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Assays

TR-FRET
BASED ASSAYS
– getting better with age
By Dr John Comley Despite having been available for nearly 15 years,TR-FRET based assays are
still the preferred fluorescent assay format for many screeners. A recent
survey ranked Cisbio’s TR-FRET product (HTRF®) as the most frequently used
generic assay technology. GPCR second messengers and kinases remain the
most popular TR-FRET applications, aided by the launch of several new assay
kits addressing these popular applications over the past year.The typical
screener today has familiarity with at least eight different assay technologies,
and 40% of them have plan to narrow their portfolio of assay technologies.
Prospects for the continued use of TR-FRET based assays, however, remain
optimistic as: 1) survey respondents appear to be choosing TR-FRET over
alternative assay technologies for its sensitivity and ease of use, while the main
consideration for alternatives to TR-FRET was price; 2) TR-FRET assays have
attained a level of maturity and acceptance in the scientific community;
3) there exists in the Pharma and Biotech a broad and highly experienced base
of TR-FRET users; and 3) TR-FRET based assay technology is still actively
evolving as evidenced by the increasing innovation of key vendors and
suppliers.These developments include enhanced stability and performance of
existing FRET pairs; increased use of red-shifted Alexa Fluor© as acceptor
dyes; better assay development flexibility by direct covalent labelling of
substrates or the use of antibody independent formats; the wider use of
terbium complexes, including using GFP as acceptor; and a new approach using
Europium chelate dyed nano-particles.Taken as a whole the diversity of
TR-FRET based assay offerings looks set to continue increasing over the
coming year.

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Assays

T
he market place for assay reagent technolo- assay reagent offerings available today, Cisbio
gies is saturated with numerous alternative HTRF and PerkinElmer LANCE have the greatest
approaches for the assay of the common market share and were ranked as being moderate-
targets like kinases and GPCRs. It is therefore ly used (as opposed to being used exclusively) by
important to recognise what influences end users respondents. In comparison, Upstate KinEASE,
to select a particular assay reagent technology. In GE Healthcare (Amersham) TR-FRET (Eu
October 2005 HTStec undertook a market study Chelate +CyDye), Invitrogen LanthaScreen and
focused on assay reagents with the particular PerkinElmer TruPoint were less popular (used
objective of understanding and documenting cur- infrequently) with survey respondents. The rela-
rent perceptions of and interest in TR-FRET (Time tive use of commercial TR-FRET based assay kits
Resolved-Fluorescence Resonance Energy is summarised in Figure 4. Not surprisingly GPCR
Transfer) based assays. second messenger and kinase assays featured most
highly, with greatest interest shown in cyclic AMP,
The TR-FRET principle followed by the various kinase offerings and then
The FRET principle is based on the transfer of IP-one. In contrast, other available assays kits
energy between two fluorophores, a donor and were less popular. It is of interest that about one in
an acceptor. When the two entities come close five respondents indicated they did not purchase
enough to each other, excitation of the donor by commercial assay kits, and presumably developed
a light source triggers an energy transfer their own TR-FRET assays using standard tool-
towards the acceptor, which in turn emits specif- box reagents.
ic fluorescence at a given wavelength. Molecular
interactions between biomolecules can be
Figure 1
assessed by coupling each partner with a fluo-
FRET Principle (explained in
rescent label and detecting the level of energy text)
transfer (Figure 1). Time resolved FRET utilises
long-lived fluorophores combined with detec-
tion on a time-resolved fluorescence basis which
allows for the minimisation of background
prompt fluorescence interferences (mainly com-
pounds and proteins present in biological fluids
or serum that are naturally fluorescent) which
are short-lived compared to the long-lived labels
used (Figure 2) For more information it is rec-
ommended that the reader consults the detailed
technical descriptions and reviews of the tech-
nology which can be found on most TR-FRET
vendor’s websites, eg www.htrf-assays.com or
www.invitrogen.com/LanthaScreen.

Interest in alternative TR-FRET

offerings and assays
TR-FRET based assays have been around since the
early 1990s and are probably the most well
known of the generic fluorescent assay technolo-
gies available today. HTStec’s survey found TR-
FRET was still the preferred assay technology of
40% of survey respondents, which is a high level
of acceptance considering the diversity and num-
ber of competitive offerings. Furthermore, Cisbio’s
TR-FRET product (HTRF®) was ranked the most
frequently used generic assay technology, closely
followed by PerkinElmer’s LANCE and Molecular Figure 2: The fluorescence lifetime of most conventional fluorophores can be 100ns or less.
Typical TR-FRET fluorophores (eg Lanathanide chelates or cryptates) exhibit a relatively long-
Devices’ FLIPR assay kits. The relative use of TR- lived fluorescence lifetime between 200µs to 1500µs.The advantage of such long lived
FRET and related assay technologies are present- emissions is the ability to use time-resolved fluorescence to discriminate from background
ed in Figure 3. Of the commercial TR-FRET based emissions i.e. sample during the blue counting window

