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BACKGROUND OF THE INVENTION
The present invention relates to the field of ligand/receptor interaction
evaluation. More specifically, in one embodiment the invention provides a
novel system for the determination of the binding affinity of a receptor
to a surface-bound, high-density ligand array and vice versa (ligand to
bound receptor). Due to the nature of the immobilized array, this system
also permits measurements of both on- and off-rates of receptor binding.
In a particular embodiment, the present invention provides a method and
device for quantitation of binding affinity utilizing fluorescence
intensity data from labelled receptors bound to peptides which have been
synthesized or immobilized on a solid support. In another embodiment, the
present invention provides a method for determining the fidelity of
synthesis of peptides by comparing the fluorescence intensity of a
receptor bound to an array to the fluorescence intensity of the receptor
bound to an array of known purity and composition.
Techniques have recently been introduced for synthesizing large arrays of
different peptides and other polymers on solid surfaces. For example, in
Pirrung et al., PCT Publication No. WO 90/15070, incorporated herein by
reference for all purposes, a technique is disclosed for generating arrays
of peptides and other materials using, for example, light-directed,
spatially-addressable synthesis techniques. See also U.S. patent
application Ser. No. 07/624,120 (Fodor et al.) which discloses, among
other things, a method of gathering fluorescence intensity data, various
photoprotective groups, masking techniques, and automated techniques for
performing light-directed, spatially-addressable synthesis techniques.
Because of their relationship to semiconductor fabrication techniques,
these methods have come to be referred to as "Very Large Scale Immobilized
Polymer Synthesis," "VLSIPS.TM." synthesis technique. Such techniques have
met with substantial success in, for example, screening various ligands
such as peptides to determine their relative binding affinity to a
receptor such as an antibody.
While meeting with substantial success, such techniques generally provide a
qualitative estimate of binding affinity for a ligand-receptor pair as
well as the relative order of affinities for a multi-ligand substrate.
Other techniques of measuring binding affinity provide only indirect
indications of binding affinity.
It is desirable to provide a direct method and device for more precisely
determining the binding affinity of a receptor to a specific ligand
substrate synthesized in a multi-ligand array.
SUMMARY OF THE INVENTION
A novel method and device for quantitatively evaluating the binding
affinity of a receptor/ligand substrate is provided by virtue of the
present invention. The method and device are particularly suited for
application to solid-phase synthesis techniques such as the VLSIPS.TM.
technique, as well as bead or pin-based techniques.
According to one preferred embodiment of the invention, an array of
ligands, particularly polymers such as peptides, is formed on a substrate
at predetermined locations. The ligands are exposed to a receptor such as
an antibody or series of antibodies, one or more of which are labelled
with a fluorescent label, such as fluorescein. Fluorescence intensity data
at or near steady-state or equilibrium are gathered from the substrate at
a variety of locations within the predetermined substrate pattern,
providing a measure of receptor binding to the surface-bound ligands. The
process is repeated with a series of different receptor concentrations.
Based on the fluorescence intensity data for the solutions of varying
concentrations, it becomes possible to extract quantitative information
regarding the binding affinity of the receptor to various ligand
substrates. The data obtained from substrate-bound ligand data herein
closely approximate solution behavior of the ligand/receptor pair.
In one embodiment, the invention provides a method of determining the
binding affinity of a receptor/ligand pair. The method includes the steps
of exposing a labelled receptor to one or more ligands on the surface of a
substrate to a labelled receptor at a first solution concentration of
receptor; exposing the ligands on the surface of a substrate to the
labelled receptor at a second receptor concentration; measuring indicia
related to the number of receptor molecules bound to the ligands; and
based on the indicia related to the number of receptor molecules bound to
the ligands, determining a binding affinity of the ligands to the
receptor.
