U.S. patent application number 13/272350 was filed with the patent office on 2012-06-21 for reagent storage in an assay device.
This patent application is currently assigned to MESO SCALE TECHNOLOGIES, LLC. Invention is credited to Sudeep Kumar, George Sigal, Michael Tsionsky.
Application Number | 20120157332 13/272350 |
Document ID | / |
Family ID | 45938967 |
Filed Date | 2012-06-21 |
United States Patent
Application |
20120157332 |
Kind Code |
A1 |
Kumar; Sudeep ; et
al. |
June 21, 2012 |
Reagent Storage in an Assay Device
Abstract
The invention relates to methods for conducting binding assays
in an assay device that includes one or more storage and use zone.
The storage zones of the assay device are configured to house one
or more reagents used in an assay conducted in the use zone of the
device.
Inventors: |
Kumar; Sudeep;
(Gaithersburg, MD) ; Sigal; George; (Rockville,
MD) ; Tsionsky; Michael; (Derwood, MD) |
Assignee: |
MESO SCALE TECHNOLOGIES,
LLC
Gaithersburg
MD
|
Family ID: |
45938967 |
Appl. No.: |
13/272350 |
Filed: |
October 13, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61455112 |
Oct 14, 2010 |
|
|
|
Current U.S.
Class: |
506/9 ; 422/547;
422/552; 422/554; 436/501 |
Current CPC
Class: |
G01N 33/54386 20130101;
G01N 33/5306 20130101; G01N 2446/00 20130101; G01N 2458/30
20130101; C12Q 1/6823 20130101; C12Q 1/6834 20130101; C12Q 1/6832
20130101 |
Class at
Publication: |
506/9 ; 422/547;
422/552; 436/501; 422/554 |
International
Class: |
C40B 30/04 20060101
C40B030/04; G01N 33/566 20060101 G01N033/566; B01L 3/00 20060101
B01L003/00 |
Claims
1. An assay device comprising (a) a storage zone comprising a
surface-reagent complex confined to said storage zone, said
surface-reagent complex comprising (i) a reagent linked to a first
targeting agent; and (ii) a surface linked to a second targeting
agent, wherein said reagent and said surface are linked, in said
surface-reagent complex, via a releasable binding interaction
between said first and second targeting agents; and (b) one or more
use zones each configured to use said reagent in an assay for an
analyte of interest in a sample.
2. The assay device of claim 1 wherein said one or more use zones
each comprise an additional reagent used in said assay.
3. The assay device of claim 1 wherein said reagent is a binding
reagent that binds a component of said assay conducted in said use
zone.
4. The assay device of claim 3 wherein said binding reagent binds
said analyte.
5. The assay device of claim 2 wherein said additional reagent
binds said analyte.
6. The assay device of claim 4 wherein said binding reagent binds a
complex formed between said additional reagent and said
analyte.
7. The assay device of claim 6 wherein said one or more use zones
each comprise a solid support and said additional reagent is bound
to said solid support.
8. The assay device of claim 1 wherein said reagent comprises a
detectable label.
9. The assay device of claim 8 wherein said detectable label is an
ECL label.
10. The assay device of claim 1 wherein said storage zone and said
one or more use zones are in fluidic communication along a fluid
path.
11. The assay device of claim 1 wherein said one or more use zones
each comprise two or more assay regions each configured to use said
reagent in one or more assays conducted with said sample in said
assay device.
12. The assay device of claim 11 wherein a first assay region of
said one or more use zones is configured to conduct an assay for a
first analyte of interest in said sample and an additional assay
region in said one or more use zones is configured to conduct an
assay for an additional analyte of interest in said sample.
13. The assay device of claim 11 wherein a first assay region of
said one or more use zones is configured to conduct a first assay
for said analyte of interest in said sample and an additional assay
region of said one or more use zones is configured to conduct a
second assay for said analyte of interest in said sample.
14. The assay device of claim 11 wherein each of said two or more
assay regions comprise an additional reagent used in said
assay.
15. The assay device of claim 14 wherein said additional reagent is
an additional binding reagent.
16. The assay device of claim 11 wherein said one or more use zones
each comprise an array of said two or more assay regions.
17. The assay device of claim 1 wherein said surface is a
particle.
18. The assay device of claim 1 wherein said surface is roughened
such that the surface area accessible to a component capable of
binding to said surface is at least three-fold larger than the
surface area of a flat surface.
19. The assay device of claim 1 wherein said surface is roughened
such that the surface area accessible to a component capable of
binding to said surface is at least two-fold larger than the
surface area of a flat surface.
20. The assay device of claim 1 wherein said surface comprises a
composite material including exposed particles distributed in a
matrix.
21. The assay device of claim 20 wherein said composite material
comprises carbon particles, graphitic particles, or carbon
nanotubes.
22. The assay device of claim 20 wherein said composite is
etched.
23. The assay device of claim 20 wherein said surface comprises one
or more indentations and/or raised features.
24. The assay device of claim 1 wherein said surface comprises a
hydrogel.
25. The assay device of claim 1 wherein said reagent is released
from said surface-reagent complex by subjecting said storage zone
to increased or decreased temperature, pH changes, an electric
potential, a change in ionic strength, competition, and
combinations thereof.
26. The assay device of claim 25 wherein said reagent is released
by subjecting said storage zone to increased temperature.
27. The assay device of claim 26 wherein said increased temperature
exceeds the melting temperature of said binding interaction.
28. The assay device of claim 1 wherein said assay device is a
cartridge.
29. The assay device of claim 1 wherein said assay device is a
multi-well assay plate and said use zone is positioned within a
well of said assay plate.
30. The assay device of claim 29 wherein said storage zone is
located on a supplemental surface of said well that does not
overlap with said use zone.
31. The assay device of claim 1 wherein said device is configured
to conduct a multiplexed measurement.
32. A method of conducting an assay for an analyte of interest in a
sample, wherein said method is conducted in an assay device
comprising: (a) a storage zone comprising a surface-reagent complex
confined to said storage zone, said surface-reagent complex
comprising (i) a reagent linked to a first targeting agent; and
(ii) a surface linked to a second targeting agent, wherein said
reagent and said surface are linked, in said surface-reagent
complex, via a releasable binding interaction between said first
and second targeting agents; and (b) one or more use zones each
configured to use said reagent in an assay for an analyte of
interest in a sample; said method comprising: (x) introducing said
sample to said one or more use zones; (y) subjecting said storage
zone to a condition that releases said reagent from said
surface-reagent complex; (z) transferring said reagent from said
storage zone to said one or more use zones; and (xx) conducting
said assay in said one or more use zones with said reagent.
33. The method of claim 32 wherein said condition is selected from
increased or decreased temperature, pH changes, applying an
electrical potential, a change in ionic strength, competition, and
combinations thereof.
34. The method of claim 32 wherein said condition comprises
increasing the temperature of said storage zone.
35. The method of claim 34 wherein said increased temperature
exceeds the melting temperature of said binding interaction.
36. The method of claim 32 wherein said reagent is a binding
reagent that binds said analyte and said method further comprises
detecting the amount of analyte bound to said binding reagent in
said one or more use zones.
37. The method of claim 36 wherein said reagent comprises a
detectable label and said detecting step comprises detecting the
presence or absence of said detectable label in said one or more
use zones.
38. The method of claim 37 wherein said detectable label is an ECL
label and said detecting step comprises detecting
electrochemiluminescence emitted in said one or more use zones.
39. The method of claim 32 wherein said storage zone and said one
or more use zones are in fluidic communication along a fluid path
and said transferring step (z) comprises transferring said reagent
from said storage zone via said fluid path to said one or more use
zones.
40. The method of claim 32 wherein said use zone comprises two or
more assay regions each configured to use said reagent in one or
more assays conducted with said sample in said assay device, said
method further comprising the step of conducting a plurality of
assays in said one or more use zones with said reagent.
41. The method of claim 40 wherein said one or more use zones each
comprise a first assay region configured to conduct an assay for a
first analyte of interest in said sample and an additional assay
region configured to conduct an assay for an additional analyte of
interest in said sample, said method comprising: (x) introducing
said sample to said one or more use zones; (y) subjecting said
storage zone to a condition that releases said reagent from said
surface-reagent complex; (z) transferring said reagent from said
storage zone to said first assay region and said second assay
region; (xx) conducting said assays in said first and second assay
regions, respectively.
42. The method of claim 41 wherein said assays are conducted
simultaneously.
43. The method of claim 41 wherein said assays are conducted
sequentially.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims benefit of U.S. Provisional
Application No. 61/455,112, filed Oct. 14, 2010 and reference is
made to U.S. application Ser. No. 12/757,685, filed Apr. 9, 2010,
the disclosures of which are incorporated herein by reference in
its entirety.
FIELD OF THE INVENTION
[0002] Improved methods for conducting binding assays are provided.
These methods include a pre-concentration step to improve assay
performance, for example, by increasing the concentration of
analyte in the sample and/or by reducing the concentration of
extraneous materials that may be present in the sample which may
hinder the performance of the binding assay.
BACKGROUND OF THE INVENTION
[0003] A substantial body of literature has been developed
concerning techniques that employ binding reactions, e.g.,
antigen-antibody reactions, nucleic acid hybridization and
receptor-ligand reactions, for the sensitive measurement of
analytes of interest in samples. The high degree of specificity in
many biochemical binding systems has led to many assay methods and
systems of value in a variety of markets including basic research,
human and veterinary diagnostics, environmental monitoring and
industrial testing. The presence of an analyte of interest may be
measured by directly measuring the participation of the analyte in
a binding reaction. In some approaches, this participation may be
indicated through the measurement of an observable label attached
to one or more of the binding materials.
[0004] There is always a desire to improve binding assays and
devices used to conduct binding assays by increasing the signal
obtained from a binding event, reducing non-specific binding,
and/or improving measurement accuracy and precision, especially
when analyzing complex biological samples.
SUMMARY OF THE INVENTION
[0005] The invention provides an assay device including (a) a
storage zone comprising a surface-reagent complex confined to the
storage zone, the surface-reagent complex comprising (i) a reagent
linked to a first targeting agent; and (ii) a surface linked to a
second targeting agent, wherein the reagent and the surface are
linked, in the surface-reagent complex, via a releasable binding
interaction between the first and second targeting agents; and (b)
one or more use zones each configured to use the reagent in an
assay for an analyte of interest in a sample. The assay device of
the invention may include one or more storage zones and/or one or
more use zones. Additionally, the storage zone may also include two
or more surface-reagent complexes, each including a distinct assay
reagent that may be used in an assay conducted in the one or more
use zones. For example, the storage zone also includes a second
surface-reagent complex confined to the storage zone, the second
surface-reagent complex comprising (i) a second reagent linked to a
third targeting agent; and (ii) a second surface linked to a fourth
targeting agent, wherein the second reagent and the second surface
are linked, in the second surface-reagent complex, via a second
releasable binding interaction between the third and fourth
targeting agents; and the one or more use zones are further
configured to use the second reagent in the assay. The use zones
may each comprise two or more assay regions each configured to use
the reagent(s) stored in the storage zone in one or more assays
conducted with a sample in the assay device.
[0006] The device may be used to conduct a plurality of assays for
one or more analytes present in the sample, e.g., a first assay
region of the one or more use zones are each configured to conduct
an assay for a first analyte of interest in the sample and an
additional assay region in the one or more use zones is configured
to conduct an assay for an additional analyte of interest in the
sample. Alternatively, a first assay region of the one or more use
zones is configured to conduct a first assay for the analyte of
interest in the sample and an additional assay region of the one or
more use zones is configured to conduct a second assay for the
analyte of interest in the sample.