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Assays

Figure 3
Relative use of TR-FRET and related assay technologies

Cisbio – HTRF
PerkinElmer – LANCE
Molecular Devices – IMAP
Upstate – KinEASE
Amersham – TR-FRET (Eu Chelate + CyDye)
Invitrogen – LanthaScreen
PerkinElmer – TruPoint
1.00 1.50 2.00 2.50 3.00 3.50
© HTStec 2005 Mean rank (where 1 = don’t use, 3 = moderate use and 5 = exclusive use
Figure 4

A typical TR-FRET screen

Relative use of commercial TR-FRET based assay kits The survey uncovered that the typical TR-FRET
screen today (2005) has an average volume of
Cyclic AMP
Tyrosine Kinases 40uL, is undertaken in a regular or low-volume
Serine/Threonine Kinases 384-well plate, generates 551,000 data points and
Protein Kinase 12% are cell-based. The average annual budget for
IP-One TR-FRET based assay reagents was
Other Assays
$145,000/screening lab in 2005, and this repre-
Beta-Secretase
Prostaglandin E2 sented 16% of respondents total assay reagent
GST and 6HIS check kits budget. The majority of the TR-FRET reagent
Caspase 3 budget was spent on standard toolbox reagents
Cytokines (IL-8) (48%) and off-the-shelf assay kits (29%) (Figure
Cytokines (IL-6) 5). From this data the global Pharma/Biotech mar-
Cortisol
ket for TR-FRET based assay reagents was esti-
Insulin
Human Fc check kit mated to be around $50 million in 2005.
Helicase
Cyclic GMP Why TR-FRET is preferred?
Caspase 6 As indicated earlier, for some common target types
Ubiquitin
users are spoilt for choice with respect to assay
Histamine
Heparanose technologies. It therefore comes as no surprise that
Caspase 8 the typical assay technologist in Pharma and
N/A – we don't purchase kit assays Biotech today is familiar with and a skilled user of
0 10 20 30 40 50 60 70 at least eight different assay technologies.
© HTStec 2005
% respondents using assay However, around 40% of these labs now have
plans to try to limit the number of different assay
Figure 5 technologies used. The main explanations given for
planning to limit the number of different technolo-
gies used include: 1) to develop greater technology
Breakdown of TR-FRET reagent budget
expertise in house; 2) to increase reagents con-
Other Assay development sumption to get better price (volume discount); and
6% services (by vendor) 3) the possibility to profile a compound in various
Custom labelling of assay 2%
reagents (by vendor)
assays using the same technology. The basis for
15% purchasing decisions between several very similar
Standard toolbox reagent offerings is not always obvious, is it there-
reagents (to develop your fore significant that the relative ranking of criteria
own customer assays) by which TR-FRET based assays were chosen over
48%
Off-the-shelf alternative assay technologies (Figure 6) appears to
assay kits suggest TR-FRET’s sensitivity and ease of use are
© HTStec 2005 29%
most important. In direct contrast, the main reason

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Assays

Figure 6
Why TR-FRET is chosen over alternatives
Sensitivity
Ease of use
Ease of assay development
Z’ Factors
Signal-to-noise ratio
High tolerance to compound interference
Number of steps
Price
Toolbox reagents available
Dynamic range
Broad range of applications
Interference corrections
Compatibility with my existing readers
Availability of custom labelling service
Tolerance to solvents (DMSO)
Large off-the-shelf assay kit offering
Preferred vendor agreement
0 10 20 30 40 50 60 70 80 90 100
© HTStec 2005 Comparative interest normalised relative to first choice decision factor ( =100%)

Figure 7
Why alternatives are chosen over TR-FRET
Price
Sensitivity
Ease of assay development
Ease of use
High tolerance to compound interference
Toolbox reagents available
Broad range of applications
Signal-to-noise ratio
Compatibility with my existing readers
Dynamic range
Z’ Factors
Preferred vendor agreement
Tolerance to solvents (DMSO)
Number of steps
Interference corrections
Large off-the-shelf assay kit offering
Availability of custom labelling service

0 10 20 30 40 50 60 70 80 90 100
© HTStec 2005 Comparative interest normalised relative to first choice decision factor ( =100%)

why alternatives assay technologies were chosen the lowest quote from a shortlist of their preferred
over TR-FRET based assays was predominantly TR-FRET assay alternatives. However, like all
price (Figure 7) and it is increasingly common to available assay technologies TR-FRET assays are
hear that screening groups are shopping around for not without their limitations (Figure 8) and the

