Multimodality-Ultra HTS (Curr Pharma Biotec)

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

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

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 lessreagents is changing the way of drug discovery. Searching for hits using high throughputscreening 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 scintillationcounters is becoming limiting since only 12 wells can be counted at a time. Chargedcoupled device (CCD) camera based instruments, that image the whole plate in oneexposure, speed up detection and are compatible with any microplate footprint.Researchers are also demanding a choice of detection methods, including fluorescence, luminescence andradioactivity, and require imagers suitable for all applications. LEADseeker Homogenous Imaging Systemis a multi-modality platform offering imaging technology and assay toolboxes for radiometric, fluorescent andluminescent 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 thissystem include enzyme assays, receptor binding and molecular interactions.

INTRODUCTION

The drive for assay miniaturization and themove towards a choice of detection methods forhigh throughput screening (HTS) is changing theway researchers are detecting assays. Throughputof miniaturized radioactive assays is currentlylimited by scintillation counters since they canonly count up to 12 wells at a time, resulting incount times of approximately 40 minutes per 384-well plate, causing bottlenecks in HTS. Even withthe short read times (<1 second/well) used forluminescent assays, the total time to read a wholeplate is just under 5 minutes. For shorter-livedsubstrates such as horse radish peroxidase (HRP)-luminol, the luminescence signal may changesubstantially in five minutes, adding to intra-wellvariation and normalisation problems. Theseproblems may be overcome by the use of imagersthat have the potential to image the entire plate atonce with the resolution required for any numberof wells. The exposure time on LEADseeker forluminescence applications is usually less than 1minute and more commonly less than 20 secondsper plate. Radiometric assays are commonlyimaged for 5 minutes, for fluorescent assays,

*Address correspondence to this author at the Amersham PharmaciaBiotech UK Limited, Amersham Place, Little Chalfont, Buckinghamshire,England, HP7 9NA; Tel: +44 29 20526417; Fax: +44 29 20526474; Email:[email protected]

imaging times are up to 30 seconds dependent onthe particular application. Another potential sourceof inaccuracies is the variation in sensitivitybetween the different photomultiplier tubes(PMTs) in the same instrument, resulting invariable data being observed in wells emitting thesame amount of light. This is usually overcome bynormalisation against reference plates. Despitethis, statistical errors between readings given byindividual PMTs will inevitably occur,contributing to instrument noise. Imager systemsare not subject to these problems because lightfrom all the wells in the plate is receivedsimultaneously.

LEADseeker, from Amersham PharmaciaBiotech, is a multi-modality assay platform whichis capable of imaging radiometric, fluorescent andluminescent based assays.

MULTI-MODALITY IMA GING PLATFORM

LEADseeker homogeneous imaging systemconsists 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 andZ plate mechanism and tip/tilt/rotate cameramovements for automated focusing. Theinstrument can be used in three modes: as a

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

manual system for assay development, as aworkstation (Fig. 1) containing a microplatecarousel and stacking system capable ofcontaining one hundred and twenty 384-wellplates or one hundred and thirty nine 1536-wellplates, or as a fully automated system, forintegration into automatic systems by ananthropomorphic robotic arm. In this format,LEADseeker is compatible with all major roboticsystems, including Aurora, CRS, Beckman,Zymark, Robocon and Scitech. For use with eitherof the automated systems, plate identification isvia a bar code reader.

The camera, from Spectral Instruments,contains a grade 1, thinned, back-illuminated CCDchip mounted in a light tight case. The chip isthinned to increase sensitivity to relatively lowlight output assays such as radiometric proximityassays that require the use of low energy isotopes.The chip has an imaging area of 24.6mm x24.6mm, at 1024 x 1024 pixels to enable highresolution imaging of microplates. As well asbeing sensitive to light, CCD chips are alsosensitive to heat. To reduce this thermal noise, theoperating temperature of the chip is cooled to -100°C under vacuum by a proprietary refrigerantgas. Dark noise is therefore substantially reduced(Fig. 2) and any remaining noise can mostly beattributed to electronic read-out noise.

