A homogeneous high throughput assay is described which screens compounds for enzyme inhibition, or receptor or other target binding. Inhibition (or binding) by the library compounds causes a change in the amount of an optically detectable label that is bound to suspendable cells or solid supports. The amounts of label bound to individual cells or solid supports are microscopically determined, and compared with the amount of label that is not bound to individual cells or solid supports. The degree of inhibition or binding is determined using this data. Confocal microscopy, and subsequent data analysis, allow the assay to be carried out without any separation step, and provide for high throughput screening of very small assay volumes using very small amounts of test compound.

FIELD OF THE INVENTION 
This invention relates to the high throughput screening of chemical 
compounds for interactions with target molecules. 
BACKGROUND OF THE INVENTION 
To find lead compounds for drug discovery programs, large numbers of 
compounds are often screened for their activity as enzyme inhibitors or 
receptor agonists/antagonists. Large libraries of compounds are needed for 
such screening. As a result of developments in this field, it is now 
possible to simultaneously produce combinatorial libraries containing 
hundreds of thousands of small molecules for screening. With the 
availability such libraries, however, has come a need for large scale, 
rapid screening methods. 
For example, the libraries may be contained on microbeads, each compound 
being present in a picomolar amount. Because the amount of compound is 
very small, it is advantageous to conduct the high throughput screening 
method in very small volumes, e.g., on the order of 1 .mu.l. Such assays 
can be performed in the 1536 well plate described in U.S. patent 
application Ser. No. 60/037,636 filed Feb. 18, 1997. Microassays in such 
small volumes, however, are difficult to accurately and repeatedly perform 
using conventional methods. 
Receptor binding assays used in high throughput screening typically involve 
three steps. First, a labelled ligand is incubated with a target receptor 
in the presence of compound to be tested for inhibition of ligand/receptor 
binding. Second, the receptor and ligand (and compound) are separated 
using filtration and/or washing of an immobilized receptor. Finally, the 
amount of labelled ligand bound to the receptor is quantified. This 
conventional screening is a `separations-mode` assay, i.e., one in which 
the bound ligand is physically separated from the free ligand using either 
a filtration membrane or the selective adhesion of either bound or free 
component to a surface (e.g., the surface of a microtiter plate). 
Separation, however, is time-consuming and therefore slows high throughput 
screening. It can also, if fluid handling steps employed are not 
sufficiently precise, create variations in the signal generated in the 
assay and can disturb equilibrium binding conditions. Furthermore, 
separation is difficult to automate and is potentially hazardous when 
radioactive materials are involved. These problems are particularly acute 
in assays conducted in microvolumes using small amounts of test compound. 
It is therefore advantageous in high throughput screening to distinguish 
bound and free ligands in a homogeneous assay, i.e., one that eliminates 
the need for separation. To be particularly useful in screening large 
scale combinatorial libraries, such an assay should readily permit small 
volumes, and small amounts of test compounds, to be used. 
A homogeneous assay is described in U.S. Pat. No. 4,568,649 which employs 
beads that are impregnated with a scintillant (these are commercially sold 
as Scintillation Proximity Assay beads (SPA.TM., Amersham Corp., Arlington 
Heights, Ill.)). The beads are also coated with a ligand that is capable 
of binding with radio-labelled target in a sample. When the ligand binds 
to the radio-labelled target, the scintillant on the bead is activated by 
the radiolabel. The level of light energy produced by the scintillant 
indicates the amount of bound labelled target in the sample. This method, 
however, requires handling of radioactive reagents and is somewhat limited 
in sensitivity. 
Another homogeneous assay is known in which signal is generated when 
labeled ligand and labeled target interact. One label is an energy 
donating Eu-cryptate having a long-lived fluorescent excited state and the 
other is an energy-accepting protein, allophycocyanin, having a short 
fluorescent excited state. Energy transfer occurs between the labels when 
they are less than 7 nm apart. During the assay, the Eu-cryptate is 
excited by a pulsed laser, and its fluorescent emission continually 
re-excites the allophycocyanin, whose fluorescence is measured by a time 
resolved fluorescence reader. This method, however, requires labeling of 
both the ligand and the target and is not as sensitive as some other 
commercially available assays. Also, allophycocyanin is a very large, 
multimeric protein which can affect the assay in an unpredictable manner. 
A fluorescent imaging plate reader has been used to perform optical 
screening in cell-based kinetic assays that measure membrane potential and 
intracellular calcium. The assay employs an optical method that limits the 
depth of field measured by a CCD camera to the bottom of an assay well, 
where fluorescence in a layer of live adherent cells is measured. By 
limiting the depth of field to the cell layer, background fluorescence 
from extracellular dye is reduced. Data is obtained over time measuring, 
e.g., depolarization of cells (Schroeder et al., (1996) Journal of 
Biomolecular Screening 1:75-80). This method, however, uses live cells 
that require maintenance for the period of the assay, necessitating 
complicated integrated fluid handling to trigger rapid cellular events. 
Such handling is very difficult, if not impossible, to perform in the 
microvolumes that are used in high throughput screening of small amounts 
of library compounds. Also, measurement is taken of a bulk sample, i.e., 
of the entire layer of cells and the assay does not discriminate between 
fluorescence bound to individual suspended cells and background 
fluorescence. 
It is therefore an object of the present invention to provide an assay for 
high throughput screening that does not require a separation step, i.e., 
is homogeneous. 
It is another object of the invention to avoid radioactive waste, and to 
avoid labeling of both ligand and target molecule. 
It is another object of the invention to provide an assay which is readily 
adaptable for miniaturization in microvolumes, and which is highly 
sensitive. 
It is another object of the invention to provide a high throughput 
screening method for detecting activity of small amounts of compounds, 
such as are found in combinatorial libraries of beads having picomolar 
amounts of compound thereon. 