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Assays

Figure 8
Biggest limitations of current TR-FRET based assay reagents

Sample (compound) absorbance

Donor quenching

Donor signal leaking to acceptor window

Matrix interference

Stability

1.00 1.50 2.00 2.50 3.00 3.50

© HTStec 2005
Mean rank (where 1 = not limiting and 5 = very limiting

most limiting of these was perceived by survey number of new developments and technology
respondents to be sample (compound) absorbance improvements are ongoing.
and donor quenching. The potential narrowing of
the preferred assay portfolio of screening groups TR-FRET vendors continue to evolve
appears to have benefited TR-FRET by virtue of its the technology
level of maturity and acceptance in the scientific Homogeneous Time Resolved Fluorescence
community and the broad and highly experienced (HTRF®), the TR-FRET technology developed by
user base. Together these factors may have Cisbio International (www.htrf-assays.com) bases
spawned something of a resurgence of innovative its principles on the photophysical properties of
activity among vendors whose products address Eu3+ Cryptates when they combine to different
this market. It is apparent from reading the fol- acceptors in a FRET process. The technology has
lowing vendor updates that a surprisingly large been applied to a number of biochemical assays
such as enzymatic activities (kinases, proteases,
ubiquitination, etc), protein-protein/nucleotide
interactions (receptor dimerisation, topoisomerase
etc), and immunoassays. The robustness of the
technology to various assay conditions has further
enabled the development of cell-based assays,
allowing the transition from simple biochemical
assays to high throughput functional ones, bring-
ing more pharmacological relevance at the level of
G␣ PLC PIP2
early phases of drug screening. The new GPCR
screening platform illustrates this trend. Both
cAMP and IP1 (inositol monophosphate) (Figure
9) have been successfully adapted to the same
IP3 direct assessment on cells in high density plate for-
Myo-inositol mats. The validation of the IP-One assay on Gq-
coupled receptors has already proven its relevance
E.R. on more than 50 receptors, including the screening
LiCl of inverse agonist activities for which few sensitive
IP2 HTS methods are available. These advances in
Ca2+
assay development are also supported by the
IP1 search for technological evolutions. The new d2
accumulation acceptor – a small organic dye compatible with
Eu3+ Cryptates – has been a key element in most
recently developed HTRF® assays. As a result of
Figure 9: The IP1 pathway. Gq coupled receptor activation induces IP3 release catalysed by
these optimisations, d2 based HTRF® assays
PLC. IP3 degradation occurs rapidly and leads ultimately to the production of IP1, while show a stability over several days allowing the
processing into myo-inositol can be prevented by the addition of LiCl number of screening runs per week to be increased

26 Drug Discovery World Spring 2006

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Assays

and the possible rescue of a run in the case of

instrument failure. Kinases remain the main appli-
cation of HTRF® after GPCRs. Most of current
assays developed involve phosphospecific antibod-
ies which detect in a sensitive and accurate man-
ner kinase activities. This typical format is com-
patible with all kinds of substrates and configura-
tions: small peptides to large proteins, multiple
phosphorylation sites, auto-phosphorylations.
HTRF® will soon combine with another assay
concept initiated by Upstate, the KinEASE® sys-
tem. This assay format consists of a universal
monoclonal antibody which recognises a series of
peptidic substrates of Thr/Ser kinases. So far, the
new assay comprises three different substrates
which have been validated on more than 70 dif-
ferent kinases including PKC and AKT families,
and Aurora A (see Upstate’s section below for
Figure 10: Top: In the SNAP-tag self-labelling reaction the benzyl-moiety carrying the label is
additional details). For the future, Cisbio concen- irreversibly transferred to the SNAP-tag. Bottom: SNAP-tag substrates used for the
trates its research on technologies that can bring development of HTRF® assays: BG-TBP, long lived cryptate donor, BG-647 fluorescence
more relevance and reliability during drug screen- acceptor for BG-TBP
ing phases. The goal is also to provide new HTS
functional assays on living cells, adaptable to Figure 11
existing affordable detection devices. 665nm Schematic of the high affinity
A21967 or interaction of FKBP-FRB in the
Rapamycin presence of the drug
For a TR-FRET assay based on HTRF® (Cisbio Rapamycin or analogous
International), the interacting compounds are 337nm compounds.The limited
labelled with a rare earth cryptate donor and a far distance between the two
red acceptor (allophycocyanine, Cy5, or Dy647). SNAP-tag fusion proteins
allows highly efficient energy
This is often done by epitope tagging of the relevant
transfer from the TBP-donor
proteins, (using FLAG-tag, His-tag or HA-tag), and ET to the 647-acceptor
using donor and acceptor labelled antibodies -FR
TR
against the tags. Covalys (www.covalys.com) offers
an alternative that enables direct covalent labelling
of the proteins of interest, eliminating the antibod-
ies and reducing assay complexity. Covalys’ SNAP-
tag™ is a protein tag which binds covalently to a
Figure 12
labelled substrate forming a stably labelled fusion
A:The high quantum efficiency
protein. Substrates carrying a donor cryptate (BG- (about 60%) and overall
TBP) or an acceptor-fluorophore (BG-647) suitable brightness of Lumiphore’s Tb3+
for HTRF are available (Figure 10). SNAP-tag complexes are derived from
labelling streamlines assay development and the highly efficient channelling
of excitation energy from the
reduces the size of the protein complex compared to
isophthalamide chelating
using antibodies, resulting in pronounced energy moiety to the coordinated
transfer. A model assay was developed using the Tb3+ ion. B: In a typical
interaction of FKBP with FRB. In the presence of complex, there are four
rapamycin these proteins interact with high affinity isophthalamide chelating
groups co-ordinating the metal
(Figure 11). Maximum energy transfer values
ion (green).These co-
observed for the FKBP-FRB system were very high ordinating groups are
at above 20,000 delta F%. This indicates strong covalently connected with a
interaction between the proteins and effective ener- use of the assay a Z’ value above 0.7 was found, scaffold that confers high
gy transfer between donor and acceptor. Inhibition indicating a very rugged system. SNAP-tag labelling complex stability in aqueous
solutions (scaffold not shown)
studies using ascomycin gave a sigmoidal displace- works equally well in crude lysates, allowing the
ment curve with a 50% inhibition at about 10nM, direct labelling of delicate proteins that tend to lose
in agreement with literature values. For repetitive binding activity during purification. The ease of the

