U.S. patent application number 10/060639 was filed with the patent office on 2002-10-03 for method of screening for specific binding interactions.
Invention is credited to Strittmatter, Warren J..
Application Number | 20020142335 10/060639 |
Document ID | / |
Family ID | 25493822 |
Filed Date | 2002-10-03 |
United States Patent
Application |
20020142335 |
Kind Code |
A1 |
Strittmatter, Warren J. |
October 3, 2002 |
Method of screening for specific binding interactions
Abstract
A method for detecting the binding of a test compound to a probe
molecule comprises providing a test compound, the test compound
having a first fluorophore bound thereto, and providing a screening
substrate. The screening substrate comprises a solid support, a
probe molecule bound to the solid support, and a second fluorophore
bound to the solid support adjacent the probe molecule. An
advantage of the invention is that this obviates the need for
binding the second fluorophore directly to the probe molecule.
Preferably, the second fluorophore is bound to the solid support by
a flexible linker group. This enables the second fluorophore to
interogate different positions on the probe molecule, which is also
bound to the solid support adjacent the linker group, enhancing the
ability of the method of the invention to detect positive binding
events (specific binding of the test compound to the probe
molecule. The first and second fluorophores together comprise the
donor and acceptor fluorophores of a fluorescence resonance energy
transfer (FRET) pair, or a "donor/acceptor pair." The test compound
is contacted to the screening substrate, and the screening
substrate illuminated with light at a wavelength that is absorbed
by the donor fluorophore. The transfer of energy from one to the
other fluorophore is then detected, with the transfer of energy
indicating the binding of the test compound to the probe.
Substrates useful for carrying out the foregoing methods are also
disclosed.
Inventors: |
Strittmatter, Warren J.;
(Durham, NC) |
Correspondence
Address: |
MYERS BIGEL SIBLEY & SAJOVEC
PO BOX 37428
RALEIGH
NC
27627
US
|
Family ID: |
25493822 |
Appl. No.: |
10/060639 |
Filed: |
January 30, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10060639 |
Jan 30, 2002 |
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08953323 |
Oct 17, 1997 |
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6348322 |
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Current U.S.
Class: |
506/7 ; 435/6.14;
435/7.9; 436/518; 506/16; 506/18 |
Current CPC
Class: |
Y10S 435/968 20130101;
Y10S 436/807 20130101; Y10S 530/812 20130101; G01N 33/542 20130101;
Y10S 530/811 20130101; Y10S 436/805 20130101 |
Class at
Publication: |
435/6 ; 435/7.9;
436/518 |
International
Class: |
C12Q 001/68; G01N
033/53; G01N 033/542; G01N 033/543 |
Claims
That which is claimed is:
1. A method for detecting the binding of a test compound to a probe
molecule, said method comprising: providing a test compound, said
test compound having a first fluorophore bound thereto; providing a
screening substrate, said screening substrate comprising a solid
support, a probe molecule bound to said solid support, and a second
fluorophore bound to said solid support adjacent said probe
molecule; wherein said first and second fluorophores together
comprise a donor and an acceptor fluorophore of a fluorescence
resonance energy transfer pair; contacting said test compound to
said screening substrate; illuminating said screening substrate
with light at a wavelength that is absorbed by said donor
fluorophore; and then detecting the transfer of energy to said
acceptor fluorophore, the transfer of energy to said acceptor
fluorophore indicating the binding of said test compound to said
probe.
2. A method according to claim 1, wherein said probe molecule is a
protein or peptide.
3. A method according to claim 1, wherein said probe molecule is a
nucleic acid.
4. A method according to claim 1, wherein said test compound is a
protein or peptide.
5. A method according to claim 1, wherein said test compound is a
nucleic acid.
6. A method according to claim 1, wherein said contacting step is
carried out by contacting a test solution containing said test
compound to said screening substrate.
7. A method according to claim 6, wherein said illuminating step is
carried out with said test solution in contact with said screening
substrate.
8. A method according to claim 1, wherein said first and second
fluorophores absorb light at different wavelengths from one
another.
9. A method according to claim 1, wherein said solid support
comprises a bead.
10. A method according to claim 1, wherein said solid support
comprises glass.
