Note: Descriptions are shown in the official language in which they were submitted.
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MICROARRAYS OF POLYPEPTIDES
FIELD OF THE INVENTION
This invention relates to methods and apparatus for fabricating microarrays of
biological samples, and the uses thereof.
BACKGROUND OF THE INVENTION
Life and development of all organisms are determined by molecular
interactions, e.g.
between DNA and proteins, proteins and proteins, or proteins and small
molecules. Among
these, protein-protein interactions play an especially important role, for
example with the
interactions between antibodies and antigens, receptors and peptide- or
protein-hormones,
enzymes and substrates or inhibitors. Many of the best-selling drugs either
act by targeting
proteins or are proteins. In addition, many molecular markers of disease,
which are the
basis of diagnostics, are proteins.
The development of techniques and reagents for high throughput protein
analysis
has been of great interest. In particular, the increasing knowledge of DNA
sequence in
organisms of interest has spurred interest in protein expression analysis.
There is now a
rapidly growing awareness of just how important proteomics is to understand
and organize
the human genome. Information about the complement of proteins present in a
cell is a key
to accelerate the discovery of medically important proteins and the genes from
which they
derive.
Genomics establishes the relationship between gene activity and particular
diseases.
However most disease processes are manifested not at the level of genes, but
at the protein
level. There is often a poor correlation between the level of activity of
different genes and
the relative abundance of the corresponding proteins. Also a protein and its
post-
translational modifications are not directly encoded for by the same gene,
therefore the
complete structure of individual proteins cannot be determined by reference to
the gene
alone.
Assays directed towards protein binding can be used for the quantitation of
protein
expression; the determination of specific interactions; to determine the
presence of ligands
for a protein, and the like. Methods of quantitating proteins in a sample by
determining
binding to a cognate antibody are known in the art.
For example, solid-phase radioimmunoassay (RIA) of antigens or antibodies in a
serum sample are well known. Catt et al. have reported such techniques on the
surtace of
plastic tubes (U.S. Pat. No. 3,646,346) and plastic discs (J. Lab. & Clin.
Med., 70: 820
(1967). In such techniques, an excess of specific antibody is first adsorbed
to a support
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surface. Then, the sample to be assayed is immunologically reacted with such
surface in a
sandwich or competitive binding technique. In the competitive binding
technique, illustrated
in U.S. Pat. No. 3,555,143, the concentration of antigen to be determined and
a known
quantity of radioactively tagged antigen are immunologically reacted with the
antibody-
adsorbed surface. The labeled antigen bound to the antibody on the surface is
then
quantitated to determine indirectly the total quantity of antigen in the
original sample. In the
sandwich technique, serum containing an unknown concentration of antigen is
immunologically reacted with the antibody-containing surface. Then in a
following step, the
bound antigen is incubated with labeled antibody and the amount of
immunologically bound,
labeled antibody is subsequently measured.
The development of high-throughput, parallel systems for protein analysis are
of
great interest, particularly where the analysis can use small amounts of
material for analysis.
Preferably such systems provide for the use of complex molecules with high
binding affinity
for their ligands, such as antibodies, protein receptors, and the like.
Literature:
Publications of interest include: Abouzied, et al., Journal of AOAC
International
77(2):495-500 (1994). Bohlander, et al., Genomics 13:1322-1324 (1992).
Drmanac, et al.,
Science 260:1649-1652 (1993). Fodor, et al., Science 251:767-773 (1991).
Khrapko, et al.,
DNA Sequence 1:375-388 (1991). Kuriyama, et al., An Isfet Biosensor, Applied
Biosensors
(Donald Wise, Ed.), Butterworths, pp. 93-114 (1989). Lehrach, et al.,
Hybridization
Fingerprinting in Genome Mapping And Sequencing, Genome Analysis, Vol 1
(Davies and
Tilgham, Eds.), Cold Spring Harbor Press, pp. 39-81 (1990). Maniatis, et al.,
Molecular
Cloning, A Laboratory Manual, Cold Spring Harbor Press (1989). Nelson, et al.,
Nature
Genetics 4:11-18 (1993). Pirrung, et al., U.S. Pat. No. 5,143,854 (1992).
Riles, et al.,
Genetics 134:81-150 (1993). Schena, M. et al., Proc. Nat. Acad. Sci. USA
89:3894-3898
(1992). Southern, et al., Genomics 13:1008-1017 (1992).
SUMMARY OF THE INVENTION
Methods are provided for forming a microarray of analyte-assay regions on a
solid
support, where each region in the array has a known amount of a selected,
analyte-specific
reagent. The method involves first loading a solution of a selected analyte-
specific reagent
in a reagent-dispensing device having an elongate capillary channel (i) formed
by spaced-
apart, coextensive elongate members, (ii) adapted to hold a quantity of the
reagent solution
and (iii) having a tip region at which aqueous solution in the channel forms a
meniscus. The
channel is preferably formed by a pair of spaced-apart tapered elements. The
microan-ay
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compositions find use in the multiplexed detection and quantitation of
ligands, e.g. antigens
or antibodies, in a miniaturized format.
In another aspect, the invention includes a substrate with a surtace having a
microarray of at least 103 distinct polynucleotide or polypeptide biopolymers
in a surtace
area of less than about 1 cm2. Each distinct biopolymer is disposed at a
separate, defined
position in said array, has a length of at least 50 subunits, and is present
in a defined
amount between about 0.1 femtomoles and 100 nanomoles.
The substrate may be used for detecting binding of ligands to a plurality of
different-
sequence, immobilized biopolymers. The substrate includes, in one aspect, a
glass support,
a coating of a polycationic polymer, such as polylysine, on said surtace of
the support, and
an array of distinct biopolymers electrostatically bound non-covalently to
said coating, where
each distinct biopolymer is disposed at a separate, defined position in a
surface array.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a side view of a reagent-dispensing device having a open-capillary
dispensing head constructed for use in one embodiment of the invention;
FIGS. 2A-2C illustrate steps in the delivery of a fixed-volume bead on a
hydrophobic
surface employing the dispensing head from FIG. 1, in accordance with one
embodiment of
the method of the invention;
FIG. 3 shows a portion of a two-dimensional array of analyte-assay regions
constructed according to the method of the invention;
FIG. 4 is a planar view showing components of an automated apparatus for
forming
arrays in accordance with the invention.
FIG. 5 shows the concentration profiles in a microarray of 110 antigens.
FIG. 6 shows the detection of protein as a ratio of the signal from two
fluorochromes,
against the dilution of the protein sample.
FIG. 7 shows graphs of the protein quantitation after dilution into serum.
FIG. 8 depicts the combinatorial detection of multiple antibodies.
