U.S. patent application number 11/746520 was filed with the patent office on 2007-10-18 for assay system using labeled oligonucleotides.
This patent application is currently assigned to Beckman Coulter, Inc.. Invention is credited to Firdous Farooqui, Daniel A. Keys, M. Parameswara Reddy.
Application Number | 20070243551 11/746520 |
Document ID | / |
Family ID | 33309467 |
Filed Date | 2007-10-18 |
United States Patent
Application |
20070243551 |
Kind Code |
A1 |
Reddy; M. Parameswara ; et
al. |
October 18, 2007 |
Assay System Using Labeled Oligonucleotides
Abstract
The present invention provides a useful system for assays that
comprises a solid support, a plurality of capture oligonucleotides
immobilized onto the solid support, and complementary
oligonucleotides attached to capture ligands. A detectable label
can be directly attached to the capture oligonucleotides or the
complementary oligonucleotides. The labeled oligonucleotides can be
detected, and used to determine the quality of the assay. A labeled
detector ligand corresponding to a target ligand can also be
independently detected apart from the labeled oligonucleotide.
Inventors: |
Reddy; M. Parameswara;
(Brea, CA) ; Keys; Daniel A.; (Irvine, CA)
; Farooqui; Firdous; (Brea, CA) |
Correspondence
Address: |
BECKMAN COULTER INC.;C/O SHELDON MAK ROSE & ANDERSON
100 East Corson Street
Third Floor
PASADENA
CA
91103-3842
US
|
Assignee: |
Beckman Coulter, Inc.
Fullerton
CA
92834-3100
|
Family ID: |
33309467 |
Appl. No.: |
11/746520 |
Filed: |
May 9, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10408626 |
Apr 7, 2003 |
7229763 |
|
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11746520 |
May 9, 2007 |
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Current U.S.
Class: |
435/6.16 ;
436/518 |
Current CPC
Class: |
B01J 2219/005 20130101;
B01J 2219/00315 20130101; B01J 2219/00497 20130101; B01J 2219/00635
20130101; B01J 2219/00722 20130101; B01J 2219/00576 20130101; B01J
2219/00527 20130101; B01J 2219/0061 20130101; B01J 2219/00585
20130101; B01J 2219/00612 20130101; C12Q 1/6834 20130101; B01J
2219/00605 20130101; B01J 2219/00659 20130101; B01J 2219/00677
20130101; B01J 2219/00574 20130101; B01J 2219/0063 20130101; B01J
2219/00596 20130101; C12Q 2563/131 20130101; C12Q 2565/549
20130101; B01J 2219/00364 20130101; C12Q 1/6834 20130101; B01J
2219/00725 20130101; B01J 2219/00387 20130101; B01J 2219/00693
20130101 |
Class at
Publication: |
435/006 ;
436/518 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; G01N 33/543 20060101 G01N033/543 |
Claims
1. An assay device comprising: a. a solid support; and b. a
plurality of capture oligonucleotides immobilized onto the solid
support, wherein at least a portion of the capture oligonucleotides
have detectable labels directly attached thereto.
2. The device of claim 1 wherein all of the capture
oligonucleotides are the same.
3. The device of claim 1 wherein at least some of the capture
oligonucleotides are different.
4. The device of claim 1 wherein all of the detectable labels are
the same.
5. The device of claim 1 wherein the detectable label is selected
from the group consisting of fluorophores, radioactive,
chemiluminescent, bioluminescent, enzyme, nephelometric,
turbidometric, and visible labels.
6. The device of claim 1 further comprising a plurality of capture
ligands attached to complementary oligonucleotides, wherein the
capture oligonucleotides and the complementary oligonucleotides
form double stranded nucleic acid duplexes.
7. An assay device comprising: a. a solid support; b. a plurality
of capture oligonucleotides immobilized onto the solid support; and
c. a plurality of capture ligands attached to complementary
oligonucleotides, wherein at least a portion of the complementary
oligonucleotides have detectable labels directly attached thereto
and the complementary oligonucleotides form double stranded nucleic
acid duplexes with the capture oligonucleotides.
8. The device of claim 7 wherein all of the capture
oligonucleotides are the same.
9. The device of claim 7 wherein at least some of the capture
oligonucleotides are different.
10. The device of claim 7 wherein the capture ligands are
antibodies.
11. The device of claim 7 wherein the capture ligands are
antigens
12. The device of claim 7 wherein all of the detectable labels are
the same.
13. The device of claim 7 wherein the detectable label is selected
from the group consisting of fluorophores, radioactive,
chemiluminescent, bioluminescent, enzyme, nephelometric,
turbidometric, and visible labels.
14. An assay kit comprising: a. a solid support; b. a plurality of
capture oligonucleotides immobilized onto the solid support wherein
at least a portion of the capture oligonucleotides have first
detectable labels directly attached thereto; and c. a plurality of
capture ligands attached to complementary oligonucleotides, the
complementary oligonucleotides being capable of hybridizing under
appropriate conditions to form double stranded nucleic acid
duplexes with the capture oligonucleotides.
15. The assay kit of claim 14 wherein all of the capture
oligonucleotides are the same.
16. The assay kit of claim 14 wherein at least some of the capture
oligonucleotides are different.
17. The kit of claim 14 wherein the capture oligonucleotides and
complementary oligonucleotides are in the form of double stranded
nucleic acid duplexes.
18. The assay kit of claim 14 further comprising: d. one or more
detector ligands having second detectable labels.
19. The assay kit of claim 18 wherein the capture ligands and
detector ligands are antibodies.
20. The assay kit of claim 18 wherein the labeled detector ligand
competes with a target ligand in binding to the capture ligand.
21. The assay kit of claim 18 wherein the detectable labels are
selected from the group consisting of fluorophores, radioactive,
chemiluminescent, bioluminescent, enzyme, nephelometric,
turbidometric, and visible labels.
22. The assay kit of claim 18 wherein the first and second
detectable labels are different fluorophores that use the same
excitation light source.
23. An assay kit comprising: a. a solid support; b. a plurality of
capture oligonucleotides immobilized onto the solid support; and c.
a plurality of capture ligands attached to complementary
oligonucleotides, wherein at least a portion of the complementary
oligonucleotides have detectable labels directly attached thereto,
and the complementary oligonucleotides being capable of hybridizing
under appropriate conditions to form double stranded nucleic acid
duplexes with the capture oligonucleotides.
24. The assay kit of claim 23 wherein all of the capture
oligonucleotides are the same.
25. The assay kit of claim 23 wherein at least some of the capture
oligonucleotides are different.
26. The assay kit of claim 23 wherein the capture oligonucleotides
and complementary oligonucleotides are in the form of double
stranded nucleic acid duplexes.
27. The assay kit of claim 23 further comprising: d. one or more
detector ligands having second detectable labels.
28. The assay kit of claim 27 wherein the capture ligands and
detector ligands are antibodies.
29. The assay kit of claim 27 wherein the labeled detector ligand
competes with a target ligand in binding to the capture ligand.
30. The assay kit of claim 27 wherein the detectable labels are
selected from the group consisting of fluorophores, radioactive,
chemiluminescent, bioluminescent, enzyme, nephelometric,
turbidometric, and visible labels.
31. The assay kit of claim 27 wherein the first and second
detectable labels are different fluorophores that use the same
excitation light source.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present Application claims the benefit of U.S. patent
application Ser. No. 10/408,626 filed on Apr. 7, 2003, the contents
of which are incorporated in this disclosure by reference in their
entirety.
BACKGROUND
[0002] The following description provides a summary of information
relevant to the present invention and is not a concession that any
of the information provided or publications referenced herein is
prior art to the presently claimed invention.