[0007] The invention also provides a multiplexed assay device
comprising (a) a storage zone comprising a surface-reagent complex
confined to the storage zone, the surface-reagent complex
comprising (i) a reagent linked to a first targeting agent; and
(ii) a surface linked to a second targeting agent, wherein the
reagent and the surface are linked, in the surface-reagent complex,
via a releasable binding interaction between the first and second
targeting agents; and (b) one or more use zones each comprising a
plurality of assay regions configured to use the reagent in a
multiplexed assay for a plurality of analytes in a sample. A first
assay region of the one or more use zones is configured to conduct
an assay for a first analyte of interest in the sample and an
additional assay region in the one or more use zones is configured
to conduct an assay for an additional analyte of interest in the
sample. In addition, the storage zone may further comprises a
second surface-reagent complex confined to the storage zone, the
second surface-reagent complex comprising (i) a second reagent
linked to a third targeting agent; and (ii) a second surface linked
to a fourth targeting agent, wherein the second reagent and the
second surface are linked, in the second surface-reagent complex,
via a second releasable binding interaction between the third and
fourth targeting agents; and the one or more use zones are further
configured to use the second reagent in the multiplexed assay.
[0008] The invention further provides a method of conducting an
assay in an assay device as described herein, including the steps:
(x) introducing the sample to the one or more use zones; (y)
subjecting the storage zone to a condition that releases the
reagent from the surface-reagent complex; (z) transferring the
reagent from the storage zone to the one or more use zones; and
(xx) conducting the assay in the one or more use zones with the
reagent. If the use zones are each configured to use a second
reagent in an assay, the method further comprises, prior to the
conducting step, subjecting the storage zone to an additional
condition that releases the second reagent from the second
surface-reagent complex; and transferring the second reagent from
the storage zone to the one or more use zones.
[0009] A method of using such an assay device may also include the
steps of (x) introducing the sample to the one or more use zones;
(i) subjecting the storage zone to a condition that releases the
reagent from the surface-reagent complex; (ii) subjecting the
storage zone to a condition that releases the second reagent from
the second surface-reagent complex; (y) transferring the reagent
from the storage zone to the first assay region; (z) transferring
the second reagent from the storage zone to the second assay
region; (xx) conducting an assay in the first assay region with the
reagent; and (yy) conducting an assay in the second assay region
with the second reagent. The transferring steps may be simultaneous
or sequential. Similarly, the conducting steps may also be
simultaneous or sequential.
[0010] In addition, the invention provides a method of using an
assay device of the invention including the steps: (x) introducing
the sample to the one or more use zones; (y) subjecting the storage
zone to a condition that releases the reagent from the
surface-reagent complex; (z) transferring the reagent from the
storage zone to the first assay region and the second assay region;
(xx) conducting the assays in the first and second assay regions,
respectively. The assays may be conducted simultaneously or
sequentially.
[0011] In another embodiment, the assay device of the invention may
be used in the conduct of an assay by (x) introducing the sample to
the one or more use zones via the storage zone; (y) adding a
diluent to the storage zone and (i) subjecting the storage zone to
a condition that releases the reagent from the surface-reagent
complex; (ii) subjecting the storage zone to an additional
condition that releases the second reagent from the second
surface-reagent complex; (z) transferring the reagent and the
second reagent from the storage zone to the first and second assay
regions; (xx) conducting the assays in the first and second assay
regions. The assays and/or transfer steps may be conducted
simultaneously and/or sequentially.
[0012] Still further, the assay device may be used in an assay by
(x) introducing the sample to the one or more use zones via the
storage zone; (y) adding a diluent to the storage zone and (i)
subjecting the storage zone to a condition that releases the
reagent from the surface-reagent complex; (ii) subjecting the
storage zone to an additional condition that releases the second
reagent from the second surface-reagent complex; (z) transferring
the reagent from the storage zone to the first assay region; (xx)
transferring the second reagent from the storage zone to the second
assay region; (yy) conducting the assays in the first and second
assay regions. The assays and/or transfer steps may be conducted
simultaneously and/or sequentially.
[0013] Moreover, the invention provides a multiplexed assay device
comprising (a) a storage zone comprising (i) a first reagent linked
to a surface in the storage zone via a first releasable binding
interaction; (ii) a second reagent linked to a second surface in
the storage zone via a second releasable binding interaction; (b) a
first use zone configured to use the first reagent in an assay for
a first analyte; and (c) a second use zone configured to use the
second reagent in an assay for a second analyte. The first
releasable binding interaction comprises a linkage between a first
targeting agent and a second targeting agent, wherein the first
targeting agent is linked to the reagent and the second targeting
agent is linked to the surface. Moreover, the reagent and the
surface are linked to form a surface-reagent complex, wherein the
surface-reagent complex is confined to the storage zone. The second
releasable binding interaction comprises a linkage between a third
targeting agent and a fourth targeting agent, wherein the third
targeting agent is linked to the second reagent and the fourth
targeting agent is linked to the second surface, and the second
reagent and the second surface are linked to form a second
surface-reagent complex, wherein the second surface-reagent complex
is confined to the storage zone. Such a multiplexed assay device
comprises a fluidic network, such that the storage zone and the
first and second use zones are in fluidic communication, wherein
the network is configured to direct fluid in the storage zone to
the first use zone, the second use zone, or both. The network is
configured to direct fluid to the first use zone and the second use
zone sequentially or simultaneously. The first and second reagents
are each confined in the storage zone to distinct regions of the
storage zone. The first and second releasable binding interactions
require the same or different conditions to release the first and
second reagents respectively, from the first and second surfaces of
the storage zone, e.g., subjecting the storage zone to increased or
decreased temperature, pH changes, an electric potential, a change
in ionic strength, competition, and combinations thereof. Moreover,
each of the first and second use zones comprise a plurality of
assay regions each configured to use the first and second reagents
in a multiplexed assay for a plurality of different analytes in a
sample.
[0014] Also provided is a method of conducting a multiplexed assay
using the multiplexed assay device described herein including (a)
introducing a sample comprising the first and second analytes to
the first and second use zones; (b) subjecting the storage zone to
a condition that releases the first reagent from the storage zone;
(c) transferring the first reagent from the storage zone to at
least one of the first and second use zones; and (d) conducting one
or more assays for at least one of the first and second analytes.
The method may also include the steps of subjecting the storage
zone to an additional condition that releases the second reagent
from the storage zone and transferring the second reagent from the
storage zone to at least one of the first and second use zones, and
optionally washing at least one of the first and second use zone
prior to the transferring step.
[0015] Also provided is a method of conducting a multiplexed assay
in a multiplexed assay device including (a) introducing a sample
comprising the first and second analytes to the first and second
use zones; (b) subjecting the storage zone to a condition that
releases the first reagent from the storage zone; (c) transferring
the first reagent from the storage zone to the first use zone; (d)
subjecting the storage zone to a condition that releases the second
reagent from the storage zone; (e) transferring the second reagent
from the storage zone to the second use zone; and (f) conducting
assays for the first and second analytes in the first and second
use zones. The method may also include washing the first and second
use zones prior to the transferring step (c), and the assays may be
conducted simultaneously or sequentially.
BRIEF DESCRIPTION OF DRAWINGS
[0016] The accompanying drawings are provided to illustrate rather
than limit the scope of the invention. Throughout the accompanying
Figures, "P" refers to a particle to which one or more moieties are
attached; "S" refers to a first solid phase; "A" refers to a target
analyte; "C" refers to contaminants; and "*" refers to a detectable
label linked to an assay component.
[0017] FIGS. 1(a)-1(e) illustrate various assay formats in which a
particle is used as an assay component.
[0018] FIGS. 2(a)-2(b) illustrate various assay formats in which a
first solid phase is used as an assay component.
[0019] FIGS. 3(a)-3(e) illustrate various assay formats in which a
particle is used as an assay component, to which a targeting agent
is linked.
[0020] FIGS. 4(a)-4(b) illustrate various assay formats in which a
first solid phase is used as an assay component, to which a
targeting agent is linked.
[0021] FIGS. 5(a)-5(b) illustrates one embodiment of the present
invention. FIG. 5(a) shows magnetic concentration of analytes using
colloids coated with anti-antibodies against the analytes and also
coated with ECL labels. Multiple antibodies may be used to bind
different analytes. FIG. 5(b) shows detection of the
analyte-colloid complexes in a sandwich immunoassay format.
[0022] FIGS. 6(a)-6(b) illustrate two alternative competitive
immunoassays according to the methods of the present invention.
[0023] FIG. 7(a)-7(c) illustrate three alternative embodiments of
an assay device include one or more storage zones and one or more
use zones. FIGS. 7(a)-(b) show an assay device including one
storage zone that houses a surface-reagent complex that supplies
reagent to use zones 1 and 2, while FIG. 7(c) shows an assay device
including multiple storage zones that each lead to a use zone. In
FIG. 7(c), sample and liquid reagent compartments in the assay
device are in fluid communication with the storage and use
zones.
[0024] FIG. 8(a)-(f) illustrate the use of an alternate assay
device of the present invention.
DETAILED DESCRIPTION OF THE INVENTION
[0025] The present invention provides improved solid phase binding
assays that include a collection, separation and/or release step.
The methods of the present invention improve assay performance by
allowing for pre-concentration of an analyte in a sample and/or by
reducing or eliminating the amount of contaminants in a sample that
may hinder the performance of the assay, e.g., by interfering with
the detection step and/or by non-specifically binding with one or
more of the components in the mixture.
(i) Samples/Analytes
[0026] Examples of samples that may be analyzed by the methods of
the present invention include, but are not limited to food samples
(including food extracts, food homogenates, beverages, etc.),
environmental samples (e.g., soil samples, environmental sludges,
collected environmental aerosols, environmental wipes, water
filtrates, etc.), industrial samples (e.g., starting materials,
products or intermediates from an industrial production process),
human clinical samples, veterinary samples and other samples of
biological origin. Biological samples that may be analyzed include,
but are not limited to, feces, mucosal swabs, physiological fluids
and/or samples containing suspensions of cells. Specific examples
of biological samples include blood, serum, plasma, feces, mucosal
swabs, tissue aspirates, tissue homogenates, cell cultures and cell
culture supernatants (including cultures of eukaryotic and
prokaryotic cells), urine, saliva, sputum, and cerebrospinal
fluid.
[0027] Analytes that may be measured using the methods of the
invention include, but are not limited to proteins, toxins, nucleic
acids, microorganisms, viruses, cells, fungi, spores,
carbohydrates, lipids, glycoproteins, lipoproteins,
polysaccharides, drugs, hormones, steroids, nutrients, metabolites
and any modified derivative of the above molecules, or any complex
comprising one or more of the above molecules or combinations
thereof. The level of an analyte of interest in a sample may be
indicative of a disease or disease condition or it may simply
indicate whether the patient was exposed to that analyte.
[0028] The assays of the present invention may be used to determine
the concentration of one or more, e.g., two or more analytes in a
sample. Thus, two or more analytes may be measured in the same
sample. Panels of analytes that can be measured in the same sample
include, for example, panels of assays for analytes or activities
associated with a disease state or physiological conditions.