Standard lens types suffer from parallax errorwhere light is distorted and shadowing can occurin wells that are not at the centre of the imagingfield. These problems can be avoided by using atelecentric lens. In LEADseeker, the telecentricBorealis™ lens is made up of multiple lens

elements and collects parallel light from samplesin all wells of a microplate. This removes parallaxerrors by collecting light equally from all wellswith no shading (Fig. 3 and Fig. 4).

In LEADseeker, as with any other CCD basedsystem, it is necessary to account for the variationin response across the chip. In order to facilitatemulti-modality operation, two correction methodsare available. The “Flat Field Correction” (FFC)method is an image correction method. A platecontaining a constant level of signal is imaged(Fig. 5a) and the differences in intensity observed

Fig. (2). Variation of dark noise (integrated optical density(IOD) units) with temperature. Noise levels are taken from 1minute exposures at a binning factor of 3x3, using an areaequivalent to one well of a 384-well plate.

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 leftshows a 96-well microplate viewed through a standard lens. The white crescent shapes around the edges of each well are thewell walls. Only in the centre of the plate can the bottoms of the wells be completely seen. With a telecentric lens (image onright), the well bottoms are completely visible in all areas of the plate.

across the whole field are then normalised by thesoftware in subsequently imaged test plates. Thishas been shown to be appropriate for bothradiometric and luminescence work. The “Well ByWell Correction” (WBWC) method is a datacorrection procedure and is recommended forfluorescence applications because of the added

complication of both excitation light delivery andcross chip detection variation. Briefly, successivecalibration plates, containing uniform and differentknown levels of signal in each plate, are imagedand the data obtained for each signal level is usedto construct a calibration curve for each separatewell (Fig. 5b). Determinations from the wells of


subsequent test plates are then related to therespective well calibration curve to determine anormalised read value.

REAGENT TOOLBOXES

As part of a multi-modality platform, assayreagents are necessary for radiometric, fluorescentand luminescent technologies. Radiometric assayssuitable for high throughput imaging are based onscintillation proximity assays (SPA) [1]. Inproximity assays, scintillant-containing beads arechemically treated on the surface to enable thecoupling of molecules such as enzyme substratesand receptor proteins. Low to medium energyisotopes, such as 3H, 125I, 33P and 35S, are used insuch assays, so that when a radiolabelled moleculebinds to a bead via interaction with a biochemicalor cellular target molecule, the energy emittedstimulates the scintillant within the bead to emitlight. This emitted light can be imaged using aCCD camera. Unbound radioactive molecules arenot close enough to stimulate the beads to emitlight. Since such molecules are not detected in theassay, there is no need to remove them from theassay, thus eliminating time-consuming separationsteps. Since CCD chips are most sensitive to lightemitted in the red region of the spectrum,europium-containing beads have been developedfor use with LEADseeker [2]. Polystyrene (PS)and yttrium oxide (YOx) beads are available withseveral bead coatings: streptavidin, wheat germagglutinin (WGA), protein A and nickel-chelatefor use with his tags.

An inherent problem of homogeneous assays iscolour quenching. The majority of compounds thatcause colour quenching are yellow or brown incolour, and therefore, absorb light in the blueregion of the spectrum. Since PMT-basedscintillation counters are most sensitive to bluelight, protocols have to be incorporated to correctfor quenching. This quenching effect is less of aproblem with red-emitting LEADseeker beads(Fig. 6).