[0003] An assay can be performed in a number of ways such as the
well-known sandwich technique and competitive technique. A variety
of specific biological binding molecules can be labeled with a
radioactive element, a fluorophore or a constituent which enters
into an enzyme reaction. Thus a sample containing suspected target
ligands can be analyzed, and the target ligand or target ligands
can be detected quantitatively by forming a complex with a labeled
anti-ligand, labeled as indicated above, and measuring the labeled
constituent in the complex to determine the quantity of the target
ligand. The anti-ligand binds to at least one site on the ligand to
form a complex. "Ligand or target ligand" and "anti-ligand," as
these terms are used herein, refer to antigens, antibodies, binding
proteins, haptens, hormone receptors, and other biological
molecules that can form a complex. The labeled anti-ligand used to
detect and measure the target ligand in the complex is referred to
herein as the "detector ligand."
[0004] In the sandwich technique mentioned above, a target ligand,
a detector ligand, and a capture ligand are used in the assay. The
detector ligand is detected quantitatively in a detector
ligand/target ligand complex to determine a quantity of the target
ligand present. The capture ligand in this technique is an
anti-ligand that binds to the target ligand. The capture ligand and
detector ligand typically bind to different sites on the target
ligand so that there is no interference between the binding of the
detector ligand to the target ligand and the capture ligand to the
target ligand.
[0005] In the sandwich technique, the target ligand binds to the
capture ligand to form a first complex. The detector ligand also
binds to the target ligand in the complex to form a second complex
in the sandwich, and the labeled constituent in the sandwiched
ligands is detected quantitatively to deduce the quantity of target
ligand present. Detection can be performed by: measuring
radioactivity where the detector ligand is radioactive; measuring
fluorescent light where there is a fluorescent label on the
detector ligand; or spectrophotometrically where an optical density
or wavelength change occurs through an enzyme reaction, or through
fluorescent quenching. Detection may require separation of the
sandwiched ligands from unbound ligands and this is generally done
by separating the capture ligand attached or immobilized onto a
surface from a solution containing unbound detector ligands.
[0006] The quantity of target ligand is deduced from the quantity
of detector ligand detected, because the two quantities are
generally directly proportional to each other in the sandwich
technique. Parallel tests against known standards are employed for
calibration.
[0007] The quantity of target ligand can be determined as an
inverse proportion using the competitive technique previously
mentioned above. With this technique, the capture ligand is
contacted either simultaneously or sequentially with a target
ligand and a known, limiting quantity of detector ligand. When the
target ligand and detector ligand bind to the capture ligand, the
quantity of detector-ligand detected in a binary complex with the
capture ligand is inversely proportional to the amount of target
ligand present. In this technique, the target ligand and detector
ligand bind to the same site or sites in close proximity to each
other to create competition.
[0008] The two techniques discussed above may be represented as
follows where C designates the capture ligand, T represents the
target ligand, and D represents the detector ligand. TABLE-US-00001
Sandwich Competitive 1. C + T CT 1. C + T CT + C sequential 2. CT +
D CTD 2. (CT + C) + D CT + CD 1. C + T + D CD + CT simultaneous
[0009] In addition to the sandwich and competitive assays described
above, a variety of other techniques for assays are known, and
include the following assays.
[0010] A method of using two different ligands tagged with two
different tagging constituents in an immunoassay to independently
detect and measure bound target ligand and bound receptor or
capture ligand is described in U.S. Pat. No. 4,385,126 to James H.
Chen et al.
[0011] A method of using oligonucleotides as capture agents for
capture ligands in assays is described in U.S. Pat. No. 5,648,213
to Reddy et al.
[0012] Assay reagents and kits using oligonucleotides as capture
agents in an assay array is described in U.S. Pat. No. 5,789,165 to
Oku et al.
[0013] Although these systems are useful, there is always a need
for systems with improved accuracy and lower costs. Accordingly, a
need exists for a system that: offers greater precision in
detection and quantification of capture ligands; offers greater
precision in detection and quantification of target ligands;
decreases variation arising from imprecision in the addition of
reagents; offers correction for the variation arising from various
assay manipulations; decreases the amount of binding interference
caused by random attachment or positioning of a label on capture
ligands; decreases the variability between different preparations
of the same labeled capture ligands; provides reusable components
to decrease expense in doing multiple assays; provides easy quality
control and standardization methods; and can be provided as an
assay kit.
SUMMARY
[0014] The present invention satisfies that need. The present
invention provides a system that uses labeled oligonucleotides in
assays to provide quality control, standardization, and greater
precision in detection.
[0015] An assay device according to the present invention comprises
a solid support and a plurality of capture oligonucleotides,
wherein at least a portion of the capture oligonucleotides have
detectable labels directly attached thereto immobilized onto the
solid support.
[0016] An assay kit according to the present invention comprises a
solid support; a plurality of capture oligonucleotides immobilized
onto the solid support; and a plurality of capture ligands attached
to complementary oligonucleotides, wherein at least a portion of
the complementary oligonucleotides have detectable labels directly
attached thereto. The complementary oligonucleotides in this assay
device being capable of hybridizing under appropriate conditions to
form double stranded nucleic acid duplexes with the capture
oligonucleotides.
[0017] A sandwich assay method for a target ligand according to the
present invention includes the step of providing a solid support
having a plurality of capture oligonucleotides immobilized on the
solid support. Another step is adding to the solid support a
plurality of capture ligands attached to complementary
oligonucleotides, wherein at least a portion of the complementary
oligonucleotides have detectable labels directly attached thereto.
Another step is providing conditions suitable for hybridization of
the complementary oligonucleotides and the capture oligonucleotides
to form double stranded nucleic acid duplexes. Another step is
bringing the target ligand in contact with the solid support.
Another step is adding a plurality of detector ligands having
second detectable labels to the solid support. Another step is
detecting the first detectable labels, thereby determining the
amount of immobilized capture oligonucleotide. Another step is
detecting the second detectable labels, thereby determining the
amount of the target ligand.
[0018] Another sandwich assay method for a target ligand according
to the present invention includes the step of providing a solid
support having a plurality of capture oligonucleotides immobilized
on the solid support, wherein at least a portion of the capture
oligonucleotides have detectable labels directly attached thereto.
Another step is adding to the solid support a plurality of capture
ligands attached to complementary oligonucleotides. Another step is
providing conditions suitable for hybridization of the
complementary oligonucleotides and the capture oligonucleotides to
form double stranded nucleic acid duplexes. Another step is
bringing the target ligand in contact with the solid support.
Another step is adding a plurality of detector ligands having
second detectable labels to the solid support. Another step is
detecting the first detectable labels, thereby determining the
amount of immobilized capture oligonucleotide. Another step is
detecting the second detectable labels, thereby determining the
amount of the target ligand.
[0019] A competitive assay method for a target ligand according to
the present invention includes the step of providing a solid
support having a plurality of capture oligonucleotides immobilized
on the solid support. Another step is adding to the solid support a
plurality of captures ligands attached to complementary
oligonucleotides, wherein at least a portion of the complementary
oligonucleotides have first detectable labels directly attached
thereto. Another step is providing conditions suitable for
hybridization of the complementary oligonucleotides and the capture
oligonucleotides to form double stranded nucleic acid duplexes.
Another step is adding the target ligand to the solid support,
wherein the target ligand competes with the detector ligand in
binding to the capture ligand. Another step is adding a plurality
of detector ligands having second detectable labels to the solid
support. Another step is detecting the first detectable labels,
thereby determining the amount of immobilized capture
oligonucleotide. Another step is detecting the second detectable
labels, thereby determining the amount of the target ligand.
[0020] Another competitive assay method for a target ligand
according to the present invention includes the step of providing a
solid support having a plurality of capture oligonucleotides
immobilized on the solid support, wherein at least a portion of the
capture oligonucleotides have first detectable labels directly
attached thereto. Another step is adding to the solid support, a
plurality of capture ligands attached to complementary
oligonucleotides. Another step is providing conditions suitable for
hybridization of the complementary oligonucleotides and the capture
oligonucleotides to form double stranded nucleic acid duplexes.