DETAILED DESCRIPTION OF THE INVENTION
Methods and compositions are provided for forming a microarray of polypeptide
regions on a solid support, where each region in the array has a known amount
of a selected
polypeptide. A robotic printer is used to deposit microdrops of protein
solutions onto a
derivatized planar surtace substrate, where the derivatized surtace binds the
polypeptide,
e.g. poly-lysine, and the like. The substrate with a surface having a microan-
ay is spotted at
a high density, usually of at least 103 distinct polypeptide in a surtace area
of less than about
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1 cm2. Each distinct polypeptide is present in a defined amount between about
0.1
femtomoles and 100 nanomoles. Any polypeptide can be used, although generally
the
polypeptide will be at least about 50 amino acids in length.
The microarrays are widely used in quantitative and analytical methods for the
detection and quantitation of proteins, or compounds that interact with
proteins, such as
polynucleotides, hormones, vitamins and other co-factors, etc. Typically a
sample
comprising ligands that are suspected of binding to a polypeptide immobilized
on the
microarray are added to the microarray under conditions that allow specific
binding between
the polypeptide and the ligand. The unbound sample is washed from the
microarray, and
the bound ligand is detected by any suitable method, e.g. through the use of
detectable
labels present on the ligand, or provided in a second, detecting step. Sample
consumption
is much lower than traditional immunoassays due to the highly parallel and
miniaturized
format of the present invention. The quantitative measurement of many
components in
parallel allows diagnosis and recognition of physiological and phenotypic
characteristics of a
sample to be based on a multidimensional pattern of expression, rather than
simply a few
parameters.
In one embodiment of the invention, comparative fluorescence is used to
monitor the
presence of bound ligands to the microarray. The use of comparative
fluorescence
measurements allows greater precision across a wide range of ligand
concentrations and
binding affinities, as compared to methods that measure the absolute amount of
bound
ligand.
In one embodiment of the invention, the biopolymers are polypeptides, e.g.
antigens,
antibodies, receptors, etc., that have functional binding properties imparted
by the three-
dimensional structure of the polypeptide, which structure is frequently
dependent on contacts
made between non-contiguous amino acid residues, such as disulphide bonds
between
cysteine residues, hydrophobic pockets, and the like. Such binding properties
include the
specific binding between a protein receptor and one or more of its naturally
occurring
ligands, for example cytokines and cytokine receptors, hormones and hormone
receptors,
chemokines and chemokine receptors, etc., including a range of protein and
polypeptide
molecules that provide for specific interactions within a biological system.
DNA binding
proteins, e.g. nuclear hormone receptors; transcription factors, etc. may be
provided on a
microarray, where the proteins retain the ability to specifically define their
cognate DNA
motif. Microarrays that maintain binding properties of antigen specific
immunological
receptors are of particular interest, which receptors include antibodies, T
cell antigen
receptors, and major histocompatibility complex proteins.
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These and other objects and features of the invention will become more fully
apparent when the following detailed description of the invention is read in
conjunction with
the accompanying figures.
Definitions
Unless indicated othenrvise, the terms defined below have the following
meanings:
"Ligano"' refers to one member of a ligand/anti-ligand binding pair. The
ligand may
be, for example, one of the nucleic acid strands in a complementary,
hybridized nucleic acid
duplex binding pair; an effector molecule in an effector/receptor binding
pair; or an antigen in
1o an antigen/antibody or antigen/antibody fragment binding pair.
"Anti-ligano"' refers to the opposite member of a ligand/anti-ligand binding
pair. The
anti-ligand may be the other of the nucleic acid strands in a complementary,
hybridized
nucleic acid duplex binding pair; the receptor molecule in an
effector/receptor binding pair; or
an antibody or antibody fragment molecule in antigen/antibody or
antigen/antibody fragment
binding pair, respectively.
"Analyte" or "analyte molecule" refers to a molecule, typically a
macromolecule, such
as a polynucleotide or polypeptide, whose presence, amount, and/or identity
are to be
determined. The analyte is one member of a ligand/anti-ligand pair.
"Analyte-specific assay reagent' refers to a molecule effective to bind
specifically to
an analyte molecule. The reagent is the opposite member of a ligand/anti-
ligand binding pair.
An "array of regions on a solid support" is a linear or two-dimensional array
of
preferably discrete regions, each having a finite area, formed on the surface
of a solid
support.
A "microarray' is an array of regions having a density of discrete regions of
at least
about 100/cmz, and preferably at least about 1000/cm2. The regions in a
microarray have
typical dimensions, e.g., diametErs, in the range of befinreen about 10-250
p.m, and are
separated from other regions in the array by about the same distance.
A support surface is "hydrophobic" if a aqueous-medium droplet applied to the
surface does not spread out substantially beyond the area size of the applied
droplet. That
is, the surtace acts to prevent spreading of the droplet applied to the
surface by hydrophobic
interaction with the droplet.
A "meniscus" means a concave or convex surface that forms on the bottom of a
liquid in a channel as a result of the surface tension of the liquid.
"Distinct biopolymers", as applied to the biopolymers forming a microarray,
means an
array member which is distinct from other array members on the basis of a
different
biopolymer sequence, and/or different concentrations of the same or distinct
biopolymers,
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and/or different mixtures of distinct or different-concentration biopolymers.
Thus an array of
"distinct polynucleotides" means an array containing, as its members, (i)
distinct
polynucleotides, which may have a defined amount in each member, (ii)
different, graded
concentrations of given-sequence polynucleotides, and/or (iii) different-
composition mixtures
of two or more distinct polynucleotides.
"Cell type" means a cell from a given source, e.g., a tissue, or organ, or a
cell in a
given state of differentiation, or a cell associated with a given pathology or
genetic makeup.
Method of Microarray Formation
This section describes a method of forming a microarray of analyte-assay
regions on
a solid support or substrate, where each region in the array has a known
amount of a
selected, analyte-specific reagent.
FIG. 1 illustrates, in a partially schematic view, a reagent-dispensing device
10 useful
in practicing the method. The device generally includes a reagent dispenser 12
having an
elongate open capillary channel 14 adapted to hold a quantity of the reagent
solution, such
as indicated at 16, as will be described below. The capillary channel is
formed by a pair of
spaced-apart, coextensive, elongate members 12a, 12b which are tapered toward
one
another and converge at a tip or tip region 18 at the lower end of the
channel. More
generally, the open channel is formed by at least two elongate, spaced-apart
members
adapted to hold a quantity of reagent solutions and having a tip region at
which aqueous
solution in the channel forms a meniscus, such as the concave meniscus
illustrated at 20 in
FIG. 2A. The advantages of the open channel construction of the dispenser are
discussed
below.
With continued reference to FIG. 1, the dispenser device also includes
structure for
moving the dispenser rapidly toward and away from a support surface, for
effecting
deposition of a known amount of solution in the dispenser on a support, as
will be described
below with reference to FIGS. 2A-2C. In the embodiment shown, this structure
includes a
solenoid 22 which is activatable to draw a solenoid piston 24 rapidly
downwardly, then
release the piston, e.g., under spring bias, to a normal, raised position, as
shown. The
dispenser is carried on the piston by a connecting member 26, as shown. The
just-
described moving structure is also referred to herein as dispensing means for
moving the
dispenser into engagement with a solid support, for dispensing a known volume
of fluid on
the support.