To miniaturise low signal 384-well plate assaysand develop assays in 1536-well plates withreduced quantities of reagents, the detection limitof the imager is important. Using tritium-labelledbiotin bound to LEADseeker imaging beads, theminimum detectable limit (MDL, defined as 3 xbackground standard deviation) can be calculatedusing PS and YOx beads in 384 and 1536-wellplates (Fig. 7). In 384-well plates for yttrium oxidebeads, the MDL is 215dpm, which is equivalent to2.5fmol biotin at the specific activity of40Ci/mmol. In 1536-well plates, the MDL is54dpm, equivalent to 625amol biotin at thespecific activity of 40Ci/mmol. For PS beads, theMDL’s are higher (430dpm, 5fmol and 215dpm,2.5fmol for 384 and 1536-well plates,respectively), due in part to the inherently lowerlight output of these beads, but also to the smallamount of background phosphorescence that isapparent in the images. Detection limits of theseorders should make it possible to image assayswith very low signal levels in 384-well plates andto miniaturise many assays into 1536-well plateformats with reduced quantities of reagents.

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. TheFFC accounts for this variation in subsequent test images. b) An example of a WBWC calibration curve derived for a singlewell of a 384-well plate, constructed using a range of three Cy3 fluor concentrations and a buffer blank.


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 concentrationsrange from 0 to 3mg/ml in a tripling dilution series from bottom to top. The false colour images show that YOx and PS beadsdo not alter in intensity in the presence of tartrazine, but that YSi and PVT SPA beads are both quenched. All four bead typesare 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 0.1mgstreptavidin coated PS or YOx bead in 10mM Hepes, pH 7.5. The final volume in 384-wells was 20µl and the plate was imagedby coincidence averaging, 2 x 10 minute exposures, using a binning factor of 3x3. For 1536-well plates, the total volume was4µl and a binning factor of 2 x 2 was used.

An attractive alternative to the use ofradioactivity in high-throughput screening hasbeen the use of fluorescence technologies [3].These approaches necessitate the labelling of oneor more of the assay components with afluorescent species. This in itself generallyrequires that the fluor contain an active group forconjugation purposes and also that it has otherchemical/physical properties that are compatiblewith the biological assay. Such reagents are

available from several suppliers (such asMolecular Probes), other examples include thefamily of CyDye fluors and quenchers availablefrom Amersham Pharmacia Biotech (Table 1).

Cyanine dyes were first synthesised in the mid1800’s for the photographic industry as spectralsensitisers or desensitisers for silver halideemulsion films [6]. However, in recent years,interest in these dyes for high technology


applications such as optical data storage [7] andfluorescent labelling probes for biological studies[8] has increased. They are characterised by aresonance structure comprising a polymethinechain bearing two terminal nitrogen atoms and anoverall positive charge. Modification of theheterocycle and/or methine chain allows for dyesto be synthesised which have a range of excitationand emission wavelengths [9], with a Stokes’ shiftof approximately 15nm. Presently, CyDye probeshave excitation and emission wavelengths rangingbetween 560nm-700nm (Fig. 8).

Cyanine dyes have large extinction coefficients,fluorescent lifetimes in the region of 1-3ns and aregenerally detected away from the naturalautofluorescence of biomolecules. They have alsobeen found to be insensitive to many relevantchemical environments such as pH and DMSO.However, in some circumstances, significantexcitation energy can be lost through the dyesinterconverting into a number of stereoisomericconformations and also by other non-fluorescentpathways, such as rotational and translationalmodes of vibration [10]. If the relaxation pathwaysof the molecule could be restricted, the preferred

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

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

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

Quantum yield

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 - - - -


route would more likely be via fluorescence. Cy3B(Fig. 9) contains a tricyclic system across thepolymethine chain which fixes the chromophore,preventing flexibility within the molecule.Consequently, it is found to be approximately 3times as intense on labelling of a biologicalconjugate when compared with Cy3. In addition,the molecule has an increased fluorescencelifetime of 2.8ns compared with Cy3 (which has alifetime of <0.3ns). This increase in lifetimesuggests the potential for use in fluorescencepolarization assays as a replacement forfluorescein.