Another step is adding the target ligand onto the solid support,
wherein the target ligand competes with the detector ligand in
binding to the capture ligand. Another step is adding a plurality
of detector ligands having second detectable labels onto the solid
support. Another step is detecting the first detectable labels,
thereby determining the amount of immobilized capture
oligonucleotide. Another step is detecting the second detectable
labels, thereby determining the amount of the target ligand
[0021] In a preferred embodiment of the assay methods (i.e.
sandwich and competitive assays), the first and second detectable
labels are fluorophores that use the same excitation light source
wavelength. The fluorophores typically have different emission
wavelengths. The operator of the assay can use the same excitation
light source, and determine the quality of the assay's capture
components, labeled oligonucleotides immobilized on the solid
support, and the concentration of the target ligand, by
independently measuring the emission wavelength of each
fluorophore.
[0022] In a preferred embodiment of the assay methods (i.e.
sandwich and competitive assays), the steps of detecting the first
detectable labels and detecting the second detectable labels are
simultaneous.
DRAWINGS
[0023] These features, aspects and advantages of the present
invention will become better understood with regard to the
following description, appended claims and accompanying figures
where:
[0024] FIG. 1 shows label detection using the invention where a CCD
camera picture detects and illuminates different concentrations of
fluorescent label Cy3 directly attached to 30-base capture
oligonucleotides immobilized onto the bottom surface of a
microtiter in a printed 3.times.3 array pattern.
[0025] FIG. 2 depicts a sandwich assay of the invention using a
plurality of immobilized capture oligonucleotides, wherein at least
a portion having directly attached fluorescent label Cy3; an
oligonucleotide-tagged antibody, wherein the oligonucleotide is
complementary to the capture oligonucleotide; an analyte, and a
PBXL-1 tagged detection antibody.
[0026] FIG. 3 depicts a sandwich assay of the invention using
immobilized capture oligonucleotides; a Cy3 labeled
oligonucleotide-tagged antibody, wherein the oligonucleotide is
complementary to the capture oligonucleotide; an analyte; and a
PBXL-1 tagged detection antibody.
[0027] FIG. 4 shows label detection in a sandwich assay using IL-8
antigen as the target ligand with the invention where a CCD camera
picture detects and illuminates different concentrations of
fluorescent label PBXL-1 corresponding to detector ligand bound in
complexes on the surface of a microtiter in a printed 3.times.3
array pattern.
[0028] FIG. 5 shows label detection in a sandwich assay using IL-8
antigen as the target ligand with the invention where a CCD camera
picture detects and illuminates the fluorescent label Cy5.5
directly attached to capture oligonucleotides immobilized onto a
microtiter plate, as shown on the left side of the figure, and
concentrations of fluorescent label PBXL-1 in bound IL-8 detector
antibodies, as shown on the right side.
[0029] FIG. 6 shows label detection in a sandwich assay using the
invention where a CCD camera picture detects and illuminates the
fluorescent label Cy5.5 directly attached to complementary
oligonucleotides attached to antibodies, as shown on the left side
of the figure, and concentrations of fluorescent label PBXL-1 in
bound detector antibodies for IL-2, IL-8, IL-12, Interferon-gamma,
FGF-basic, and GMCSF antigens, as shown on the right side.
[0030] FIG. 7 depicts a competitive assay of the invention using
immobilized complementary oligonucleotides; a Cy3 labeled
oligonucleotide-tagged antibody, wherein the oligonucleotide is
complementary to the capture oligonucleotide; a target analyte; and
a competing PBXL-1 labeled analyte.
DESCRIPTION
[0031] The following discussion describes embodiments of the
invention and several variations of these embodiments. This
discussion should not be construed, however, as limiting the
invention to these particular embodiments. Practitioners skilled in
the art will recognize numerous other embodiments as well.
[0032] The present invention provides a system that uses capture
oligonucleotides in assays to provide quality control,
standardization, and greater precision in detection of target
ligands. The use of capture oligonucleotides also yields efficient
and easy manufacturing, and quality control methods that far exceed
prior systems in ease of use for manufacturer and consumer.
[0033] As used herein, the phrase "directly attached" or "directly
attaching" in referring to attaching a label to a capture or
complementary oligonucleotide means that the label is attached to
the oligonucleotide, and not to a ligand or anti-ligand which can
also be attached to the oligonucleotide.
[0034] As used herein, the term "capture oligonucleotide" means an
oligonucleotide immobilized or attached to a solid support which
can bind a complementary oligonucleotide attached to a capture
ligand.
[0035] As used herein, the terms "complementary oligonucleotide" or
"oligonucleotide complementary to a capture oligonucleotide" refers
to a nucleotide sequence attached to a capture ligand that
hybridizes to the capture oligonucleotide under conditions suitable
for hybridization thereby forming double stranded nucleic acid
duplexes.
[0036] The present invention's use of detectable labels directly
attached to capture oligonucleotides or to complementary
oligonucleotides provides important innovations not taught in any
of the references discussed in the background section.
[0037] The invention's use of labeled oligonucleotides is
significant. In the invention, the label is directly attached to
the capture oligonucleotide or the complementary oligonucleotide,
and can be easily detected by a manufacturer or customer without
the need or presence of a capture ligand. This provides an
effective method of determining the quality of the assay device. In
addition, oligonucleotide arrays are more stable than the antibody
arrays that can be used as a labeled capture ligand, and
oligonucleotides have a longer shelf life. Therefore, a user of the
present invention can have a readily available labeled
oligonucleotide assay device on the shelf, and an easily verifiable
quality control system in place.
[0038] Another benefit of the present invention is that the
proximity and orientation of labels on an oligonucleotide can be
more easily and precisely controlled. For example, using directly
attached labels on oligonucleotides in the invention bound to a
surface, the distance and orientation (and therefore the
interaction) of a fluorescent dye to the surface is uniform, and
can be easily controlled, resulting in more uniform and consistent
fluorescence. In contrast, using labels attached to an antibody
without an oligonucleotide as an intermediary link, the distance
and orientation of the fluorescent labels to the surface is random,
resulting in variable and inconsistent fluorescence (due to
interference of the surface with the fluorescence emission).
[0039] Other important beneficial differences are that: [0040] 1.
Oligonucleotides can be used to label a variety of classes of
molecules, such as antibodies, nucleic acids, lectins, cell-surface
receptors, etc. A single array of first (unlabeled)
oligonucleotides, complementary to the labeled
oligonucleotide-complexes, can be used as a universal substrate to
generate a self-assembling array of these different classes of
molecules. [0041] 2. The fluorescence characteristics of labels may
be affected by the secondary and tertiary conformations and
structures of different proteins. This risk is minimized or
eliminated by labeling through oligonucleotides. [0042] 3. A
fluorescent label can be attached to either the immobilized capture
oligonucleotide or to its complementary oligonucleotide-antibody
complex.
[0043] The invention's use of directly attached labels on
oligonucleotides does not interfere with the performance,
sensitivity, or dynamic range of detection of the target ligand in
an assay, while providing a convenient method to monitor the
quality of an array from manufacture throughout the actual assay
procedure. The CCD camera picture from FIG. 4 shows the invention
does not interfere with the performance, sensitivity, or dynamic
range of an assay.
[0044] The CCD camera picture of FIG. 4 shows that the
concentration of labeled oligonucleotides did not have an
appreciable effect on detection of the IL-8 analyte regardless of
the concentration of IL-8 analyte. The detection of IL-8 analyte
was consistent. In FIG. 4, columns A-E on the top of the figure
correspond to the respective concentrations of Cy3 labeled
oligonucleotides from 6 .mu.M, 4 .mu.M, 2 .mu.M, 1 .mu.M, to 0
.mu.M. In FIG. 4, the concentration of IL-8 analyte, the target
ligand, ranged from 0 pg/ml to 1000 pg/ml as identified on the
right side of the figure. The intensity of the fluorescent
emissions or brightness of the spots correlated to detection of the
IL-8 analyte.
[0045] The protocol for the IL 8 assay shown in FIG. 4 is described
below:
[0046] Step I. 140 ng/well of antibody-oligonucleotide conjugate in
casein buffer were added, and the plate was shook at 37.degree. C.
for 1 hour. The plate washed with wash buffer (0.02% Tween 20 in
1.times. Tris Buffer Saline) 3 times.