SUMMARY OF THE INVENTION 
The present invention relates to a high throughput assay for rapidly 
screening a plurality of compounds. The assay determines the degree of 
inhibition by the compounds of a ligand/receptor interaction, or of an 
enzyme catalyzed reaction, or the degree of binding of library compounds 
to a target molecule. Inhibition (or binding) by the library compounds 
causes a change in the amount of an optically detectable label that is 
bound either to suspendable cells or to suspendable solid supports. The 
degree of inhibition (or binding) is determined by measuring, by 
microscopy, the amounts of label that are bound to individual cells or 
solid supports. These amounts are compared with the amount of label that 
is not bound to individual cells or solid supports (i.e., background 
signal). The degree of inhibition or binding is determined using this 
data. Preferably, measurement is performed using a confocal microscope. 
The assay is homogeneous, i.e., no separation step is required to remove 
unbound label, since the amount of bound label is distinguished by 
scanning of the individual cells or solid supports. The method allows 
exceptional sensitivity and high throughput to be obtained in assays using 
small volumes, and small amounts of test compound.

DETAILED DESCRIPTION OF THE INVENTION 
All patent applications, publications, or other references that are listed 
herein are hereby incorporated by reference. 
The present invention allows high throughput screening of compounds, such 
as those found in combinatorial libraries, to determine active drug 
candidates. The method allows substantial reduction in the time required 
for screening such libraries over that required by non-heterogeneous 
methods. It also eliminates the need for disposal of washed reactants and 
is highly adaptable to performance in micro-volume assay vessels. In 
addition, the method improves over conventional homogeneous assay methods 
in its higher signal to noise ratio and in requiring use of only one 
label. It provides a sensitive assay that allows successful screening of 
very small amounts of compounds derived from microbead libraries, thereby 
allowing more assays using those microbeads, and faster, more efficient 
screening. 
The method of the invention involves measurement of the amounts of bound 
and free signal in the assay by microscopy. This can be carried out, for 
example, by sequentially viewing different depths in the sample using a 
conventional microscope employing a narrow depth of focus. According to 
the preferred method of the invention, however, confocal microscopy is 
employed to individually determine the amount of bound signal to 
individual cell sized particles. 
Confocal microscopy confines detection of an illuminated object, or sample, 
to a thin object plane. A view of a "slice" of the object, or sample, is 
obtained. This is achieved, for example, by placing a spacial filter, such 
as a pinhole, in the image plane located between the objective lens and a 
detector. Only light emitted from a narrow region near the object plane 
converges through the spacial filter. Light from other planes is blocked 
by the filter. Images are obtained of the object plane, e.g., by scanning, 
in sequence, the points in the field of view, to obtain the "slice". 
Confocal microscopy using laser scanning is particularly preferred for use 
in the invention. A suitable laser scanning microscope is sold as 
"IMAGN/2000" by Biometric Imaging Inc. (Mountain View, Calif.). Laser 
scanning microscopes are also described in U.S. Pat. Nos. 5,556,764 and 
5,547,849. These microscopes are conventionally used to analyze blood 
within a capillary tube to determine the number of cells labelled by 
fluorescent antibodies. 
Non-laser scanning confocal microscopes are well known and can also be used 
to practice the invention. For example, confocal microscopes using 
spinning Nipkow disks, or similar arrangements can be used, if desired. 
Such microscopes are described, e.g., in Dixon (1996) Nature, 383:760; 
Juskaitis et al. (1996) Nature 383:804; Petran et al. (1968) J. Opt. Soc. 
Am. 58, 661; and Xiao et al. (1988) Appl Phys. Lett. 53:716. 
Useful confocal microscopes are also described, for example, in U.S. Pat. 
Nos. 5,032,720; 5,120,953; 5,260,578; 5,304,810; 5,283,684; 5,351,152, and 
5,162,946. 
According to the method of the invention, data obtained by confocal 
microscopy are analyzed to determine the difference between signal 
associated with individual suspended cells or solid supports, and 
background signal, and to obtain a measure of inhibition or binding. 
Conventional confocal microscopy for, e.g., counting CD4+ cells, is not 
concerned with this determination, or with high throughput screening of 
compounds. 
Any desired optically detectable label can be used in the present 
invention, including fluorescent labels and chemiluminescent labels. If 
the label is chemiluminescent, it is preferred that it generate a 
short-lived signal. Enzymes that produce a visible color change in the 
presence of appropriate substrate, such as horse radish peroxidase and 
alkaline phosphatase, can also be used. Fluorescent labels are referred 
and are described below with respect to preferred embodiments of the 
invention. Other optically detectable labels, however, may be substituted 
in these embodiments if desired. 
In one embodiment, the assay screens compounds for inhibition of binding of 
a fluorescently labeled ligand to a target molecule contained on the 
surface of either suspendable cells or solid supports. The degree of 
binding inhibition is determined by measuring, through confocal 
microscopy, amounts of labeled ligand bound to individual cells or 
supports in the presence of the compounds. 
In this embodiment, a suspended cell-membrane bound receptor can be 
contacted with fluorescently labelled ligand, in the presence of library 
compounds to be screened for inhibition of receptor binding. (The term 
"receptor" is used herein to encompass receptor domains as well as whole 
receptors.) As an example, a library of compounds to be screened for 
inhibition of the binding of IL-8 to its cell surface receptor is 
contacted with a suspension of cells bearing the receptor in the presence 
of fluorescently labelled IL-8. When examined by confocal microscopy, 
cells bound to labeled IL-8 appear as regions of increased fluorescence on 
a background of relatively constant "free" label. The amount of cell 
associated fluorescence is less in an assay where active compound inhibits 
binding of ligand to receptor. 