The dispensing device just described is carried on an arm 28 that may be moved
either linearly or in an x-y plane to position the dispenser at a selected
deposition position,
as will be described.
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FIGS. 2A-2C illustrate the method of depositing a known amount of reagent
solution
in the just-described dispenser on the surface of a solid support, such as the
support
indicated at 30. The support is a polymer, glass, or other solid-material
support having a
surtace indicated at 31.
In one general embodiment, the surface is a relatively hydrophilic, i.e.,
wettable
surface, such as a surface having native, bound or covalently attached charged
groups.
One such surface described below is a glass surface having an absorbed layer
of a
polycationic polymer, such as poly-I-lysine.
In another embodiment, the surface has or is formed to have a relatively
hydrophobic
character, i.e., one that causes aqueous medium deposited on the surface to
bead. A
variety of known hydrophobic polymers, such as polystyrene, polypropylene, or
polyethylene
have desired hydrophobic properties, as do glass and a variety of lubricant or
other
hydrophobic films that may be applied to the support surface.
Initially, the dispenser is loaded with a selected analyte-specific reagent
solution,
such as by dipping the dispenser tip, after washing, into a solution of the
reagent, and
allowing filling by capillary flow into the dispenser channel. The dispenser
is now moved to a
selected position with respect to a support surtace, placing the dispenser tip
directly above
the support-surface position at which the reagent is to be deposited. This
movement takes
place with the dispenser tip in its raised position, as seen in FIG. 2A, where
the tip is
typically at least several 1-5 mm above the surface of the substrate.
Wth the dispenser so positioned, solenoid 22 is now activated to cause the
dispenser tip to move rapidly toward and away from the substrate surface,
making
momentary contact with the surface, in effect, tapping the tip of the
dispenser against the
support surtace. The tapping movement of the tip against the surface acts to
break the
liquid meniscus in the tip channel, bringing the liquid in the tip into
contact with the support
surface. This, in tum, produces a flowing of the liquid into the capillary
space between the
tip and the surface, acting to draw liquid out of the dispenser channel, as
seen in FIG. 2B.
FIG. 2C shows flow of fluid from the tip onto the support surface, which in
this case is
a hydrophobic surface. The figure illustrates that liquid continues to flow
from the dispenser
onto the support surtace until it forms a liquid bead 32. At a given bead
size, i.e., volume,
the tendency of liquid to flow onto the surtace will be balanced by the
hydrophobic surface
interaction of the bead with the support surface, which acts to limit the
total bead area on the
surface, and by the surface tension of the droplet, which tends toward a given
bead
curvature. At this point, a given bead volume will have formed, and continued
contact of the
dispenser tip with the bead, as the dispenser tip is being withdrawn, will
have little or no
effect on bead volume.
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For liquid-dispensing on a more hydrophilic surtace, the liquid will have less
of a
tendency to bead, and the dispensed volume will be more sensitive to the total
dwell time of
the dispenser tip in the immediate vicinity of the support surface, e.g., the
positions
illustrated in FIGS. 2B and 2C.
The desired deposition volume, i.e., bead volume, formed by this method is
preferably in the range 2 pl (picoliters) to 2 nl (nanoliters), although
volumes as high as 100
nl or more may be dispensed. It will be appreciated that the selected
dispensed volume will
depend on (i) the "footprint" of the dispenser tip, i.e., the size of the area
spanned by the tip,
(ii) the hydrophobicity of the support surface, and (iii) the time of contact
with and rate of
withdrawal of the tip from the support surtace. In addition, bead size may be
reduced by
increasing the viscosity of the medium, effectively reducing the flow time of
liquid from the
dispenser onto the support surface. The drop size may be further constrained
by depositing
the drop in a hydrophilic region surrounded by a hydrophobic grid pattern on
the support
surface.
In a typical embodiment, the dispenser tip is tapped rapidly against the
support
surface, with a total residence time in contact with the support of less than
about 1 msec,
and a rate of upward travel from the surface of about 10 cm/sec.
Assuming that the bead that forms on contact with the surface is a
hemispherical
bead, with a diameter approximately equal to the width of the dispenser tip,
as shown in FIG.
2C, the volume of the bead formed in relation to dispenser tip width (d) is
given in Table 1
below. As seen, the volume of the bead ranges between 2 pl to 2 nl as the
width size is
increased from about 20 to 200 ~,m.
TABLE 1
d Volume (nl)
20 p,m 2 x 10'
50 ~m 3.1 x 10''
100 p,m 2.5 x 10-'
200 p 2
At a given tip size, bead volume can be reduced in a controlled fashion by
increasing
surface hydrophobicity, reducing time of contact of the tip with the surface,
increasing rate of
movement of the tip away from the surtace, and/or increasing the viscosity of
the medium.
Once these parameters are fixed, a selected deposition volume in the desired
pl to nl range
can be achieved in a repeatable fashion.
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After depositing a bead at one selected location on a support, the tip is
typically
moved to a corresponding position on a second support, a droplet is deposited
at that
position, and this process is repeated until a liquid droplet of the reagent
has been deposited
at a selected position on each of a plurality of supports.
The tip is then washed to remove the reagent liquid, filled with another
reagent liquid
and this reagent is now deposited at each another array position on each of
the supports. In
one embodiment, the tip is washed and refilled by the steps of (i) dipping the
capillary
channel of the device in a wash solution, (ii) removing wash solution drawn
into the capillary
channel, and (iii) dipping the capillary channel into the new reagent
solution.
From the foregoing, it will be appreciated that the tweezers-like, open-
capillary
dispenser tip provides the advantages that (i) the open channel of the tip
facilitates rapid,
efficient washing and drying before reloading the tip with a new reagent, (ii)
passive capillary
action can load the sample directly from a standard microwell plate while
retaining sufficient
sample in the open capillary reservoir for the printing of numerous arrays,
(iii) open
capillaries are less prone to clogging than closed capillaries, and (iv) open
capillaries do not
require a pertectly faced bottom surface for fluid delivery.
A portion of a microarray 36 formed on the surtace 38 of a solid support 40 in
accordance with the method just described is shown in FIG. 3. The array is
formed of a
plurality of analyte-specific reagent regions, such as regions 42, where each
region may
include a different analyte-specific reagent. As indicated above, the diameter
of each region
is preferably between about 20-200 p.m. The spacing between each region and
its closest
(non-diagonal) neighbor, measured from center-to-center (indicated at 44), is
preferably in
the range of about 20-400 pm. Thus, for example, an array having a center-to-
center
spacing of about 250 pm contains about 40 regions/cm or 1,600 regions/cm2.
After
formation of the array, the support is treated to evaporate the liquid of the
droplet forming
each region, to leave a desired array of dried, relatively flat regions. This
drying may be
done by heating or under vacuum.