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Assays

assays have also been described recently, but lack

of low-cost, versatile reagents has limited their
commercial availability. A joint venture between
Echelon Biosciences (www.echelon-inc.com) and
Lumiphore (www.lumiphore.com) has resulted in
a sensitive TR-FRET assay for important lipid
kinases. Lumiphore has exclusive licence to spe-
cific lanthanide-complex technology from the
University of California, Berkeley which it has
further developed and commercialised. These
highly luminescent terbium (Tb3+) complexes
(Figure 12) have a quantum yield of approxi-
mately 60%, making them the brightest lan-
thanide complexes available for commercial
time-resolved luminescence applications in aque-
ous environments. A new macrocyclic Tb3+ com-
plex, with two peak emission lines at 490nm and
Figure 13: The PI3K TR-FRET assay is sensitive (with inhibition curves in the low nanomolar
545nm, has proven particularly useful in lumi-
range), and specific (the PI(3,4,5)P3 product shows greater than 70 times lower EC50 value nescence resonance energy transfer (LRET)
compared to substrate) applications as a donor with fluorescein and rho-
damine dyes as well as phycobiliprotein and R-
phycoerythrin as acceptors. Soon to be released
SNAP-tag labelling approach, obviating the need to Eu-complexes will enable multiplex assays when
use antibodies, and the possibility to work from used in combination with Tb. Furthermore, the
complex samples, make this a valuable tool for the excitation maxima of these dyes are between
development of HTRF assays. 340-350nm which is sufficiently red-shifted to
avoid undesirable excitation of biological sam-
Phosphoinositide kinases phosphorylate the lipid ples or plastics. Echelon has incorporated
head of phosphatidylinositol at the 3’, 4’, or 5’ Lumiphore dyes into its proprietary PI3K screen-
position; and the lipid products of these enzymes ing reagents and assay platforms. The initial
act as cell-signalling molecules essential in multi- PI3K assay is competitive with the enzymatic
ple aspects of cell growth and survival. A partic- product, PI(3,4,5)P3, competing with a fluores-
ular lipid kinase, phosphatidylinositol-3-kinase cent PI(3,4,5)P3 tracer for binding to a specific
(PI3K), is an established drug target for cancer Tb-conjugated binding protein. Exceptional sig-
therapeutics with exciting promise. The first nal to background binding is observed, and the
assays for PI3K were radioactive with laborious assay is both sensitive and specific (Figure 13).
HPLC or TLC separations. Non-radioactive Because this assay measures directly the enzy-
matic product of PI3K, it avoids the limitations
of popular ATP depletion kinase assays and
allows researchers the opportunity to screen for
both active site and allosteric inhibitors. Further
lipid kinase and phosphatase assays, including
diabetes targets SHIP2 and PTEN, are planned
for release later this year; as well as, Tb-labelled
secondary antibodies and additional generic
assay reagents to enable researchers flexible
design of custom applications.

GE Healthcare (www.amershambiosciences.com)
also provides a range of fluorescence assay solu-
tions based on TR-FRET. GE’s offering uses
europium (TMT) chelates as donors (Eu3+ with
terpyridine-bis(methylenamine) tetra acetic acid).
The 3-amino group of the europium (TMT) chelate
Figure 14: An example of a GE Healthcare’s TR-FRET format assay is readily converted to the isothiocyanate, thereby