This embodiment is schematically depicted in FIG. 1. FIGS. 1A and 1C show a 
cell with attached receptors (`Y`), as they bind to fluorescently-labelled 
ligands (solid square flagged with `F`). In FIG. 1C, 
fluorescently-labelled ligands are displaced by library compound (open 
triangles). FIGS. 1B and 1D schematically indicate fluorescence collected 
from sections of samples with and without active library compounds, 
respectively. Presence of a "spot" relative to background fluorescence 
indicates bound ligand, while the background fluorescence itself results 
from free ligand. Confocal microscopy allows measurement of individual 
cell-sized "spots" of bound fluorescence. The amount of free fluorescent 
ligand can be simultaneously determined. In FIG. 1B, the cells are 
significantly more fluorescent than the background and therefore show up 
as more intense spots. In FIG. 1D, the effect of displacement by an active 
library compound is illustrated. The same cells have become dimmer or 
indistinguishable from background as a consequence of ligand displacement. 
Thus, the loss of `bound` signal indicates an active molecule in a 
high-throughput screening assay. Since receptor binding assays are 
typically conducted with an excess of ligand, the amount of background 
fluorescence does not normally change. 
In the assay of the invention, the amount of fluorescence associated with 
individual cells (or solid supports) in the assay is totaled. This total 
provides a measure of the amount of binding between ligand and target. 
This amount is compared to the amount of free fluorescence to arrive at a 
value indicative of the activity of the library compound (the amount may 
increase or decrease for active drug candidates, depending on the way in 
which the assay is set up). Specific methodologies for arriving at 
particular values indicating binding (or inhibition of binding) are 
described below. While background fluorescence can be individually 
measured in each assay, this may not be necessary where the background 
fluorescence is relatively constant. 
The method of the invention is particularly advantageous in increasing the 
level of signal to background noise. By eliminating signal from label 
contained in solution outside of the "slice" containing the measured 
particles or cells, the background noise is significantly reduced. The 
ratio of signal to background noise was found to be about fifteen times 
lower in a commercially available conventional heterogeneous (i.e., 
separation-based) assay using .sup.125 I labeled ligand than in the method 
of the invention. 
The method is especially effective in measuring fluorescence for samples 
that have been allowed to settle. Confocal microscopy allows accurate 
measurement of a "slice" or "section" of liquid in a container. Thus, 
measurement can be taken of, e.g., the bottom 10% of the sample where 
fluorescence bound to the cells or solid particles is concentrated. This 
is not possible in prior art assays using conventional optical detection 
since such assays do not eliminate signal from the volume above the 
settled cells or solid particles. Elimination of this signal accounts, in 
part, for the very high signal to noise ratio achieved by the method of 
the invention. 
Preferably, the suspended cells (or suspended solid supports) are allowed 
to settle for about 10 minutes or more, so that more than about 75% of the 
cells or supports are contained in less than about 25% of the volume of 
the assay container, i.e., a cell or solid support layer forms on the 
bottom. Most preferably, more than 90% of the cells or supports are 
allowed to settle in less than about 10% of the volume of the container. 
In one preferred embodiment, the thickness of the layer of cells or 
supports is about the same as the thickness of the confocal object plane. 
The time required for settling is a function of column height, and so is 
higher, e.g., for samples in 96 well plates than for samples in 1536 well 
plates. 
Any desired combination of ligand and receptor can be employed in the assay 
of the invention to test for active inhibitors. Non-limiting examples of 
ligands and their cell based receptors include neurokinin and NK2R cells, 
and IL-8 and IL-8B/CHO cells. These receptors and ligands are discussed 
further in the Examples below. 
Other examples of ligands and receptors include, but are not limited to, 
insulin/insulin receptor, bradykinin/bradykinin receptor, 
erythropoietin/EPO receptor, and leptin/Ob receptor. Cell lines for 
performing these assays are available. For example, IM-9 (ATCC CCL-159) 
constitutively expresses human insulin receptor. IMR-90 (ATCC CRL 7931) 
constitutively expresses bradykinin B2 receptor and can be stimulated with 
interleukin-1.delta. to produce bradykinin B1 receptor. Cells that express 
EPO receptor are described by Kitamura et al., J. Cellular Physiology 
140:323-334 (1989). Cells expressing leptin receptor (i.e., OB receptor) 
are described by Tartaglia et al., Cell 83:1263-1271 (1995). 
In another embodiment of the invention, a target molecule is bound, e.g. 
via a biotin/avidin association, to suspendable solid supports, and 
library compound and fluorescently labeled ligand in solution are 
contacted with the supports. Active compound causes a decrease in 
support-associated fluorescence by displacing fluorescent ligand from the 
target, and the presence and/or potency of the test compound is 
quantitated. The brightness of fluorescence of the supports diminishes in 
proportion to the potency of the test compound. This method is 
schematically depicted in FIG. 2. 
"Suspendable solid support" is intended to refer to any solid support 
capable of being suspended in a liquid. The support should be small enough 
so it does not block optical access to the rest of the solution upon 
settling to the bottom of the assay well. On the other hand, the support 
should be large enough so that it does not remain in suspension for an 
extended period of time after the assay components are combined. The 
preferred supports are less than about 50 .mu.m in diameter, most 
preferably less than 10 .mu.m in diameter. The diameter of the supports is 
preferably less than, although not significantly less than, the thickness 
of the confocal object plane. The supports are preferably greater than 1 
.mu.m in diameter, so that the suspension does not require centrifugation 
to condense the supports to the bottom of the assay container. 
A preferred suspendable support is a 6.2 .mu.m bead made of polystyrene and 
commercially available from Spherotech (Libertyville, Ill.). Such beads 
are avidin coated, typically containing 10.sup.6 binding sites per bead. 
Any suitable suspendable solid support, however, can be employed, 
including cellulose beads, controlled pore-glass beads, silica gels, and 
other types of polystyrene beads (optionally cross-linked with 
divinylbenzene and optionally grafted with polyethylene glycol and 
optionally functionalized with amino, hydroxy, carboxyl, or halo groups). 