In some cases, it is desired to first rehydrate the droplets containing the
analyte
reagents to allow for more time for adsorption to the solid support. It is
also possible to spot
out the analyte reagents in a humid environment so that droplets do not dry
until the arraying
operation is complete.
Automated Apparatus for Forming Arrays
In another aspect, the invention includes an automated apparatus for forming
an
array of analyte-assay regions on a solid support, where each region in the
array has a
known amount of a selected, analyte-specific reagent.
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The apparatus is shown in planar, and partially schematic view in FIG. 4. A
dispenser device 72 in the apparatus has the basic construction described
above with
respect to FIG. 1, and includes a dispenser 74 having an open-capillary
channel terminating
at a tip, substantially as shown in FIGS. 1 and 2A-2C.
The dispenser is mounted in the device for movement toward and away from a
dispensing position at which the tip of the dispenser taps a support surface,
to dispense a
selected volume of reagent solution, as described above. This movement is
effected by a
solenoid 76 as described above. Solenoid 76 is under the control of a control
unit 77 whose
operation will be described below. The solenoid is also referred to herein as
dispensing
means for moving the device into tapping engagement with a support, when the
device is
positioned at a defined array position with respect to that support.
The dispenser device is carried on an arm 74 which is threadedly mounted on a
worm screw 80 driven (rotated) in a desired direction by a stepper motor 82
also under the
control of unit 77. At its left end in the figure screw 80 is carried in a
sleeve 84 for rotation
about the screw axis. At its other end, the screw is mounted to the drive
shaft of the stepper
motor, which in tum is carried on a sleeve 86. The dispenser device, worm
screw, the two
sleeves mounting the worm screw, and the stepper motor used in moving the
device in the
"x" (horizontal) direction in the figure form what is referred to here
collectively as a
displacement assembly 86.
The displacement assembly is constructed to produce precise, micro-range
movement in the direction of the screw, i.e., along an x axis in the figure.
In one mode, the
assembly functions to move the dispenser in x-axis increments having a
selected distance in
the range 5-25 pm. In another mode, the dispenser unit may be moved in precise
x-axis
increments of several microns or more, for positioning the dispenser at
associated positions
on adjacent supports, as will be described below.
The displacement assembly, in tum, is mounted for movement in the "y"
(vertical)
axis of the figure, for positioning the dispenser at a selected y axis
position. The structure
mounting the assembly includes a fixed rod 88 mounted rigidly between a pair
of frame bars
90, 92, and a worm screw 94 mounted for rotation between a pair of frame bars
96, 98. The
3o worm screw is driven (rotated) by a stepper motor 100 which operates under
the control of
unit 77. The motor is mounted on bar 96, as shown.
The structure just described, including worm screw 94 and motor 100, is
constructed
to produce precise, micro-range movement in the direction of the screw, i.e.,
along a y axis
in the figure. As above, the structure functions in one mode to move the
dispenser in y-axis
increments having a selected distance in the range 5-250 Vim, and in a second
mode, to
CA 02366123 2001-09-24
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move the dispenser in precise y-axis increments of several microns (wm) or
more, for
positioning the dispenser at associated positions on adjacent supports.
The displacement assembly and structure for moving this assembly in the y axis
are
referred to herein collectively as positioning means for positioning the
dispensing device at a
selected array position with respect to a support.
A holder 102 in the apparatus functions to hold a plurality of supports, such
as
supports 104 on which the ,microarrays of reagent regions are to be formed by
the
apparatus. The holder provides a number of recessed slots, such as slot 106,
which receive
the supports, and position them at precise selected positions with respect to
the frame bars
on which the dispenser moving means is mounted.
As noted above, the control unit in the device functions to actuate the two
stepper
motors and dispenser solenoid in a sequence designed for automated operation
of the
apparatus in forming a selected microarray of reagent regions on each of a
plurality of
supports.
The control unit is constructed, according to conventional microprocessor
control
principles, to provide appropriate signals to each of the solenoid and each of
the stepper
motors, in a given timed sequence and for appropriate signaling time. The
construction of
the unit, and the settings that are selected by the user to achieve a desired
array pattern, will
be understood from the following description of a typical apparatus operation.
Initially, one or more supports are placed in one or more slots in the holder.
The
dispenser is then moved to a position directly above a well (not shown)
containing a solution
of the first reagent to be dispensed on the support(s). The dispenser solenoid
is actuated
now to lower the dispenser tip into this well, causing the capillary channel
in the dispenser to
fill. Motors 82, 100 are now actuated to position the dispenser at a selected
array position at
the first of the supports. Solenoid actuation of the dispenser is then
effective to dispense a
selected-volume droplet of that reagent at this location. As noted above, this
operation is
effective to dispense a selected volume preferably between 2 pl and 2 nl of
the reagent
solution.
The dispenser is now moved to the corresponding position at an adjacent
support
and a similar volume of the solution is dispensed at this position. The
process is repeated
until the reagent has been dispensed at this preselected corresponding
position on each of
the supports.
Where it is desired to dispense a single reagent at more than two array
positions on
a support, the dispenser may be moved to different array positions at each
support, before
moving the dispenser to a new support, or solution can be dispensed at
individual positions
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on each support, at one selected position, then the cycle repeated for each
new array
position.
To dispense the next reagent, the dispenser is positioned over a wash solution
(not
shown), and the dispenser tip is dipped in and out of this solution until the
reagent solution
has been substantially washed from the tip. Solution can be removed from the
tip, after
each dipping, by vacuum, compressed air spray, sponge, or the like.
The dispenser tip is now dipped in a second reagent well, and the filled tip
is moved
to a second selected array position in the first support. The process of
dispensing reagent at
each of the corresponding second-array positions is then carried out as above.
This process
is repeated until an entire microarray of reagent solutions on each of the
supports has been
formed.
Microarrav Substrate
This section describes embodiments of a substrate having a microarray of
biological
polymers carried on the substrate surface, in particular a microarray of
distinct polypeptides
bound on a glass slide coated with a polycationic polymer is described.
A substrate is formed according to another aspect of the invention, and
intended for
use in detecting binding of labeled ligands to one or more of a plurality
distinct biopolymers.
In one embodiment, the substrate includes a glass substrate having formed on
its surface, a
coating of a polycationic polymer, preferably a cationic polypeptide, such as
poly-lysine or
poly-arginine. Formed on the polycationic coating is a microarray of distinct
biopolymers,
each localized at known selected array regions, such as regions.
The slide may be coated by placing a uniform-thickness film of a polycationic
polymer, e.g., poly-I-lysine, on the surface of a slide and drying the film to
form a dried
coating. The amount of polycationic polymer added is sufficient to form at
least a monolayer
of polymers on the glass surface. The polymer film is bound to surface via
electrostatic
binding between negative silyl-OH groups on the surface and charged amine
groups in the
polymers. Poly-1-lysine coated glass slides may be obtained commercially,
e.g., from Sigma
Chemical Co. (St. Louis, Mo.).