Certain such panels include panels of cytokines and/or their
receptors (e.g., one or more of TNF-alpha, TNF-beta, IL1-alpha,
IL1-beta, IL2, IL4, IL6, IL-10, IL-12, IFN-y, etc.), growth factors
and/or their receptors (e.g., one or more of EGF, VGF, TGF, VEGF,
etc.), drugs of abuse, therapeutic drugs, vitamins, pathogen
specific antibodies, auto-antibodies (e.g., one or more antibodies
directed against the Sm, RNP, SS-A, SS-alpha, J0-1, and Scl-70
antigens), allergen-specific antibodies, tumor markers (e.g., one
or more of CEA, PSA, CA-125 II, CA 15-3, CA 19-9, CA 72-4, CYFRA
21-1, NSE, AFP, etc.), markers of cardiac disease including
congestive heart disease and/or acute myocardial infarction (e.g.,
one or more of Troponin T, Troponin I, myoglobin, CKMB,
myeloperoxidase, glutathione peroxidase, .beta.-natriuretic protein
(BNP), alpha-natriuretic protein (ANP), endothelin, aldosterone,
C-reactive protein (CRP), etc.), markers associated with hemostasis
(e.g., one or more of Fibrin monomer, D-dimer,
thrombin-antithrombin complex, prothrombin fragments 1 & 2,
anti-Factor Xa, etc.), markers of acute viral hepatitis infection
(e.g., one or more of IgM antibody to hepatitis A virus, IgM
antibody to hepatitis B core antigen, hepatitis B surface antigen,
antibody to hepatitis C virus, etc.), markers of Alzheimers Disease
(alpha-amyloid, beta-amyloid, A.beta. 42, A.beta. 40, A.beta. 38,
A.beta. 39, A.beta. 37, A.beta. 34, tau-protein, etc.), markers of
osteoporosis (e.g., one or more of cross-linked Nor C-telopeptides,
total deoxypyridinoline, free deoxypyridinoline, osteocalcin,
alkaline phosphatase, C-terminal propeptide of type I collagen,
bone-specific alkaline phosphatase, etc.), markers of fertility
state or fertility associated disorders (e.g., one or more of
Estradiol, progesterone, follicle stimulating hormone (FSH),
lutenizing hormone (LH), prolactin, hCG, testosterone, etc.),
markers of thyroid disorders (e.g., one or more of thyroid
stimulating hormone (TSH), Total T3, Free T3, Total T4, Free T4,
and reverse T3), and markers of prostrate cancer (e.g., one or more
of total PSA, free PSA, complexed PSA, prostatic acid phosphatase,
creatine kinase, etc.). Certain embodiments of invention include
measuring, e.g., one or more, two or more, four or more or 10 or
more analytes associated with a specific disease state or
physiological condition (e.g., analytes grouped together in a
panel, such as those listed above; e.g., a panel useful for the
diagnosis of thyroid disorders may include e.g., one or more of
thyroid stimulating hormone (TSH), Total T3, Free T3, Total T4,
Free T4, and reverse T3).
[0029] The methods of the present invention are designed to allow
detection of a wide variety of biological and biochemical agents,
as described above. In one embodiment, the methods may be used to
detect pathogenic and/or potentially pathogenic virus, bacteria and
toxins including biological warfare agents ("BWAs") in a variety of
relevant clinical and environmental matrices, including and without
limitation, blood, sputum, stool, filters, swabs, etc. A
non-limiting list of pathogens and toxins that may be analyzed
(alone or in combination) using the methods of the present
invention is Bacillus anthracia (anthrax), Yersinia pestis
(plague), Vibrio cholerae (cholera), Francisella tularensis
(tularemia), Brucella spp. (Brucellosis), Coxiella burnetii (Q
fever), orthopox viruses including variola virus (smallpox), viral
encephalitis, Venezuelan equine encephalitis virus (VEE), western
equine encephalitis virus (WEE), eastern equine encephalitis virus
(EEE), Alphavirus, viral hemorrhagic fevers, Arenaviridae,
Bunyaviridae, Filoviridae, Flaviviridae, Ebola virus,
staphylococcal enterotoxins, ricin, botulinum toxins, Clostridium
botulinum, mycotoxin, Fusarium, Myrotecium, Cephalosporium,
Trichoderma, Verticimonosporium, Stachybotrys, glanders, wheat
fungus, Bacillus globigii, Serratia marcescens, yellow rain,
trichothecene mycotoxins, Salmonella typhimurium, aflatoxin,
Xenopsylla cheopis, Diamanus montanus, alastrim, monkeypox,
Arenavirus, Hantavirus, Lassa fever, Argentine hemorrhagic fevers,
Bolivian hemorrhagic fevers, Rift Valley fever virus, Crimean-Congo
virus, Hanta virus, Marburg hemorrhagic fevers, yellow fever virus,
dengue fever viruses, influenza (including human and animal strains
including H5N1 avian influenza), human immunodeficiency viruses I
and II (HIV I and II), hepatitis A, hepatitis B, hepatitis C,
hepatitis (non-A, B or C), Enterovirus, Epstein-Barr virus,
Cytomegalovirus, herpes simplex viruses, Chlamydia trachomatis,
Neisseria gonorrheae, Trichomonas vaginalis, human papilloma virus,
Treponema pallidum, Streptococcus pneumonia, Haemophilus
influenzae, Mycoplasma pneumoniae, Chlamydophila pneumoniae,
Legionella pneumophila, Staphylococcus aureus, Moraxella
catarrhalis, Streptococcus pyogenes, Clostridium difficile,
Neisseria meningitidis, Klebsiella pneumoniae, Mycobacterium
tuberculosis, coronavirus, Coxsackie A virus, rhinovirus,
parainfluenza virus, respiratory syncytial virus (RSV),
metapneumovirus, and adenovirus.
(ii) Binding Reagents
[0030] The skilled artisan in the field of binding assays will
readily appreciate the scope of binding agents and companion
binding partners that may be used in the present methods. A
non-limiting list of such pairs include (in either order)
oligonucleotides and complements, receptor/ligand pairs,
antibodies/antigens, natural or synthetic receptor/ligand pairs,
amines and carbonyl compounds (i.e., binding through the formation
of a Schiff's base), hapten/antibody pairs, antigen/antibody pairs,
epitope/antibody pairs, mimitope/antibody pairs, aptamer/target
molecule pairs, hybridization partners, and intercalater/target
molecule pairs.
[0031] The binding assays of the methods of the present invention
may employ antibodies or other receptor proteins as binding
reagents. The term "antibody" includes intact antibody molecules
(including hybrid antibodies assembled by in vitro re-association
of antibody subunits), antibody fragments and recombinant protein
constructs comprising an antigen binding domain of an antibody (as
described, e.g., in Porter, R. R. and Weir, R. C. J. Cell Physiol.,
67 (Suppl); 51-64 (1966) and Hochman, 1. Inbar, D. and Givol, D.
Biochemistry 12: 1130 (1973)), as well as antibody constructs that
have been chemically modified, e.g., by the introduction of a
detectable label.
[0032] Binding reagents and binding partners that are linked to
assay components to enable the attachment of these assay components
to each other or to solid phases may be described herein as
"targeting agents". For targeting agents that work in pairs, e.g.,
antigen-antibody, oligonucleotides-complement, etc., one targeting
agent of the pair may be referred to herein as the first targeting
agent, whereas the companion targeting agent may be referred to as
the second targeting agent. In certain embodiments, these targeting
agents are selected based on the reversibility of their binding
reactions. In particular, targeting agent pairs may be selected,
e.g., because under a first set of conditions the pair will bind to
form a binding complex which, under a second set of conditions, can
be caused to dissociate to break apart the complex, e.g, by
subjecting bound targeting agent pairs to increased or decreased
temperature, changes in chemical environment or assay buffer (e.g.,
ionic strength changes, pH changes, addition of denaturants,
changes in light or electrical potential, etc.), adding competing
binding reagents that compete with one targeting agent for binding
to another targeting agent, and combinations thereof. Suitable
conditions may be derived through routine experimentation. There
are many well-established cleavable chemical linkers that may be
used that provide a covalent bond that may be cleaved without
requiring harsh conditions. For example, disulfide containing
linkers may be cleaved using thiols or other reducing agents,
cis-diol containing linkers may be cleaved using periodate,
metal-ligand interactions (such as nickel-histidine) may be cleaved
by changing pH or introducing competing ligands. The terms "cleave"
or "cleaving" are also used herein to refer to processes for
separating linked assay components that do not require breaking
covalent bonds, e.g., there are many well-established reversible
binding pairs and conditions that may be employed (including those
that have been identified in the art of affinity chromatography).
By way of example, the binding of many antibody-ligand pairs can be
reversed through changes in pH, addition of protein denaturants or
chaotropic agents, addition of competing ligands, etc.
[0033] The targeting agents may be pairs of oligonucleotides
comprising complementary sequences. The preferred length is
approximately 5 to 100 bases, preferably, approximately, 10 to 50
bases, and more preferably approximately 10 to 25 bases. In
addition, the targeting oligonucleotides sequences need not be
identical in length and in certain embodiments it may be beneficial
to provide one targeting oligonucleotide sequence that is longer
than its binding partner, e.g., by up to 25 bases, or up to 15
bases, or up to 10 bases. Known methods that are commonly employed
for strand separation employ i) temperatures above the melting
temperature for the complex, ii) use an alkaline pH of 11 (or
higher) or a low pH; iii) use high ionic strength and/or iv) use
nucleic acid denaturants such as formamide. Other methods for
strand separation include the use of helicase enzymes such as Rep
protein of E. coli that can catalyse the unwinding of the DNA, or
binding proteins such as 32-protein of E. coli phage T4 that act to
stabilize the single-stranded form of DNA. In specific embodiments,
dissociation of complementary nucleic acid strands is accomplished
by exposing the strands to elevated temperature greater than
60.degree. C.
[0034] The methods of the present invention may be used in a
variety of assay devices and/or formats. The assay devices may
include, e.g., assay modules, such as assay plates, cartridges,
multi-well assay plates, reaction vessels, test tubes, cuvettes,
flow cells, assay chips, lateral flow devices, etc., having assay
reagents (which may include targeting agents or other binding
reagents) added as the assay progresses or pre-loaded in the wells,
chambers, or assay regions of the assay module. These devices may
employ a variety of assay formats for specific binding assays,
e.g., immunoassay or immunochromatographic assays. Illustrative
assay devices and formats are described herein below. In certain
embodiments, the methods of the present invention may employ assay
reagents that are stored in a dry state and the assay devices/kits
may further comprise or be supplied with desiccant materials for
maintaining the assay reagents in a dry state. The assay devices
preloaded with the assay reagents can greatly improve the speed and
reduce the complexity of assay measurements while maintaining
excellent stability during storage. The dried assay reagents may be
any assay reagent that can be dried and then reconstituted prior to
use in an assay. These include, but are not limited to, binding
reagents useful in binding assays, enzymes, enzyme substrates,
indicator dyes and other reactive compounds that may be used to
detect an analyte of interest. The assay reagents may also include
substances that are not directly involved in the mechanism of
detection but play an auxiliary role in an assay including, but not
limited to, blocking agents, stabilizing agents, detergents, salts,
pH buffers, preservatives, etc. Reagents may be present in free
form or supported on solid phases including the surfaces of
compartments (e.g., chambers, channels, flow cells, wells, etc.) in
the assay modules or the surfaces of colloids, beads, or other
particulate supports.
[0035] In one embodiment, assay reagents may be provided in an
assay device that includes one or more regions or zones used for
reagent storage. These storage zones may include the reagent bound
to a surface within the storage zone, such that the reagent is
confined within that zone until it is subjected to conditions
sufficient to release the reagent for use elsewhere in the device.
For example, the storage zone may include a surface-reagent complex
comprising a reagent linked to a first targeting agent and a
surface linked to a second targeting agent, wherein the reagent and
the surface are linked in the surface-reagent complex, via a
releasable binding interaction between the first and second
targeting agents. In this embodiment, the reagent is released from
the surface-reagent complex and the storage zone by subjecting the
storage zone to conditions sufficient to disrupt the releasable
binding interaction between the first and second targeting agents.