A further understanding of the nature and advantages of the inventions
herein may be realized by reference to the remaining portions of the
specification and the attached drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is an overall flow diagram illustrating a process for determining
the relative binding affinity of a ligand and a receptor;
FIG. 2 illustrates a device and process for forming diverse polymer
sequences on a substrate;
FIGS. 3a, 3b, 3c, and 3d illustrate a device and method for gathering
fluorescence intensity data as a function of location on a substrate;
FIG. 4 illustrates an isotherm of YGGFL peptide data and, specifically,
plots the ratio of fluorescence intensity to receptor concentration versus
fluorescence intensity;
FIG. 5 illustrates an isotherm of YGGFL peptide (synthesized as a free
peptide and then immobilized on a surface) information which specifically
plots the normalized fluorescence intensity versus receptor concentration;
FIG. 6 illustrates YGGFL and YGTFL isotherms; and
FIG. 7 illustrates kinetic antibody binding data to a YGGFL substrate.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
Contents
I. Glossary
II. Detailed Description
III. Examples
IV. Conclusion
I. Glossary,
The following terms are intended to have the following general meanings as
they are used herein:
1. Ligand: A ligand is a molecule that is recognized by a particular
receptor. Examples of ligands that can be investigated by this invention
include, but are not restricted to, agonists and antagonists for cell
membrane receptors, toxins and venoms, viral epitopes, hormones (e.g.,
steroids etc.), hormone receptors, peptides, enzymes, enzyme substrates,
cofactors, drugs, lectins, sugars, oligonucleotides, nucleic acids,
oligosaccharides, proteins, and monoclonal antibodies.
2. Monomer: A member of the set of small molecules which are or can be
joined together to form a polymer. The set of monomers includes but is not
restricted to, for example, the set of common L-amino acids, the set of
D-amino acids, the set of synthetic and/or natural amino acids, the set of
nucleotides and the set of pentoses and hexoses. The particular ordering
of monomers within a polymer is referred to herein as the "sequence" in
the polymer. As used herein, monomers refers to any member of a basis set
for synthesis of a polymer. For example, dimers of the 20 naturally
occurring L-amino acids form a basis set of 400 monomers for synthesis of
polypeptides. Different basis sets of monomers may be used at successive
steps in the synthesis of a polymer. Furthermore, each of the sets may
include protected members which are modified after synthesis. The
invention is described herein primarily with regard to the analysis of
molecules containing sequences of monomers such as amino acids, but could
readily be applied in the analysis of other polymers. Such polymers
include, for example, both linear and cyclic polymers of nucleic acids,
polysaccharides, phospholipids, and peptides having either .alpha.-,
.beta.-, or .omega.-amino acids, heteropolymers in which a known drug is
covalently bound to any of the above, polynucleotides, polyurethanes,
polyesters, polycarbonates, polyureas, polyamides, polyethyleneimines,
polyarylene sulfides, polysiloxanes, polyimides, polyacetates, or other
polymers which will be apparent upon review of this disclosure. Such
polymers are "diverse" when polymers having different monomer sequences
are formed at different predefined regions of a substrate. Methods of
cyclization and polymer reversal of polymers are disclosed in copending
application Ser. No. 796,727, filed on the same date as the present
application, entitled "POLYMER REVERSAL ON SOLID SURFACES," incorporated
herein by reference for all purposes.
3. Peptide: A polymer in which the monomers are alpha amino acids and which
are joined together through amide bonds and alternatively referred to as a
polypeptide. In the context of this specification it should be appreciated
that the amino acids may be the L-optical isomer or the D-optical isomer.
Peptides are two or often more amino acid monomers long, and often more
than 20 amino acid monomers long. Standard abbreviations for amino acids
are used (e.g., P for proline). These abbreviations are included in
Stryer, Biochemistry, Third Ed., 1988, which is incorporated herein by
reference for all purposes. Small case and large case letters are utilized
to differentiate the D-isomer and L-isomer, respectively.
4. Receptor: A molecule that has an affinity for a given ligand. Receptors
may be naturally-occurring or manmade molecules. Also, they can be
employed in their unaltered state or as aggregates with other species.
Receptors may be attached, covalently or noncovalently, to a binding
member, either directly or via a mediating linker. Examples of receptors
which can be employed by this invention include, but are not restricted
to, antibodies, cell membrane receptors, monoclonal antibodies and
antisera reactive with specific antigenic determinants (such as on
viruses, cells or other materials), drugs, polynucleotides, nucleic acids,
peptides, cofactors, lectins, sugars, polysaccharides, cells, cellular
membranes, and organelles. Receptors are sometimes referred to in the art
as anti-ligands. As the term receptors is used herein, no difference in
meaning is intended. A "ligand receptor pair" is formed when two
macromolecules have combined through non-covalent molecular recognition to
form a complex.