11. A screening substrate useful for the high throughput screening
of molecular interactions, said substrate comprising: a solid
support having a surface portion, said surface portion comprising a
plurality of discreet known regions; a plurality of different probe
molecules bound to said surface portion, with different probe
molecules positioned on said surface portion in different ones of
said discrete known regions; and a plurality of the same
fluorophores connected to said surface portion, said fluorophores
positioned on said surface portion in all of said plurality of
discreet known regions.
12. A screening substrate according to claim 11, wherein said probe
molecules are proteins or peptides.
13. A screening substrate according to claim 11, wherein said probe
molecules are nucleic acids.
14. A screening substrate according to claim 11, wherein said
fluorophores are bound to said solid support by a linking
group.
15. A screening substrate according to claim 14, wherein said
linking group is a polymer, said polymer having a plurality of said
second fluorophores conjugated thereto at separate locations along
the length thereof.
16. A screening substrate according to claim 15, wherein said
fluorophores are conjugated to said polymer linking group at
separate locations along the length thereof.
17. A screening substrate according to claim 11, wherein said solid
support comprises glass.
18. A method for detecting the binding of a test compound to a
probe molecule, said method comprising: providing a test compound,
said test compound having a first fluorophore bound thereto;
providing a screening substrate according to claim 11, wherein said
fluorophore bound to said surface portion of said solid support
comprises a second fluorophore; wherein said first and second
fluorophores together comprise the donor and acceptor fluorophores
of a fluorescence resonance energy transfer pair; contacting said
test compound to said screening substrate; illuminating said
screening substrate with light at a wavelength that is absorbed by
said donor fluorophore; and then detecting the transfer of energy
to said acceptor fluorophore in one of said discreet known regions,
the transfer of energy to said acceptor fluorophore indicating the
binding of said test compound to the probe bound to that discreet
known region.
19. A method according to claim 18, wherein said contacting step is
carried out by contacting a test solution containing said test
compound to said screening substrate.
20. A method according to claim 19, wherein said illuminating step
is carried out with said test solution in contact with said
screening substrate.
21. A method according to claim 18, wherein said second
fluorophores are conjugated to said solid support by a polymer
linking group, said polymer linking group having a plurality of
said second fluorophores conjugated thereto at separate locations
along the length thereof so that energy transfer between said first
and second fluorophores upon binding of the test compound to the
probe molecule is enhanced.
22. A mixture useful for the high throughput screening of molecular
interactions, said mixture comprising: a plurality of discreet
solid supports each having a surface portion, each such support
having a different probe molecule bound to the surface portion
thereof, and a plurality of the same fluorophores bound to the
surface portion of each of said solid supports.
23. A mixture according to claim 22, wherein said probe molecules
are proteins or peptides.
24. A mixture according to claim 22, wherein said probe molecules
are nucleic acids.
25. A mixture according to claim 22, wherein said fluorophores are
bound to said solid support by a linking group.
26. A mixture according to claim 25, wherein said linking group is
a polymer, said polymer having a plurality of said second
fluorophores conjugated thereto at separate locations along the
length thereof.
27. A mixture according to claim 26, wherein a plurality of said
fluorophores are conjugated to said polymer linking group at
separate locations along the length thereof.
28. A mixture according to claim 22, wherein each of said discreet
solid supports has a different tag component bound thereto.
29. A mixture according to claim 22, wherein each of said discreet
solid supports comprises a bead having a diameter of 10 to 2000
.mu.m.
30. A method for detecting the binding of a test compound to a
probe molecule, said method comprising: providing a test compound,
said test compound having a first fluorophore bound thereto;
providing a mixture according to claim 20, wherein said fluorophore
bound to said surface portion of said solid support is a second
fluorophore; wherein said first and second fluorophores together
comprise the donor and acceptor fluorophores of a fluorescence
resonance energy transfer pair; contacting said test compound to at
least one of said discreet solid supports; illuminating said
discreet solid support with light at a wavelength that is absorbed
by said donor fluorophore; and then detecting the transfer of
energy to said acceptor fluorophore from said discreet solid
support, the transfer of energy to said acceptor fluorophore
indicating the binding of said test compound to the probe bound to
that discreet solid support.
31. A method according to claim 30, wherein said contacting step is
carried out by contacting a test solution containing said test
compound to at least one of said discreet solid supports.
32. A method according to claim 31, wherein said illuminating step
is carried out with said test solution in contact with said
discreet solid support.