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Assays

allowing labelling of primary amine groups on a

range of biomolecules. The CyDye fluor Cy5 is an Emission at 730nm
excellent choice of acceptor for europium (TMT)
chelates in TR-FRET format assays. This is
because Cy5 is small compared to traditional Excitation at 340nm
acceptors such as allophycocyanin and can help
reduce steric hindrance. CyDye fluors are also
unaffected by amino acid residues such as trypto-
phan. This means there will be no change in fluo-
rescence signal when labelling proteins or peptides
with CyDye fluors. GE’s offering includes a range
of TRF and CyDye generic reagents (donors and
acceptors) and europium (TMT) isothiocyanate
(Figure 14).
Eu-nanoparticle
Peptide
Europium(III)-chelate dyed nanoparticles are being Streptavidin AlexaFluor680-labelled anti-phospho antibody
developed by Hidex (www.hidex.com) for use as
labels in immunoassays. Nanoparticles have high Biotin
specific activity, because several thousands of Phosphorylated amino acid
europium(III)-chelates are embedded inside a single
polystyrene shell. High label content is possible,
Figure 15: Principle of nanoparticle-based kinase assay. Biotinylated peptides bind to
since lanthanide chelates do not suffer from self- streptavidin coated nanoparticle. If the peptide is phosphorylated, acceptor-labelled antibody
quenching phenomenon. Nanoparticles have been recognising phosporylation site binds to peptide.The europium(III)-chelates in proximity of
used as labels in heterogeneous immunoassays and the acceptor labels (marked with dark orange) participate to the energy transfer
also as donors in homogeneous TR-FRET based
assays. In TR-FRET based assays using europi-
um(III)-chelate dyed nanoparticles as donors the completely avoided by using a very small nanopar-
energy is transferred from a large number of excit- ticle (eg 47nm diameter) and by spectral resolu-
ed europium chelates via non-radiant dipole-dipole tion: using an acceptor molecule, which can be
interactions to a single acceptor molecule during measured at near-infrared area (eg at 730nm) the
the long lifetime emission of the europium chelates. emission caused by europium(III)-chelates is at
Thus the measured signal from a single acceptor is minimum. The polystyrene shell also protects the
increased compared to traditional donor-acceptor europium(III)-chelates from unwanted matrix
pairs. The increased background signal caused by effects, eg assay buffer may contain heavy metals
emission of europium(III)-chelate can be almost without causing any quenching effect on the

Figure 16
Invitrogen’s LanthaScreen™
TR-FRET format uses terbium
as the donor species, allowing
acceptors such as fluorescein
or GFP to be used. In kinase
assays, physiologically relevant
protein substrates can be
prepared as GFP fusions,
simplifying the assay
development process

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Assays

fluorescence of a particle. Homogeneous assays

using europium(III)-chelated dyed nanoparticles as
donors have been employed in high throughput
screening. For example immunoassay for 17␤-
estradiol has been used to screen inhibitors for
enzyme 17␤-hydroxysteroid dehydrogenase type 1
(17␤-HSD-1). 17␤-HSD-1 catalyses a reversible
reduction of estrone to 17␤-estradiol and its activ-
ity is increased, eg in some breast cancer patients.
Several HTS applications using the nanoparticle
labels are currently under development by Hidex
for example a nanoparticle-based kinase inhibitor
screening assay will be commercialised this year.
The assay uses streptavidin coated europium(III)-
chelate dyed nanoparticles with 47nm diameter.
The substrate peptide is biotinylated and
antibody specific for phosphorylation is labelled
with AlexaFluor 680 label (Figure 15). The assay
has a high specific signal level, is robust and
easily automated.
By using terbium rather than europium as the
donor species in the FRET pair, Invitrogen’s
LanthaScreen™ technology (www.invitrogen.com/
LanthaScreen) maintains the advantages of a time-
resolved, ratiometric format, but adds flexibility in
terms of the types of acceptors that can be used. In
addition to simplifying TR-FRET kinase assays by
allowing the use of substrates that are directly
labelled with fluorescein (rather than relying on
biotinylated substrates that then must be developed
by an addition of streptavidin-APC), physiological-
ly relevant protein substrates can be prepared as
GFP fusions, with GFP serving as the FRET accep-
tor for terbium (Figure 16). This strategy provides
a facile method to assay kinases that require a
docking site on the substrate for efficient phospho-
rylation to take place, as is the case with many
MAP kinase pathway members such as p38␣ or
RAF. Labelling kinase substrate as a GFP fusion has
other practical advantages, such as batch-to-batch
consistency that is not possible when a substrate
protein is randomly labelled through accessible
amino groups, and lower cost compared to using an
acceptor-labelled antibody. The strategy of using
GFP as an acceptor in a TR-FRET assay also has
been applied recently to protease assays.
Deubiquitinating enzymes (DUBs) are proteases
that cleave ubiquitin from specific target proteins,
often playing a role in cellular processes by ‘rescu-
ing’ ubiquitinated proteins from proteosomal
degradation. Inhibition of DUBs that are involved
in the rescue of oncogenic proteins is emerging as a
target for pharmaceutical intervention.
Importantly, DUBs require the presence of an intact
ubiquitin protein for efficient proteolysis, and