As described herein, those conditions may include but are not
limited to, subjecting the storage zone to increased or decreased
temperature, light, altering the pH of that zone, applying an
electrical potential, changes in ionic strength, adding a
competitor, and combinations thereof.
[0036] The surface to which the second targeting agent, and
thereby, the reagent, is linked, may be any solid support that can
be incorporated within or confined to the storage zone. For
example, the surface may be the surface of one or more particles,
as described herein, present in the storage zone. Alternatively,
the surface is a surface of the storage zone, for example, a
surface of a compartment, channel, conduit, well, etc., within the
storage zone. Preferably, the storage zone surface is roughened or
includes one or more raised features or indentations that increase
the relative surface area within the storage zone available to hold
surface-reagent complexes. In one embodiment, the storage zone
surface includes surface area-enhancing features that increase the
surface area, such that the surface area accessible to a component
capable of binding to that surface is at least two-fold larger than
the surface area of a flat surface. In a preferred embodiment, the
surface area accessible for binding is at least three-fold larger
than the surface area of a flat surface. The high surface area
support can be provided by roughening a surface or otherwise
providing three dimensional texture to a surface. A variety of
established approaches for preparing roughened or textured surfaces
will be available to one skilled in the art. Included in these
approaches is the production of surfaces with high aspect ratio
features such as arrays of columns that are prepared through
conventional machining, micro-machining or lithography (e.g.,
approaches using LIGA or other micro-fabrication technologies as
described in U.S. Pat. Nos. 5,707,799 and 5,952,173) or injection
molding.
[0037] The storage zone surface may also include a composite
material comprising exposed particles distributed in a matrix. The
composite material may include, but is not limited to, carbon
particles, graphitic particles, or carbon nanotubes. Optionally,
the composite may be etched (e.g., by chemical or plasma etching)
to expose more particles and increase the surface roughness. In one
specific example, the surface is provided by a printed carbon ink.
In another embodiment, the storage zone surface may include a
porous support that provides an enhanced surface area through the
surface area available in its pores. Such porous supports include
porous membranes (such as filtration membranes and lateral flow
membranes) and gels. Preferred gels include hydrogels. A number of
suitable hydrogels are well established as supports for reagents,
as are chemistries for linking reagents to hydrogels, for
applications such as affinity chromatography, solid phase synthesis
of biological polymers and binding assays, in applications.
Examples of such hydrogels include, but are not limited to,
polymers of sugars (polysaccharides), acrylic acid, acrylates,
acrylamides, ethylene glycol, propylene glycol. The hydrogels may
be cross-linked and/or may be co-polymers of different monomer
components.
[0038] An assay device that incorporates a storage zone for
reagents also includes a use zone configured to use those reagents
in an assay conducted in that device. Therefore, once the reagent
is released from the surface-reagent complex, free reagent is
available for use in an assay conducted in the use zone. Free
reagent is transferred from the storage zone to the use zone,
wherein it can participate in an assay for an analyte of interest.
That assay may involve one or more additional reagents present in
the use zone or otherwise supplied to the use zone. In one
embodiment, the use zone may include one or more additional
reagents bound to a solid support within the use zone and/or dried
on a surface of the use zone. In a specific embodiment, the reagent
is a binding reagent capable of binding an analyte of interest in a
sample, and the use zone includes an additional reagent, bound to a
solid support within the use zone, wherein that additional reagent
also binds the analyte of interest. In this embodiment, the analyte
present in the sample binds to the surface of the use zone via the
additional reagent, as well as to the free reagent transferred from
the storage zone to form a sandwich complex. The binding reagent
may include a detectable label, e.g., an ECL label, and the analyte
may be detected in the use zone by detecting the presence or
absence of the label, e.g., via measuring electrochemiluminescence
emitted in the use zone. The sample may be introduced to the use
zone directly or the sample is first introduced to the storage zone
and thereafter, the sample flows from the storage zone to the use
zone. The reagent may be released prior to contacting the storage
zone with sample or after the storage zone is contacted with
sample. In one embodiment, sample is introduced to the storage
zone, which is then subjected to conditions required for release of
the reagent from the surface-reagent complex. Thereafter, the
sample and the free reagent are optionally incubated prior to
transferring the sample-reagent mixture to the use zone.
[0039] In a preferred embodiment, the storage zone and the use zone
are in fluidic communication along a fluid path. For example, the
assay device may be a cartridge and the storage zone and the use
zone are positioned within the cartridge along a fluid path.
Examples of this embodiment are shown in FIG. 7(a)-(c). In FIG.
7(a), the assay device includes a storage zone and at least two use
zones and each of the storage zones and use zones are in fluid
communication. The use zones may be configured in the assay device
in series, as shown in FIG. 7(a) or in parallel, as shown in FIG.
7(b). FIG. 7(c) shows yet another configuration of an assay device
including multiple storage and use zones. In the embodiment shown
in FIG. 7(c), the storage and use zones are also in fluidic
communication with sample and/or reagent compartments within the
assay device.
[0040] Another embodiment is shown in FIG. 8. The assay device of
FIG. 8 includes a storage zone including a first surface-reagent
complex and a second surface-reagent complex and at least two use
zones, wherein the storage zone and the use zone are in fluidic
communication via a fluidic network. Sample is introduced into a
compartment of the device in panel (a) and the fluidic network
carries that sample to the use zones, as shown in panel (b). Panels
(b) and (c) also shows that diluents can be passed through the
storage zone (under conditions that do not release the
surface-reagent complexes) and carried through the fluidic network
to the use zones to provide an optional wash of the use zones.
Diluent is then passed through the storage zone while subjecting
the storage zone to a condition that releases the first reagent,
which is then carried to the fluidic network in to use zone 1, as
shown in panel (d). The second reagent is then released by a second
set of conditions and carried, via flow of diluents through the
microfluidic network, to use zone 2, as shown in panel (e).
Finally, the use zones are optionally washed to remove excess
reagent, as shown in panel (f).
[0041] In one embodiment, the storage zone and use zones are
included within a fluidic network further comprising one or more
vent ports in fluidic communication with the storage and use zones
(directly or through vent conduits) so as to allow the
equilibration of fluid in the zones with the atmosphere or to allow
for the directed movement of fluid into or out of a specified zone
by selectively sealing, opening (to atmospheric pressure) or
applying positive or negative pressure to specific vent ports.
[0042] In another embodiment, the assay device is a multi-well
assay plate and the use zone is positioned within a well of the
plate, while the storage zone is located on a supplemental surface
of the well that does not overlap with the use zone.
[0043] In a further embodiment, the assay device may include one or
more surface-reagent complexes in the storage zone. In the
embodiment depicted in FIG. 8, for example, the storage zone
includes a first surface-reagent complex (as described above) and
also includes a second surface-reagent complex confined to the
storage zone, the second surface-reagent complex including (i) a
second reagent linked to a third targeting agent; and (ii) a second
surface linked to a fourth targeting agent, wherein the second
reagent and the second surface are linked, in the second
surface-reagent complex, via a second releasable binding
interaction between the third and fourth targeting agents; and the
use zone is further configured to use the second reagent in the
assay. The various reagents stored within the storage zone may be
used in one or more assays conducted in the use zone, or each of
the reagents stored within the storage zone may be used in each of
the assays conducted in the use zone. The reagents stored within
the storage zone may be selectively released, i.e., one of the
reagents may be released from the surface-reagent complex
composition by a first set of conditions that differ from a second
set of conditions used to release another reagent stored in the
storage zone.
[0044] Additionally, the use zone may include two or more assay
regions each configured to use the reagents stored within the
storage zone in one or more assays conducted with a single sample
in the device. In one embodiment, the use zone includes a first
assay region configured to conduct an assay for a first analyte of
interest in a sample and the use zone may also include an
additional assay region configured to conduct an assay for an
additional analyte of interest that may also be present in the
sample. Alternatively, the first assay region in the use zone may
be used to conduct a first assay for an analyte, while another
assay region in the use zone may be used to conduct a second assay
for the same analyte. Still further, the assay device may include a
plurality of use zones each configured to use the reagents stored
within the storage zone in one or more assays conducted with a
single sample in the device. Each use zone may include one or more
assay regions as described above. Moreover, the assay device may
include a plurality of storage zones, e.g., for each use zone there
is a corresponding storage zone. Various configurations of an assay
device including multiple use zones and/or storage zones are shown
in FIG. 7(a)-(c) and FIG. 8.
[0045] As described above, a storage zone may include a plurality
of different reagents as surface-reagent complexes. In one
embodiment different reagents are held in the storage zone by
different releasable binding reactions that are cleaved under
different conditions. Therefore, by subjecting each defined region
of the storage zone to the appropriate conditions, each reagent is
selectively released from the storage zone. The different reagents
may be in surface-reagent complexes that are inter-mixed or held in
distinct regions of the storage zone. As described herein, those
conditions may include but are not limited to, subjecting the
region to increased or decreased temperature, light, altering the
pH of that region, changing the ionic strength, applying an
electrical potential, adding a competitor, and combinations
thereof. By using binding reactions cleaved under different
conditions, it is possible to selectively release one reagent at a
time from surface-reagent complexes in the storage zone. For
example, one reagent may be selectively released by heating while
another may be selective released by changing pH or one reagent may
be selectively released using a first competitor while another may
be selectively released using a second competitor. In another
embodiment, different reagents may be released one a time using
different releasable binding reactions that require increasingly
stringent cleavage conditions (such as increasing temperature,
increasing or decreasing pH, increasing competitor concentration,
increasing levels of light, increasing or decreasing ionic
strength, etc.). For example, a first reagent may be released at a
first temperature level and a second reagent may be subsequently
released at a second higher temperature level.
[0046] In another embodiment, the storage zone may include a
plurality of defined spatial regions, at least two of the different
regions holding different reagents in surface-reagent complexes
that hold the reagents through releasable binding interactions as
described above. In this embodiment, cleavage of a reagent in a
specific spatial region can be carried out by applying cleavage
conditions (such as applying light, temperature, electrical
potential, etc.) in a manner that confines the cleavage condition
to the specific spatial region of interest. In this embodiment,
releasable binding interactions used for holding different reagents
can be the same or different, because release of individual
reagents can be directed by application of the cleavage condition
to defined region. In a preferred embodiment, the device is
configured for a multiplexed assay measurement and the device
includes (a) a storage zone comprising a surface-reagent complex
confined to the storage zone, the surface-reagent complex including
(i) a reagent linked to a first targeting agent; and (ii) a surface
linked to a second targeting agent, wherein the reagent and the
surface are linked, in the surface-reagent complex, via a
releasable binding interaction between the first and second
targeting agents; and (b) a use zone comprising a plurality of
assay regions configured to use the reagent in a multiplexed assay
for a plurality of analytes in a sample. The storage zone may
further comprise a second surface-reagent complex confined to the
storage zone, the second surface-reagent complex including (iii) a
second reagent linked to a third targeting agent; and (iv) a second
surface linked to a fourth targeting agent, wherein the second
reagent and the second surface are linked, in the second
surface-reagent complex, via a second releasable binding
interaction between the third and fourth targeting agents; and the
use zone is further configured to use the second reagent in the
multiplexed assay. In this regard, the use zone comprises two or
more assay regions each configured to use the reagent and the
second reagent in one or more assays conducted with the sample in
the assay device, and this configuration of assay device enables
the conduct of a plurality of assays in the use zone with the
reagent and optionally, a second reagent.
[0047] The use zone may include a first assay region configured to
conduct an assay for a first analyte of interest in the sample and
an additional assay region configured to conduct an assay for an
additional analyte of interest in the sample, and an assay in such
a device comprises the following steps:
[0048] (x) introducing the sample to the use zone via the storage
zone;
[0049] (y) introducing a diluent to the storage zone;
[0050] (z) subjecting the storage zone to a condition that releases
the reagent from the surface-reagent complex;
[0051] (xx) transferring the reagent from the storage zone to the
first assay region and the second assay region; and
[0052] (yy) conducting the assays in the first and second assay
regions, respectively.