33. A method according to claim 30, wherein said second
fluorophores are conjugated to said solid support by a polymer
linking group, said polymer linking group having a plurality of
said second fluorophores conjugated thereto at separate locations
along the length thereof so that energy transfer between said first
and second fluorophores upon binding of the test compound to the
probe molecule is enhanced.
34. A method according to claim 30, wherein said illuminating and
detecting steps are sequentially repeated with sequential ones of
said discreet solid supports.
35. A method according to claim 34, wherein said illuminating and
detecting steps are sequentially repeated in a flow sorting
apparatus.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to methods and apparatus used
to screen for specific binding interactions by fluorescent
techniques. The methods are particularly useful for high throughput
screening of such interactions in combinatorial libraries,
including chip-based libraries, pin-based libraries, and split-pool
libraries.
BACKGROUND OF THE INVENTION
[0002] The development of techniques for the screening of large
numbers of different molecules for possible biological activity has
become a important in developing new drugs. Known as "combinatorial
chemistry" techniques, these techniques typically involve the
random generation of compounds to be screened. Combinatorial
chemistry techniques are thus dramatically different from "rational
drug" design techniques, where specific structures for new
compounds are designed based on a knowledge of other active
compounds or the target site of the compound. Instead,
combinatorial chemistry relies on the large numbers of candidate
compounds generated, and the rapid screening of those compounds, to
provide a sufficient probability of identifying active
compounds.
[0003] Numerous different combinatorial chemistry techniques are
known. For example, U.S. Pat. No. 5,445,934 to Fodor et al.
describes a substrate in which large numbers of different polymers,
such as nucleotides or peptides, are affixed to discreet regions of
that substrate for the screening thereof for biological activity.
In another example, U.S. Pat. No. 5,565,324 to Still et al. reviews
"split-pool" combinatorial libraries, and describes split pool
libraries encoded with chemical tags.
[0004] The majority of work in developing new combinatorial
chemistry techniques has focused on the methods for generating the
combinatorial library, on the substrate structures for carrying the
members of the library, or on the different structures from which
the library can be comprised. Comparatively little attention has
been devoted to developing new methods for detecting binding events
within a combinatorial library. Accordingly, there is a need for
new detecting methods that can be employed in conjunction with
combinatorial chemistry techniques.
SUMMARY OF THE INVENTION
[0005] A method for detecting the binding of a test compound to a
probe molecule comprises providing a test compound, the test
compound having a first fluorophore bound thereto, and providing a
screening substrate. The screening substrate comprises a solid
support, a probe molecule bound to the solid support, and a second
fluorophore bound to the solid support adjacent the probe molecule.
An advantage of the invention is that this obviates the need for
binding the second fluorophore directly to the probe molecule.
[0006] Preferably, the second fluorophore is bound to the solid
support by means of a flexible linker group. This enables the
second fluorophore to interogate different positions on the probe
molecule, which is also bound to the solid support adjacent the
linker group. As discussed in greater detail below, this enhances
the ability of the method of the invention to detect positive
binding events (specific binding of the test compound to the probe
molecule).
[0007] The first and second fluorophores together comprise the
donor and acceptor fluorophores of a fluorescence resonance energy
transfer (FRET) pair, or a "donor/acceptor pair." In preferred
embodiments, the first and second fluorophores are different, the
first and second fluorophores emit light at different wavelengths
from one another, and the second fluorophore absorbs light at a
wavelength emitted by the first fluorophore.
[0008] The test compound is contacted to the screening substrate,
and the screening substrate illuminated with light at a wavelength
that is absorbed by the donor fluorophore (which may be either the
first or second fluorophore). The transfer of energy from one to
the other fluorophore (that is, from the donor to acceptor
fluorophore) is then detected (for example, by detecting the
presence or absence of light emitted by the acceptor fluorophore)
with the transfer of energy indicating the binding of the test
compound to the probe.
[0009] The method is useful in diagnostic assays for the detection
of a particular compound, or in high through-put, or combinatorial,
screening of multiple compounds. The method can be employed as a
competition assay, where the test compound competes for binding
with a target compound, where the binding of the target compound
indicates the absence of the test compound.