Another common assay approach, particularlyin the field of protease cleavage, is the use of adual labelled peptide substrate, incorporating botha fluorescent “Donor” and non-fluorescent energyacceptor, or “Quencher” [3]. In order to optimisesuch an approach with the CyDye fluors (as thedonor), a family of CyDye quenchers (or “dark”dyes) have been developed (Fig. 10).

The presence of a dinitrobenzyl moiety mayallow for orientation of the benzyl ring in relationto the chromophore which could allow for either

collision quenching or the generation of radicalanion pairs [11]. From a range of hybridizationexperiments (data not shown) it has been foundthat use of these CyDye quenchers with a CyDyedonor can be compared to and considered moreefficient than some other donor dyes with Dabcylquencher systems.

A further alternative to radiometric assays is theuse of chemiluminescence. Chemiluminescentreactions generally involve the formation of ametastable intermediate which upondecomposition, gives out visible light. Achemiluminescent molecule serves as the substratefor an enzyme, typically horseradish peroxidase oralkaline phosphatase, that is usually linked to abiological entity such as streptavidin. This can beused to report on the binding of streptavidin to abiotinylated moiety, such as a peptide oroligonucleotide.

Light output profiles of chemiluminescentsubstrates vary considerably with the enzyme-substrate combination in use. The light outputprofile of horseradish peroxidase reachesmaximum level quickly but also deterioratesquickly. Alkaline phosphatase substrates tend toreach a maximum level after 20-40 minutes butmay then persist at or near this level for severalhours. This is illustrated in the data shown in (Fig.11).

Short-lived substrates such as HRP-luminol areideally suited for imaging since the whole plate isimaged in 20-60 seconds. This results in less errordue to signal decay compared to counting on aPMT based system where counting a plate maytake as long as five minutes. Wavelength shiftersmay also be incorporated in reaction mixtures that

Fig. (10). Cyanine probes: Quenchers.

Fig. (9). Cyanine probes: Cy3B (N-hydroxysuccinimide(NHS) ester).

N

O

N

C O

O

N

O

O

HO3S

N+

HO3S

N

O2N

O O

N O

NO2

SO3H

N+

HO3S

N

O O

NO O

NO2

NO2

SO3H

A. CY5Q (NHS ester) B. CY7Q (NHS ester)


alter the wavelength of emitted light from(typically) 420nm to 500-600nm. Theseparticularly suit the sensitivity range of CCD-based detection systems, which have bettersensitivity towards the red region of the spectrum[12]. Substrates that naturally emit longerwavelength light have been reported [13].

ASSAY APPLICATIONS SUITABLE FORIMAGING

Imaging systems can be used to study a rangeof applications including enzyme assays, receptorbinding studies and molecular interactions.

PROTEASE ASSAYS

An example of a protease assay suitable forimaging utilises the metallo-endoproteinase, Asp-N. Asp-N is often used for peptide mapping andprotein sequencing. The enzyme cleavages an 8-mer peptide on the N-terminal side of aspartic acid(D) [14]. The assay has been configured in aquench format in which the N-terminus of thepeptide has been labelled with Cy3B and the C-terminus with Cy5Q. Therefore, prior to proteasecleavage the intact peptide cannot generate afluorescent signal at Cy3B emission wavelengths(following excitation with light at Cy3Bwavelengths) due to quenching by Cy5Q.Proteolytic digestion of the peptide releases thequench effect, so excitation at Cy3B wavelengthsresults in the concomitant emission at Cy3B

wavelengths (Fig. 12). This assay format has alsobeen adapted for use with other proteases.