A suitable microarray substrate is also made through chemical derivatization
of glass.
Silane compounds with appropriate leaving groups on a terminal Si will
covalently bond to
glass surfaces. A derivatization molecule can be designed to confer the
desired chemistry to
the surface of the glass substrate. An example of such a bifunctional reagent
is amino-
propyl-tri(ethoxy)silane, which reacts with glass surfaces at the
tri(ethoxy)silane portion of
the molecule while leaving the amino portion of the molecule free. Surfaces
having terminal
amino groups are suitable for adsorption of biopolymers in the same manner as
poly-lysine
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WO 00/63701 PCT/US00/10171
coated slides. The identity of the terminal surface group can be modified by
further chemical
reaction. For example, reaction of the terminal amine in the above example
with
glutaraldehyde results in a terminal aldehyde group. Further layers of
modification may be
applied to achieve the desired reactivity before spotting the microarray, such
as by
application of a Protein A or Protein G solution to the silynated glass.
Additional surfaces
that bind polypeptides are nitrocellulose-coated glass slides, available
commercially from
Schleicher and Schuell, and protein-binding plastics such as polystyrene.
The spotted polypeptides may be attached by either adsorption or covalent
bonding.
Adsorption occurs through electrostatic, hydrophobic, Van der Waals, or
hydrogen-bonding
interactions between the spotted polypeptide and the array substrate. Simple
application of
the polypeptide solution to the surface in an aqueous environment is
sufficient to adsorb the
polypeptide. Covalent attachment is achieved by reaction of functional groups
on the
polypeptide with a chemically activated surface. For example, if the surface
has been
activated with a highly reactive electrophilic group such as an aldehyde or
succinimide
group, unmodified polypeptides react at amine groups, as at lysine residues or
the terminal
amine, to form a covalent bond.
To form the microarray, defined volumes of distinct biopolymers are deposited
on the
polymer-coated slide, as described in Section II. According to an important
feature of the
substrate, the deposited biopolymers remain bound to the coated slide surtace
non-
covalently when an aqueous sample is applied to the substrate under conditions
that allow
binding of labeled ligands in the sample to cognate binding partners in the
substrate array.
In a preferred embodiment, each microarray contains at least 103 distinct
polynucleotide or polypeptide biopolymers per surface area of less than about
1 cmz. In one
embodiment, the microarray contains 400 regions in an area of about 16 mm2, or
2.5 x 103
regions/cm2. Also in a preferred embodiment, the biopolymers in each
microarray region are
present in a defined amount between about 0.1 femtomoles and 100 nanomoles (in
the case
of polynucleotides). As above, the ability to form high-density arrays of this
type, where
each region is formed of a well-defined amount of deposited material, can be
achieved in
accordance with the microarray-forming method described in Section II.
Also in a preferred embodiment, the biopolymers have lengths of at least about
50
units, e.g. amino acids, nucleotides, etc., i.e., substantially longer than
polymers which can
be formed in high-density arrays by various in situ synthesis schemes.
The polypeptide biopolymers may comprise polypeptides from any source.
Polypeptides of interest include those isolated from cells or other biological
sources,
synthesized polypeptides, including synthesized peptides and peptides selected
from
combinatorial libraries, polypeptides synthesized from recombinant nucleic
acids, and the
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like. In one embodiment, the polypeptides are isolated from phage display
libraries or clones
(see Huse et al. (1989) Science. 1989 246(4935):1275-81; Winter et al. (1994)
Annu Rev
Immunol. 12:433-55; Clackson et al. (1991) Nature 352(6336):624-8). Usually
the
polypeptides on each discrete region of the array will be substantially pure.
Uses of the Microarrays
Arrays of whole cells, peptides, enzymes, antibodies, antigens, receptors,
ligands,
phospholipids, polymers, drug congener preparations or chemical substances can
be
fabricated by the means described in this invention for large scale screening
assays in
medical diagnostics, drug discovery, molecular biology, immunology and
toxicology.
Microarrays of immobilized polypeptides prepared in accordance with the
invention
can be used for large scale binding assays in numerous diagnostic and
screening
applications. The multiplexed measurement of quantitative variation in levels
of large
numbers of proteins allows the recognition of patterns defined by several to
many different
proteins. One can simultaneously assess many physiological parameters and
disease-
specific patterns.
One embodiment of the invention involves the separation, identification and
characterization of proteins present in a biological sample. For example, by
comparison of
disease and control samples, it is possible to identify "disease specific
proteins". These
proteins may be used as targets for drug development or as molecular markers
of disease.
Polypeptide arrays are used to monitor the expression levels of proteins in a
sample
where such samples may include biopsy of a tissue of interest, cultured cells,
microbial cell
populations, biological fluids, including blood, plasma, lymph, synovial
fluid, cerebrospinal
fluid, cell lysates, culture supernatants, amniotic fluid, etc., and
derivatives thereof. Of
particular interest are clinical samples of biological fluids, including blood
and derivatives
thereof, cerebrospinal fluid, urine, saliva, lymph, synovial fluids, etc. Such
measurements
may be quantitative, semi-quantitative, or qualitative. Where the assay is to
be quantitative
or semi-quantitative, it will preferably comprise a competition-type format,
for example
between labeled and unlabeled samples, or between samples that are
differentially labeled.
Assays to detect the presence of ligands to the immobilized polypeptides may
be
performed as follows, although the methods need not be limited to those set
forth herein.
Samples, fractions or aliquots thereof are added to a microarray comprising
bound
polypeptide. Samples may comprise a wide variety of biological fluids or
extracts as
described above. Preferably, a series of standards, containing known
concentrations of
control ligand(s) is assayed in parallel with the samples or aliquots thereof
to serve as
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controls. The incubation time should be sufficient for ligand molecules to
bind the
polypeptides. Generally, from about 0.1 to 3 hr is sufficient, usually 1 hr
sufficing.
After incubation, the insoluble support is generally washed of non-bound
components. Generally, a dilute non-ionic detergent medium at an appropriate
pH, generally
7-8, is used as a wash medium. From one to six washes may be employed, with
sufficient
volume to thoroughly wash non-specifically bound proteins present in the
sample.
In order to detect the presence of bound ligands, a variety of methods may be
used.
These fall into three general groups. The ligand itself may be labeled with a
detectable
label, and the amount of bound label directly measured. Alternatively, the
labeled sample
may be mixed with a differentially labeled, or unlabeled sample in a
competition assay. In
yet another embodiment, the sample itself is not labeled, but a second stage
labeled reagent
is added in order to quantitate the amount of ligand present.
