST antibody which specifically binds to a p65-
GST tagged protein, followed by interaction with
NF-κB dsDNA 5’ end-labelled on the coding
strand with Cy5. The FRET signal generated when
the three components are bound together can be
disrupted with an excess of unlabelled NF-κB-
specific dsDNA (Fig. 24).
Assays were carried out in black opaque 384-
well microplates by adding 70nM Cy3-anti-GST
antibody to 20nM NF-κB-p65-GST protein in
10mM HEPES buffer, pH7, containing 0.2mM
EDTA, 20mM NaOAc, 0.05% (v/v) IPEGAL,
5mM DTT and 1mg/ml BSA. The final assay
A Multi-Modality Assay Platform
volume was 30µl. Assays were incubated for 60
minutes at room temperature. Following
incubation, 100nM Cy5 labelled dsDNA
(containing the NF-κB consensus binding
sequence) was added together with various
concentrations of unlabelled specific or non-
specific competitor dsDNA sequence. Reactions,
in 50µl total assay volume, were incubated for a
further 60 minutes before imaging on LEADseeker
for 90 seconds at 540nm excitation and 690nm
emission wavelengths (Fig. 25). A WBWC
method was used with 4 standard concentrations of
between 0 and 200nM of a Cy3-Cy5 carboxyl dye
designed for calibration purposes.
100
80
%B/Bo
60
40
20
0 With this range of applications and full
-8 -7 -6 -5
log 10[competitor DNA] (M)
Fig. (25). NF-κB competition binding assays. Competition for
binding of the Cy5 labelled dsDNA to the NF-κB protein in the
presence of either unlabelled specific (n) or non-specific (s)
competitor dsDNA sequence is shown. Values are means ±
SEM (n=3) and are expressed as %B/Bo derived by standard
data analysis. For reference, maximum signal:background levels
achieved were in the order 1.7 - 1.8:1 (expressed as specific
total FRET signal above the background signal observed in the
presence of a 500-fold excess of specific inhibitor) and the IC50
value determined for the specific sequence competitor was
115nM.
CONCLUSION
The progress towards ultra-high throughout
screening (uHTS) has driven a move away from
conventional plate counting technology to imaging
systems capable of imaging entire plates. Systems
capable of detecting radiometric, fluorescent and
luminescent signals offer researchers a choice of
detection methods expanding the range of
applications suitable for uHTS.
Current Pharmaceutical Biotechnology, 2000, Vol. 1, No. 3 279
Using new europium containing beads, 384 and
1536-well radiometric proximity assays can be
read in 10 minutes or less. Due to the emission
characteristics of the beads, the effect of colour
quenching on signal output, a problem
traditionally associated with homogeneous assays,
is greatly reduced.
The sensitivity of CCD chips to light emitted in
the red region of the spectrum makes them ideal
for the detection of cyanine dyes, such as CyDyes.
In addition, CyDye fluors are particularly suited as
fluorescent probes since their red to near infrared
absorption and emission maxima (500-800nm) are
easily distinguishable from the shorter wavelength
endogenous or autofluorescence associated with
biological samples and equipment. Novel
developments have resulted in the development of
CyDye probes with either enhanced or reduced
fluorescence characteristics for use in FRET
applications.
The use of imagers in chemiluminescent assays
results in a significant reduction in statistical
variation across microplates as the time required to
analyse a complete microplate is considerably less
than for PMT-based luminometers.
automation of imager systems such as
LEADseeker, true ultra-high throughput screening
is an achievable goal.
ABBREVIATIONS
[3H]5-CT = [3H]5-Carboxamidotryptamine
AP = Alkaline phosphatase
ATP = Adenosine 5’-triphosphate
BMBP = Biotinylated myelin basic protein
CCD = Charged coupled device
ds DNA = Double stranded DNA
Erk-1 = Extracellular signal-regulated
kinase 1
FFC = Flat field correction
FRET = Fluorescence resonance energy
transfer
GST = Glutathione-S-transferase
280 Current Pharmaceutical Biotechnology, 2000, Vol. 1, No. 3 Fowler et al.