[0047] Step II. IL-8 antigen from 1000-0 pg/ml per well in casein
buffer was added and reacted at 37.degree. C. for 1 hour and washed
with wash buffer 3 times. The concentrations of antigen used in the
assay were: 0 pg/ml, 5 pg/ml, 100 pg/ml, and 1000 pg/ml.
[0048] Step III. Biotinylated antibody (purchased from R & D
Systems, 614 McKinley Place N.E. Minneapolis, Minn. 55413) 50 ng
per well was added, incubated at 37.degree. C. for 1 hour, and
washed 3 times.
[0049] Step IV. Streptavidin PBXL-1 (purchased from Martek, 6480
Dobbin Road, Columbia, Md. 21045) was added (1 mg dissolved in 1 ml
of water) 1:150 dilution, 50 .mu.l/well and incubated at 37.degree.
C. for 1 hour and washed 3 times with wash buffer. 50 .mu.l of wash
buffer was kept in each well and imaged using a CCD camera. The CCD
camera collected the fluorescent emission at 575 nm that correlated
to loading or immobilizing of capture oligonucleotides onto the
plate, and the fluorescent emission at 675 nm correlated to the
amount of detector antibody bound to the plate.
[0050] In the invention, capture oligonucleotides are bound or
immobilized onto a solid support. Solid supports capable of having
capture oligonucleotides immobilized onto the surface include, but
are not limited to polypropylene, polystyrene, glass,
nitrocellulose, polyvinylidene fluoride ("PVDF"), and nylon. The
solid supports used in the invention may take different forms such
as bead, plate, film, or other structures.
[0051] Complementary oligonucleotides for the capture
oligonucleotides can be directly attached to a variety of different
molecules including antigens, antibodies, binding proteins,
haptens, hormone receptors, hormones, lectins, carbohydrates,
metabolites, drugs, enzyme substrates, and viral proteins for use
in the present invention. Complementary oligonucleotides have been
directly attached to antibodies to human IL-1b, IL-2, IL-4, IL-6,
IL-8, IL-10, IL-12, VEGF, FGF-basic, IFNg, GMCSF, TNF, and used in
assays for the invention. The methods for attachment of
complementary oligonucleotides to the above antibodies are well
known to one of ordinary skill in the art. See e.g., U.S. Pat. No.
5,648,213 (Reddy).
[0052] In one aspect of the invention, conditions must be suitable
to permit the complementary oligonucleotides to hybridize to
capture oligonucleotides to form nucleic acid duplexes. The
conditions conducive to the formation of nucleic acid duplexes are
well known to one of ordinary skill in the arts (as described, for
example, in Sambrook and Russell, Molecular Cloning: A Laboratory
Manual, 3.sup.rd Edition, Cold Spring Harbor Laboratory Press, New
York, or Current Protocols in Molecular Biology, edited by
Frederick Ausubel, John Wiley and Sons Publishing, New York,
1987).
[0053] Capture and complementary oligonucleotides used in the
present invention range from about 15 to about 45 base
oligonucleotides. The capture and complementary oligonucleotides
preferably each have about 20 to about 30 base oligonucleotides. In
one aspect of the present invention a pair of capture and
complementary oligonucleotides can be used to capture a single
specific target ligand. For example, a pair of capture and
complementary oligonucleotides can be used in a well a microtiter
plate to capture a specific target ligand in that well.
[0054] In another aspect of the invention, an array of different
capture and complementary oligonucleotide can be used to capture
different target ligands. When using more than one pair of capture
and complementary oligonucleotides, the preferred sequences are
selected from a group of pairs that do not have substantial cross
hybridization. Preferred pairs include: TABLE-US-00002 TABLE 1 SEQ
ID NO 1: 5' - GCTTACCGAA TACGGCTTGG AGAACCTATC - 3' SEQ ID NO 2: 5'
- GATAGGTTCT CCAAGCCGTA TTCGGTAAGC - 3' SEQ ID NO 3: 5' -
GCGTGGTCCG CGATCTTCCT ACGATTGATG - 3' SEQ ID NO 4: 5' - CATCAATCGT
AGGAAGATCG CGGACCACGC - 3' SEQ ID NO 5: 5' - TTTGAGGTTT CGGAGCGTTC
CGTGCATCGC - 3' SEQ ID NO 6: 5' - GCGATGCACG GAACGCTCCG AAACCTCAAA
- 3' SEQ ID NO 7: 5' - CTCATGAAGG CCGTCGGGAA ATTCCAAGTT - 3' SEQ ID
NO 8: 5' - AACTTGGAAT TTCCCGACGG CCTTCATGAG - 3' SEQ ID NO 9: 5' -
TGGATTCCGT TATCACCATT TGGACCCTGC - 3' SEQ ID NO 10: 5' - GCAGGGTCCA
AATGGTGATA ACGGAATCCA - 3' SEQ ID NO 11: 5' - TACGCTCCCA GTGTGATCAC
CAAAGCTTAC - 3' SEQ ID NO 12: 5' - GTAAGCTTTG GTGATCACAC TGGGAGCGTA
- 3' SEQ ID NO 13: 5' - AGACTGAACT ACCGGCGGTT GCACTACTAA - 3' SEQ
ID NO 14: 5' - GCCAAACACG CCCAATCACT GTTACATGTC - 3' SEQ ID NO 15:
5' - TTAGTAGTGC AACCGCCGGT AGTTCAGTCT - 3' SEQ ID NO 16: 5' -
GACATGTAAC AGTGATTGGG CGTGTTTGGC - 3' SEQ ID NO 17: 5' - GCTTGACGTC
TACCACCGTG AACATAAGGA - 3' SEQ ID NO 18: 5' - TCCTTATGTT CACGGTGGTA
GACGTCAAGC - 3' SEQ ID NO 19: 5' - TCGCCAACGT AGCTGTGCTA CAGTTGATTC
- 3' SEQ ID NO 20: 5' - GAATCAACTG TAGCACAGCT ACGTTGGCGA - 3' SEQ
ID NO 21: 5' - GCAGCGGCTA AACCTTGAGA TCGAATGGAA - 3' SEQ ID NO 22:
5' - TTCCATTCGA TCTCAAGGTT TAGCCGCTGC - 3' SEQ ID NO 23: 5' -
TGCGATATAC TCCATGCCTC TCTTGGCGGA - 3' SEQ ID NO 24: 5' - TCCGCCAAGA
GAGGCATGGA GTATATCGCA - 3'
For example, an array selected from the above different sequences
can be used in a well of a microtitier plate when more than one
target ligand is captured in the well.
[0055] A variety of different types of detectable labels can be
directly attached to the capture or complementary oligonucleotides
and used in the present invention. Those detectable labels include
but are not limited to fluorophores, radioactive, chemiluminescent,
bioluminescent, enzyme, nephelometric, turbidometric, and visible
labels. Examples of fluorophores that can be used in the invention
include but are not limited to rhodamine 110, rhodal, fluorescein,
coumarin, and derivatives of rhodamine 110, rhodal, or fluorescein.
Cyanine dyes such as Cy2, Cy3, Cy5, Cy5.5, and Cy7. Examples of
radioactive labels that can be used in the invention include but
are not limited to .sup.32P, .sup.33P, .sup.35S, .sup.3H, and
.sup.125I. Examples of chemiluminescent labels that can be used in
the invention include but are not limited to acridinium esters,
ruthenium complexes, metal complexes, oxalate ester--peroxide
combination. Examples of enzyme labels that can be used in the
invention include but are not limited to alkaline phosphatase,
horseradish peroxidase, beta-galactosidase. Examples of visible
labels that can be used in the invention include but are not
limited to thiopeptolides, anthroquinone dyes, nitro blue
tetrazolium, ortho-nitrophenol .beta.-D-galacto-piranoside (ONPG).
The same type of labels, discussed above, can also be used on
detector ligands.