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Assays

Figure 17
Invitrogen’s LanthaScreen™
TR-FRET format has recently
been used to incorporate GFP
as an acceptor in substrates
for deubiquitinating enzymes
(DUBs). By genetically
encoding the acceptor species
and attaching the terbium-
donor species downstream of
the site of proteolysis, a
physiologically relevant
substrate that contains the
full-length ubiquitin (that is
required for efficient
proteolysis) is prepared, and
the assay can be continuously
monitored to detect DUB
activity or inhibition

chemically synthesised peptides have not been procedure: initiate the kinase reaction, add the
reported as efficient substrates for DUB activity. By Binding Solution and read. With IMAP TR-FRET,
expressing a DUB substrate that contains an N-ter- the Binding Solution includes the novel Tb-donor
minal GFP fused to ubiquitin that contains a ter- already bound to the binding entities. Upon addi-
bium-labelled cysteine proximal to the site of pro- tion of the Binding Solution, the phosphorylated
teolysis, a substrate is prepared that shows a high population of the fluorescently labelled kinase
degree of FRET that is reduced in the presence of substrate (FAM or TAMRA) binds as usual to the
DUB activity (Figure 17). This strategy highlights a binding entities and is brought into close proxim-
key advantage of using GFP as the acceptor, as it ity to the Tb donor, thus producing resonance
would be extremely difficult to site-specifically energy transfer (Figure 19). Due to the long life-
incorporate two distinct labels using chemical time of Tb fluorescence, the detection can be run
labelling strategy. Again, the directly labelled for- in time-resolved mode, which virtually eliminates
mat provides for specific advantages over other for- fluorescence interference from assay components
mats in that the reaction can be monitored in real
time, providing valuable kinetic information for
elucidating mechanisms of enzyme activity or inhi-
bition. In addition to a DUB substrate, Invitrogen
has also recently launched terbium- and fluores-
cein-labelled ubiquitin, allowing for the monitoring
of mono- or polyubiquitination processes in either
a real-time kinetic or an endpoint format.
The latest addition to the IMAP® platform for
kinases, phosphatases and phosphodiesterases
from Molecular Devices (www.moldev.com) is
IMAP’s TR-FRET detection mode. This new
detection system uses all the components of the
IMAP fluorescence polarisation (FP) system plus a
IMAP terbium (Tb) labelled TR-FRET donor (‘Tb
donor’, Figure 18) consisting of a phosphate-con-
Figure 18: Principle of the IMAP TR-FRET assay. Upon phosphorylation by the target kinase,
taining linker, a sensitiser and a Tb complex com- fluorescent substrate binds to the trivalent metal entities of the IMAP binding reagent.This
bined into one molecule. Just like IMAP FP, this binding brings the substrate into close proximity of the Tb donor that is pre-bound, allowing
assay is a simple, homogeneous ‘mix and read’ energy transfer to occur

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Assays

or compounds in a screen. IMAP TR-FRET com-

bines all the advantages of IMAP – an antibody-
free, stable, generic, homogeneous format – with
the advantages of TR-FRET for detection of phos-
phorylation. These advantages include 1) reduced
minimum percentage substrate conversion and
flexibility of substrate concentration (Figure 20);
2) increased substrate size, such as proteins, com-
pared to FP detection mode; 3) low background
due to time-resolved detection and 4) ratiometric
read-out. These features make the assay well suit-
ed for HTS and applicable to a wide variety of
kinases. IMAP TR-FRET provides further advan-
tages over existing TR-FRET technologies. First, it
is truly antibody independent. Therefore the
search for a compatible antibody is eliminated,
which accelerates the assay development process.
Figure 19: Calibration curve using BLK/Lyntide (5FAM-EFPIYDFLPAKKK-NH2).
Second, the signal is extremely stable. IMAP TR-
Unphosphorylated and phosphorylated Blk/Lyntide were diluted to 0.3µM, 1µM, and 3µM in
IMAP reaction buffer.They were then mixed at varying ratios as indicated in graph by % FRET’s novel Tb donor is remarkably stable,
phosphorylation and 20µL were pipetted/well (white 384 well plate). 60µL of Binding Solution requiring no additives, such as fluoride, to achieve
with Tb-donor was added.The plate was read in TR-FRET mode after overnight incubation stability and signal intensity to last more than 24
hours. IMAP TR-FRET maintains the solid per-
formance of the IMAP platform. Its signal stabili-
ty allows customers to more efficiently schedule
their screens compared with alternative TR-FRET
detection methods. The IMAP Platform, with its
flexible Progressive Binding System, various
Substrate Finder plates, validated FAM or
TAMRA labelled substrates (Figure 20), and the
FP and TR-FRET detection modes accelerates the
transitions from assay development to screening
and hit evaluation for virtually any kinase, phos-
phatase and phosphodiesterase with precision
and reproducibility.