Additional supports include grafted co-poly beads, poly-acrylamide beads, 
latex beads, dimethylacrylamide beads (optionally cross-linked with 
N,N.sup.1 -bis-acryloyl ethylene diamine), glass particles coated with 
hydrophobic polymers, etc., (i.e., having a rigid or semi-rigid surface). 
Divinylbenzene-crosslinked, polyethyleneglycol-grafted polystyrene type 
beads can be used, such as TentaGel S-NH.sub.2 .RTM. beads (Rapp Polymere, 
Tubingen, Germany). 
The solid support can be coated with any desired target, including, but not 
limited to, hydrolases (including proteases, esterases, nucleases), 
ligases (DNA or RNA based) and transpeptidases, as well as binding 
proteins such as antibodies, and DNA-binding proteins, and domains of 
those proteins. 
The target (or ligand) coated on the solid support may be bound thereto by 
any desired means. It may, for example, be biotinylated and then 
non-covalently linked to a streptavidin coated support. It is also 
possible to bind the target (or ligand) to antibodies (which are specific 
for the target) that have been coated on the support. Covalent linkages 
are also known in the art. 
The support may also be coated with a recombinantly produced receptor, or 
receptor binding domain. This is particularly advantageous for receptors 
or domains that are not normally expressed on the cell surface. For 
example, nuclear receptors, such as steroid receptors, are advantageously 
expressed recombinantly, and employed in the microbead assay of the 
invention. One such receptor is human recombinant estrogen receptor 
(Alexis Biochemicals, San Diego, Calif.). For a cell surface receptor, 
however, it is preferred to use suspended cells expressing the receptor as 
opposed to beads having the receptor bound thereto. 
It is also possible, for example, to coat the suspendable solid supports 
with ligand, and perform the assay of the invention with labelled receptor 
in solution, in the presence of compounds to be screened for inhibition of 
ligand/receptor binding. 
It is also possible, according to the invention, to incubate fluorescently 
labelled library compounds in solution with suspended cells or solid 
supports, and measure the binding between said compounds and cells or 
supports in the absence of ligand. In other words, the assay provides a 
direct measure of binding between the compounds and target molecule on the 
cells or supports, without the need to add a ligand that is displaced by 
the compounds. 
In another embodiment, a library of compounds is assayed for inhibition of 
an enzyme catalyzed reaction and the amounts of fluorescence bound to 
individual suspendable solid supports measured to determine the degree of 
inhibition. For example, in one such assay, the amount of fluorescence 
bound to a microbead in the presence of inhibitory compounds is greater 
than for non-inhibitory compounds. The amounts of fluorescence bound to 
individual beads are determined by confocal microscopy. Using this type of 
assay, inhibition can be determined of a protease, such as cathepsin D, 
which cleaves fluorescently labelled substrate bound to the solid support. 
For cathepsin D, the substrate can be a peptide, e.g. 
lys-pro-ile-glu-phe-phe-arg-leu, linked at one end to the microbead and at 
the other end to the fluorescent label Cy-5; either linkage can be 
accomplished using a spacer such as gamma aminobutyric acid. 
It is also possible, using this type of assay, to determine inhibition of 
endonuclease cleavage of fluorescently labelled oligonucleotide. The 
endonuclease is placed in solution with library compound and suspendable 
solid supports that are coated with fluorescently labelled oligonucleotide 
substrate. Upon cleavage of the substrate, fluorescently labeled product 
is released from the supports. The amount of fluorescence that remains 
bound to the bead increases where active inhibitor is present. 
In another assay for enzyme inhibition, both enzyme and fluorescently 
labelled substrate are incubated in solution with test compound and 
microbeads coated with a ligand. The ligand (such as an antibody) 
specifically binds to the reaction product of the enzyme catalyzed 
reaction, the reaction product retaining the fluorescent label. For 
example, inhibitors of tyrosine kinase can be determined in an assay in 
which kinase and fluorescently labelled peptide substrate are in solution. 
The peptide substrate contains a tyrosine amino acid in the middle of its 
sequence, and the reaction product contains phosphotyrosine. The assay 
solution contains suspendable microbeads coated with antibody for the 
phosphotyrosine containing reaction product. Successful inhibition by a 
library compound results in a decrease in fluorescence bound to beads as 
compared with controls. 
In assays of the invention in which inhibition of an enzyme catalyzed 
reaction is determined, the inhibitory compounds can inhibit by any 
mechanism. For example, they can inhibit by binding to enzyme, binding to 
substrate, binding to a complex of enzyme and substrate, or binding to a 
complex of enzyme and product. 
Preferably, the assay of the invention is performed using a microtiter 
plate having microvolume containers, such as the 1536 well plate described 
in U.S. patent application Ser. No. 60/037,636 filed Feb. 18, 1997. A 
confocal scanning microscope sequentially scans the bottom of each well in 
the microtiter plate. 
The method can also be carried out in conventional 96 well microtiter 
plates, or in any other container or on any surface capable of holding 
liquid samples and of being scanned by a confocal microscope. Examples 
include 12-well, 24-well, 384-well, 864-well plates, and 
microscope-slides. 
In the embodiment of the invention in which receptor-bearing cells are 
employed, the desired density of cells will preferably be between about 
100 and 1000 cells per microliter in a 1536 well plate and between about 
30 to 300 cells per microliter in a conventional 96 well plate. Typically, 
it is believed necessary to measure signals from at least 100 to 1000 
cells per sample to obtain a statistically relevant result. The optimum 
density can be determined using these concentrations as a guideline, as 
well as the size of the particular cells employed, and by measuring the 
signal provided in assays using known inhibitors of the ligand/receptor 
interaction. The area scanned can be limited to reduce scanning time and 
thereby increase throughput, as long as the number of cells measured is 
sufficient. 