Examples of labels that permit direct measurement of ligand binding include
radiolabels, such as 3H or 'Z51, fluorescers, dyes, beads, chemilumninescers,
colloidal
particles, and the like. Suitable fluorescent dyes are known in the art,
including fluorescein
isothiocyanate (FITC); rhodamine and rhodamine derivatives; Texas Red;
phycoerythrin;
allophycocyanin; 6-carboxyfluorescein (6-FAM); 2',T-dimethoxy-4',5'-dichloro-6-
carboxyfluorescein (JOE); 6-carboxy-X-rhodamine (ROX); 6-carboxy-2',4',7',4,7-
hexachlorofluorescein (HEX); 5-carboxyfluorescein (5-FAM); N,N,N',N'-
tetramethyl-6-
carboxyrhodamine (TAMRA); sulfonated rhodamine; Cy3; CyS; etc. Preferably the
compound to be labeled is combined with an activated dye that reacts with a
group present
on the ligand, e.g. amine groups, thiol groups, aldehyde groups, etc.
Particularly where a second stage detection is performed, for example by the
addition
of labeled antibodies that recognize the ligand, the label can be a covalently
bound enzyme
capable of providing a detectable product signal after addition of suitable
substrate.
Examples of suitable enzymes for use in conjugates include horseradish
peroxidase,
alkaline phosphatase, malate dehydrogenase and the like. Where not
commercially
available, such antibody-enzyme conjugates are readily produced by techniques
known to
those skilled in the art. The second stage binding reagent may be any compound
that binds
the ligands with sufficient specificity such that it can be distinguished from
other components
present. In a preferred embodiment, second stage binding reagents are
antibodies specific
for the ligand, either monoclonal or polyclonal sera, e.g. mouse anti-human
antibodies, etc.
For an amplification of signal, the ligand may be labeled with an agent such
as biotin,
digoxigenin, etc., where the second stage reagent will comprise avidin,
streptavidin, anti
digoxigenin antibodies, etc. as appropriate for the label.
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Microan-ays can be scanned to detect binding of the ligands, e.g. by using a
scanning
laser microscope, by fluorimetry, a modified ELISA plate reader, etc. For
example, a
scanning laser microscope may perform a separate scan, using the appropriate
excitation
line, for each of the fluorophores used. The digital images generated from the
scan are then
combined for subsequent analysis. For any particular array element, the ratio
of the
fluorescent signal with one label is compared to the fluorescent signal from
the other label
DNA, and the relative abundance determined.
The microarrays and methods of detecting ligands may be used for a number of
screening, investigative and diagnostic assays. In one application, an array
of antibodies is
bound to total protein from an organism to monitor protein expression for
research or
diagnostic purposes. Labeling total protein from a normal cell with one color
fluorophore and
total protein from a diseased cell with another color fluorophore and
simultaneously binding
the two samples to the same array allows for differential protein expression
to be measured
as the ratio of the two fluorophore intensities. This two-color experiment can
be used to
monitor expression in different tissue types, disease states, response to
drugs, or response
to environmental factors.
In screening assays, for example to determine whether a protein or proteins
are
implicated in a disease pathway or are correlated with a disease-specific
phenotype,
measurements may be made from cultured cells. Such cells may be experimentally
manipulated by the addition of pharmacologically active agents that act on a
target or
pathway of interest. This application is important for elucidation of
biological function or
discovery of therapeutic targets.
For many diagnostic and investigative purposes it is useful to measurement
levels of
ligands, e.g. protein ligands, in blood or serum. This application is
important for the
discovery and diagnosis of clinically useful markers that correlate with a
particular diagnosis
or prognosis. For example, by monitoring a range of antibody or T cell
receptor specificities
in parallel, one may determine the levels and kinetics of antibodies during
the course of
autoimmune disease, during infection, through graft rejection, etc.
Alternatively, novel
protein markers associated with a disease of interest may be developed through
comparisons of normal and diseased blood sample, or by comparing clinical
samples at
different stages of disease.
In another embodiment of the invention, the polypeptide arrays are used to
detect
post-translational modifications in proteins, which is important in studying
signaling pathways
and cellular regulation. Post-translational modifications can be detected
using antibodies
specific for a particular state of a protein, such as phosphorylated,
glycosylated,
famesylated, etc.
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The detection of these interactions between ligands and polypeptides can lead
to a
medical diagnosis. For example, the identity of a pathogenic microorganism can
be
established unambiguously by binding a sample of the unknown pathogen to an
array
containing many types of antibodies specific for known pathogenic antigens.
EXPERIMENTAL
It is to be understood that this invention is not limited to the particular
methodology,
protocols, cell lines, animal species or genera, and reagents described, as
such may vary. It
is also to be understood that the terminology used herein is for the purpose
of describing
particular embodiments only, and is not intended to limit the scope of the
present invention
which will be limited only by the appended claims.
As used herein the singular forms "a", "and", and "the" include plural
referents unless
the context clearly dictates otherwise. All technical and scientific terms
used herein have the
same meaning as commonly understood to one of ordinary skill in the art to
which this
invention belongs unless clearly indicated otherwise.
The following examples are put forth so as to provide those of ordinary skill
in the art
with a complete disclosure and description of how to make and use the subject
invention,
and are not intended to limit the scope of what is regarded as the invention.
Efforts have
been made to ensure accuracy with respect to the numbers used (e.g. amounts,
2o temperature, concentrations, etc.) but some experimental errors and
deviations should be
allowed for. Unless otherwise indicated, parts are parts by weight, molecular
weight is
average molecular weight, temperature is in degrees centigrade; and pressure
is at or near
atmospheric.
EXAMPLE 1
Antibodv and Antigen Microarrays
A set of antibody and antigen pairs with which highly controlled experiments
could be
performed was assembled, using 115 different ligand/anti-ligand pairs.
Methods
Array preparation: Antibody solutions were prepared at 100-200 p.g/mL in a
PBS/0.02% sodium azide buffer without glycerol. The antibodies were spotted
onto glass
slides treated with poly-I-lysine. The slides are derivitized by the following
procedure. Place
slides in slide racks, then racks in chambers. Prepare cleaning solution by
dissolving 70 g
NaOH in 280 mL ddH20, then adding 420 mL 95% ethanol. Total volume is 700 mL
(= 2 X
350 mL); stir until completely mixed. Pour solution into chambers with slides;
cover
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chambers with glass lids. Mix on orbital shaker for 2 hr. Quickly transfer
racks to fresh
chambers filled with ddH20. Rinse vigorously by plunging racks up and down.
Repeat
rinses 4X with fresh ddH20 each time. Prepare polylysine solution: 70 mL poly-
L-lysine +
70 mL tissue culture PBS in 560 mL water. Transfer slides to polylysine
solution and shake
15 min. - 1 hr. Transfer rack to fresh chambers filled with ddH20. Plunge up
and down 5X to
rinse. Centrifuge slides on microtiter plate carriers for 5 min. @ 500 rpm.
Dry slide racks in
45° C vacuum oven for 10 min.