Specific examples of receptors which can be investigated by this invention
include but are not restricted to:
a) Microorganism receptors: Determination of ligands which bind to
receptors, such as specific transport proteins or enzymes essential to
survival of microorganisms, is useful, as in the discovery of a new class
of antibiotics. Of particular value would be antibiotics against
opportunistic fungi, protozoa, and those bacteria resistant to the
antibiotics in current use.
b) Enzymes: For instance, determination of the binding site of enzymes such
as the enzymes responsible for cleaving neurotransmitters is useful. Also
of value would be determination of ligands which bind to certain receptors
to modulate the action of the enzymes which cleave the different
neurotransmitters is useful in the development of drugs which can be used
in the treatment of disorders of neurotransmission.
c) Antibodies: For instance, the invention may be useful in investigating
the ligand-binding site on the antibody molecule which combines with the
epitope of an antigen of interest; determining a sequence that mimics an
antigenic epitope may lead to the-development of vaccines in which the
immunogen is based on one or more of such sequences or may lead to the
development of related diagnostic agents or compounds useful in
therapeutic treatments such as for autoimmune diseases (e.g., by blocking
the binding of the "self" antibodies). For purposes of the present
invention, "antibody" includes a whole antibody or an antibody fragment
(Fab or (Fab).sub.2).
d) Nucleic Acids: Sequences of nucleic acids may be synthesized to
establish DNA or RNA binding sequences.
Catalytic Polypeptides: Polymers, preferably polypeptides, which are
capable of promoting a chemical reaction involving the conversion of one
or more reactants to one or more products. Such polypeptides generally
include a binding site specific for at least one reactant or reaction
intermediate and an active functionality proximate to the binding site,
which functionality is capable of chemically modifying the bound reactant.
Catalytic polypeptides and others are described in, for example, PCT
Publication No. WO 90/05746, WO 90/05749, and WO 90/05785, which are
incorporated herein by reference for all purposes.
f) Hormone receptors: For instance, the receptors for insulin and growth
hormone. Determination of the ligands which bind with high affinity to a
receptor is useful in the development of, for example, an oral replacement
of the daily injections which diabetics must take to relieve the symptoms
of diabetes, and in a replacement for the scarce human growth hormone
which can only be obtained from cadavers or by recombinant DNA technology.
Other examples are the vasoconstrictive hormone receptors; determination
of those ligands which bind to a receptor may lead to the development of
drugs to control blood pressure.
g) Opiate receptors: Determination of ligands which bind to the opiate
receptors in the brain is useful in the development of less-addictive
replacements for morphine and related drugs.
5. Substrate: A material having a rigid or semi-rigid surface. In many
embodiments, at least one surface of the substrate will be substantially
flat, although in some embodiments it may be desirable to separate
synthesis regions physically for different polymers with, for example,
wells, raised regions, etched trenches, or the like. According to other
embodiments, small beads, such as beads with peptides on the surface
(which, in some embodiments, may be released upon completion of the
synthesis), are employed. In other embodiments, however, the substrate
need only be semi-soluble, i.e., a substrate, such a polyethylene glycol
(PEG) that is a solid in some but not all solvents.
6. Protective Group: A material which is bound to a monomer unit and which
may be selectively removed therefrom.
7. Predefined Region: A predefined region is a localized area on a
substrate which is, was, or is intended to be used for formation of a
selected polymer and is otherwise referred to herein in the alternative as
a "selected" region. The predefined region may have any convenient shape,
e.g., circular, rectangular, elliptical, wedge-shaped, etc. For the sake
of brevity herein, "predefined regions" are sometimes referred to simply
as "regions." In some embodiments, a predefined region and, therefore, the
area upon which each distinct polymer sequence is synthesized is smaller
than about 1 cm.sup.2 or less than 1 mm.sup.2. In preferred embodiments
the regions have an area less than about 10,000 .mu.m.sup.2 or, more
preferably, less than 100 .mu.m.sup.2 and may, in some embodiments be
less than 10 .mu.m.sup.2. Within these regions, the polymer synthesized
therein is preferably synthesized in a substantially pure form.