[0010] Since the acceptor fluorophore is excited only when the
donor fluorophore is in close proximity thereto, an advantage of
the present invention is that there need not be a step of
separating the test compound from the solid support (for example,
an intervening washing step) to distinguish nonspecific binding
from specific binding. For example, the entire process can be
carried out in liquid, particularly aqueous, phase, by contacting a
solution containing the test compound to the solid support, with
the detecting step carried out while that solution is still in
contact with the solid support.
[0011] The foregoing and other objects and aspects of the present
invention are explained in detail below.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1A schematically illustrates the method of the present
invention, where the test compound binds to the probe compound and
light is emitted at a second wavelength .lambda..sub.c.
[0013] FIG. 1B schematically illustrates the method of the present
invention, where the test compound does not bind to the probe
compound.
[0014] FIG. 2 illustrates a screening substrate carrying multiple
different probes useful for carrying out the present invention.
[0015] FIG. 3 illustrates a mixture of screening substrates, each
carrying a different probe, useful for carrying out the method of
the present invention.
DETAILED DESCRIPTION OF THE INVENTION
[0016] Methods of the present invention are schematically
illustrated in FIG. 1A and FIG. 1B. In overview, a test compound 11
has a first fluorophore 12 bound thereto, and a screening substrate
comprising a solid support 15 has a plurality of probe molecules 10
bound thereto. A second fluorophore 13 is bound to a linker 14,
which linker is in turn bound to the solid support. The screening
substrate is illuminated with light at a wavelength .lambda..sub.a
that is absorbed by the first fluorophore 12, which as illustrated
is a donor fluorophore. The transfer of energy to the second
fluorophore 13, here the acceptor fluorophore, is detected, here by
detecting light at a second wavelength .lambda..sub.c. As shown in
FIG. 1A, light is emitted at the second wavelength .lambda..sub.c
when the test compound 11, binds to the probe 10. As shown in FIG.
1B, light is not emitted at wavelength .lambda..sub.c when the test
compound 11 is not bound to the probe 10.
[0017] Any suitable solid support can be used to carry out the
present invention, and numerous different solid supports are well
known to those skilled in the art. Examples of suitable materials
from which the solid support may be formed include cellulose,
pore-glass, silica gel, polystyrene, particularly polystyrene
cross-linked with divinylbenzene, grafted copolymers such as
polyethyleneglycol/polystyrene, polyacrylamide, latex,
dimethylacrylamide, particularly cross-linked with
N,N'bis-acrylolyl ethylene diamine and comprising
N-t-butoxycarbonyl-beta- -alanyl-N'acrylolyl hexamethylene diamine,
composites such as glass coated with a hydrophobic polymer such as
cross-linked polystyrene or a fluorinated ethylene polymer to which
is grafted linear polystyrene, and the like. Thus the term "solid
support" includes materials conventionally considered to be
semi-solid supports. General reviews of useful solid supports that
include a covalently-linked reactive functionality may be found in
Atherton et al., Prospectives in Peptide Chemistry, Karger, 101-117
(1981); Amamath et al., Chem. Rev. 77: 183 (1977); and Fridkin, The
Peptides, Vol. 2, Chapter 3, Academic Press, Inc., pp 333-363
(1979). The solid support may take any suitable form, such as a
bead or microparticle, a tube, a plate, a microtiter plate well, a
glass microscope cover slip, etc.
[0018] The present invention can be used with probe molecules, or
libraries (where groups of different probe molecules are employed),
of any type. In general, such probe molecules are organic
compounds, including but not limited to that may be used to carry
out the present include oligomers, non-oligomers, or combinations
thereof. Non-oligomers include a wide variety of organic molecules,
such as heterocyclics, aromatics, alicyclics, aliphatics and
combinations thereof, comprising steroids, antibiotics, enzyme
inhibitors, ligands, hormones, drugs, alkaloids, opioids,
benzodiazepenes, terpenes, prophyrins, toxins, catalysts, as well
as combinations thereof. Oligomers include peptides (that is,
oligopeptides) and proteins, oligonucleotides (the term
oligonucleotide also referred to simply as "nucleotide, herein)
such as DNA and RNA, oligosaccharides, polylipids, polyesters,
polyamides, polyurethanes, polyureas, polyethers, poly (phosphorus
derivatives) such as phosphates, phosphonates, phosphoramides,
phosphonamides, phosphites, phosphinamides, etc., poly (sulfur
derivatives) such as sulfones, sulfonates, sulfites, sulfonamides,
sulfenamides, etc., where for the phosphorous and sulfur
derivatives the indicated heteroatom for the most part will be
bonded to C, H, N, O or S, and combinations thereof. Numerous
methods of synthesizing or applying such probe molecules on solid
supports (where the probe molecule may be either covalently or
non-covalently bound to the solid support) are known, and such
probe molecules can be made in accordance with procedures known to
those skilled in the art. See, e.g., U.S. Pat. No. 5,565,324 to
Still et al., U.S. Pat. No. 5,284,514 to Ellman et al., U.S. Pat.