[0056] Methods for attachment of detectable labels to
oligonucleotides are well known to one of ordinary skill in the
arts. For example, such methods are described in Yang and Millar,
Methods in Enzymology, Vol. 278, pages 417-444, 1997.
[0057] The system of the present invention can be used in the
creation of assay devices, in quality control in the manufacturing
process, in quality control in the customer's laboratory, and as a
final quality control during the detection of the target ligand in
the assay.
[0058] An assay device according to the present invention comprises
a solid support and a plurality of capture oligonucleotides,
wherein at least a portion of the capture oligonucleotides have
detectable labels directly attached thereto immobilized onto the
solid support. Embodiments of the assay device can include an assay
device where: all of the capture oligonucleotides are the same; or
all of the detectable labels are the same; or wherein the
detectable label is selected from the group consisting of
fluorophores, radioactive, chemiluminescent, bioluminescent,
enzyme, nephelometric, turbidometric, and visible labels.
[0059] An assay kit according to the present invention comprises a
solid support; a plurality of capture oligonucleotides immobilized
onto the solid support; and a plurality of capture ligands attached
to complementary oligonucleotides, wherein at least a portion of
the complementary oligonucleotides have detectable labels directly
attached thereto. The complementary oligonucleotides in this assay
device being capable of hybridizing under appropriate conditions to
form double stranded nucleic acid duplexes with the capture
oligonucleotides. Embodiments of this assay kit can include an
assay device where: all of the capture oligonucleotides are the
same; or where the capture ligands are antibodies; or wherein all
of the detectable labels are the same; or wherein the capture
oligonucleotides and complementary oligonucleotides are in the form
of double stranded nucleic acid duplexes.
[0060] A sandwich assay method for a target ligand according to the
present invention includes the step of providing a solid support
having a plurality of capture oligonucleotides immobilized on the
solid support. Another step is adding to the solid support of a
plurality of capture ligands attached to complementary
oligonucleotides, wherein at least a portion of the complementary
oligonucleotides have detectable labels directly attached thereto.
Another step is providing conditions suitable for hybridization of
the complementary oligonucleotides and the capture oligonucleotides
to form double stranded nucleic acid duplexes. Another step is to
bring the target ligand in contact with the solid support. Another
step is adding a plurality of detector ligands having second
detectable labels to the solid support. Another step is detecting
the first detectable labels, thereby determining the amount of
immobilized capture oligonucleotide. Another step is detecting the
second detectable labels, thereby determining the amount of the
target ligand.
[0061] Another sandwich assay method for a target ligand according
to the present invention includes the step of providing a solid
support having a plurality of capture oligonucleotides immobilized
on the solid support, wherein at least a portion of the capture
oligonucleotides have detectable labels directly attached thereto.
Another step is adding to the solid support a plurality of capture
ligands attached to complementary oligonucleotides. Another step is
providing conditions suitable for hybridization of the
complementary oligonucleotides and the capture oligonucleotides to
form double stranded nucleic acid duplexes. Another step is
bringing the target ligand in contact with the solid support.
Another step is adding a plurality of detector ligands having
second detectable labels to the solid support. Another step is
detecting the first detectable labels, thereby determining the
amount of immobilized capture oligonucleotide. Another step is
detecting the second detectable labels, thereby determining the
amount of the target ligand.
[0062] Embodiments of the sandwich assay described can include:
wherein the capture ligands and the detector ligands are
antibodies; or wherein the capture ligands and the detector ligands
are antigens; or wherein the step of detecting the first and second
detectable labels is by quantitatively measuring each label; or
wherein the step of adding a plurality of detector ligands occurs
before the step of providing conditions suitable for hybridization;
or wherein the detectable label is selected from the group
consisting of fluorophores, radioactive, chemiluminescent,
bioluminescent, enzyme, nephelometric, turbidometric, and visible
labels.
[0063] A competitive assay method for a target ligand according to
the present invention includes the step of providing a solid
support having a plurality of capture oligonucleotides immobilized
on the solid support. Another step is adding to the solid support a
plurality of capture ligands attached to complementary
oligonucleotides, wherein at least a portion of the complementary
oligonucleotides have first detectable labels directly attached
thereto. Another step is providing conditions suitable for
hybridization of the complementary oligonucleotides and the capture
oligonucleotides to form double stranded nucleic acid duplexes.
Another step is adding the target ligand to the solid support,
wherein the target ligand competes with the detector ligand in
binding to the capture ligand. Another step is adding a plurality
of detector ligands having second detectable labels to the solid
support. Another step is detecting the first detectable labels,
thereby determining the amount of immobilized capture
oligonucleotide. Another step is detecting the second detectable
labels, thereby determining the amount of the target ligand.
[0064] Another competitive assay method for a target ligand
according to the present invention includes the step of providing a
solid support having a plurality of capture oligonucleotides
immobilized on the solid support, wherein at least a portion of the
capture oligonucleotides have first detectable labels directly
attached thereto. Another step is adding to the solid support, a
plurality of capture ligands attached to complementary
oligonucleotides. Another step is providing conditions suitable for
hybridization of the complementary oligonucleotides and the capture
oligonucleotides to form double stranded nucleic acid duplexes.
Another step is adding the target ligand onto the solid support,
wherein the target ligand competes with the detector ligand in
binding to the capture ligand. Another step is adding a plurality
of detector ligands having second detectable labels onto the solid
support. Another step is detecting the first detectable labels,
thereby determining the amount of immobilized capture
oligonucleotide. Another step is detecting the second detectable
labels, thereby determining the amount of the target ligand
[0065] In a preferred embodiment of any of the assay methods
described above (i.e. sandwich assay or competitive assay), the
first and second detectable labels are fluorophores that use the
same excitation light source wavelength. The fluorophores typically
have different emission wavelengths. The operator of the assay can
use the same excitation light source, and determine the quality of
the assay's capture components, labeled oligonucleotides
immobilized on the solid support, and the concentration of the
target ligand, by independently measuring the emission wavelength
of each fluorophore.
[0066] In a preferred embodiment of any of the assay methods
described above (i.e. sandwich assay or competitive assay), the
steps of detecting the first detectable labels and detecting the
second detectable labels are simultaneous.
[0067] Preferred embodiments of sandwich assays of the invention
are depicted in FIGS. 2 and 3. In both embodiments, Cy3 labeled
oligonucleotides and PBXL-1 labeled detection antibody are the
first and second detectable labels, and these respective labels use
the same excitation wavelength of 550 nm. However, the Cy3 label
directly attached to the oligonucleotides has an emission
wavelength of 575 nm, whereas the PBXL-1 label has an emission
wavelength of 675 nm. Therefore, when an excitation wavelength of
550 nm is used, detection and measurement of both labels can result
because each label has a different emission wavelength.
[0068] The difference between the embodiments shown in FIGS. 2 and
3 is which oligonucleotide has the directly attached Cy3 label. In
FIG. 2, the Cy3 label is directly attached to the capture
oligonucleotide immobilized on the surface, whereas the
complementary oligonucleotide attached to the antibody (capture
ligand) does not have the Cy3 label. In FIG. 3, the reverse
situation occurs wherein the Cy3 label is directly attached to the
complementary oligonucleotide attached to an antibody, and the
capture oligonucleotide immobilized on the surface is unlabeled. In
FIGS. 2 and 3, the same detector ligand, a detection antibody
labeled with PBXL-1, is depicted.
[0069] FIG. 7 depicts a preferred embodiment of a competitive assay
of the present invention using immobilized capture
oligonucleotides; a Cy3 labeled oligonucleotide-tagged antibody,
wherein the oligonucleotide is complementary to the capture
oligonucleotide; a target analyte; and a competing PBXL-1 labeled
analyte. In FIG. 7, the detector ligand is called the "competing
labeled analyte", and the "target ligand" is called the target
analyte.