Assays were carried out in black opaque 384-well or 1536-well microplates by adding substratepeptide, Cy3B-YVADAPVK-Cy5Q to a finalassay concentration of 100nM in a volume of 50µl(384-well assay) or 5µl (1536-well assay) 50mMTris buffer, pH8, containing 0.005% (v/v)Tween™ 20. To 384-well assays, 5µl Asp-Nenzyme at 1µg/ml was added, resulting in a finalassay volume of 55µl. In 1536-well assays, 0.5µlof enzyme at 1µg/ml was added, resulting in afinal volume of 5.5µl. In no enzyme control wells,the volume was made up using assay buffer.Following incubation at room temperature forbetween 0 and 60 minutes, imaging was performedon LEADseeker at 540nm excitation and 570nmemission wavelengths; exposure times of 60seconds (384-well assay) and 90 seconds (1536-well assay) were used (Fig. 13). A WBWC methodwas employed with four standard concentrationsof between 0 and 100nM Cy3B.

KINASE ASSAYS

Protein kinases play an important role in thecontrol of many of the key steps in cellularprocesses, such as signal transduction and cellcycle control [15]. Due to the nature of theenzymes, kinases are inherently suitable forradiometric proximity based screening assays.Incubation with [γ-33P] adenosine 5’-triphosphate(ATP) and a kinase, under the appropriate assay

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 ambienttemperature.


conditions, results in the incorporation of theradiolabelled phosphate in the peptide or proteinsubstrate. Use of a biotinylated, glutathione-S-transferase (GST) or his-tagged substrate allowscapture onto streptavidin, glutathione or nickelchelate beads, respectively. The close proximity ofbound [33P] to the scintillant containing beadscauses the emission of light that is detected by theimaging system. A schematic example of aproximity kinase assay using streptavidin coatedbeads is shown in (Fig. 14).

Assays of the mitogen-activated protein (MAP)kinase enzyme, extracellular signal-regulatedkinase 1 (Erk-1) have been imaged in 384 [16] and1536-well format. A MAP kinase time course wasperformed in 1536-well format using 12.5pmolesbiotinylated myelin basic protein (bMBP), 20nCi[γ-33P]ATP, 5pmoles unlabelled ATP, 0.05µg Erk-1 in 50mM MOPS, pH 7.2, containing 5mMMgCl2. The reaction volume was 5µl. Assays wereincubated for increasing times, up to 60 minutes,at 37°C, prior to the addition of a ‘stop’ solutioncontaining 500µM unlabelled ATP, 5mM EDTA,

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

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 for1536-well assay) and are expressed as nominal signal units. For reference, maximum signal:background levels achieved werein the order 12:1 (384-well assay) and 14:1 (1536-well assay), expressed as the total Cy3B signal obtained after extensivecleavage (approximately 3 hours) above that of the uncleaved (no enzyme) peptide control.


0.1% (v/v) Triton X-100 and 50µg streptavidincoated PS LEADseeker beads in PBS. The finalvolume was 8µl. Plates were centrifuged for 10minutes at 1000 x g to reduce non-proximityeffects. Medium energy isotopes are incompatiblewith the use of PS beads in suspension due to theirrelatively long path length producing higherbackgrounds. These limitations can be overcomeby settling or centrifuging beads, or floating themby the addition of caesium chloride. Plates werethen imaged for 5 minutes. The effect of time onErk-1 kinase activity was shown by a signalincrease. Images using PS beads were obtained(Fig. 15) and used to construct a typical timecourse graph (Fig. 16). Linear phosphorylation ofbMBP was observed up to approximately 30minutes.

Using this assay protocol, the inhibitory effectsof staurosporine, a non-specific inhibitor of proteinkinases [17], on Erk-1 has been studied.Staurosporine, dissolved in DMSO, was added toassays covering concentrations of 0.01µM to50µM. The effect of staurosporine on Erk-1activity was shown by a signal decrease withincreasing staurosporine concentrations. From atypical inhibition curve (Fig. 17) an IC50 value of4.1µM was obtained.

Other kinases suitable for radiometric imaginginclude p56lck, which is critical for T-cellactivation [18]. Assays for this enzyme have beenperformed in both 384 and 1536-well format tolook at the effect of a range of inhibitors [19].

Fig. (15). Image of an Erk-1 kinase time course (n=3). A 5 minute image was taken. Non specific binding (NSB) wasdetermined in the absence of enzyme.