[0053] The conducting step of each assay may be performed
simultaneously or sequentially.
[0054] Alternatively, an assay method may include an incubation
step between the sample and free reagent before the mixture of
sample and free reagent is introduced to the use zone. Such a
method would include the following steps:
[0055] (x) introducing the sample to the storage zone;
[0056] (y) subjecting the storage zone to a condition that releases
the reagent from the surface-reagent complex, and optionally
incubated the sample with the free reagent in the storage zone;
[0057] (z) transferring the mixture formed in (y) from the storage
zone to the first assay region and the second assay region; and
[0058] (xx) conducting the assays in the first and second assay
regions, respectively.
[0059] Still further, the use zone may include a first assay region
configured to conduct an assay for a first analyte of interest in
the sample and an additional assay region in the use zone
configured to conduct an assay for an additional analyte of
interest in the sample, and an assay in such a device may
comprise:
[0060] (x) introducing the sample to the use zone via the storage
zone;
[0061] (y) introducing a diluent to the storage zone and [0062] i)
subjecting the storage zone to a condition that releases the
reagent from the surface-reagent complex; [0063] ii) subjecting the
storage zone to an additional condition that releases a second
reagent from a second surface-reagent complex;
[0064] (z) transferring the reagent and the second reagent from the
storage zone to the first and second assay regions; and
[0065] (xx) conducting the assays in the first and second assay
regions.
[0066] The conducting step of each assay may be performed
simultaneously or sequentially. Likewise, the transfer of the
reagent and the second reagent may be done simultaneously or
sequentially.
[0067] Alternatively, an assay method using a device that includes
a first assay region configured to conduct an assay for a first
analyte of interest in the sample and an additional assay region in
the use zone configured to conduct an assay for an additional
analyte of interest in the sample may also include an incubation
step, i.e.,
[0068] (x) introducing the sample to the storage zone; [0069] i)
subjecting the storage zone to a condition that releases the
reagent from the surface-reagent complex; [0070] ii) subjecting the
storage zone to an additional condition that releases a second
reagent from a second surface-reagent complex; [0071] (iii)
incubating the storage zone with the free reagent and free second
reagent formed in steps (x)(i) and (x)(ii);
[0072] (y) transferring the mixture formed in step (x)(iii) from
the storage zone to the first and second assay regions; and
[0073] (z) conducting the assays in the first and second assay
regions.
[0074] In one specific embodiment, the assay device is a cartridge,
such as that described in copending application Ser. No.
61/284,276, filed Dec. 16, 2009, the disclosure of which is
incorporated herein by reference. As shown, e.g., in FIG. 9 of U.S.
Ser. No. 61/284,276, a cartridge may include various compartments,
i.e., a sample chamber, an assay reagent chamber, waste chambers,
and detection chambers, as well as a fluidic network that connects
various compartments and/or fluid ports/vents. The storage zone may
be incorporated within, e.g., a reagent chamber, and likewise, the
use zone may be included within, e.g., the detection chamber.
Additionally or alternatively, an additional storage chamber may be
incorporated within the cartridge described therein.
[0075] In another specific embodiment, the assay device is a
multi-well assay plate, such as that described in co-pending
application Ser. No. 11/642,970, filed Dec. 21, 2006, the
disclosure of which is incorporated herein by reference. The assay
plate may include a plate body with a plurality of wells defined
therein, wherein the plurality of wells includes a binding surface
having a capture reagent immobilized therein, and an additional
reagent located on a surface of the plate or well that does not
overlap with the binding surface. In one embodiment, the additional
reagent is located on a reagent storage shelf positioned on a wall
of a well. Alternatively, an assay plate may include assay wells
that are connected to dedicated reagent spaces located in the
regions between the assay wells. In such an embodiment, a reagent
space may be in fluidic communication with the surrounding wells
via e.g., a notch. In addition, suitable assay plates are described
in U.S. patent application Ser. No. 11/642,970, the disclosure of
which is incorporated herein by reference.
(iii) Solid Phases
[0076] A wide variety of solid phases are suitable for use in the
methods of the present invention including conventional solid
phases from the art of binding assays. Solid phases may be made
from a variety of different materials including polymers (e.g.,
polystyrene and polypropylene), ceramics, glass, composite
materials (e.g., carbon-polymer composites such as carbon-based
inks). Suitable solid phases include the surfaces of macroscopic
objects such as an interior surface of an assay container (e.g.,
test tubes, cuvettes, flow cells, cartridges, wells in a multi-well
plate, etc.), slides, assay chips (such as those used in gene or
protein chip measurements), pins or probes, beads, filtration
media, lateral flow media (for example, filtration membranes used
in lateral flow test strips), etc.
[0077] Suitable solid phases also include particles (including but
not limited to colloids or beads) commonly used in other types of
particle-based assays e.g., magnetic, polypropylene, and latex
particles, materials typically used in solid-phase synthesis e.g.,
polystyrene and polyacrylamide particles, and materials typically
used in chromatographic applications e.g., silica, alumina,
polyacrylamide, polystyrene. The materials may also be a fiber such
as a carbon fibril. Microparticles may be inanimate or
alternatively, may include animate biological entities such as
cells, viruses, bacterium and the like.
[0078] The particles used in the present method may be comprised of
any material suitable for attachment to one or more binding
partners and/or labels, and that may be collected via, e.g.,
centrifugation, gravity, filtration or magnetic collection. A wide
variety of different types of particles that may be attached to
binding reagents are sold commercially for use in binding assays.
These include non-magnetic particles as well as particles
comprising magnetizable materials which allow the particles to be
collected with a magnetic field. In one embodiment, the particles
are comprised of a conductive and/or semiconductive material, e.g.,
colloidal gold particles.
[0079] The microparticles may have a wide variety of sizes and
shapes. By way of example and not limitation, microparticles may be
between 5 nanometers and 100 micrometers. Preferably microparticles
have sizes between 20 nm and 10 micrometers. The particles may be
spherical, oblong, rod-like, etc., or they may be irregular in
shape.
[0080] The particles used in the present method may be coded to
allow for the identification of specific particles or
subpopulations of particles in a mixture of particles. The use of
such coded particles has been used to enable multiplexing of assays
employing particles as solid phase supports for binding assays. In
one approach, particles are manufactured to include one or more
fluorescent dyes and specific populations of particles are
identified based on the intensity and/or relative intensity of
fluorescence emissions at one or more wave lengths. This approach
has been used in the Luminex xMAP systems (see, e.g., U.S. Pat. No.
6,939,720) and the Becton Dickinson Cytometric Bead Array systems.
Alternatively, particles may be coded through differences in other
physical properties such as size, shape, imbedded optical patterns
and the like.
[0081] In certain embodiments of assays of the invention, particles
may have a dual role as both i) a solid phase support used in an
analyte concentration, collection and/or separation step and ii) as
a detectable label or platform for detectable labels in a
measurement step. In one example, a method of conducting a binding
assay may comprise contacting a sample comprising an analyte with a
particle linked to a first binding reagent that binds that analyte
to form a complex comprising the analyte bound to the first binding
reagent. The complex is then collected by collection of the
particle (via magnetic collection, centrifugation, gravity
sedimentation, etc.) and some or all of the unbound components of
the sample are separated from the complex by removing some or all
of the sample volume and, optionally, washing the collected
particles. The complex is then released by resuspending the
particles in the original or a new liquid media. The complex on the
particle is then contacted with a second binding reagent bound to a
solid phase, the second binding reagent binding the complex so as
to bring the complex and particle to a surface of the solid phase.
The amount of analyte in the sample is measured by measuring the
amount of analyte bound to the solid phase, which in turn is
measured by measuring the amount of particles bound to the solid
phase (either by directly measuring the particles or by measuring
detectable labels in or on the particles by, e.g., the measurement
approaches described below).
[0082] The invention also includes assay methods that employ
magnetic particles as detectable labels or as platforms for
detectable labels in a binding assay. Advantageously, when using
magnetic particles as a label or a label platform, a magnetic field
can be applied to speed the kinetics for the binding of i) assay
components linked to a magnetic particle to ii) binding reagents
immobilized on a solid phase.
[0083] Accordingly, one embodiment is a method for conducting a
binding assay comprising
[0084] (a) contacting (i) a sample comprising a target analyte with
(ii) a magnetic particle linked to a first binding reagent that
binds the target analyte and thereby forms a complex comprising the
target analyte bound to the first binding reagent;
[0085] (b) contacting a solution comprising the complex with a
second binding reagent bound to a solid phase, wherein the second
binding reagent binds to the complex;
[0086] (c) applying a magnetic field to concentrate the particles
near to the solid phase and thereby accelerating the rate of
binding between the complex and the second binding reagent and
[0087] (d) measuring the amount of the analyte bound to the solid
phase.
[0088] Optionally, such a method may also include, prior to step
(b), collection and release steps as described elsewhere in this
application so as to pre-concentrate the analyte and/or remove
interferents from the sample. The magnetic particles used in such
method are, preferably, between 10 nm and 10 um in diameter, more
preferably between 50 nm and 1 um. The step of applying a magnetic
field may be achieved through the use of permanent or
electromagnets, e.g., by placing the magnet on the opposite side of
the solid phase relative to the second binding reagent. Optionally,
the magnet or magnetic field is translated and/or rotated along the
solid phase so as to move the particles along the binding surface
and allow the particles to interrogate the surface for available
binding sites. Alternatively, or in conjunction with movement of
the magnet/field, the magnetic field is intermittently removed and,
while the magnetic field is removed, the particles are resuspended
(e.g., by mixing) and then reconcentrated on the solid phase
(thereby, allowing for allowing the particles to change rotational
orientation on the surface and allowing them to interrogate
additional areas on the surface. The method may also include a
washing step, prior to the measuring step, to remove unbound
particles. During such a washing step, the magnetic field is
removed to allow for non-bound particles to be washed away.
Alternatively, a magnetic field above the surface can be used to
pull unbound particles away from the surface. The magnetic reaction
acceleration approach may also be applied to multiplexed assay
methods, as described elsewhere in this application, e.g., the
solid phase may include an array of a plurality of different second
binding reagents for use in array-based multiplexed
measurements.
(iv) Collection and Release
[0089] Collection, as used herein, refers to the physical
localization of a material in a mixture. Collection includes the
localization of a material through binding reactions or adsorption.
For example, a material in a mixture may be collected on a solid
phase by adsorption of the material on the solid phase or by
binding of the material to binding reagents on the solid phase.
Collection is not, however, limited to localization at a solid
phase and may also include techniques in the art for localizing
materials at a location/volume within a larger fluid volume, for
example, localization of materials through the use of optical
tweezers (which use light to manipulate microscopic objects as
small as a single atom, wherein the radiation pressure from a
focused laser beam is able to trap small particles), electric or
magnetic fields, focused flow, density gradient centrifugation,
etc.
[0090] Certain embodiments of the invention include the collection
of microparticles or materials that are bound to microparticles.
Suitable collection methods include the many methods known in the
art of microparticle-based assays that achieve localization of
microparticles from a suspension. These include sedimentation under
gravity or by centrifugation, filtration onto a filter or porous
membrane, localization (of magnetizable particles) by application
of a magnetic field, binding or adsorption of the particles to a
macroscopic solid phase, use of optical tweezers, etc.