If suspendable supports are employed in the preferred size of about 6 
.mu.m, the preferred density of supports will generally be in the same 
preferred range as for cells. The density varies depending on the size of 
the supports and the amount of target affixed to each support. 
The signal detected according to the invention, is preferably generated by 
a fluorescent label. The label can be attached to a ligand which binds to 
a receptor or other target molecule. It is also possible to use labelled 
receptor (or other target molecule) in certain embodiments of the 
invention. Also, if desired, a "secondary labelling" approach can be used 
in which labelled antibody probes, e.g., for unlabelled ligand, receptor, 
or target molecule. 
It is also possible to conduct an assay according to the invention in which 
library compounds themselves are fluorescently labelled. For example, a 
library of compounds that are primary amines can be labelled with an 
amine-specific fluorescent label (e.g., monofunctional Cy5-NHS ester). The 
compounds can then be tested for direct binding to a target molecule on a 
cell or suspended support. The amount of bound fluorescence correlates to 
the degree of binding. 
Fluorescent labels suitable for use in the invention are well known and 
include cyanine dyes such as Cy-5, Cy-5.5, and Cy7 (Amersham Corp.), 
fluorescein, rhodamine and Texas red. In the embodiment of the invention 
employing cells, it is preferred that the fluorescent label fluoresce at a 
relatively high wavelength, i.e., higher than about 450 nm, to avoid 
interference from cell originating fluorescence and fluorescence 
originating from glass and plastic containers. The labels most preferably 
fluoresce above 600 nm, and at less than about 800 mn. Labels that excite 
at about 400 nm can avoid photobleaching caused by near-UV light. 
Non-fluorescent labels can also be used in embodiments of the invention 
described above. In one embodiment using the chemiluminescence generating 
label luciferase, a receptor is coated on suspendable solid supports. The 
coated supports are incubated with luciferase conjugated ligand, 
luciferase substrate, and compound to be tested. In the absence of 
inhibition of receptor/ligand binding, the luciferase becomes associated 
with the suspendable solid supports, and the chemiluminescent signal that 
results from luciferase's enzymatic action concentrates around the 
supports. In the presence of inhibitory compounds, the signal associated 
with the supports decreases. 
In a separate embodiment of the invention, the assay is performed to 
determine the degree of binding to a treated surface of an insuspendable 
solid support. The support can be a container, or vessel, itself, such as 
the bottom of a microtiter plate. Alternately, the insuspendable support 
can be, e.g., a disc. In the embodiment in which the support is the bottom 
of the well of the microtiter plate, the plate is coated with a target 
molecule, and then exposed to labelled ligand. A confocal section 
including the bottom layer of the plate is measured for optically 
detectable signal. Free signal is measured in other confocal sections that 
do not include the bottom of a microtiter plate. The signal bound to the 
support can then be calculated. This method is advantageous in that 
scanning can be rapidly performed. Individual cells/beads do not need to 
be identified, resulting in higher throughput. Use of a thin confocal 
object plane is preferred to exclude signal emanating from above the 
coated plate, and to maintain a high signal to noise ratio. In one 
embodiment of this method, the confocal object plane is less than about 10 
.mu.m. 
It is preferred that the compounds assayed in the high throughput method of 
the invention be derived from combinatorial libraries on polymer beads. By 
synthesizing sufficient compound on each bead for a few assays, compound 
handling is reduced or eliminated. Such beads, e.g., can contain on the 
order of 100 picomoles of compound per bead, and tests are often performed 
at concentrations of about 1 .mu.M. With such beads, a test volume of 1 
.mu.l is advantageous since it is possible to use one bead for up to about 
one hundred tests. 
Preferably, the library compounds are eluted from the beads and evaporated 
to dryness in microtiter plates in preparation for the assay. Compounds on 
beads can be released by photocleavage, or another type of cleavage. 
Cleavage of photocleavable linkers is preferred. Such linkers, and methods 
for their cleavage, are described in Barany et al. (1985) J. Am. Chem. 
Soc. 107:4936. Examples of other linkers and the relevant cleavage 
reagents are described in WO 94/08051. 
Using combinatorial libraries prepared on beads, the identity of active 
compounds is preferably determined using the encoding system described in 
WO 94/08051, and in U.S. patent applications Ser. Nos. 08/436,120 and 
08/239,302 (which correspond to WO 95/30642). In this system, chemical 
tags encoding the identities of the compounds are applied to the solid 
supports. The identity of the compound on a given support can be 
determined by detaching the chemical tags from the support, identifying 
the tags by, e.g., gas chromatography, and correlating the identities of 
tags with the identity of the compound. Once an active compound is 
identified, the corresponding bead (which had contained the compound) can 
be examined, and the identity of the compound determined by releasing the 
tags and decoding by this method. 
When several large libraries are available for testing, it may be 
advantageous to "scout" each library by placing more than one test 
compound in each assay container. Assay containers having an active 
compound can be further investigated by individually evaluating each of 
the plurality of compounds present in such containers. Screening at "high 
density" in this manner allows one to statistically evaluate the number 
and potency of active compounds in each library. Libraries which contain 
the most active compounds can be more thoroughly tested. If the proportion 
of active compounds screened in the assay is high, a second assay of the 
active compounds may be performed at lower concentrations to select only 
the most active compounds to choose those that should be further 
evaluated. 
The invention is illustrated by the following examples, which are not 
intended to limit the scope of the invention. 
EXAMPLE 1 
Screening of Compounds for Receptor Binding 
The invention was demonstrated using confocal microscopy apparatus and 
software originally intended for fluorescent cytometry. The data obtained 
were further analyzed to provide values indicative of the amount of 
binding between ligand and a cell surface receptor. 
1) Synthesis of Ligands 
(a) Cy5-labelled Neurokinin-A (NKA). 