The antibodies and antigens were prepared in a 384-well microtitre plate
containing
at least three wells each of 110 different antibodies or antigens. A 16-tip
print head on the
1o arrayer spotted the plate three times for a total of 1152 spots, with 9-12
duplicate spots per
antibody or antigen. The spacing befinreen spots was 375 micrometers. The
arrays were
sealed in an airtight container. They can be stored at 4° C for short
term storage (~1 month)
or frozen for longer storage.
The back sides of the slides were marked with a diamond scribe or indelible
marker
to delineate the location of the spots. To remove unbound protein, the arrays
were dunked
several times in PBS/3% non-fat milk/0.1 % Tween-20, and transferred
immediately to a
solution of PBS/3% non-fat milk, and let block overnight at 4° C. The
milk solution was first
centrifuged (10 minutes at 10000 x g) to remove particulate matter.
After blocking, the slides were dunked and thoroughly agitateed for one minute
each
in three consecutive room temperature washes of 0.2X PBS to remove the unbound
milk
protein. The arrays remained in the last wash until application of the protein
mixture.
Sample preparation: Protein solutions were prepared in a 0.1 carbonate or
phosphate buffer at pH 8.0, using up to ~15 ~g protein per array (when using
25 ~L per
array) at a concentration such that after mixing with the dye solution (see
below), the final
protein concentration is 0.2-2 mg/mL.
NHS-ester activated Cy-dyes (Amersham, catalog # PA23001 (Cy3) and PA25001
(Cy5)) were dissolved in a 0.1 M pH 8.0 carbonate buffer so that the final
concentration of
the dye after mixing with the protein solution was 100-300 p.M. (Each vial of
dye contains
200 nmols.) The dye and protein solutions were mixed, and allowed to react in
the dark at
room temperature for 45 minutes. The reference protein solution was mixed with
the Cy3
dye solution, and the test protein solution with the Cy5 dye solution. The
reactions were
quenched by adding enough 1 M pH 8 tris or glycine to each so that at least a
200-fold
excess of quencher:dye concentration was achieved.
Each mix was loaded into a microconcentrator having the appropriate molecular
weight cutoff. A 3000 D cutoff captures most proteins while still removing the
dye. If smaller
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proteins are not important, the 10000 D cutoff is faster. The mix was
centrifuged according
to the microconcentrator instructions. The 10000 D microcon typically requires
20 minutes
and the 3000 D microcon requires 80 minutes of centrifugation at 10000 x g and
room
temperature. After centrifuging, 3% milk blocker was added to either the Cy5
or Cy3-labeled
protein mixes. (The milk must first be centrifuged to remove particulate
matter: 10 minutes at
10000 x g.) Add 25 pL milk for each array to be generated from the protein
mix. PBS was
added to each microcon to a 500 8L volume, and centrifuged again. The
concentrated
samples were collected into a small volume (~5 p.L) of PBS to prevent drying
and
precipitation.
The Cy3-labeled reference protein solution was distributed to the appropriate
Cy5-
labeled test protein solutions, and PBS added to each mix to achieve a volume
of 25 p,L per
array. Particulate matter or precipitate was removed by 1) filtering with a
0.45 pm spin filter,
or 2) centrifuging 10 minutes at 14000 x g and pipetting out the supernatant.
Detection: Each array was removed individually from the PBS wash. Without
allowing the array to dry, 25 pL of the dye-labeled protein solution was
placed over the spots
(within the marked boundaries), with a cover slip placed over the protein
solution. The cover
slip has dimensions at least '/< inch longer than the dimensions of the array.
The arrays
were placed in a sealed humidification chamber with a layer of PBS under the
arrays, and
incubated at 4° C for approximately two hours. Each array was briefly
dunked in PBS to
remove the protein solution and the cover slip, and transfered immediately to
a slide rack in
a PBS/0.1 % Tween-20 solution. After all the arrays have been racked in the
PBS/Tween
solution, they were washed on an orbital shaker for ~20 minutes at room
temperature. The
arrays were transferred to a new rack (to minimize Tween carryover) in a PBS
solution and
rocked gently for 5-10 minutes, then transferred to wash solutions of PBS,
HZO, and H20 for
five minutes each of gentle agitation. The arrays were then spin-dried and
scanned.
Analysis: The fluorescence intensity at each spot reflects the level of
binding to that
particular protein. The relative concentration between proteins in
differentially dye-labeled
pools is determined by comparing the fluorescence intensities between the
color channels at
each spot. The following method is used to determine relative concentrations.
The location of each analyte spot on the array is outlined using "gridding"
software,
such as GenePix or ScanAlyze, which places a boundary around each spot on the
array.
The fluorescence signal from each spot is determined as the average or median
of
the pixel intensities within the boundary outlined using the gridding
software. Each color
channel is treated independently. Optionally statistical methods are used to
reject "outlier"
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pixels within the circle, i.e. pixels that have intensities significantly
outside the average pixel
intensity.
The background is subtracted from the signal. The background may be determined
as 1 ) the median or average of pixel intensities from the local area around
each spot, or as
2) the median or average or pixel intensities from within certain spots or
areas determined to
be non-binding background areas. Statistical methods may be used to reject
outlier pixels in
the background.
The relative binding at each spot between proteins in the separately labeled
pools is
equivalent to the ratio of fluorescence intensities in the two color channels.
In order for the
ratio to reflect the true relative concentrations, the background-subtracted
signal from one of
the color channels must be multiplied by a normalization factor. The
normalization factor
may be determined by selecting spots for which the true concentrations are
known and
calculating the factor that most accurately returns the true color ratio.
Alternatively, if no
control spots are used, one may assume that the average binding across every
spot on the
array is roughly equal for the two protein pools. A normalization factor is
then calculated that
gives an average color ratio of one for all the spots on the array.
Once all arrays have been normalized and color ratios have calculated, changes
in
protein concentration from array to array may be compared. Interpretation is
simplest if the
same reference pool is used for each experiment.
Results:
To test the specificity, quantitation, and limits of detection of the protein
array, six
mixes of antigens were made in which the concentration of each protein varied
uniquely
across the mixes. For example, one protein changed from high to low
concentration, another
from low to high, and another from low to high to low. The concentrations
varied three
orders of magnitude over the whole set. This set of six mixes was detected at
various
concentrations and in various levels of fetal calf serum (FCS) background. The
ability to
reconstruct the actual concentration changes from the data indicated the level
of
performance of the microarrays.
Microarrays were constructed containing 6 to 9 duplicate spots from each
antibody.
Figure 5 presents a series of these arrays generated from the set of six
unique protein mixes
(labeled with the red-fluorescing dye Cy5) compared against a reference mix
(labeled with
the green-fluorescing dye Cy3) containing an equal amount of each protein. For
each spot
on the array, the red/green ratio was calculated and then plotted as a
function of dilution.