No. 5,445,934 to Fodor et al. (the disclosures of all United States
patents cited herein are to be incorporated herein by reference in
their entirety).
[0019] Test compounds used to carry out the present invention may
be of any type, including both oligomers or non-oligomers of the
types described above in connection with probe molecules above.
Again, such test compounds are known and can be prepared in
accordance with known techniques. Fluorophores can be bound to, or
conjugated to, such test compounds in accordance with known
techniques, preferably by covalently joining the fluorophore to the
test compound. Such covalent joining includes the preparation of a
fusion protein, such as a fusion protein of a test compound and a
green fluorescent protein.
[0020] First and second fluoropohores that can be used to carry out
the present invention can be selected based on the physical
properties thereof, as is kown in the art of fluorescence resonance
energy transfer (FRET), the two being selected so that they
together comprise the donor and acceptor fluorophores of an FRET
pair. Either the first or the second fluorophore can serve as the
donor fluorophore, with the other serving as the acceptor
fluorophore.
[0021] FRET is a distance-dependent interaction between the
electronic excited states of two fluorophores in which excitation
is transferred from a donor molecule to an acceptor molecule
without emission of a photon. In general, the primary conditions
for FRET are (i) that the donor and acceptor molecules be in close
proximity to one another (typically 1 or 10 to 100 or 200
Angstroms); (ii) that the absorption spectrum of the acceptor
overlap the fluorescence emission spectrum of the donor; and (iii)
that the donor and acceptor transition dipole orientations be
approximately or essentially parallel. Examples of suitable donor
and acceptor pairs include:
[0022] fluorescein and tetramethylrhodamine;
[0023] 5-(2'-aminoethyl) aminonaphthalene-1-sulfonic acid (EDANS)
and fluorescein;
[0024] EDANS and 4-(4'-dimethylaminopheylazo)benzoic acid
(DABCYL);
[0025] fluorescein and fluorescein; and
[0026] 4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a
diaza-5-indacene-3-propioni- c acid (BODIPY FL) and BODIPY FL.
[0027] See generally R. Haugland, Handbook of Fluorescent Probes
and Research Chemicals (Sixth Ed. 1995). Abbreviations that are
standard in the art are used herein. Note also that the present
invention can be carried out when the donor and acceptor
fluorophores are the same, with FRET being detected by the
resulting fluorescence emission. Biophys. J. 69, 1565 (1995). As
indicated above, one, or both, of the fluorophores can be a green
fluorescent protein, and it is particularly advantageous to employ
green fluorescent protein as the fluorophore when the test compound
is a protein or peptide by preparing a fusion protein of the test
compound and a green fluorescent protein.
[0028] Linking groups used to immobilize the fluorophore to the
solid support carry out the present invention are, in general,
polymers, including both water soluble polymers and water insoluble
polymers (solubility referring to solubility when the linking group
is free, rather than bound to the solid support). The polymers are
elongate flexible chains of repeating monomeric units, and carry or
contain functional groups along the chain length therof to which
fluorophores can be conjugated. Numerous polymers that can be
functionalized so that a fluorophore can be conjugated thereto,
typically by a covalent bond, are known, and will be readily
apparent to those skilled in the art. Examples include, but are not
limited to, polysaccharides such as dextran, polyvinyl alcohol,
polypeptides such as polylysine, and polyacrylic acid. The
fluorophore may be bound to the linking group in any conformation
or position, including to the free chain end thereof. However, in
preferred embodiments of the invention, a plurality of fluorophores
are conjugated to the polymer chain of the linking group at
separate, spaced-apart locations along the length thereof. This
enables excitation of the fluorophores even when the probe molecule
binds to the target in different orientations, thereby enhancing
the ability of the system to detect positive binding events.