[0091] Release, as used herein, refers to delocalization of a
previously collected material. Materials that are held at a
localized position through chemical bonds or through specific or
non-specific binding interactions may be allowed to delocalize by
breaking the bond or interaction so that the materials may diffuse
or mix into the surrounding media. There are many well-established
cleavable chemical linkers that may be used that provide a covalent
bond that may be cleaved without requiring harsh conditions. For
example, disulfide containing linkers may be cleaved using thiols
or other reducing agents, cis-diol containing linkers may be
cleaved using periodate, metal-ligand interactions (such as
nickel-histidine) may be cleaved by changing pH or introducing
competing ligands. Similarly, there are many well-established
reversible binding pairs that may be employed (including those that
have been identified in the art of affinity chromatography). By way
of example, the binding of many antibody-ligand pairs can be
reversed through changes in pH, addition of protein denaturants or
chaotropic agents, addition of competing ligands, etc. Other
suitable reversible binding pairs include complementary nucleic
acid sequences, the hybridization of which may be reversed under a
variety of conditions including changing pH, increasing salt
concentration, increasing temperature above the melting temperature
for the pair and/or adding nucleic acid denaturants (such as
formamide). Such reversible binding pairs may be used as targeting
agents (as described above), e.g., a first targeting agent may be
linked to a first binding reagent that binds an analyte, a second
targeting agent may be linked to a solid phase, and a binding
interaction of the first and second targeting agents may be used to
reversibly immobilize the first binding reagent on the solid
phase.
[0092] Release also includes physical delocalization of materials
by, for example, mixing, shaking, vortexing, convective fluid flow,
mixing by application of magnetic, electrical or optical forces and
the like. Where microparticles or materials bound to microparticles
have been collected, such physical methods may be used to resuspend
the particles in a surrounding matrix. Release may simply be the
reverse of a previous collection step (e.g., by any of the
mechanisms described above) or collection and release could proceed
by two different mechanisms. In one such example, collection of
materials (such as an analyte or a complex comprising an analyte)
bound to a particle can be achieved by physical collection of the
particle. The materials are then released by cleaving a bond or
reversing a binding reaction holding the material on the particle.
In a second such example, materials (such as an analyte of a
complex comprising an analyte are collected on a surface through a
binding interaction with a binding reagent that is linked to the
surface. The material is then released by breaking a bond or a
second binding interaction linking the binding reagent to the
surface.
[0093] Collection followed by release may be used to concentrate
and/or purify analytes in a sample. By collecting in a first volume
and releasing into a second smaller volume, an analyte in a sample
may be concentrated. Through concentration, it is often possible to
significantly improve the sensitivity of a subsequent measurement
step. By collecting from a sample and removing some or all of the
uncollected sample, potential assay interferents in the sample may
be reduced or eliminated. Optionally, removal of the unbound sample
may include washing a collected material with and releasing the
collected material into defined liquid reagents (e.g., assay or
wash buffers) so as to provide a uniform matrix for subsequent
assay steps.
(iv) Measurement Methods
[0094] The methods of the invention can be used with a variety of
methods for measuring the amount of an analyte and, in particular,
measuring the amount of an analyte bound to a solid phase.
Techniques that may be used include, but are not limited to,
techniques known in the art such as cell culture-based assays,
binding assays (including agglutination tests, immunoassays,
nucleic acid hybridization assays, etc.), enzymatic assays,
colorometric assays, etc. Other suitable techniques will be readily
apparent to one of average skill in the art. Some measurement
techniques allow for measurements to be made by visual inspection,
others may require or benefit from the use of an instrument to
conduct the measurement.
[0095] Methods for measuring the amount of an analyte include label
free techniques, which include but are not limited to i) techniques
that measure changes in mass or refractive index at a surface after
binding of an analyte to a surface (e.g., surface acoustic wave
techniques, surface plasmon resonance sensors, ellipsometric
techniques, etc.), ii) mass spectrometric techniques (including
techniques like MALDI, SELDI, etc. that can measure analytes on a
surface), iii) chromatographic or electrophoretic techniques, iv)
fluorescence techniques (which may be based on the inherent
fluorescence of an analyte), etc.
[0096] Methods for measuring the amount of an analyte also include
techniques that measure analytes through the detection of labels
which may be attached directly or indirectly (e.g., through the use
of labeled binding partners of an analyte) to an analyte. Suitable
labels include labels that can be directly visualized (e.g.,
particles that may be seen visually and labels that generate an
measurable signal such as light scattering, optical absorbance,
fluorescence, chemiluminescence, electrochemiluminescence,
radioactivity, magnetic fields, etc). Labels that may be used also
include enzymes or other chemically reactive species that have a
chemical activity that leads to a measurable signal such as light
scattering, absorbance, fluorescence, etc. The use of enzymes as
labels has been well established in in Enzyme-Linked ImmunoSorbent
Assays, also called ELISAs, Enzyme ImmunoAssays or EIAs. In the
ELISA format, an unknown amount of antigen is affixed to a surface
and then a specific antibody is washed over the surface so that it
can bind to the antigen. This antibody is linked to an enzyme, and
in the final step a substance is added that the enzyme converts to
a product that provides a change in a detectable signal. The
formation of product may be detectable, e.g., due a difference,
relative to the substrate, in a measurable property such as
absorbance, fluorescence, chemiluminescence, light scattering, etc.
Certain (but not all) measurement methods that may be used with
solid phase binding methods according to the invention may benefit
from or require a wash step to remove unbound components (e.g.,
labels) from the solid phase. Accordingly, the methods of the
invention may comprise such a wash step.
[0097] In one embodiment, an analyte(s) of interest in the sample
may be measured using electrochemiluminescence-based assay formats,
e.g. electrochemiluminescence (ECL) based immunoassays. The high
sensitivity, broad dynamic range and selectivity of ECL are
important factors for medical diagnostics. Commercially available
ECL instruments have demonstrated exceptional performance and they
have become widely used for reasons including their excellent
sensitivity, dynamic range, precision, and tolerance of complex
sample matrices. Species that can be induced to emit ECL
(ECL-active species) have been used as ECL labels, e.g., i)
organometallic compounds where the metal is from, for example, the
noble metals of group VIII, including Ru-containing and
Os-containing organometallic compounds such as the
tris-bipyridyl-ruthenium (RuBpy) moiety and ii) luminol and related
compounds. Species that participate with the ECL label in the ECL
process are referred to herein as ECL coreactants. Commonly used
coreactants include tertiary amines (e.g., see U.S. Pat. No.
5,846,485), oxalate, and persulfate for ECL from RuBpy and hydrogen
peroxide for ECL from luminol (see, e.g., U.S. Pat. No. 5,240,863).
The light generated by ECL labels can be used as a reporter signal
in diagnostic procedures (Bard et al., U.S. Pat. No. 5,238,808,
herein incorporated by reference). For instance, an ECL label can
be covalently coupled to a binding agent such as an antibody,
nucleic acid probe, receptor or ligand; the participation of the
binding reagent in a binding interaction can be monitored by
measuring ECL emitted from the ECL label. Alternatively, the ECL
signal from an ECL-active compound may be indicative of the
chemical environment (see, e.g., U.S. Pat. No. 5,641,623 which
describes ECL assays that monitor the formation or destruction of
ECL coreactants). For more background on ECL, ECL labels, ECL
assays and instrumentation for conducting ECL assays see U.S. Pat.
Nos. 5,093,268; 5,147,806; 5,324,457; 5,591,581; 5,597,910;
5,641,623; 5,643,713; 5,679,519; 5,705,402; 5,846,485; 5,866,434;
5,786,141; 5,731,147; 6,066,448; 6,136,268; 5,776,672; 5,308,754;
5,240,863; 6,207,369; 6,214,552 and 5,589,136 and Published PCT
Nos. WO 99/63347; WO 00/03233; WO 99/58962; WO 99/32662; WO
99/14599; WO 98/12539; WO 97/36931 and WO 98/57154, all of which
are incorporated herein by reference.
[0098] The capture/collection and release methods of the invention
may be applied to singleplex or multiplex formats where multiple
assay measurements are performed on a single sample. Multiplex
measurements that can be used with the invention include, but are
not limited to, multiplex measurements i) that involve the use of
multiple sensors; ii) that use discrete assay domains on a surface
(e.g., an array) that are distinguishable based on location on the
surface; iii) that involve the use of reagents coated on particles
that are distinguishable based on a particle property such as size,
shape, color, etc.; iv) that produce assay signals that are
distinguishable based on optical properties (e.g., absorbance or
emission spectrum) or v) that are based on temporal properties of
assay signal (e.g., time, frequency or phase of a signal).
(v) Assay Formats
[0099] One embodiment of the present invention employs a specific
binding assay, e.g., an immunoassay, immunochromatographic assay or
other assay that uses a binding reagent. The immunoassay or
specific binding assay according to one embodiment of the invention
can involve a number of formats available in the art. The
antibodies and/or specific binding partners can be labeled with a
label or immobilized on a surface. Thus, in one embodiment, the
detection method is a binding assay, e.g., an immunoassay,
receptor-ligand binding assay or hybridization assay, and the
detection is performed by contacting an assay composition with one
or more detection molecules capable of specifically binding with an
analyte(s) of interest in the sample.
[0100] In one embodiment, the assay uses a direct binding assay
format. An analyte is bound to a binding partner of the analyte,
which may be immobilized on a solid phase. The bound analyte is
measured by direct detection of the analyte or through a label
attached to the analyte (e.g., by the measurements described
above).
[0101] In one embodiment, the assay uses a sandwich or competitive
binding assay format. Examples of sandwich immunoassays performed
on test strips are described in U.S. Pat. No. 4,168,146 to Grubb et
al. and U.S. Pat. No. 4,366,241 to Tom et al., both of which are
incorporated herein by reference. Examples of competitive
immunoassay devices suitable for use with the present methods
include those disclosed in U.S. Pat. No. 4,235,601 to Deutsch et
al., U.S. Pat. No. 4,442,204 to Liotta, and U.S. Pat. No. 5,208,535
to Buechler et al., all of which are incorporated herein by
reference.
[0102] In a sandwich assay, analyte in the sample is bound to a
first binding reagent and a second labeled binding reagent and the
formation of this "sandwich" complex is measured. In a solid phase
sandwich assay, the first binding reagent is immobilized on a solid
phase and the amount of labeled antibody on the solid phase, due to
formation of the sandwich complex, is then measured. The signal
generated in a sandwich assay will generally have a positive
correlation with the concentration of the analyte. Various
configurations of sandwich assays that use the methods of the
present invention are shown in FIGS. 1-4. In one embodiment, e.g.,
in FIG. 1(a), the assay includes contacting a sample comprising a
target analyte with a particle or solid phase linked to a first
binding reagent that binds the target analyte, thereby forming a
complex comprising the target analyte bound to the first binding
reagent. The complex is collected, separated and released, as
described herein, and then a sandwich is formed by contacting the
complex with an additional binding reagent (e.g., a second binding
reagent). As shown in FIG. 1(a) and FIG. 1(b), the particle or
solid phase may or may not be cleaved from the complex prior to
contacting the complex with an additional binding reagent.
[0103] In a competitive assay, unlabelled analyte in the test
sample is measured by its ability to compete with labeled or
immobilized analyte. In the example of competitive assays employing
labeled analytes, the unlabeled analyte in a sample blocks the
ability of the labeled analyte to bind a binding reagent by
occupying the binding site. Thus, in a competitive assay, the
signal generated has an inverse correlation with the concentration
of analyte in a sample. FIGS. 6(a) and 6(b) show the use of the
methods of the present invention in a two step competitive format.
As in FIG. 1(a), the analyte of interest in the sample is
pre-concentrated. Labeled analyte bound to a solid support is
incubated with the pre-concentrated analyte complex. FIGS. 6(a) and
6(b) serve to illustrate how the methods of the present invention
may be used in a competitive assay format. The skilled artisan will
understand that alternate configurations of a competitive
immunoassay may be achieved using the methods of the present
invention without undue experimentation.