[0070] As one of ordinary skill in the art will know, there are
several types of fluorescent or other labels that can be used in
this manner. For example, the following pairs of labels can be
used: fluorescein and rhodamine; Cy3 and Cy5; PBXL-1 and Cy5.5; and
fluorescein and PBXL-1.
[0071] The assay devices, method of manufacturing, and assays for
target ligands can use the same capture oligonucleotides and
complementary oligonucleotides or combinations of different capture
oligonucleotides and their respective complementary
oligonucleotides. One of ordinary skill in the art will know how to
prepare a variety of assays, including but not limited to sandwich
and competitive assays, according to the present invention.
[0072] A variety of different permutations of the invention is
contemplated, and not meant to be limited by this disclosure. The
present invention is not limited to the preferred embodiments
described in this section. The embodiments are merely exemplary,
and one skilled in the art will recognize that many others are
possible in accordance with this invention. Having now generally
described the invention, the same will be more readily understood
through references to the following examples, which are provide by
way of illustration, and are not intended to be limiting of the
present invention, unless so specified.
EXAMPLE 1
Immobilizing Capture Oligonucleotides with Directly Attached Cy3
Label onto a Microtiter Plate, and Detecting the Directly Attached
Cy3 Label
[0073] A. Synthesis of 5'-Dye-Label 3'-Amino Capture
Oligonucleotides with Directly Attached Cy3 Labels
[0074] A 30-mer oligonucleotide was synthesized on a
3'-amino-modifier C7 CPG (Glen Research Part # 20-2957-01,
purchased from Glen Research, Sterling, Va.) on 1 .mu.mole scale
(coupling efficiency .apprxeq.98%) on ABI 394 (purchased from
Applied Biosystems, 850 Lincoln Center Drive, Foster City, Calif.
94404) using A.sup.pac, G.sup.ipr-pac, C.sup.ac and T
phosphoramdites. At the 5' end, dye phosphoramidite Cy3 (purchased
from Glen research, Sterling, Va.) was coupled using the standard
protocol recommended by the supplier of the reagents.
[0075] Next, the oligonucleotide was cleaved and deprotected using
ammonia for 24 hours at room temperature and purified on polypak
cartridges (Glen Research, Sterling, Va.). The dye labeled
oligonucleotide was analyzed on CE (Beckman Pace 5000, with SSDNA
gel kit).
[0076] B. Immobilization & Detection of Capture
Oligonucleotides Attached to Microtiter Plates
[0077] The activation and chemical derivitization of polypropylene
microtiter plates is described in patent application Ser. No.
10/033,308 filed Oct. 24, 2001, incorporated in its entirety by
reference herein. The application describes the immobilization of
amino oligonucleotides used in this example.
[0078] In brief, the wells of polypropylene microtiter plates were
aminated by radio frequency plasma discharge treatment under an
ammonia atmosphere using a Plasma Science Model PS0150 RFPD
generator system. The aminated plates were then succinylated by
treatment with 0.1 M succinic anhydride in 0.1 M sodium acetate for
20 hrs, followed by 3 washes with 0.1 M sodium acetate and 3 washes
with isopropyl alcohol. Succinylated plates were then reacted for 2
hrs with 250 mM triazole in acetonitrile containing 3%
triethylamine, followed by 3 washes with acetonitrile. Capture
oligonucleotides were chemically coupled to the surface of the
activated plates by depositing approximately 10 nl spots of 20
.mu.M solution of the capture oligonucleotides containing about 0
to about 6 .mu.M of Cy3-labeled oligonucleotides, onto the bottom
surface of a microtiter plate in a 3.times.3 microarray pattern. A
Biomek 2000 high-density replicating tool (Beckman Coulter,
Fullerton, Calif.) or a ProSys Gantry System (Cartesian
Technologies, Irvine, Calif.) were used to deposit the spots in
microarrays.
[0079] The plates were incubated 16 hr at room temperature in a
humidified chamber. The plates were then `quenched` by incubating
in a 1% casein solution in carbonate buffer, pH 9.3, for 1 hour to
bind to any amino-reactive groups remaining on the plate surface.
The plates were then washed with water, followed by a wash with TE
buffer (10 mM Tris, pH 7.5, 1 mM EDTA).
[0080] The microarrays were illuminated with a 550 nm light source
and visualized with a charge coupled device camera ("CCD camera").
The CCD camera used was a Photometrics CoolSNAP camera (Roper
Scientific, Tucson, Ariz.) mounted with a 575 nm emission filter.
FIG. 1 shows the CCD camera picture illustrating the emissions from
labels of the capture oligonucleotides having concentrations of 6
.mu.M, 4 .mu.M, 2 .mu.M, 1 .mu.M, and 0 .mu.M of Cy3-labeled
oligonucleotides identified by Columns A, B, C, D, and E
respectively.
EXAMPLE 2
Immobilizing Capture Oligonucleotides with Directly Attached Cy3.5,
or Cy5, or Cy5.5 Labels onto a Microtiter Plate, and Detecting the
Directly Attached Cy3.5, or cy5, or cy5.5 Labels
[0081] In other experiments, the same procedure described in
Example 1 was used except that Cy3.5 or Cy5 or Cy5.5 labels were
used in place of Cy3 labels. At the 5' end, Cy3.5 or Cy5 or Cy5.5
labels (purchased from Glen research, Sterling, Va.) were coupled
using the standard protocol recommended by the supplier of the
reagents. The Cy3.5, or Cy5, or Cy5.5 labels were subsequently
detected using the CCD camera similar to the manner described in
Example 1. The difference being that each label's specific
excitation wavelength and emission wavelength for replaced the Cy3
wavelengths identified in Example 1.
EXAMPLE 3
Immobilizing Capture Oligonucleotides onto a Microtiter Plate,
Hybridizing Complementary Oligonucleotides with Directly Attached
Labels, and Detecting the Directly Attached Labels in the
Duplexes
[0082] A. Synthesis of Unlabeled 5'-3'-amino Capture
Oligonucleotide.
[0083] Example 1 (above) described the basic procedure for
synthesis of amino-oligonucleotide. That procedure was used in this
example except that unlabeled nucleotides were used for the
terminal base in place of a label.
[0084] B. Immobilization Of Capture Amino Oligonucleotide onto a
Microtiter Plate
[0085] The immobilization of capture oligonucleotides onto a
microtiter plate as used in this example is the procedure described
under Example 1. The plates were then `quenched` by incubating in a
1% casein solution in carbonate buffer, pH 9.3, for 1 hour to bind
to any amino-reactive groups remaining on the plate surface. The
plates were then washed with water, followed by a wash with TE
buffer (10 mM Tris, pH 7.5, 1 mM EDTA).
[0086] C. Synthesis Of Labeled Complementary Oligonucleotide
[0087] Example 1 (above) described the basic procedure for
synthesis of Cy3 dye labeled amino-oligonucleotides that was also
used in this example.
[0088] D. Mixing the Immobilized Capture Oligonucleotides with
Complementary Oligonucleotides Under Suitable Conditions to Form
Nucleic Acid Duplexes
[0089] 50 .mu.l of 10 nM complementary oligonucleotides in casein
buffer was added to the immobilized capture oligonucleotides. This
step was performed under conditions typical for a normal
immunoassay binding step (i.e. moderate salt and temperature
conditions), partly to protect the structure and integrity of the
antibody portion of the complex, and partly for ease of use.
[0090] E. Detection of Complementary Oligonucleotides to Determine
Immobilized Capture Oligonucleotides
[0091] The microarrays were illuminated with a 550 nm light source
and visualized with a CCD camera. The CCD camera used was a
Photometrics CoolSNAP camera (Roper Scientific, Tucson, Ariz.)
mounted with a 575 nm emission filter.
EXAMPLE 4
A Sandwich Assay Using Cy3 Labeled Capture Oligonucleotides, IL-8
Antibodies Conjugated to Complementary Oligonucleotides, Detector
Ligands with PBXL-1 Labels, and IL-8 Antigens
[0092] The procedure described in Example 1 was used to synthesize
and immobilize arrays of capture oligonucleotide with directly
attached Cy3 labels onto a microtiter plate. The complementary
oligonucleotides were synthesized by the same procedure as the
labeled oligonucleotides except that unlabeled nucleotides were
used for the terminal base, and attached to the capture
antibody.