[0029] Linking groups are covalently or non-covalently bound to the
solid support in accordance with known techniques. For example,
polymer linking groups can be covalently grafted onto a substrate
polymer by any suitable reaction chemistry, such as free radical or
condensation chemistry (e.g., polyacrylic acid linking groups may
be covalently grafted onto a polypropylene substrate). In general,
the same techniques used to prepare linking groups that are used to
link probe molecules to solid supports for combinatorial chemistry
can be used as the linking groups for the fluorophores herein. See,
e.g., U.S. Pat. No. 5,624,711 to Sundberg et al. ("Derivatization
of Solid Supports and Methods for Oligomer Synthesis)(the
disclosure of which is incorporated by reference herein in its
entirety).
[0030] The density of the linking groups on the solid support is
such that one or more linking groups as described above, carrying
one or more fluorophores, is positioned on the substrate a distance
that will enable fluorescence resonance energy transfer between a
first fluorophore on a test compound that binds to the probe and a
second fluorophore on the linker bound to the solid support
adjacent the probe. Where multiple different probes are on the
substrate, each active probe will have such a spatial relationship
with the linkers on the substrate. Typically, the density of the
linking groups is sufficient so that the distance between one or
more linking groups and the adjacent probe is not greater than 200
or 300 Angstroms from the probe molecule or molecules. The length,
or average length, of the linking groups is not critical so long as
it is sufficient to position fluorophores bound to the linking
group effectively for FRET to or from fluorophores that are bound
to test compounds that in turn bind to the probe molecules as
described herein.
[0031] Where multiple different probe molecules are desired to be
tested, a screening substrate useful for the high throughput
screening of molecular interactions, such as in "chip-based" and
"pin-based" combinatorial chemistry techniques, can be prepared in
accordance with known techniques. As schematically illustrated in
FIG. 2, the substrate comprises a solid support 15 having a surface
portion, with the surface portion comprising a plurality of
discreet known regions (A through X). A plurality of different
probe molecules (not shown) are bound to the surface portion, with
different probe molecules positioned on the surface portion in
different ones of the discrete known regions. All can be prepared
in accordance with known techniques. See, e.g., U.S. Pat. No.
5,445,934 to Fodor et al., U.S. Pat. No. 5,288,514 to Ellman, and
U.S. Pat. No. 5,624,711 to Sundberg et al. In addition a plurality
of the same fluorophores 13 are connected to or conjugated the
surface portion, preferably by covalent bonding, with the
fluorophores positioned on the surface portion in all of the
plurality of discreet known regions (for clarity, fluorophores 13
are shown only in regions M through X). Connection of the
fluorophores to the solid support is carried out in accordance with
known techniques, or variations thereof that will be apparent to
those skilled in the art, and can be carried out with linking
groups as described above.
[0032] Methods for detecting the binding of a test compound to a
probe molecule employing a screening substrate as described in
connection with FIG. 2 above comprise, in general, providing a test
compound 11, the test compound having a first fluorophore 12 bound
thereto. A screening substrate as described above is provided,
where the fluorophore bound to the surface portion of the solid
support serves as a second fluorophore. The test compound is
contacted to the screening substrate (typically by providing the
test compound in a liquid, usually aqueous, solution), and the
screening substrate illuminated with light at a wavelength that is
absorbed by the first fluorophore. The presence or absence of light
emitted by the second fluorophore in one of the discreet known
regions is then detected, the emission of light from the second
fluorophore indicating the binding of the test compound to the
probe bound to that discreet known region.
[0033] In the alternative, screening of libraries of probe
molecules may be carried out with mixtures of solid supports as
used in "split-pool" combinatorial chemistry techniques. As
schematically illustrated in FIG. 3, such mixtures comprise a
plurality of discreet solid supports 15 each having a surface
portion, each such support having a different probe molecule 10
bound to the surface portion thereof; and a plurality of the same
fluorophores (not shown) bound to the surface portion of each of
the solid supports (again, the second fluorophore is preferably
bound the solid support by means of a linker group). Such mixtures
can be prepared in accordance with procedures known in the art, and
tag components can be added to the discreet solid supports in
accordance with procedures known in the art. See, e.g., U.S. Pat.