8. Substantially Pure: A polymer is considered to be "substantially pure"
within a predefined region of a substrate when it exhibits characteristics
that distinguish it from other predefined regions. Typically, purity will
be measured in terms of biological activity or function as a result of
uniform sequence. Such characteristics will typically be measured by way
of binding with a selected ligand or receptor. Preferably the region is
sufficiently pure such that the predominant species in the predefined
region is the desired sequence. According to preferred aspects of the
invention, the polymer is 5% pure, more preferably more than 10% pure,
preferably more than 20% pure more preferably more than 80% pure, more
preferably more than 90% pure, more preferably more than 95% pure, where
purity for this purpose refers to the ratio of the number of ligand
molecules formed in a predefined region having a desired sequence to the
total number of molecules formed in the predefined region.
II. Detailed Description
FIG. 1 is an overall flow chart illustrating a process for determining the
binding affinity of a receptor to a ligand and, in particular, a process
for determining the binding affinities of a receptor to an array of
ligands synthesized on a substrate. All operations herein are preferably
performed at constant and substantially atmospheric pressure and ambient
temperature, although in some embodiments it will be of interest to
determine binding affinity under a variety of conditions and the
experiment will be repeated at, for example, different temperatures.
As shown at step 102, an array or arrays of ligands such as peptides are
synthesized on a support, or fully synthesized polymers are immobilized on
a support. The method may be sequentially performed on one array or may be
done simultaneously on a series of duplicate arrays. In some embodiments,
duplicate arrays are synthesized on separate substrates.
The method of forming the arrays will vary from one application to another.
According to preferred embodiments, the ligands are formed according to
the light-directed synthesis techniques of Pirrung et al., WO 90/15070,
previously incorporated by reference for all purposes. Other methods will
be used according to some embodiments such as the "pin" method described
in Geysen, or the method described in copending application Ser. No.
796,243, filed on the same day as the present application, and
incorporated herein by reference for all purposes. Whether the ligand is
synthesized on or presynthesized and then immobilized on the surface is
not essential to the present invention, however.
At step 104 the array or arrays of polymers are exposed to a receptor at a
first solution concentration [R].sub.1. According to some embodiments the
receptor is marked with an appropriate label such as a fluorescein tag.
Fluorescent tags are preferred because the measurements herein are usually
taken with a receptor solution in contact with the immobilized ligands to
ensure that the immobilized ligand are in equilibrium with the receptor.
By "equilibrium" herein it is intended to mean that the amount of receptor
bound to the ligand does not change appreciably over time. Confocal
microscopy provides one preferred method of distinguishing between surface
receptor fluorescence and background fluorescence from bulk solution
receptors, due to the limited depth of field of a confocal microscope.
Because the microscope cannot completely exclude fluorescence from bulk
solution, contributions of the bulk fluorescence can be eliminated by
comparing areas of derivatized and underivatized substrates. This method
for eliminating background fluorescence may not be accurate at high
receptor concentrations, when non-specific receptor binding to
underivatized substrates becomes non-negligible.
In various embodiments, the substrate and first receptor (either labelled
or unlabelled) are exposed to a second labelled receptor which binds to
the first receptor, preferably at multiple locations. In this embodiment,
it is not necessary to label the first receptor. This procedure can be
used to provide signal amplification and, therefore, an improved
signal-to-noise ratio from the data.
At step 106 the array of the polymers is exposed to the receptor, for a
time sufficient to allow receptor-ligand interaction, preferably to
equilibrium conditions, and at step 108 the fluorescence intensity of the
receptor at each of the concentrations [R].sub.n is determined for each of
the ligands on each array. The process can be repeated for each receptor
concentration by repeating steps 104 to 108, as indicated by step 107.
It will be recognized that while the invention is illustrated herein by way
of a serial process in which the receptor concentration is varied while
contacting a single array of polymers, other embodiments will find wide
application. For example, in preferred embodiments, a plurality of
duplicate (or near duplicate) arrays can be synthesized in different
regions on a substrate, and by simultaneously contacting all of the arrays
with receptor solutions of different concentrations it becomes possible to
reduce substantially the time required for performing the process. In
embodiments where a single array of polymers is utilized, it is preferred
that the receptor concentration be increased during the process, rather
than decreased, because bound receptor may be difficult to remove.
At step 110 this information is fit to analysis equations or otherwise
processed for determination of the binding affinity of the receptor to the
various ligands on the substrate and at step 112 the K.sub.d values, among
others, are extracted. In a most preferred embodiment the data are used to
determine binding affinity by determining the slope of a best fit line
through a plot having, on the y-axis, the ratio of fluorescence intensity
to receptor concentration and, on the x-axis, the fluorescence intensity.