(vi) Specific Embodiments
[0104] In one embodiment, a method is provided for conducting a
binding assay comprising contacting a sample comprising a target
analyte, A, and which may also contain various sample contaminants
as shown in FIG. 1(a), with a particle linked to a first binding
reagent that binds the target analyte and thereby forms a complex
comprising the target analyte bound to the first binding reagent.
Once the sample is mixed with the particle to form the complex, the
complex is collected. This collection step may involve accumulation
of the complex at a surface, e.g., by centrifugation of the
particles, allowing the particles to rise or settle under gravity,
filtering the particles onto a filtration media, magnetically
collecting the particles (in the case of magnetic particles), etc.
Alternatively, the collection step may involve accumulation of the
complex within a defined volume within the sample, e.g., by holding
the particles in this defined volume through the use of optical
tweezers or focused flow. Optionally, the unbound components of the
sample are then separated from the complex, e.g., by removing all
or part of the non-collected components and/or by washing the
collected complex with an additional assay medium or wash buffer.
Thereafter, the complex is released, e.g., resuspended into the
assay medium, and the complex is contacted with a second binding
reagent bound to a solid phase, wherein the second binding reagent
binds to the complex. The amount of analyte is detected by
measuring the amount of a detectable label linked to an assay
component bound to the solid phase. The detectable label may be
linked to the first binding reagent, an optional third binding
reagent, if one is used in the assay format, the particle or an
additional assay component that is comprised within or bound to the
complex.
[0105] A variety of approaches are provided for conducting the
collection and release steps described above and for providing the
labeled reagent. FIG. 1(a) shows a method with the following steps:
(i) a first binding reagent linked to a particle binds to the
analyte to form a complex, (ii and iii) the complex is collected
and released by collection and resuspension of the particle during
which steps the analyte may be concentrated and/or separated from
contaminants in the sample, (iv) the complex binds to a second
binding reagent on a solid phase and (v) the complex is contacted
with a labeled third binding reagent that binds the analyte in the
complex such that it can be detected. FIG. 1(b) shows a method
similar to the one in FIG. 1(a), except that the complex is
released in step (iii) by cleaving the first binding reagent from
the particle instead of simply resuspending the particle. FIGS.
1(c) and 1(d) show methods similar to the one in FIG. 1(a) except
that that the label is attached to (or incorporated within) the
particle (FIG. 1(c)) or attached to the first binding reagent (FIG.
1(d)) and the step of contacted the complex with a labeled third
binding reagent is omitted. Alternatively, if the particle is
measured directly (e.g., by direct visual observation of the
particle), the label may be omitted. FIG. 1(e) shows a method
similar to the one in FIG. 1(b) except that the label is attached
to the first binding reagent and the step of contacting the complex
with a labeled third binding reagent is omitted.
[0106] The measuring step may comprise any suitable method of
measuring the presence of a detectable label in a sample (see the
Measurement Methods section), e.g., optical absorbance,
fluorescence, phosphorescence, chemiluminescence, light scattering
or magnetism. In one embodiment, the detectable label is an
electrochemiluminescent label and the measuring step comprises
measuring an ECL signal and correlating that signal with an amount
of analyte in the sample. Thus, the measuring step may further
comprise contacting the complex with an electrode and applying a
voltage waveform to the electrode to generate ECL.
[0107] The methods described in FIGS. 1(a)-1(e) may be applied to
multiplex measurements for multiple analytes in a sample. In such
methods, the first, second and third binding reagents (if present)
may be selected to bind multiple analytes (e.g., the use of poly-dT
as a binding reagent to capture multiple mRNAs in a sample through
the common poly-dA tail sequence) or, alternatively, the methods
may employ a plurality of different first binding reagents, second
binding reagents and/or third binding reagents to bind to the
multiple analytes. To allow for independent measurement of
different analytes, such multiplex methods employs at least one of
the group consisting of i) a plurality of different first binding
reagents, ii) a plurality of second binding reagents and iii) a
plurality of third binding reagents (the different reagents within
(i), (ii) or (iii) being selected for their ability to
preferentially bind a target analyte relative to other target
analytes). Where a plurality of first binding reagents are used,
individual particles may be attached to mixtures of the different
first binding reagents or, alternatively, the particles may be
prepared so that individual particles are attached to only one type
of first binding reagent (e.g., such that an individual particle
preferentially binds one of the target analytes relative to other
target analytes).
[0108] The multiplex methods may use a variety of approaches for
independently measuring different analytes. In one embodiment, a
plurality of labeled binding reagents with different preferences
for target analytes may be used (e.g., a plurality of different
labeled third binding reagents as in FIGS. 1(a) and 1(b), a
plurality of different labeled first binding reagents as in FIG.
1(e) or a plurality of different labeled first binding
reagent-particle conjugates as in FIGS. 1(c) and 1(d)). The labels
on the different labeled reagents (or, alternatively, the particles
in the particle conjugates) are selected to provide distinguishable
assay signals such that the different labeled reagents and,
therefore, the different target analytes, can be measured
independently. In another embodiment, a plurality of second binding
reagents with different preferences for target analytes may be
used. The different second binding reagents may be patterned into
different discrete binding domains on one or more solid phases
(e.g., as in a binding array) such that assay signals generated on
the different binding domains and, therefore, the different
analytes, can be measured independently (e.g., by independently
addressing binding domains on electrode arrays or by independently
measuring light emitted from different binding domains in a
luminescence assay). Alternatively, the different second binding
reagents may be coupled to different coded beads (as described in
the Solid Phases section) to allow for the different analytes to be
measured independently.
[0109] In an alternative embodiment, a method of conducting a
binding assay is provided as shown in FIGS. 2(a)-2(b), which
comprises contacting a sample comprising a target analyte with a
first solid phase, S, linked to a first binding reagent that binds
the target analyte and forms a complex comprising the target
analyte bound to the first binding reagent. Once the sample is
contacted with the first solid phase, the unbound components of the
sample are separated from the complex, the complex is released from
the solid phase into the assay medium and the first solid phase is
removed from the first binding reagent. Thereafter, the released
complex is contacted with a second solid phase comprising a second
binding reagent that binds to the complex, and the amount of
analyte bound to the second solid phase is quantified. The
detectable label may be linked to the first binding reagent, an
optional third binding reagent, if one is used in the assay format,
the particle or an additional assay component that is comprised
within or bound to the complex. In FIG. 2(a), the label is attached
to a third binding reagent (and the method includes the step of
contacting the complex with the third binding reagent), whereas the
label is attached to the first binding reagent in FIG. 2(b).
[0110] As described for FIG. 1, the methods described in FIG. 2 may
also be extended to multiplex measurements, e.g., by employing at
least one of the group consisting of i) a plurality of different
first binding reagents, ii) a plurality of second binding reagents
and iii) a plurality of third binding reagents (the different
reagents within (i), (ii) or (iii) being selected for their ability
to preferentially bind a target analyte relative to other target
analytes).
[0111] The invention also provides a method of conducting a
multiplexed binding assay for a plurality of analytes that includes
contacting (i) a sample with (ii) one or more first solid phases
linked to one or more first binding reagents that bind the analytes
to form complexes comprising the analytes bound to the first
binding reagents. The unbound components of the sample are,
optionally, separated from the complexes. The complexes are
released and then contacted with a plurality of binding domains
comprising second binding reagents that bind to the complexes,
wherein each binding domain comprises a second binding reagent that
binds to a complex comprising a secondary target analyte.
Thereafter, the amount of analyte bound to the binding domains is
measured.
[0112] According to another embodiment, a multiplexed assay may
comprise the acts of contacting at least a portion of a sample with
one or more binding surfaces comprising a plurality of binding
domains, immobilizing one or more analytes on the domains and
measuring the analytes immobilized on the domains. In certain
embodiments, at least two of the binding domains differ in their
specificity for analytes of interest. In one example of such an
embodiment, the binding domains are prepared by immobilizing, on
one or more surfaces, discrete domains of binding reagents that
bind analytes of interest. Optionally, the sample is exposed to a
binding surface that comprises an array of binding reagents.
Optionally, the surface(s) may define, in part, one or more
boundaries of a container (e.g., a flow cell, well, cuvette, etc.)
which holds the sample or through which the sample is passed. The
method may also comprise generating assay signals that are
indicative of the amount of the analytes in the different binding
domains, e.g., changes in optical absorbance, changes in
fluorescence, the generation of chemiluminescence or
electrochemiluminescence, changes in reflectivity, refractive index
or light scattering, the accumulation or release of detectable
labels from the domains, oxidation or reduction or redox species,
electrical currents or potentials, changes in magnetic fields,
etc.
[0113] Assays of certain embodiments of the invention may employ
targeting agents to link the target analyte with a binding reagent
in the assay medium. Such assay formats are illustrated in FIGS.
3(a)-3(e) and FIGS. 4(a)-4(b), which are analogous to FIGS.
1(a)-1(e) and FIGS. 2(a)-2(b), except that the binding of analyte
to a first binding reagent on a solid phase/particle takes place
through two steps: (i(a)) contacting the first binding reagent
linked to a first targeting agent to a particle (or other solid
phase) linked to a second targeting agent that binds to the first
targeting agent (thus attaching the first binding reagent to the
particle or other solid phase) and (i(b)) contacting the first
binding reagent with a sample comprising a target analyte that
binds the first binding reagent. Step i(a) may occur before step
i(b) (as shown in the figures) or the two steps may occur in the
reverse order or concurrently. Steps i(a) and i(b) may both be
carried out during the conduct of an assay or, alternatively, the
first binding reagent may be supplied to the user pre-bound to the
solid phase through the targeting agents (e.g., if the targeting
agents were pre-bound during manufacturing), in which case step
i(a) may be omitted.
[0114] Thus, in one embodiment, the method includes contacting a
sample comprising a target analyte with a particle linked to a
first binding reagent that binds the target analyte, wherein the
first binding reagent is linked to a first targeting agent and the
particle is linked to a second targeting agent, and the first
binding reagent and the particle are linked via a binding reaction
between the first and second targeting agents to form a complex
comprising the target analyte bound to the first binding reagent
(see e.g., FIG. 3(a)). The complex is then collected and unbound
components in the sample are separated from the complex. The
complex is released and the released complex is contacted with a
second binding reagent bound to a solid phase, wherein the second
binding reagent binds to the complex. The amount of analyte bound
to the solid phase is measured. As in the embodiments described
above and illustrated in FIGS. 1(a)-1(e), the detectable label may
be attached to various assay components in the medium, e.g., to a
third binding reagent, as in FIGS. 3(a)-3(b), to the particle, as
in FIG. 3(c), or to the first binding reagent, as in FIGS.
3(d)-3(e). Moreover, the complex is optionally cleaved from the
particle prior to the detection step, as in FIGS. 3(b) and
3(d).
[0115] In one embodiment, the assay may include (a) contacting a
sample comprising a target analyte with a first solid phase linked
to a first binding reagent that binds the target analyte, wherein
the first binding reagent is linked to a first targeting agent and
the first solid phase is linked to a second targeting agent, and
the first binding reagent and the first solid phase are linked via
a binding reaction between the first and second targeting agents to
form a complex comprising the target analyte bound to the first
binding reagent (see e.g., FIGS. 4(a)-4(b)). The complex is then
collected and unbound components in the sample are separated from
the complex. The complex is released, e.g., resolubilized, and the
first solid phase is removed. The released complex is contacted
with a second binding reagent bound to a second solid phase,
wherein the second binding reagent binds to the complex. The amount
of analyte bound to the second solid phase is measured. The
detectable label may be attached to any suitable assay component,
e.g., the first binding reagent, as in FIG. 4(b), or the third
binding reagent, as in FIG. 4(a).