HRP = Horse radish peroxidase [3] Pope, A. J.; Haupts, U. M., and Moore, K. J. (1999)
Drug Discovery Today, 4(8), 350-362.
HTS = High throughput screening [4] Mujumdar, R. B.; Ernst, L. A.; Mujumdar, S. R.;
Lewis, C. J., and Waggoner, A. S. (1993) Bioconjug.
IOD unit = Integrated optical density unit Chem., 4(2), 105-111.

MAP kinase = Mitogen-activated protein kinase [5] Mujumdar, S. R.; Mujumdar, R. B.; Grant, C. M., and
Waggoner, A. S. (1996) Bioconjug. Chem., 7(3), 356-
MDL = Minimum detectable limit 362.

[6] Sturmer, D. M. (1948) in The Theory of the

MIP-1α = Macrophage inflammatory Photographic Process, (James, T. H., Ed.)
protein Macmillan, NY,

NHS = N-hydroxysuccinimide [7] Matsui, F. (1990) in Infrared Absorbing Dyes,

(Matsuoka, M., Ed.) Plenum Press, NY, pp.117.
NSB = Non specific binding [8] Waggoner, A. (1993) Patent Number US5268486,
PBS = Phosphate buffered saline [9] Tyutyulkov, N. (1991) in Polymethine Dyes Structure
and Properties, St. Kliment Ohridski University
PMT = Photomultiplier tube Press, Sofia,

PS = Polystyrene [10] Lincoln, L. L. (1972) Patent Number US03679427.

[11] Lieberwirth, U. (1997) J. Fluorescence, 7((Suppl.)),

PVT = Polyvinyl toluene 595-615.
RSD = Relative standard deviation [12] Akhavan-Tafti, H.; Schaap, A. P.; Arghavani, Z.;
DeSilva, R.; Eickholt, R. A.; Handley, R. S.;
SD = Standard deviation Schoenfelner, B. A.; Sugioka, K., and Sugioka, Y.
(1994) J. Biolumin. Chemilumin., 9(3), 155-164.
SEM = Standard error of the mean [13] Akhavan-Tafti, H. (1999) Patent Number US5965736
SPA = Scintillation proximity assays [14] Drapeau, G. R. (1979) J. Biol. Chem., 255 839-840.

UHTS = Ultra-high throughput screening [15] Krebs, E. G. (1994) Trends Biochem. Sci., 19(11),
439-439.
WBWC = Well by well correction
[16] Fowler, A.; Harvey, M.; Cox, A.; Jessop, R. A.;
Looker, M. R.; Davies, I.; Morris, J.; Santos, A.;
WGA = Wheat germ agglutinin Turner, J., and Price-Jones, M. (1998) Genetic
Engineering News, 18(21), 34-36.
Yox = Yttrium oxide
[17] Lawrence, D. S. and Nui, J. (1998) Pharmacol. Ther.,
Ysi = Yttrium silicate 2 81-114.

[18] Straus, D. B. and Weiss, A. (1992) Cell, 70 585-593.

ACKNOWLEDGEMENT
The authors wish to thank their colleagues for
supplying the data used in this manuscript.
[19] Beveridge, M.; Park, Y-W.; Hermes, J. D.; Brophy,
G., and Santos, A. (1999). Poster presented at the
IBC's 4th Annual International Exposition and
Symposium on Drug Discovery Technology, Boston,
MA.

REFERENCES [20] Zhang, J.; Chung, T. D. Y., and Oldenburg, K. R.

(1999) Journal of Biomolecular Screening, 4(2), 67-
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[2] Bell, C. D. and Howse, J. H. C. (1996) Patent Rghei, N., and Bell, P. (1999). Poster presented at the
Number EP556005.
A Multi-Modality Assay Platform Current Pharmaceutical Biotechnology, 2000, Vol. 1, No. 3 281

5th Annual Conference of The Society for Annual International Exposition and Symposium on
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[22] To, Z. P.; Bonhaus, D. W.; Eglen, R. M., and [24] Mercurio, F. and Manning, A. M. (1999) Curr. Opin.
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