In LANCE™, PerkinElmer (www.perkinelmer.com)

offers one of the widest TR-FRET toolboxes avail-
able on the market enabling assays to be quickly
developed for a vast range of drug targets especially
enzymes such as kinases. TR-FRET based assays are
eminently suited for the increased productivity and
performance demanded today by customers, and
uHTS applications can be readily adapted on to
microplate imagers such as PerkinElmer’s
ViewLux™ enabling greatly improved precision and
throughput. PerkinElmer’s recently launched
LANCE cAMP cell based assay for analysis and
screening of GPCR drug targets is an excellent
example of applying new innovation to a trusted
screening platform, which is based on TR-FRET
chemistry (Figure 21). LANCE cAMP relies on use
Figure 20: IMAP TR-FRET Assay for Abl kinase using TAMRA and FAM labelled Abltide (Fl-
of the proprietary Europium chelate dyes, a unique-
KKGEAIYAAPFA-NH2) Indicated amounts of Abl (Upstate) were incubated for 1h RT in
20µL IMAP reaction buffer in the presence of 100µM ATP and 1µM of either FAM or ly high performing antibody against cAMP, and was
TAMRA labelled Abltide. 60µL of Binding Solution was added and TR-FRET was read after an first to market with next generation higher per-
incubation of 3h forming small red-shifted light emission acceptor

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Assays

Figure 21
LANCE cAMP Assay Principle.
Light pulse at 340nm excites
the Europium chelate of the
Eu-SA/b-cAMP tracer.The
energy emitted from the
chelate is transferred to the
Alexa-labelled antibodies
bound to the tracer, generating
a TR-FRET signal at 665nm.
Residual energy from the
chelate will produce light at
615nm. cAMP of a sample
competes with the tracer for
antibody binding sites and
causes signal reduction

dyes such as Alexa Fluor© 647. These new enhance- cell based assay solutions, utilising state of the art
ments offer significant improvements in assay per- refinements to its existing TR-FRET technologies,
formance such as robustness, sensitivity, stability, such as seen with LANCE cAMP and TruPoint.
ease of automation and also permit simpler assay
development for tough target classes such as G␣␫- Upstate’s (www.upstate.com) original KinEASE®
coupled GPCRs. Another unique TR-FRET range of FP (fluorescence polarisation) assay for-
approach PerkinElmer has taken was the develop- mats have now been extended to include the
ment of its proprietary TruPoint™ assay system HTRF® format in partnership with Cisbio. The
which is ideal for the analysis of many different platform consists of a choice of three biotinylated
enzyme targets such as protease and helicases. peptides, a proprietary monoclonal antibody
TruPoint again utilises the high energy emission labelled with Eu3+ Cryptate (Eu[K]) and strepta-
Europium chelate dyes but now in conjunction with vidin-XL665. A schematic version of the assay is
a europium dye quencher (Figure 22). TruPoint displayed in Figure 23. Each peptide contains a com-
offers an extremely robust assay solution with mon C-terminal phosphoserine epitope with three
enhanced sensitivity, often requiring far less enzyme different N-terminal sequences allowing access to a
than competitive technologies, can be run using cell large range of kinases. The monoclonal antibody
lysates (eg caspase 3 assay), and in addition a posi- binds to the phosphorylated serine (the epitope) in
tive signal production (increase) format yields a the biotinylated peptide which in turn is bound to
greatly reduced incidence of false positives. Overall streptavidin-XL665 allowing FRET to occur.
PerkinElmer is committed to continue expansion in Increasing kinase activity produces more signal and
the area of high value functional biochemical and is proportional to the amount of kinase added to the
well. As can be seen in Figure 24, MST was titrated
with all three peptides, signal was observed with all
three peptides; with substrate three being the opti-
Europium
Quenching Dye mal choice. Representative kinase/substrate pairs are
Chelate Dye
shown in Figure 24 demonstrating excellent
signal/background ratios at low concentration of
kinase for all three conditions. HTRF KinEASE®
615nm has been initially validated with 70 kinases and
determined the optimal peptide for each kinase. It is
Caspase 3
Peptide Caspase 3 highly likely that many more kinases will phospho-
Substrate rylate one or more of the three peptides. It is also
possible to vary the N-terminal sequence to gain
access to even more kinases, maybe in excess of 100.
Figure 22: The TruPoint caspase-3 assay principle is based on dequenching of the Europium
Hence Upstate and Cisbio bring to the marketplace
chelate dye signal upon cleavage of the caspase 3 peptide substrate and removal of the a universal serine/threonine kinase assay system util-
quenching dye from proximity to the Europium ising just one labelled monoclonal antibody.