It will of course be recognized that in most embodiments, a line will not
actually be mechanically "plotted" and, instead, such operations will be
conducted in an appropriately programmed digital computer, using discrete
logic, or the like. To faciltate comparison to other experiments, one can
plot normalized fluorescence intensity on the y axis or receptor
concentration on the x axis. A "normalized" plot has all intensity scaled
to saturation intensity.
The exact sequence of steps described above can be varied without departing
from the scope of the invention herein. For example, after exposing the
polymer array to the receptor at a first concentration, it may be
desirable to extract the fluorescence intensity information for the array
at this initial concentration before proceeding with different
concentrations. Based on information from the array exposed to the
receptor at the first concentration, it may become evident that the
receptor in question does not bind in any appreciable way to certain
ligands. Accordingly, in subsequent steps, a ligand array may be
synthesized which eliminates the ligands without appreciable binding
affinity. The subsequent arrays may have other attributes, such as
increased synthesis area for the ligands of primary interest.
In some embodiments, one or several fully pre-synthesized ligands are
immobilized on the surface of the substrate in selected regions. For
instance, such peptides can serve as standards for fidelity of synthesis
of other polymers. These pre-synthesized ligands can be duplicates of one
or more of the peptides which have been synthesized on the substrate. By
comparison of the binding affinity of these pre-synthesized ligands to the
ligands which are synthesized on the substrate directly, it will be
possible to determine if the synthesized peptides are of less than desired
purity, and to estimate the degree of impurity of the substrate
synthesized ligands.
FIG. 2 schematically illustrates a preferred embodiment of a reactor system
200 for synthesizing polymers on the prepared substrate. It will be
recognized that the system shown in FIG. 2 is only illustrative of the
systems for synthesizing the polymers for use herein. For example, the
polymers may be synthesized on a substrate in some embodiments using a
channel block for directing the flow of reactants over a substrate in
accordance with the techniques disclosed in copending application Ser. No.
796,243, entitled "VERY LARGE SCALE IMMOBILIZED POLYMER SYNTHESIS USING
MECHANICALLY DIRECTED FLOW PATHS," previously incorporated herein by
reference for all purposes.
The reactor system includes a body 202 with a cavity 204 on a surface
thereof. In preferred embodiments the cavity 204 is between about 50 and
1000 .mu.m deep with a depth of about 500 .mu.m preferred. The bottom of
the cavity is preferably provided with an array of ridges 206 which extend
both into the plane of the Figure and parallel to the plane of the Figure.
The ridges are preferably about 50 to 200 .mu.m deep and spaced at about 2
to 3 mm. The purpose of the ridges is to generate turbulent flow for
better mixing. The bottom surface of the cavity is preferably
light-absorbing so as to prevent reflection of impinging light.
A substrate 212 is mounted above the cavity 204. The bottom surface of the
substrate is attached to a photoremovable protective group, such as NVOC,
nitroveratryloxy carbonyl with or without an intervening spacer molecule.
The substrate is preferably transparent to a wide spectrum of light, but
in some embodiments is transparent only at a wavelength at which the
protective group may be removed (such as near UV in the case of NVOC). The
substrate in some embodiments is a conventional microscope glass slide or
cover slip. The substrate is preferably as thin as possible, while still
providing adequate physical support. Preferably, the substrate is less
than about 1 mm thick, more preferably less than 0.5 mm thick, more
preferably less than 0.1 mm thick, and most preferably less than 0.05 mm
thick. In alternative preferred embodiments, the substrate is quartz or
silicon.
The substrate and the body serve to seal the cavity except for an inlet
port 208 and an outlet port 210. The body and the substrate may be mated
for sealing in some embodiments with one or more gaskets or O-rings.
According to a preferred embodiment, the body is provided with two
concentric gaskets and the intervening space is held at vacuum to ensure a
leak-proof seal of the substrate to the gaskets. A mask 228 is placed on
or near the substrate 212.