Figure 6 presents plots of the log of the red-to-green ratio (R/G) versus
dilution for eight of
the antigens. The ideal slope, calculated as the log of the concentration
ratio of the proteins,
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is shown as a straight solid line decreasing from 1.5 to -1.5. The other lines
on the graph
represent duplicate spots on the array. The slopes of the experimental data
are very similar
to the ideal slope over the six concentrations tested, indicating that these
antibodies
detected the cognate antigens specifically and quantitatively. Deviations from
ideal slope
appear to occur systematically between the duplicate spots, suggesting that
the largest error
in quantitation occurred in pipetting or data reduction rather than in random
variability in the
system.
The detection of a specific protein is limited not only by concentration, but
also by the
concentration of background proteins. To determine how well specific proteins
can be
detected in high protein background, the set of unique protein mixes was
spiked into varying
amounts of FCS before dye labeling. FCS concentrations 10 times greater and
100 times
greater than the antigen mix concentration were used. Figure 7 shows the
effect of protein
background on quantitation for the proteins IgG and flag. Without the serum
background,
accurate quantitation is observed for both proteins over the entire
concentration range,
which was from 120 ng/mL to 120 pg/mL. At the 10x serum concentration, the
flag protein
still shows accurate quantitation, but IgG shows slight deviation from the
ideal slope at the
high and low limits. At the 100x serum concentration, both proteins exhibit
marked
deviations from the ideal slope. The partial concentrations (the antigen
concentration
divided by the total protein concentration) ranged from 4 x 10'5 to 4 x 10'$
for the 100x serum
trial. Thus the partial concentration detection limit is ~2 x 10'~ for flag
and ~2 x 10'' for IgG
using these antibodies. These partial concentrations are in a physiological
range for many
clinically interesting blood serum proteins. The results of this type of
analysis for each
antigen tested are presented in the table below. Antibodies were classified
according to the
presence of accurate quantitation over the entire range for all of the low
background trial and
at least part of the higher background trials (++), They were classified as
(+) if they showed
accurate quantitation for most of the low background trial. Many of the
antibodies showed
either no signal or non-specific signal.
In a second mode of detection, antigens were spotted onto the array to detect
labeled antibodies. Figure 7 presents an example of specific detection of
antibodies in four
unique mixes. A combinatorial labeling scheme was employed that enabled
identification of
specific antigen/antibody binding. An analysis similar to that described above
was carried
out to classify the binding specificit'~ of antigens on the microarray. The
results of that
analysis are presented in the table below along with the antibody array
results. According to
this analysis, the protein array works at least as well or better using
spotted antigens as
compared to spotted antibodies.
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Antibody Antigen
Antibody/antigen array array
++ ++
+ +
- -
Part.
conc.
limit
Anti-AIM-1 x 1.OOE-06 x
Anti-HCG x x
Anti-MAP4 x x
Anti-Pert x x
Anti-Flag (new) x x
Anti-Alpha HCG x 4.OOE-08 x
Anti-Fc. IgG x 1.OOE-07 x
Anti-Flag (old) x
Anti-Human IgG x
Anti-Mint2 x x
Anti-Sin x
Anti-SOD x x
Anti-ABR x 6.OOE-05 x
Anti-AKAP-KL x x
Anti-Dematin x 1.OOE-04 x
Anti-Dlg x x
Anti-DSIF x x
Anti-FI N 13 x x
Anti-H DAC3 x x
Anti-HIF-1 alpha x x
Anti-ICH-IL x x
Anti-IGF2R x x
Anti-Kanadaptin x x
Anti-La x x
Anti-LAIR-1 x x
Anti-LAP2 x x
Anti-MEKK3 x x
Anti-Mint1 x x
Anti-MST3 x x
Anti-p19 Skp1 x x
Anti-p38 gamma x x
Anti-Rab4 x x
Anti-TEF-1 x x
Anti-ZO-1 x x
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Anti-Tropomyosin x
Anti-Alkaline x x
phosphatase
Anti-cTnl x 4.OOE-04 x
Anti-DFF45 x x
Anti-Fibronectin x x
Anti-GOK x x
Anti-GS15 x x
Anti-Insulin x x
Anti-LAT x x
Anti-MAD-3 x
Anti-mGIuR1 x x
Anti-MST1 x x
Anti-Myoglobin x x
(ResGen)
Anti-Myoglobin x
(Sigma)
Anti-Neuroglycan x x
C
Anti-PSA 2F5 x x
Anti-PSA F5 x
Anti-Rad50 x x
Anti-RBC x x
Anti-Rim x x
Anti-ROCK-1 x x
Anti-SRPK1 x x
Anti-VLA-3alpha x x
Anti-Adaptin x x
alpha
Anti-Bax x x
Anti-Calretinin x x
Anti-c-Cbl x x
Anti-Clathrin x x
H
Anti-DEK x x
Anti-DGKO x x
Anti-Efp x x
Anti-erg2 x x
Anti-h H R23B x x
Anti-Kalinin x x
B1
Anti-PUNTS x x
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Anti-RNCAM x x
Anti-SRP54 x x
Anti-TFII-I x x
Anti-TIF2 x x
Anti-TSP-1 x x
Anti-VHR x x
Anti-AKAP149 x x
Anti-alpha acid gly coprotein x x
(AGP)
Anti-Annexin II x x
Anti-ARNT1 x x
Anti-Brm x x
Anti-Calmodulin x x
Anti-Calnexin x x
Anti-CaM K IV x x
Anti-CAS x
Anti-CLA-1 x x
Anti-CRP x x
Anti-Cyclin A x
Anti-DNA pol delta x x
Anti-eIF-5 x x
Anti-ERp72 x x
Anti-ESA x x
Anti-G3VP x x
Anti-Gelsolin x x
Anti-Hsp70 x x
Anti-Hsp90 x x
Anti-IAK-1 x x
Anti-IQGAP1 x x
Anti-KA P3A x x
Anti-Ki-67 x x
Anti-LRP x x
Anti-MEK5 x x
Anti-Neurabin x x
Anti-Numb x x
Anti-PARP x x
24
CA 02366123 2001-09-24
WO 00!63701 PCT/US00/10171
Anti-Pax-5 x x
Anti-PDI x x
Anti-P13-K p170 x x
Anti-rSec8 x x
Anti-SIRPalpha1 x x
Anti-Smad4 x x
Anti-TAF-172 x x
Anti-TZAR x x
Anti-Transportin x x
Anti-Utrophin x x
it is apparent from the above experimental data and descriptions that the
subject
methods provide a useful method for constructing a microarray comprising
immobilized
polypeptides. The polypeptides retain the binding specificity, and are useful
in the detection
and quantitation of ligands that bind to polypeptides, including proteins and
fragments
thereof, peptides, nucleic acids, factors and co-factors, and the like.
All publications and patent applications cited in this specification are
herein
incorporated by reference as if each individual publication or patent
application were
specifically and individually indicated to be incorporated by reference.
Although the foregoing invention has been described in some detail by way of
illustration and example for purposes of clarity of understanding, it will be
readily apparent to
those of ordinary skill in the art in light of the teachings of this invention
that certain changes
and modifications may be made thereto without departing from the spirit or
scope of the
appended claims.