[0093] The protocol for the attachment of oligonucleotide to
antibody, discussed in U.S. Pat. No. 5,648,213, which is
incorporated by reference herein, was used for this example. For
the IL-8 assay, the target ligand was IL-8 antigen, the capture
ligand was an IL-8 antibody conjugated to an oligonucleotide that
is complementary to capture oligonucleotides of an immobilized
array, and the detector ligands were IL-8 antibodies labeled with
PBXL-1 (a fluorescent dye purchased from Martek Biosciences Corp.;
Columbia, Md.). The detector ligands, IL-8 antibodies, were labeled
with PBXL-1 through a biotin-streptavidin interaction by mixing:
IL-8 detection antibodies that were covalently coupled to biotin
(as supplied by the manufacturer, R & D Systems); and PBXL-1
that was supplied as a streptavidin conjugate supplied by the
manufacturer, Martek.
[0094] 50 .mu.l of 10 nM of IL-8 antibody conjugated to an
oligonucleotide that is complementary to capture oligonucleotides
in casein buffer was added to the immobilized capture
oligonucleotides. This step was performed under conditions typical
for a normal immunoassay binding step (i.e. moderate salt and
temperature conditions), partly to protect the structure and
integrity of the antibody portion of the conjugate, and partly for
ease of use.
[0095] Concentrations of IL-8 antigen ranging from 1000-0 pg/ml per
well in casein buffer were added and reacted at 37.degree. C. for 1
hour and washed with wash buffer 3 times. The concentrations of
IL-8 antigen used in the assay were: 0 pg/ml, 5 pg/ml, 100 pg/ml,
and 1000 pg/ml.
[0096] Wells were incubated with 50 ng biotinylated IL-8 antibody
for 1 hr at 37.degree. C., washed 3 times with wash buffer, then
incubated with streptavidin-PBXL-1 for 1 hr at 37.degree. C., and
washed 3 times with wash buffer.
[0097] The plates were illuminated with a single excitation light
source of 550 nm. The plates were visualized with a CCD camera
mounted with a 675 nm emission filter to detect bound IL-8 detector
antibodies. The plates were also visualized with a CCD camera
mounted with a 575 nm emission filter to detect the immobilized
capture oligonucleotides.
[0098] FIG. 4 shows the CCD camera picture illustrating the
emissions from PBXL-1 labels of the bound IL-8 detector antibodies
for each concentrations of IL-8 antigen. On the right side of the
picture is the IL-8 antigen concentration, and top columns A, B, C,
D, and E of the picture respectively correspond to 6 .mu.M, 4
.mu.M, 2 .mu.M, 1 .mu.M, and 0 .mu.M of Cy3 labeled capture
oligonucleotides. Whereas, FIG. 1 shows the CCD camera picture
illustrating the emissions from Cy3 labeled capture
oligonucleotides from the same plate used in this example.
EXAMPLE 5
A Sandwich Assay Using Cy5.5 Labeled Capture Oligonucleotides, IL-8
Antibodies Conjugated to Complementary Oligonucleotides, Detector
Ligands with PBXL-1 Labels, and IL-8 Antigens
[0099] The procedure described in Example 1 was used to synthesize
and immobilize arrays of capture oligonucleotide with directly
attached Cy5.5 label, instead of the Cy3 label used in Example 1,
onto a microtiter plate. The complementary oligonucleotides were
synthesized by the same method as the labeled oligonucleotides
except that unlabeled nucleotides were used for the terminal base,
and attached to the capture antibody.
[0100] The protocol for attachment of complementary oligonucleotide
to IL-8 antibody for the IL-8 assay was discussed in U.S. Pat. No.
5,648,213, which is incorporated by reference herein, and was used
for this example. The target ligand was IL-8 antigen, the capture
ligand was an IL-8 antibody conjugated to an oligonucleotide that
is complementary to capture oligonucleotides of an immobilized
array, and the detector ligands were IL-8 antibodies labeled with
PBXL-1 (a fluorescent dye purchased from Martek Biosciences Corp.;
Columbia, Md.). The detector ligands, IL-8 antibodies, were labeled
with PBXL-1-1 through a biotin-streptavidin interaction by mixing:
IL-8 detector antibodies that were covalently coupled to biotin (as
supplied by the manufacturer, R & D Systems); and PBXL-1 that
was supplied as a streptavidin conjugate supplied by the
manufacturer, Martek.
[0101] 50 .mu.l of 10 nM of IL-8 antibody conjugated to an
oligonucleotide that is complementary to capture oligonucleotides
in casein buffer was added to the immobilized capture
oligonucleotides. This step was performed under conditions typical
for a normal immunoassay binding step (i.e. moderate salt and
temperature conditions), partly to protect the structure and
integrity of the antibody portion of the conjugate, and partly for
ease of use.
[0102] Concentrations of IL-8 antigen ranging from 250-0 pg/ml per
well in casein buffer were added and reacted at 37.degree. C. for 1
hour and washed with wash buffer 3 times. The concentrations of
IL-8 antigen used in the assay were: 0 pg/ml, 1 pg/ml, 2 pg/ml, 5
pg/ml, 10 pg/ml, 25 pg/ml, 100 pg/ml, and 250 pg/ml.
[0103] Wells were incubated with 50 ng biotinylated IL-8 antibody
for 1 hr at 37.degree. C., washed 3 times with wash buffer, then
incubated with streptavidin-PBXL-1 for 1 hr at 37.degree. C., and
washed 3 times with wash buffer.
[0104] The plates were illuminated using a light source with 680 nm
excitation filter and visualized with a CCD camera mounted with a
715 nm emission filter to detect Cy5.5 emission of immobilized
capture oligonucleotides. The plates were illuminated using a light
source with 550 nm excitation filter and visualized with a CCD
camera mounted with a 675 nm emission filter to detect PBXL-1
emission of bound IL-8 detector antibodies.
[0105] FIG. 5 shows the CCD camera picture illustrating the
emissions from the Cy5.5 labels of the capture oligonucleotides on
the left side, and emissions from PBXL-1 labels of the bound IL-8
detector antibodies on the right side.
EXAMPLE 6
A Sandwich Assay Using Capture Oligonucleotides; Cy5.5 Labeled
Complementary Oligonucleotides Conjugated to Antibodies; Detector
Ligands with Pbxl-1, and IL-2, IL-8, IL-12, Interferon-Gamma,
FGF-Basic, and GMCSF Antigens as Target Ligands
[0106] A. Synthesis of Unlabeled 5'-3'-amino Capture
Oligonucleotides
[0107] Example 1 (above) described the basic procedure for
synthesis of amino-oligonucleotide. That procedure was used in this
example except that unlabeled nucleotides were used for the
terminal base in place of a label.
[0108] B. Synthesis of Labeled Complementary Oligonucleotide
[0109] Example 3 (above) described the basic procedure for
synthesis of dye labeled amino-oligonucleotides that was also used
in this example. In this example, Cy5.5 was the directly attached
label, for complementary oligonucleotides, used instead of Cy3
label.
[0110] C. Seven Different Capture/Complementary Oligonucleotide
Pairs Were Prepared and Used from a List of Preferred
Oligonucleotide Pairs
[0111] A different capture and complementary oligonucleotide
sequence was used for each of the target ligands tested in this
example: IL-2, IL-8, IL-12, Interferon-gamma, FGF-basic, GMCSF
antigens, and an internal control antigen, used in this example.
Seven different preferred pairs of capture and complementary
oligonucleotides were used in this example. The seven pairs were
selected from a list of preferred sequence pairs because the
sequences do not substantially cross hybridize when used together
in an assay. The list of preferred pair was previously
identified.