Neurokinin-A (HKTDSFVGLM) was purchased from Cambridge Research 
Biochemicals (PP-05-0826A), and monofunctional Cy5 dye was purchased from 
Amersham (as the Fluorolink.TM. conjugation kit, cat. # A25001). NKA (1 
mg) was dissolved in 0.88 mL bicarbonate buffer (100 mM NaHCO.sub.3, pH 
9.3), and Cy5 dye (about 1 mg) was dissolved in 0.13 mL bicarbonate 
buffer. The two solutions were mixed and incubated for 1.5 hr at room 
temperature, then transferred to 5.degree. C. for an additional 17 hrs. 
The conjugate was purified by HPLC (gradient 20-40% CH.sub.3 CN in H.sub.2 
O, 0.1% TFA, on a Vydac.RTM. analytical C18 column, R.sub.f 8.9 min @ 1.5 
mL/min) to yield 157 mol (18% theoretical yield). Identity of the ligand 
was verified by competition vs. receptor and mass spectrometry. 
(b) Cy5-labelled IL-8. 
Human interleukin-8, Ser72.fwdarw.Cys mutant (hIL-8(S72C)) was cloned from 
human cDNA using PCR techniques and sequenced to confirm the mutation and 
the sequence. Monofunctional Cy5-iodoacetamide was obtained from Amersham. 
To 500 nM Cy5-iodoacetamide was added 200 nmol (200 .mu.l of a 1 mM stock) 
of hIL-8(S72C) in 20 mM sodium phosphate, pH 6.5, 400 mM NaCl. The tube 
was vortexed and placed in the dark at ambient temperature. HPLC analysis 
indicated that after 48 hours the reaction was complete. The product was 
purified (2.times.100 .mu.l injections) from unreacted Cy5-iodoacetamide 
and oxidized hIL-8(S72C) by HPLC. Sample was reconstituted in 50 mM sodium 
phosphate, pH 7.2 at an estimated concentration of 400 .mu.M. A more 
accurate concentration was then determined from a fluorescence spectrum of 
an aliquot of this sample. Further characterization indicated that 
hIL(S72C)-Cy5 had a mobility similar to wild type IL-8 on a 16% SDS-PAGE 
analysis and displayed a K.sub.i of 2 nM in a conventional .sup.125 
I!IL-8 ligand displacement assay. 
2) Cell lines 
(a) Cells expressing neurokinin-2 receptor (NK2R/CHO) were obtained. Cells 
expressing NK2R are well known, and readily obtained by those skilled in 
the art. For example, NK2R expressing cells are described in the following 
references: Arkinstall, S., M. Edgerton, et al. (1995). "Co-expression of 
the neurokinin NK2 receptor and G-protein components in the fission yeast 
Schizosaccharomyces pombe." FEBS Lett 375(3): 183-7; Bradshaw, C. G., K. 
Ceszkowski, et al. (1994). "Synthesis and characterization of selective 
fluorescent ligands for the neurokinin NK2 receptor." J Med Chem 37(13): 
1991-5; Grisshammer, R., J. Little, et al. (1994). "Expression of rat NK-2 
(neurokinin A) receptor in E. coli." Receptors Channels 2(4): 295-302; 
Lundstrom, K., A. Mills, et al. (1995). "High-level expression of G 
protein-coupled receptors with the aid of the Semliki Forest virus 
expression system." J Recept Signal Transduct Res 15(1-4): 617-30; and 
Turcatti, G., K. Nemeth, et al. (1996). "Probing the structure and 
function of the tachykinin neurokinin-2 receptor through biosynthetic 
incorporation of fluorescent amino acids at specific sites." J Biol Chem 
271(33): 19991-8. 
b) Cells expressing IL-8A and IL-8B receptor (IL-8A/CHO and IL-:8B/CHO) 
were obtained as follows: 
CHO IL-8A and IL-8B cell lines were prepared by cationic lipopolyamine 
(lipofectamine, GIBCO BRL) mediated transfection of CHO-K1 cells (ATCC) 
with pCDNAIII plasmids encoding the sequences of human IL-8A or IL-8B. 
Cells were cultured under G418 selection (1 mg/ml) in DMEM, 10% fetal 
bovine serum, 2 mM L-glutamine, and 2% non-essential amino acids and 
clonal lines expressing the highest receptor levels were maintained for 
use in these experiments. The human IL-8A receptor cDNA was cloned from 
HL-60 cell (ATCC) mRNA. (Clones encoding IL-8A are described in Ahuja et 
al., (1992) Nature Genetics 2:31.) First-strand cDNA was synthesized using 
M-MLV reverse transcriptase (Promega Riboclone.TM. cDNA synthesis system) 
and IL-8A cDNA was amplified by polymerase chain reaction using primers, 
5'CCGAATTCGACATGTCAAATATTACAGATCC3' and 5'GCTCTAGATCAGAGGTTGGAAGAGAC3'. 
The PCR product was digested with EcoRI+Xbal and ligated into 
EcoRI/Xbal/calf intestinal phosphatase-digested pcDNA3 vector 
(Invitrogen). The DNA sequence of one candidate was confirmed using the 
Promega Silver Sequence.TM. method. To generate the human IL-8B expression 
clone, an approximately 1.8 kb cDNA fragment was recloned from pBluescript 
clone BS-p3 (Murphy and Tiffany, (1991) Science, 253:1280) into the pcDNA3 
vector, using EcoRI and Xhol. 