No. 5,565,324 to Still et al.
[0034] Methods of using mixtures of solid supports as shown in FIG.
3 above comprise providing a test compound, the test compound
having a first fluorophore bound thereto, and providing a mixture
of solid supports as described above. The test compound is then
contacted to at least one of the discreet solid supports (typically
by contacting the support to or immersing the support in a liquid,
usually aqueous, solution containing the test compound), and the
discreet solid support illuminated with light at a wavelength that
is absorbed by the first fluorophore. The presence or absence of
light emitted by the second fluorophore is then detected from the
discreet solid support, the emission of light from the second
fluorophore indicating the binding of the test compound to the
probe bound to that discreet solid support.
[0035] An advantage of using mixtures of discreet solid supports in
methods as described above is that the method can be carried out in
solution or in a liquid phase, without the need for a step of
separating unbound test compound from the solid support. Further,
by passing the discreet solid supports continuously through a
conduit, which conduit is associated with a fluorescent detector,
while exciting the first fluorophore, and then separating those
discreet solid supports where the second fluorophore emits light
from those that do not (the separating step illustrated by arrows
20, 21), the method can advantageously be carried out in a
fluorescent sorter in accordance with known techniques. See
generally M. Melamed et al., An Historical Review of the
Development of Flow Cytometers and Sorters, in Flow Cytometry and
Sorting, pgs 1-9 (2d Ed. 1990). Such apparatus is generically
referred to as flow sorting apparatus herein.
[0036] In various embodiments set forth above, the present
invention has been explained with reference to the first
fluorophore being the donor fluorophore, the first and second
fluorophores being different, and the transfer of energy to the
acceptor fluorophore being determined by the emission of light
therefrom. While this is one preferred embodiment of the invention,
it should be understood that either the first or the second
fluorophore can be the donor, and that (where the donor and the
acceptor are the same) the transfer of energy to the acceptor
fluorophore can be still be detected by means such as detecting a
characteristic fluorescence emission.
[0037] The present invention is explained in greater detail in the
following non-limiting Examples, in which "M" means molar, "nm"
means nanometers, and ".mu.L" means microliters.
EXAMPLE 1
Assay of Protein:Protein Binding by Fluorescence Resonance Energy
Transfer
[0038] The purpose of this assay is to determine the domain of
laminin recognized by a monoclonal anti-laminin antibody.
[0039] First, prepare synthetic peptides of 10 amino acids each
which span the primary sequence of laminin. Then, each peptide is
deposited into individual wells of a 96 well microtiter plate,
along with fluorescein-bound dextran (10.sup.-5M). After incubation
for one hour remove the unbound peptide and the unbound dextran.
Next, conjugate the anti-laminin antibody with
tetramethylrhodamine, add 10.sup.-5M of the bound antibody to each
well, and incubate the plate at room temperature for two hours. In
a spectrofluorometer, assay each well for antibody-peptide
interaction by exciting at 495 nm (the excitation frequency for
fluorescein) and measuring emission at 570 nm (the emission
frequency for rhodamine).
EXAMPLE 2
Assay of Protein:Protein Binding with Human:Anti-Human IgG
[0040] In a 96 well microtiter plate place a Bio-Rad cellulose
paper insert into each well. Add 5 .mu.l of 10.sup.-6 M
rhodamine-bound dextran, and 1 .mu.l of either rat or human IgG
(equal to 32 picomoles) to each well.
[0041] Wash each well once with 100 .mu.L of buffer, and then add 5
.mu.L FITC-bound anti-human IgG, incubate at room temperature, and
aspirate the media.
[0042] Fluorescence is then measured in a Perkin-Elmer LS50
spectrofluorometer, with an excitation frequency of 495 nm (the
excitation frequency of FITC) and an emission frequency of 570 nm
(the emission frequency of rhodamine). Results in fluorescence
units for three different runs on two separate days are given in
Table 1 below.
1TABLE 1 In well: Rat IgG Human IgG Day 1: 783 1000 897 973 817 979
Day 2: 490 521 525 572 457 538 Note the consistently higher levels
of emissions for human IgG as compared to rat IgG in all runs.
[0043] The foregoing is illustrative of the present invention, and
is not to be construed as limiting thereof. The invention is
defined by the following claims, with equivalents of the claims to
be included therein.
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