Fig. (14). Schematic concept of a kinase proximity assay using streptavidin coated beads.


An alternative method to study kinase activity,which removes the need for radioactivity, is theuse of chemiluminescence. Mouse anti-phosphotyrosine antibody immobilised ontomicroplates can be used to capture biotinylatedtyrosine phosphopeptide. These capturedphosphopeptides can be detected by reaction withstreptavidin conjugated to alkaline phosphatase. Aschematic diagram illustrating the assay format isshown in (Fig. 18).

This assay format has been used with twophosphopeptides, tyrosine kinase biotinylatedphosphopeptide 2 (Biotin-EGPWLEEEEEA[pY]GWMDF-amide) and tyrosine kinase biotinylatedphosphopeptide 3 (Biotin-RRLIEDAE[pY]AARG-amide, both from Pierce). Black 384-well

microplate wells were coated with 50µl/wellmouse anti-phosphotyrosine antibody at 4.6 µg/mlin 0.05M Na2CO3/NaHCO3 buffer, pH 9.6 for 3hours at ambient temperature and washed threetimes with 75µl/well phosphate buffered saline(PBS)/0.05% (v/v) Tween 20 wash buffer. Wellswere treated with 75µl/well 0.2% (w/v) I-Block™(Tropix Inc.) in PBS/Tween 20 blocking buffer for1 hour at ambient temperature, drained overabsorbent paper, then sealed with adhesive filmand stored at 4°C until required.

Dilutions (50µl/well) of tyrosine kinasebiotinylated phosphopeptides 2 or 3 in 0.2%I-Block in PBS/Tween 20 were added to platewells and incubated for 30 minutes at ambienttemperature, then washed with 3 x 50µl wash

Fig. (18). Schematic representation of chemiluminescent immunoassay format.

Fig. (16). Erk-1 time course. An increase in enzyme activityover time was seen as an increase in integrated opticaldensity units. Values are means ± standard deviation (SD)(n=3).

Fig. (17). Inhibition of Erk-1 kinase activity bystaurosporine. Values are means ± S.D. (n=4).


buffer. Streptavidin-enzyme conjugate in blockingbuffer (50µl/well) was added to plate wells,incubated (30 minutes, ambient), then washed with3 x 75µl/well wash buffer, followed by 2 x 75µl20mM Tris, 1mM MgCl2, pH 9.5 assay buffer).LumiPhos 530 streptavidin-alkaline phosphataseconjugate was used for chemiluminescentdetection at 50µl/well.

For photomultiplier instrument-based detection(i.e. Wallac 1450 MicroBeta™), the instrumentwas used in chemiluminescence mode with a counttime of 0.2 seconds per well, in order to reduce thetotal plate acquisition time. For CCD imaging,LEADseeker was used to record chemil-uminescence from plates with exposures of 30seconds or less with 2 x 2 binning. Data from bothPMT- and CCD-based systems was recorded 2hours after the time of addition of chemil-uminescent substrate. Similar detection sensitivitywas demonstrated in 96 (data not shown), 384(Fig. 19) and 1536-well formats (data not shown).

The same plate read on different instrumentsgave information on the relative performance ofLEADseeker and a PMT-based plate reader(Wallac MicroBeta), as shown in (Fig. 20). Thisdata is also summarised in (Table 2).

Using LEADseeker, the time required to imagea complete microplate is considerably less than

that taken for PMT-based luminometers eventhough in the latter systems, the time required perwell is shorter. Furthermore, the statisticalvariation observed across the wells of a microplateis significantly reduced. This is partly due to theconstantly changing luminescence output andpartly due to variation between photomultipliersreading different areas of the plate. This problemis worse for the shorter-lived HRP-basedsubstrates because of the greater variability ofoutput.