Fluid from storage 218 is pumped through the inlet port into the cavity by
way of a pump 216 which may be, for example, a model no. B-120-S made by
Eldex Laboratories. Selected fluids are circulated into the cavity by the
pump, through the cavity, and out the outlet for recirculation or
disposal. The reactor may be subjected to ultrasonic radiation and/or
other method of stirring to aid in agitation in some embodiments.
For the sake of a compact system, a reflective mirror 222 may be provided
for directing light from a light source 224 onto the substrate. Light
source 224 may be, for example, a Xe(Hg) light source manufactured by
Oriel and having model no. 66024. This form of lithography is referred to
herein as proximity printing. As will be apparent from this disclosure,
projection printing and the like may also be used according to some
embodiments.
Light from the light source is permitted to reach only selected locations
on the substrate as a result of the mask 228. Mask 228 may be, for
example, a glass slide having etched chrome thereon. The mask 228 in one
embodiment is provided with a grid of transparent locations and opaque
locations and, in the case of a contact printing process, is preferably
placed so that the side printed with the opaque regions is placed in
direct contact with the substrate. Such masks may be manufactured by, for
example, Photo Sciences, Inc. Light passes freely through the transparent
regions of the mask, but is reflected from or absorbed by other regions.
Therefore, only selected regions of the substrate are exposed to light.
In operation, the substrate is placed on the cavity and sealed thereto. All
operations in the process of preparing the substrate are carried out in a
room or enclosure lit primarily or entirely by light of a wavelength
outside of the light range at which the protective group is removed. For
example, in the case of NVOC, the room should be lit with a conventional
dark room light which provides little or no UV light. All operations are
preferably conducted at about room temperature.
A first wash solution for deprotection (without a monomer) is circulated
through the cavity. The solution preferably, in the case of amino acid
coupling, is of 5 mM sulfuric acid in dioxane solution which serves to
keep exposed amino groups protonated and decreases their reactivity with
photolysis by-products. Absorptive materials such as N,N-diethylamino
2,4-dinitrobenzene, for example, may be included in the deprotection fluid
which serves to absorb light and prevent reflection and unwanted
photolysis.
The slide is, thereafter, positioned in a light raypath from the mask such
that first locations on the substrate are illuminated and, therefore,
deprotected. In preferred embodiments the substrate is illuminated for
between about 1 and 15 minutes with a preferred illumination time of about
10 minutes at 10-20 mW/cm.sup.2 with 365 .mu.m light. The slides are
neutralized (i.e., brought to a pH of about 7) after photolysis with, for
example, a solution of di-isopropylethylamine (DIEA) in methylene chloride
for about 5 minutes.
The first monomer is then placed at the first locations on the substrate.
After irradiation, the slide is removed, treated in bulk to add the first
monomer, and then reinstalled in the flow cell. Alternatively, a fluid
containing the first monomer, preferably also protected by a protective
group, is circulated through the cavity by way of pump 216. If, for
example, it is desired to attach the amino acid Y to the substrate at the
first locations, the amino acid Y (bearing a protective group on its
.alpha.-nitrogen), along with reagents used to render the monomer
reactive, and/or a carrier, is circulated from a storage container 218,
through the pump, through the cavity, and back to the inlet of the pump.
As the solution containing the monomer to be attached is circulated through
the cavity, the amino acid or other monomer will react at its carboxy
terminus with amino groups on the regions of the substrate which have been
deprotected. Of course, while the invention is illustrated by way of
circulation of the monomer through the cavity, the invention could be
practiced by way of removing the slide from the reactor and submersing it
in an appropriate monomer solution.
After addition of the first monomer, the solution containing the first
amino acid is then purged from the system. After circulation of a
sufficient amount of the DMF dimethylformamide methylene chloride such
that removal of the amino acid can be assured (e.g., about 50.times. times
the volume of the cavity and carrier lines), the mask or substrate is
repositioned, or a new mask is utilized such that second regions on the
substrate will be exposed to light and the light is engaged for a second
exposure. This will deprotect second regions on the substrate and the
process is repeated until the desired polymer sequences have been
synthesized.
The process is repeated in preferred embodiments herein to synthesize
substrates having duplicate peptide sequence arrays, or subarrays thereof.
According to techniques described herein, some embodiments of the
invention provide for synthesis of duplicate "copies" of the various
polymer sequence arrays on a single substrate in various regions. These
various regions are then selectively exposed to receptor solutions of
differing concentrations. Indicia related to the number of receptor
molecules bound to the substrate are then measured. According to preferred
embodiments the indicia are obtained by measurement f fluorescent
intensity of a substrate exposed to fluorescently labelled receptor.