3) Assay 
(a) Binding of Cy5-NKA to NK2R/CHO cells 
A culture of NK2R/CHO cells, near confluence, was washed with 12 mL 
DPBS(Mg.sup.+2 & Ca.sup.+2 free), trypsinized by adding trypsin (2 mL/T-25 
flask), incubating at 37.degree. C. for about 5 min, then quenching with 
10 mL media. Cells were then counted, and diluted to the desired final 
concentration of cells (5,000 cells/25 .mu.L). The assay was set up with 
the following components: Buffer (1.times. BSS+0.2% BSA, containing 
thiorphan and bacitracin) containing varying concentrations of Cy5-NKA 
(0.76 to 136 nM final) 10 .mu.L; Cells, 40 .mu.L. The plate was covered 
and wrapped in aluminum foil to protect from light and left to shake for 1 
hr at room temperature. For each sample, 25 .mu.L was removed, placed in 
an IMAGN 2000.TM. capillary, and read using IMAGN.TM. software (Biometric 
Imaging, Inc.) The sample was allowed to settle until the cells rested on 
the bottom of the well. The time require for settling was typically 10 
minutes. 
(b) Displacement of Cy5-NKA bound to NK2R/CHO cells by SR48,968. 
SR-48,968 is a known inhibitor of the binding of NKA neurokinin 2 receptor. 
Setup and assay were performed as in (a), at a fixed concentration of 
Cy5-NKA of 5 .mu.M, and diluting stock concentration of SR-48,968 (stock 1 
mM, 1.6% DMSO, in BSS) from 88 nM to 8.8 pM in Cy5-NKA-containing buffer. 
(c) Displacement of Cy5-IL-8 bound to cells by unlabelled IL-8. 
A culture of IL-8B/CHO cells, near confluence, was trypsinized by adding 
trypsin (2 mL/T-25 flask), incubating at 37.degree. C. for about 1 min, 
then quenching with 4 mL media. Cells were then counted, and diluted to 
the desired final concentration (15,000 cells/25 .mu.L). The assay was set 
up in a 96-well microliter plate (100 .mu.L total volume), consisting of 
the following components: Buffer (1.times. BSS) 30 .mu.L; Cells, 25 .mu.L; 
Background fluor (Tris-Cy5, 60 .mu.M), 25 .mu.L; hIL-8(S72C)-Cy5(20 nM), 
10.mu.L; unlabelled IL-8 (various concentrations, diluted in BSS), 10 
.mu.L. The plate was covered and wrapped in aluminum foil to protect from 
light and left to shake for 1 hr at room temperature. For each sample, 85 
.mu.L was removed and placed in an IMAGN 2000.TM. capillary, and read 
using IMAGN software. 
4) Data Analysis 
The data that results from the IMAGN system consists of a tab delimited 
text file that contains information about each cell that has been 
identified in the field. The IMAGN system provides position, shape, and 
intensity data (for two channels, Cy5 and Cy5.5) for each cell (or 
cell-sized object) in the imaging field, as well as baseline information. 
(In a typical analysis using this type of equipment, cells are identified 
by two or more contiguous pixels having intensities significantly greater 
than the baseline signal.) Statistical parameters (e.g., standard 
deviations) of the data are also tabulated. This data can be analyzed to 
give a scalar value for the sample which provides a measure of the amount 
of binding. Three possible ways to perform this further analysis are 
described below. 
(a) Mean Fluorescence Intensity. 
This value is derived from the data table as the mean value across all 
cells of "MaMO". Channel 0 (zero) is the Cy5 fluorescence channel, and the 
MaMO value is calculated as the peak Cy5 fluorescence (corresponding to 
fluorescence bound to the cell) with the minimum (baseline fluorescence) 
value subtracted out. "MaMO" is tabulated for each cell in the field. 
This analysis is particularly useful for assays that involve high fluor 
concentrations and relatively high levels of occupation of cell surface 
receptors. It may not be advantageous where all cells cannot be detected, 
e.g., at low fluor concentrations or low levels of occupation of cell 
surface receptors. Analysis of F value (explained below) can provide more 
accurate data under these circumstances. Specifically, more weakly 
fluorescent cells (by virtue of their size and/or idiosyncratic binding 
characteristics) will `disappear` first in the imaging field when being 
displaced by a competing ligand. ("Disappearance", in this sense, means 
that the pixels comprising the cell's image are not significantly higher 
in intensity than the baseline image.) Two changes are observed 
experimentally when challenging a fixed fluorescent ligand concentration 
with a competitor. First, the overall intensity of the cells decreases 
(i.e., the average MaMO value decreases). Second, the number of observable 
cells decreases, since the weaker cells vanish into the background. The 
F-value measures the total fluorescence in the cell layer and therefore 
provides a more accurate measurement when all cells cannot be counted. 
(b) F-value. 
The F-value is determined by multiplying the MFI by the total number of 
cells loaded into the well (as opposed to the number of fluorescent cells 
counted), and dividing by 1000. This value provides the total fluorescence 
of the cell layer, and includes cells whose fluorescence is so weak as to 
be undetectable in a given scan by the confocal microscope. The F-value is 
believed to generally provide a more accurate measure of binding in a 
given sample. 
(c) Percent of Control. 
Percent of control analysis can be performed using values obtained from 
either an MFI or F-value analysis, and results in normalization of this 
data. It is calculated according to the following formula for the relevant 
value. "Max" and "min" refer to the maximum and minimum fluorescence for 
the relevant value. 
EQU % Control=((Value-Min)/(Max-Min)).times.100 
The MFI calculated for binding of Cy5-NKA to NK2R/CHO cells is shown in 
FIG. 3. These results show a maximum MFI of 7,720 and a Kd (dissociation 
constant) of 3.9 nM. 
Results calculated as a percent of control for displacement of Cy5-NKA by 
SR48.968 are shown in FIG. 4. An IC.sub.50 of 0.575 nM was determined and 
an estimated Ki of 0.29 nM. An R.sup.2 value of 0.963 was determined, 
indicating "goodness of fit" of the experimental data to the theoretically 
determined values. 