RECEPTOR BINDING ASSAYS

Receptor binding assays have traditionally beenscreened using radiometric filter binding assays orSPA. Miniaturization of these assays is possibleusing imaging proximity assays. Receptors can beimmobilised directly onto imaging beads via anumber of coupling methods, including the use ofWGA, which binds to N-acetyl-β-D-glucosamineresidues in membranes and selects for membraneglycolipids and glycoproteins. Once immobilised,the receptor is close enough to the bead so that,should a suitably radiolabelled ligand bind to thereceptor, it will be in close enough proximity tostimulate the bead to emit light. This is shownschematically in (Fig. 21).

0

2000

4000

6000

8000

10000

12000

0 0.5 1 1.5

pmoles phosphopeptide / well

IOD

uni

ts

Phosphopeptide 2

Phosphopeptide 30

50

100

150

200

250

300

0 10 20 30

nM phosphopeptide 2 added

Sig

nal:

No

ise

LEADseeker

Microbeta

Fig. (19). Detection of biotinylated phosphopeptides bychemiluminescent immunoassay using LumiPhos 530alkaline phosphatase substrate in 384-well plates. Less than19.5 femtomoles phosphopeptide 2 or phosphopeptide 3were detected with LumiPhos 530 after 2 hours incubation.

Fig. (20). A comparison of normalised signal to noisedata taken from the same 384-well microplate read onLEADseeker (u) and on a Wallac MicroBeta plate reader(o). Signal to noise was calculated as mean signal / 3 xstandard deviation of the signal from control wells. Theerror bars shown represent relative standard deviationfrom triplicate wells. No more than 6 minutes elapsedbetween reading the microplate on the two differentinstruments.


Several receptor binding assays have beenimaged using radiolabelled ligands such as thechemokine [125I]macrophage inflammatoryprotein-1α (MIP-1α), which has a high affinity forthe CCR1 chemokine receptor and [3H]imipraminewhich binds to the serotonin transporter protein[21]. To study the 5-HT7 receptor, which plays akey role in cognitive and behavioural processes,the ligand [3H]5-carboxamidotryptamine ([3H]5-CT) has been used [22]. Assays have beenminiaturized to 384-well format usingLEADseeker. Assays contained 2.5µg cloned rat5-HT7 receptor, 14nCi [3H]5-CT and 750µg WGAcoated YOx beads. Non-specific binding wasdetermined by the addition of 40µM 5-CT. Thefinal assay volume was made up to 50µl by theaddition 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 resultedin a signal to background ratio of greater than 5:1(Fig. 22).

A displacement curve was obtained usingincreasing amounts of unlabelled 5-CT and anEC50 value of 2.6nM was determined (Fig. 23).

Another receptor type suitable for assayingusing imaging are G-protein coupled receptors.The quantification of agonist-induced activity canbe measured via increased [35S]GTPγS binding[23].

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

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

MicroBeta LEADseeker

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

38.9 0.96

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:Noisemean(signal) / 3 x S.D.(blank).

163 140 344 301

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


MOLECULAR INTERACTIONS

The study of molecular interactions are otherexamples of assays which are highly suited tofluorescent detection. An example of this is thestudy of the transcription factor, NF-κB. NF-κBhas received extensive interest over the pastdecade because of its pivotal role in cellularprocesses, such as inflammation and apoptosis[24-25]. The interaction between the p65 subunitof NF-κB and a 19 base pair double stranded (ds)DNA sequence containing the 10 base pairrecognition site for NF-κB can be examined usinga fluorescence resonance energy transfer (FRET)assay. The assay comprises of a Cy3 labelled anti-

GST antibody which specifically binds to a p65-GST tagged protein, followed by interaction withNF-κB dsDNA 5’ end-labelled on the codingstrand with Cy5. The FRET signal generated whenthe three components are bound together can bedisrupted 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-GSTantibody to 20nM NF-κB-p65-GST protein in10mM HEPES buffer, pH7, containing 0.2mMEDTA, 20mM NaOAc, 0.05% (v/v) IPEGAL,5mM DTT and 1mg/ml BSA. The final assay

Fig. (22). 5-HT7 receptor binding assay using [3H]5-CT.Values are means ± SEM (n=3)

Fig. (23). [3H]5-CT displacement curve. Values are means± SEM (n=2).