Specifically, a first derivatized substrate, or a first of a series of
duplicate arrays on a single substrate, is then exposed to a receptor of
interest in a solution at a first concentration. The receptor is labelled
with, for example, a fluorescein marker. The substrate is exposed to the
receptor via placement of a solution or suspension of the receptor in a
cavity adjacent the substrate or by contacting the entire surface of the
slide in bulk. The receptor will preferentially bind to certain regions of
the substrate which contain complementary sequences.
FIG. 3a schematically illustrates the process wherein duplicate arrays of
polymers 303 and 305, in this case the polymers A, B, and C, have been
synthesized on the substrate 212. Such polymers will, for example, be
peptides having different amino acid sequences. Each of the arrays is
exposed to a solution 306 containing a selected receptor R at a selected
concentration in a reservoir 310. In the particular example illustrated in
FIG. 3, solution 306a has a lower receptor concentration than solution
306b. The receptor is labelled with a label Fl. The various polymer arrays
are exposed to different receptor concentrations through the use of
channel block 307, which is mated to the substrate and has channels
therein which are filled with the respective receptor solutions.
As shown in FIG. 3a, the receptor binds to polymers B and C, but not in any
appreciable amount to polymer A. In the particular embodiment shown in
FIG. 3a, the receptor binds to polymer B more strongly than polymer C. The
higher receptor concentration of solution 306b results in more binding to
both polymers than in solution 306a.
Confocal microscope 309 is used to measure the fluorescent light intensity
along the surface of the substrate. Because the confocal microscope has a
limited depth of field (DOF), background fluorescence from the solution
receptor molecules can be limited, yet solution equilibrium or near
equilibrium is ensured because the solution remains in contact with the
polymer arrays. At higher concentrations of labelled receptor,
interference by the solution receptor may be minimized through the use of
increasing objective strength in the confocal microscope, such as an
increase from 10.times.to 40.times.. Alternatively, interference may be
reduced by reducing aperture size, or by labelling only a portion of the
receptor.
Antibodies are typically suspended in what is commonly referred to as a
"supercocktail," which may be, for example a solution of about 1% BSA
(bovine serum albumin), 0.5 TWEEN.TM. polyoxyethylene soritan monolaurate
in PBS (phosphate buffered saline) buffer. The antibodies are diluted with
supercocktail buffer to a final concentration of, for example, about 0.03
to 5 .mu.g/ml. It will be recognized that these concentrations are
illustrative, and find particular application for affinities in the
nano-molar range. Higher concentrations will generally be needed for lower
affinity measurements.
For purposes of increasing the signal-to-noise ratio of the system, some
embodiments of the invention provide for exposure of the substrate to a
first labelled or unlabelled receptor followed by exposure of a labelled,
second receptor (e.g., an antibody) which binds at multiple sites on the
first receptor. If, for example, the first receptor is an antibody derived
from a first species of an animal, the second receptor is an antibody
derived from a second species directed to epitopes associated with the
first species. In the case of a mouse antibody, for example, fluorescein
labelled goat antibody or antiserum which is antimouse may be used to bind
at multiple sites on the mouse antibody, providing several times the
fluorescence compared to the attachment of a single mouse antibody at each
binding site. This process may be repeated again with additional
antibodies (e.g., goat-mouse-goat, etc.) for further signal amplification.
FIG. 3b illustrates a fluorescence detection device 316 for detecting
fluorescein-labelled receptors on a substrate in greater detail. The
substrate 212 is placed on a flow-cell 329 with one or more reservoirs
310. The reservoir(s) is filled with a receptor solution at a selected
receptor concentration. In the case where multiple arrays are synthesized
on a single substrate, the flow cell isolates the receptor solutions of
different concentrations on the different arrays.
The substrate is placed under a microscope which includes one or more
objectives 308. Light (about 488 nm) from a laser 310, which in some
embodiments is a model no. 2020-05 argon ion laser manufactured by
Spectraphysics, is focused on a pinhole 331 with a lens 333 and,
thereafter, is directed at the substrate by a dichroic mirror 318 which
passes greater than about 520 nm light but reflects 488 n | | |