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Assays

Table 1: Comparison of commercial TR-FRET offerings

SUPPLIER TR-FRET TR-FRET PAIRS REMARKS

NAME OFFERING
NAME DONOR ACCEPTOR

Cisbio HTRF® Europium-Cryptate XL665 Eu3+ Cryptate stability

Broad product portfolio
Very low compound interference
d2 New small organic acceptor
Enhanced assay stability
Ultra-low miniaturisation for HTS

Echelon TRUE FRET™ Terbium TAMRA, Bodipy TMR, etc Modified for increased stability and
quantum yield
Fluorescein, Bodipy FL, etc Both assays and reagents available

GE Healthcare TR-FRET format Europium (TMT) Chelate CyDye Cy5 Small size relative to APC reduces
assays steric hindrance
CyDye unaffected by amino acid
residues like tryptophan

Hidex Proxiscreen Eu(III)-chelate dyed AlexaFluor680 Enhanced energy transfer

nanoparticle Decreased matrix effect due to robust
donor particle

Invitrogen LanthaScreen™ Terbium Fluorescein, Alexa Fluor 488 520/25 emission

Allows assay of substrates directly
labelled with fluorescein
GFP,YFP 570/10 emission
Rhodamine, Alexa Fluor 546 Allows assays using fluorescent protein
fusions of native substrates
Alexa Fluor 633 665/10 emission
Alexa Fluor 647 Allows multiplexing with green
acceptors with no spectral interference

Lumiphore LumiRay Green Terbium FAM,TMR, RPE, etc

Molecular IMAP TR-FRET Terbium 5FAM, 5TAMRA Antibody independent, select between
Devices TR-FRET or FP readout without
changing IMAP technology
Allows measurement of larger
substrates (proteins) & higher substrate
concentrations
>24 hour signal stability allows for
more flexible screening

PerkinElmer LANCE™ Europium Chelate APC High S/B (10s)

Very low compound interference
Alexa Fluor 647 Smaller size acceptor vs APC
Beneficial for competitive kinase assays
Very low compound interference
TruPoint™ Europium Chelate QSY-7 QSY-7 is a potent quencher of
europium
Extremely high S/B (up to 1000)
Suitable for hydrolysing enzymes & cell
lysates

Upstate HTRF® KinEASE, Europium-Cryptate SA-XL665 Developed in collaboration with Cisbio

4G10, PIProfiler

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Assays

Summary
Despite having been available for many years TR-
FRET based assay technology continues to evolve
and get better, with a wide range of new develop-
ments to enhance and extend this assay formats
capabilities. These developments include several
new kits addressing relevant second messenger
GPCR targets (eg cyclic AMP and IP-One); a new
universal serine/threonine kinase assay system;
the enhanced stability and performance of exist-
ing FRET pairs; increased use of red-shifted Alexa
Fluor© as acceptor dyes; better assay develop-
ment flexibility by direct covalent labelling of
substrates or the use of antibody independent for-
mats; the wider use of terbium complexes, includ-
ing using GFP as acceptor; and a new approach
using Europium chelate dyed nano-particles.
Taken as a whole a surprisingly large number of
TR-FRET pairings are now being promoted (see Figure 23: Schematic of the HTRF KinEASE® assay format.The kinase is incubated with
Table 1 for a summary of donor and acceptor compound and 1 of 3 biotinylated peptides, the reaction is started by the addition of ATP.
pairs) and the diversity of these offerings looks set The reaction is stopped by the addition of detection reagent containing Eu[K]-STK
to continue increasing over the coming year. In (monoclonal antibody) and streptavidin-XL665. Active kinase phosphorylates the peptide to
conclusion, the outlook for the sustained prefer- which the Eu[K] labelled antibody binds, streptavidin-XL665 binds to the N-terminal biotin,
hence allowing FRET.The amount of phosphorylation is proportional to the activity of the
ential use of TR-FRET based assays by screening kinase.The plate may be read after 1hr and the signal is stable >24 hr. NB these reactions
groups is positive. DDW were performed as 50µL reaction + 50µL detection giving a total of 100µL; it is
straightforward to reduce the total volume to ⬉4µL
John Comley is Managing Director of HTStec Limited
an independent market research consultancy whose
focus is on assisting clients delivering novel enabling
platform technologies (liquid handling, laboratory
automation, detection instrumentation and assay
reagent technologies) to drug discovery. Over the past
2.5 years HTStec has published 15 market reports on
drug discovery technologies and Dr Comley has
authored 13 review articles in Drug Discovery World.
Further information on accessing the market report
‘TR-FRET Based Assay Trends 2005’ can be obtained
by visiting www.htstec.com or e-mail
[email protected] to receive a free copy of the
Report’s Executive Summary and Table of Contents.

Figure 24: Examples of HTRF

MST1 Three peptides KinEASE® reactions.The left
hand panel illustrates the use
50 of the 3 peptides with MST 1,
70 substrate 1, substrate 2 and
40 60 substrate 3. An excellent
S1
Signal/Noise

50 signal/noise background ratio

Signal/Noise

30 S2 BrSK2 (h)
40 was obtained with substrates 2
S3 PAK3 (h) and 3 with substrate 1 being
20 30
IKKb (h) the weakest substrate.The
20
10 right hand panel shows
10 representative examples of a
0 0 kinase/substrate pairs
0 50 100 150 200 250 0 100 200 300 BrSK2/substrate 1,
Kinase (ng/well) final volume = 100µL Kinase (ng/well) final volume = 100µL PAK3/substrate 2 and
IKK␤/substrate 3

Drug Discovery World Spring 2006 37