[0112] D. Immobilization of Capture Amino Oligonucleotide in a
Microarray onto a Microtiter Plate
[0113] The immobilization of capture oligonucleotides onto a
microtiter plate as used in this example was the basic procedure
described under Example 1 and 3. In this example, each of the six
different capture oligonucleotides were immobilized at
predetermined spots in the microarray to correspond to the specific
antigen or target ligand.
[0114] A different antigen (not one of the six target antigens),
chicken ovalbumin, was used as an internal control, and the seventh
different capture oligonucleotide was immobilized in three corner
positions. This internal control is shown in the picture in FIG. 6
as the brightest PBXL-1 emissions at three corner positions in the
0 pg/ml and 2 pg/ml concentrations of the target antigens.
[0115] E. Preparation of Capture Ligands and Detector Ligands
[0116] The capture ligand used in this example for an IL-8 antigen
was an IL-8 antibody. The IL-8 antibody was conjugated to an
oligonucleotide that is complementary to its respective capture
oligonucleotide in immobilized microarray. The detector ligands for
IL-8 antigens were IL-8 antibodies labeled with PBXL-1 (a
fluorescent dye purchased from Martek Biosciences Corp.; Columbia,
Md.). The detector ligands, IL-8 antibodies, were labeled with
PBXL-1-1 through a biotin-streptavidin interaction by mixing: IL-8
detector antibodies that were covalently coupled to biotin (as
supplied by the manufacturer, R & D Systems); and PBXL-1 that
was supplied as a streptavidin conjugate supplied by the
manufacturer, Martek.
[0117] The capture ligands and detector ligands for the remaining
antigens were made using the same process described for the IL-8.
In place of IL-8 antibodies, the respective antibodies for IL-2,
IL-12, Interferon-gamma, FGF-basic, and GMCSF were used to create
the necessary components of the sandwich.
[0118] F. IL-8 Assay Protocol Used
[0119] The basic protocol for the IL 8 assay discussed in Example 4
was used for this example. However, in addition to IL-8 antigen,
the following target ligands were also used in this example: IL-2,
IL-12, Interferon-gamma, FGF-basic, and GMCSF antigens.
[0120] The first step in the assay was the immobilization of the
capture oligonucleotides in a predetermined microarray as described
in the section on immobilizing capture oligonucleotides.
[0121] The second step was the addition of about 50 .mu.l of 10 nM
capture ligands having complementary oligonucleotides, prepared as
described in the preceding section, in casein buffer to the
immobilized capture oligonucleotides in the wells of the microtiter
plate. This step was performed under conditions typical for a
normal immunoassay binding step (i.e. moderate salt and temperature
conditions), partly to protect the structure and integrity of the
antibody portion of the conjugate, and partly for ease of use.
[0122] The third step involved the addition of the specific
concentrations of IL-2, IL-8, IL-12, Interferon-gamma, FGF-basic,
and GMCSF antigens to the wells. The concentrations of each antigen
used in the assay were: 0 pg/ml, 2 pg/ml, 10 pg/ml, 100 pg/ml, 400
pg/ml, and 1000 pg/ml. The added antigen in casein buffer were
reacted at 37.degree. C. for 1 hour and washed with wash buffer 3
times.
[0123] The fourth step was the addition of the detector ligands for
each antigen, prepared as described in the preceding section, to
the wells of the microtiter plate.
[0124] Wells were incubated with biotinylated antibodies for each
antigen, then PBXL-1 as described in examples above.
[0125] The plates were illuminated using a light source with 680 nm
excitation filter and visualized with a CCD camera mounted with a
715 nm emission filter to detect Cy5.5 emission of immobilized
capture oligonucleotides. The plates were illuminated using a light
source with 550 nm excitation filter and visualized with a CCD
camera mounted with a 675 nm emission filter to detect PBXL-1
emission of bound IL-2, IL-8, IL-12, Interferon-gamma, FGF-basic,
and GMCSF antigens.
[0126] FIG. 6 shows the CCD camera picture illustrating the Cy5.5
emissions from the complementary oligonucleotides on the left side,
and PBXL-1 emissions from the bound detector antibodies for IL-2,
IL-8, IL-12, Interferon-gamma, FGF-basic, and GMCSF antigens on the
right side. The analyte (or antigen) concentration is identified to
the right of the picture illustrating the PBXL-1 emissions.
[0127] Having thus described the invention, it should be apparent
that numerous modifications and adaptations may be resorted to
without departing from the scope and fair meaning of the instant
invention as set forth hereinabove and as described hereinbelow by
the claims.
[0128] Although the present invention has been described in
considerable detail with reference to certain preferred versions
thereof, other versions are possible. Therefore, the spirit and
scope of the appended claims should not be limited to the
description of the preferred versions described herein.
[0129] All features disclosed in the specification, including the
claims, abstracts, and drawings, and all the steps in any method or
process disclosed, may be combined in any combination, except
combinations where at least some of such features and/or steps are
mutually exclusive. Each feature disclosed in the specification,
including the claims, abstract, and drawings, can be replaced by
alternative features serving the same, equivalent or similar
purpose, unless expressly stated otherwise. Thus, unless expressly
stated otherwise, each feature disclosed is one example only of a
generic series of equivalent or similar features.
[0130] Any element in a claim that does not explicitly state
"means" for performing a specified function or "step" for
performing a specified function, should not be interpreted as a
"means" or "step" clause as specified in 35 U.S.C. .sctn. 112.
Sequence CWU 1
1
24 1 30 DNA Artificial completely synthesized 1 gcttaccgaa
tacggcttgg agaacctatc 30 2 30 DNA Artificial completely synthesized
2 gataggttct ccaagccgta ttcggtaagc 30 3 30 DNA Artificial
completely synthesized 3 gcgtggtccg cgatcttcct acgattgatg 30 4 30
DNA Artificial completely synthesized 4 catcaatcgt aggaagatcg
cggaccacgc 30 5 30 DNA Artificial completely synthesized 5
tttgaggttt cggagcgttc cgtgcatcgc 30 6 30 DNA Artificial completely
synthesized 6 gcgatgcacg gaacgctccg aaacctcaaa 30 7 30 DNA
Artificial completely synthesized 7 ctcatgaagg ccgtcgggaa
attccaagtt 30 8 30 DNA Artificial completely synthesized 8
aacttggaat ttcccgacgg ccttcatgag 30 9 30 DNA Artificial completely
synthesized 9 tggattccgt tatcaccatt tggaccctgc 30 10 30 DNA
Artificial completely synthesized 10 gcagggtcca aatggtgata
acggaatcca 30 11 30 DNA Artificial completely synthesized 11
tacgctccca gtgtgatcac caaagcttac 30 12 30 DNA Artificial completely
synthesized 12 gtaagctttg gtgatcacac tgggagcgta 30 13 30 DNA
Artificial completely synthesized 13 agactgaact accggcggtt
gcactactaa 30 14 30 DNA Artificial completely synthesized 14
ttagtagtgc aaccgccggt agttcagtct 30 15 30 DNA Artificial completely
synthesized 15 gccaaacacg cccaatcact gttacatgtc 30 16 30 DNA
Artificial completely synthesized 16 gacatgtaac agtgattggg
cgtgtttggc 30 17 30 DNA Artificial completely synthesized 17
gcttgacgtc taccaccgtg aacataagga 30 18 30 DNA Artificial completely
synthesized 18 tccttatgtt cacggtggta gacgtcaagc 30 19 30 DNA
Artificial completely synthesized 19 tcgccaacgt agctgtgcta
cagttgattc 30 20 30 DNA Artificial completely synthesized 20
gaatcaactg tagcacagct acgttggcga 30 21 30 DNA Artificial completely
synthesized 21 gcagcggcta aaccttgaga tcgaatggaa 30 22 30 DNA
Artificial completely synthesized 22 ttccattcga tctcaaggtt
tagccgctgc 30 23 30 DNA Artificial completely synthesized 23
tgcgatatac tccatgcctc tcttggcgga 30 24 30 DNA Artificial completely
synthesized 24 tccgccaaga gaggcatgga gtatatcgca 30
* * * * *