[0116] The releasing step in the various assay formats described
herein may comprise cleaving a binding reagent from the particle
(e.g., as shown in FIG. 1(b)). This may be accomplished by any
suitable method, e.g., subjecting the complex to increased
temperature, pH changes, altering the ionic strength of the
solution, competition, and combinations thereof.
[0117] If a targeting agent is employed in the assay format, the
releasing step comprises disassociating the first and second
targeting agents, e.g., by subjecting the complex to increased
temperature, pH changes, altering the ionic strength of the
solution, competition, and combinations thereof as discussed
above.
[0118] The measuring step in the various assay formats described
herein may comprise any suitable method of measuring the presence
of a detectable label in a sample, e.g., optical absorbance,
fluorescence, phosphorescence, chemiluminescence, light scattering
or magnetism. In one embodiment, the detectable label is an
electrochemiluminescent label and the measuring step comprises
measuring an ECL signal and correlating that signal with an amount
of analyte in the sample. Thus, the measuring step may further
comprise contacting the complex with an electrode and applying a
voltage waveform to the electrode to generate ECL.
[0119] By analogy to the description of FIGS. 1 and 2, the methods
in FIGS. 3 and 4 may also be extended to multiplex measurements,
e.g., by employing at least one of the group consisting of i) a
plurality of different first binding reagents, ii) a plurality of
second binding reagents and iii) a plurality of third binding
reagents (the different reagents within (i), (ii) or (iii) being
selected for their ability to preferentially bind a target analyte
relative to other target analytes). In such multiplex methods, a
common targeting reagent pair may be used to link a plurality of
different first binding reagents to the corresponding particles or
other solid phases. Alternatively, a unique targeting reagent pair
may be used for each different first binding reagent (e.g., a
different set of complementary oligonucleotides may be used to
target each of the different first binding reagents). Such an
approach may be used to i) target different first binding reagents
to different distinguishable particles (e.g., particles bearing
distinguishable labels) or ii) enable multiplexing through the use
of a plurality of different second binding reagents, each of which
binds preferentially to a different first targeting agent (thus
preferentially binding complexes comprising one of the plurality of
analytes).
EXAMPLES
Example 1
Dual Use of Labeled Magnetic Particle to Concentrate and Detect
Analytes of Interest
[0120] As shown in FIG. 5, magnetic particles are coated with
antibodies against the analytes of interest and a large number
(e.g., greater than 100) ECL labels. By attachment of the ECL
labels to the antibodies (either before or after coating the
antibodies on the particles), very high numbers of labels can be
easily achieved. A particle of only 60 nm in diameter can support
roughly 160 antibody molecules, assuming about 50 nm.sup.2 of
surface area per antibody. Thus, attachment of only 1 label per
antibody allows labeling ratios of greater than 100 labels per
particle to be achieved for 60 nm particles. Labeling ratios of
greater than 1000 labels per particle are achieved by increasing
the number of labels per antibody and/or increasing the particle
size).
[0121] A 1 mL or greater volume of sample is combined with the
particles in a container and after incubating the mixture to allow
the antibodies to bind their respective targets, a magnetic field
is applied such that the magnetic particles collect on a surface in
the container (a variety of commercial magnetic tube holders or
probes are available for carrying out this step). The complexes are
washed with buffered saline to remove unbound components of the
sample. The magnetic field is removed and the particles are then
re-suspended in 100 uL of a suitable assay diluent, thus providing
a 10-fold or greater increase in concentration relative to the
original sample. The particle-analyte complexes are transferred to
an assay plate (e.g., a MULTI-ARRAY.RTM. 96-well assay plate, Meso
Scale Diagnostics, LLC, Gaithersburg, Md.) that includes a binding
surface comprising an array of antibody binding reagents directed
against the analytes of interest. Complexes that bind the array are
measured by ECL on a SECTOR.RTM. Imager instrument (Meso Scale
Diagnostics, LLC). The magnetic collection step provides for
improvements in assay performance by allowing for pre-concentration
of analyte into a small volume and removal of potential
interferents in the sample.
Example 2
Assay Using Antibodies Coupled to Magnetic Particles Through
Oligonucleotide Hybridization Reactions
[0122] Magnetic particles are coated with oligonucleotides and a
large number (greater than 100) ECL labels. Conjugates are formed
comprising antibodies against analytes of interest and
oligonucleotides complementary to the oligonucleotides on the
particles. The antibody conjugates and particles are subjected to
conditions sufficient to hybridize the complementary
oligonucleotide sequences (e.g., appropriate temperature, ionic
strength and denaturing conditions, as described hereinabove) and
thereby coat the antibodies on the particles. These particles are
then used to assay for analytes of interest as described in Example
1.
Example 3
Demonstration of the Release of Antibodies Coupled to Magnetic
Particles Through Oligonucleotide Hybridization Reactions
[0123] Magnetic beads (Dynalbeads.RTM. MyOne.TM.-Streptavidin C1
beads, Invitrogen Corporation) were coated with a biotinylated
oligonucleotide by the following procedure: The beads (3 mg) were
washed three times at 60.degree. C. in hybridization buffer (20 mM
Tris, 1 mM EDTA, 250 mM NaCl, 0.01% Triton-X at pH=8 and 0.1% BSA).
The beads were then coated at room temperature with 750 pmoles of a
19-mer biotinylated oligonucleotide (Oligo 1, Tm=40.degree. C.), in
1 mL of hybridization buffer, for one hour with gentle mixing. The
coated beads were washed 5.times. with hybridization buffer at
60.degree. C. and then resuspended in hybridization buffer at a
final concentration of 10 ug/mL. The magnetic beads were then
coated with labeled mouse immunoglobulin by the following
procedure: Mouse immunoglobulin (mIgG) was labeled with
Sulfo-TAG.TM. ECL labels (Meso Scale Diagnostics, LLC.) according
to the manufacturer's instructions. The protein was also labeled
with an oligonucleotide having a terminal thiol group (Oligo 2, the
complement of Oligo 1) using a bifunctional coupling reagent
(sulfosuccinimidyl 4-(N-maleimidomethyl)-1-cyclohexane carboxylate
("SMCC")) and conventional coupling protocols, e.g., protein is
reacted with the NHS-ester in SMCC to label the protein and the
resulting complex is reacted with thiolated oligonucleotides which
reacts with the maleimide group in SMCC. The labeled mIgG-oligo
conjugate (0.1 pmol) was then mixed with the oligo-coated magnetic
beads (500 ug of beads) in hybridization buffer for 1 hour at room
temperature to hybridize the complementary oligonucleotide
sequences and thereby immobilize the mIgG onto the beads. The
resulting antibody-coated beads were washed and resuspended in
hybridization buffer.
[0124] The beads were incubated under different conditions,
including incubating the suspension at room temperature for one
hour (with or without the presence of free Oligo2 as a competitor)
and incubating the suspension at 60.degree. C. for 10 min. (with or
without the presence of free Oligo2 as a competitor). The beads
were then magnetically collected and the supernatant analyzed by
ECL assay to measure the amount of labeled mIgG that was released
from the beads. To measure the labeled mIgG, the supernatant was
transferred to the well of a MULTI-ARRAY plate in which the
electrode is coated with goat anti-mouse antibodies (MULTI-ARRAY
GAM Plate, Meso Scale Diagnostics, LLC.). The plate was incubated
with shaking during which time labeled mIgG in the solution bound
to the immobilized goat anti-mouse antibodies. The wells were
washed with PBS, filled with 150 uL of Read Buffer T (Meso Scale
Diagnostics) and analyzed on a SECTOR Imager instrument.
[0125] Table 1 shows that, in the absence of competing
oligonucleotides, the linkage of the mIgG to the beads was stable
at room temperature. The mIgG could be efficiently released from
the beads by exposure to short periods of time above the melting
temperature of the Oligo 1-Oligo2 pair. The efficiency of release
could be further enhanced by addition of free Oligo2 as a
competitor.
TABLE-US-00001 TABLE 1 Efficiency of different release techniques.
Release Technique % of Released Material 1 H at RT 6% 1 H at RT
with free Oligo 23% 10 min 60 C. 50% 10 min 60 C. with Free Oligo
57%
Example 4
Assay Including Capture of Analyte Through Collection of Magnetic
Particles and Release by Denaturation of a Linkage Comprising an
Oligonucleotide Pair
[0126] Magnetic beads (Dynalbeads.RTM. MyOne.TM.-Streptavidin C1
beads, Invitrogen Corporation) were coated with biotinylated
oligonucleotides as described in Example 3. The magnetic beads were
then coated with antibodies against human TNF-alpha and IL-5 using
i) antibodies that were labeled with Sulfo-TAG and Oligo1 and ii)
the coating procedure of Example 3.
[0127] Assay Procedure with Pre-Concentration. Sample containing
human TNF-alpha or IL-5 (1 mL of sample) was combined with 200 ng
of antibody-coated beads (prepared as described above) and
incubated for 1 hr at room temperature. The beads were magnetically
collected and washed with hybridization buffer. The antibody on the
beads (including any labeled-antibody-analyte complexes that were
formed during the incubation) were released into 100 uL of a 1:20
dilution of hybridization buffer (.about.10 mM salt) at elevated
temperature (60.degree. C.), i.e., by denaturing the
oligonucleotide pairs linking the antibodies to the beads. The
resulting solution was transferred to a well of a MULTI-ARRAY
96-well plate, each well of which included an array of capture
antibodies including an anti-TNF-alpha spot and an anti-IL-5 spot.
The plate was incubated with shaking for 1 hr at room temperature
to allow the labeled-antibody-analyte complexes to bind to the
appropriate capture antibody spots. The wells were then washed
three times with PBS and then filled with 125 uL of Read Buffer T
(Meso Scale Diagnostics) and read on a SECTOR Imager instrument.
The instrument measures and reports the ECL intensity from each
array element (or "spot") in the antibody array.
[0128] Conventional Immunoassay Protocol without Pre-Concentration.
Sample containing human TNF-alpha or IL-5 (30 uL) was combined with
20 uL of a solution containing labeled (Sulfo-TAG) detection
antibodies at a concentration of 1 ug/mL. The resulting solution
was incubated for 1 hr in a well of a MULTI-ARRAY plate having
anti-TNF-alpha and anti-IL-5 spots. The wells were washed, filled
with Read Buffer T and analyzed in a SECTOR Imager instrument as
described for the protocol with collection and release.
[0129] Results. The results presented in Table 2 show that the
protocol with collection and release provided specific assay
signals for both TNF-alpha and IL-5 (signal in the presence of
analyte--signal in the absence of analyte) that were substantially
higher than those obtained using the conventional protocol, without
any substantial change in the background signal in the absence of
analyte. The enhancement in specific signal for 10 pg/mL samples
was greater than 5-fold for TNF-alpha and greater than 10-fold for
IL-5.
TABLE-US-00002 TABLE 2 Assay Analyte TNF IL-5 Concentration,
Conven- Pre- Conven- Pre- pg/mL tional Concentration tional
Concentration 0 371 398 22 27 1 530 1,095 95 451 10 3,301 18,323
723 9,831 100 31,005 75,864 8,057 48,895
[0130] The present invention is not to be limited in scope by the
specific embodiments described herein. Indeed, various
modifications of the method in addition to those described herein
will become apparent to those skilled in the art from the foregoing
description and accompanying figures. Such modifications are
intended to fall within the scope of the claims. Various
publications are cited herein, the disclosures of which are
incorporated by reference in their entireties.
* * * * *