The Hill coefficient measured was 0.996. The Hill coefficient is a measure 
of the cooperativity in binding. For a simple system, where one receptor 
binds to one ligand, the Hill coefficient is 1. For polyvalent systems, 
the Hill coefficient can vary from one, but ideally is a small integer for 
polyvalent ligands, or the reciprocal of a small integer for polyvalent 
receptors. The NKA/NK2R and IL-8/IL-8BR systems involve monovalent 
receptors and monovalent ligands. 
Results, calculated as F-values, for displacement of labelled IL-8 by 
unlabelled IL-8, are shown in FIG. 5. The graph insert provides the 
equation for a four-parameter curve fit: 
EQU y=((M1-M4)/(1+(M0/M3).sup.M2))+M4. 
where M is the maximum value, M4 is the minimum value, M3 is the midpoint 
of the curve (the apparent Kd), and M2 is the slope at the midpoint. 
Binding constants for the above results can be determined, by Scatchard 
analysis, which involves the measurement of bound ligand as a function of 
free ligand concentration. Specifically, the ratio of bound to free ligand 
is plotted versus the bound ligand concentration. A straight line results, 
whose slope is the negative reciprocal of the dissociation constant for 
the ligand, and whose x-intercept is the maximum binding value 
(B.sub.max). 
The results of these experiments demonstrate that the method of the 
invention allows precise quantitative analysis of ligand/target binding, 
and can be used for a variety of targets commonly employed in 
combinatorial library screening. The method also allows rapid detection 
and analysis in high-throughput screening. The variablity in the results 
is low and the results strongly correlate with those obtained by 
conventional methods of analysis. The method is easily performed in a 
microscopic volume. It is therefore well-suited for high-throughput 
screening in microliter samples, such as when using a 1536-well plate, and 
allows efficient use of small amounts of library compounds derived from 
coated microbeads. 
5) Comparison with conventional assay. 
In receptor binding assays, the sensitivity of the assay is limited by 
background. It is possible to compare sensitivities of different assays by 
calculating the ratios of maximum signal to background. For the assay 
described above involving IL-8 binding, the assay of the invention was 
found to provide a signal (F-value) of 6300 on a non-specific background 
of 200, for a signal-to-background ratio of 31.5. For example, .sup.125 
I!IL-8 binding analysis using a similar concentration of labelled IL-8 
results in a signal of 85,000 cpm on a non-specific background of 39,000 
cpm, for a signal-to-background ratio of 2.1. Based on these values, the 
improvement in sensitivity is about 15-fold using the assay of the 
invention. 
EXAMPLE 2 
Screening of compounds for SH2 domain binding 
A high-throughout assay is performed to determine inhibitors of binding of 
ligand to the SH2-domain of human Grb2. Grb2 is an adaptor protein, the 
SH2-domain of which binds to phosphotyrosine containing proteins and 
promotes signal transduction by causing the association of specific 
cellular proteins. Cloning of Grb2 is described in Lowenstein et al., Cell 
70:431-442 (1992). IRS-1 is a substrate for the insulin receptor (IR) 
tyrosine kinase, and binds to the SH2 domain of Grb2 after being tyrosine 
phosphorylated. (The interaction of IRS-1 with Grb2 is described in 
Skolnick et al., EMBO 12:1929-1936 (1993).) 
Preferred ligands for this assay have a dissociation constant of less than 
about 1 .mu.M. The present assay employs a synthetic ligand for the 
SH2-domain that is a phosphotyrosine containing peptide derived from the 
sequence of IRS-1 (located near the pY896 amino acid). The peptide has the 
sequence KSPGNpYVNIE-CO.sub.2 NH.sub.2. 
Streptavidin coated 6.2 .mu.m beads (Spherotech) are employed that contain 
1.times.10.sup.6 binding sites per bead. The beads are incubated with the 
SH2-domain (Src-homology 2 domain), which has been labelled at a specific 
site with biotin during expression (Schatz, P. J. (1993) Bio/Technology 
11, 1138-1143). 
Beads, ligand, and library compounds are combined, incubated and shaken in 
1536 well plates. The samples are then allowed to settle for about 10 
minutes, and the plates read by a laser scanning confocal microscope. 
Active compounds cause a decrease in the amount of bound fluorescence. 
Results are analyzed as described above in Example 1 to determine binding 
and/or inhibition constants. 
EXAMPLE 3 
Screening of compounds for EGF Receptor Kinase Domain binding 
A high throughput assay is performed to determine library compounds that 
inhibit binding of ligand to the kinase domain of the epidermal growth 
factor (EGF). This kinase phosphorylates a number of proteins and 
peptides, including the peptide RRKGSTAENAEYLRVA (the "1173" peptide). The 
tyrosine underlined in the amino acid sequence is phosphorylated by the 
kinase. 
EGF Receptor Kinase Domain is obtained from Alexis Biochemicals (San Diego, 
Calif.). The 1173 peptide is biotinylated and then incubated with 
streptavidin coated 6.2 .mu.Km microbeads (Spherotech) to affix the 
peptide to the beads. Monoclonal antibodies specific for phosphotyrosine 
are obtained from Sigma (St. Louis, Mo.) or Biogenesis (Sandown, N.H.), 
and are labelled with Cy5 following the manufacturer's directions 
(Amersham). 
The assay is conducted in 1536 well plates. The EGF kinase domain is 
incubated in the presence of the 1173 coated microbeads, 
Mg.circle-solid.ATP, Cy5 labelled anti-phosphotyrosine antibody, and 
library compound in neutral buffer. Upon phosphorylation of the 1173 
peptide by the kinase, the peptide is bound by the fluorescently labelled 
antibody, and fluorescence becomes associated with the beads. Inhibitory 
library compound prevents phosphorylation, resulting in a decrease in bead 
associated fluorescence. The wells of the plate are scanned by a laser 
scanning confocal microscope, and the resulting data further analysed as 
described in Example 1 to determine inhibition and/or binding values.