Fig. (24). Schematic representation of the NF-κB competition binding assay.


volume was 30µl. Assays were incubated for 60minutes at room temperature. Followingincubation, 100nM Cy5 labelled dsDNA(containing the NF-κB consensus bindingsequence) was added together with variousconcentrations of unlabelled specific or non-specific competitor dsDNA sequence. Reactions,in 50µl total assay volume, were incubated for afurther 60 minutes before imaging on LEADseekerfor 90 seconds at 540nm excitation and 690nmemission wavelengths (Fig. 25). A WBWCmethod was used with 4 standard concentrations ofbetween 0 and 200nM of a Cy3-Cy5 carboxyl dyedesigned for calibration purposes.

CONCLUSION

The progress towards ultra-high throughoutscreening (uHTS) has driven a move away fromconventional plate counting technology to imagingsystems capable of imaging entire plates. Systemscapable of detecting radiometric, fluorescent andluminescent signals offer researchers a choice ofdetection methods expanding the range ofapplications suitable for uHTS.

Using new europium containing beads, 384 and1536-well radiometric proximity assays can beread in 10 minutes or less. Due to the emissioncharacteristics of the beads, the effect of colourquenching on signal output, a problemtraditionally associated with homogeneous assays,is greatly reduced.

The sensitivity of CCD chips to light emitted inthe red region of the spectrum makes them idealfor the detection of cyanine dyes, such as CyDyes.In addition, CyDye fluors are particularly suited asfluorescent probes since their red to near infraredabsorption and emission maxima (500-800nm) areeasily distinguishable from the shorter wavelengthendogenous or autofluorescence associated withbiological samples and equipment. Noveldevelopments have resulted in the development ofCyDye probes with either enhanced or reducedfluorescence characteristics for use in FRETapplications.

The use of imagers in chemiluminescent assaysresults in a significant reduction in statisticalvariation across microplates as the time required toanalyse a complete microplate is considerably lessthan for PMT-based luminometers.

With this range of applications and fullautomation of imager systems such asLEADseeker, true ultra-high throughput screeningis 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-regulatedkinase 1

FFC = Flat field correction

FRET = Fluorescence resonance energytransfer

GST = Glutathione-S-transferase

-8 -7 -6 -50

20

40

60

80

100

log10[competitor DNA] (M)

%B/B

o

Fig. (25). NF-κB competition binding assays. Competition forbinding of the Cy5 labelled dsDNA to the NF-κB protein in thepresence 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 standarddata analysis. For reference, maximum signal:background levelsachieved were in the order 1.7 - 1.8:1 (expressed as specifictotal FRET signal above the background signal observed in thepresence of a 500-fold excess of specific inhibitor) and the IC50

value determined for the specific sequence competitor was115nM.


HRP = Horse radish peroxidase

HTS = High throughput screening

IOD unit = Integrated optical density unit

MAP kinase = Mitogen-activated protein kinase

MDL = Minimum detectable limit

MIP-1α = Macrophage inflammatoryprotein

NHS = N-hydroxysuccinimide

NSB = Non specific binding

PBS = Phosphate buffered saline

PMT = Photomultiplier tube

PS = Polystyrene

PVT = Polyvinyl toluene

RSD = Relative standard deviation

SD = Standard deviation

SEM = Standard error of the mean

SPA = Scintillation proximity assays

UHTS = Ultra-high throughput screening

WBWC = Well by well correction

WGA = Wheat germ agglutinin

Yox = Yttrium oxide

Ysi = Yttrium silicate

ACKNOWLEDGEMENT

The authors wish to thank their colleagues forsupplying the data used in this manuscript.

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