Note: Descriptions are shown in the official language in which they were submitted.
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ASSAY MODULES HAVING ASSAY REAGENTS AND METHODS OF
MAKING AND USING SAME
10
FIELD OF THE INVENTION
The invention relates to assay modules, such as assay plates, cartridges,
multi-
well assay plates, reaction vessels, and methods for conducting chemical,
biochemical,
and/or biological assays. The invention also relates to the incorporation of
dry reagents
into these modules and/or the use dry reagent in these methods.
BACKGROUND OF THE INVENTION
Numerous methods and systems have been developed for conducting chemical,
biochemical, and/or biological assays. These methods and systems are essential
in a
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variety of applications including medical diagnostics, food and beverage
testing,
environmental monitoring, manufacturing quality control, drug discovery, and
basic
scientific research.
Depending on the application, it is desirable that assay methods and systems
have one or more of the following characteristics: i) high throughput, ii)
high
sensitivity, iii) large dynamic range, iv) high precision and/or accuracy, v)
low cost, vi)
low consumption of reagents, vii) compatibility with existing instrumentation
for
sample handling and processing, viii) short time to result, ix) multiplexing
capability,
and x) insensitivity to interferents and complex sample matrices. It is also
desirable in
to many applications that these types of performance benefits are achieved
with assay
= formats that are easy to carry out, are amenable to automation, and/or
use stable dry
reagents. There is substantial value to new assay methods and systems with
these
characteristics.
A variety of approaches have been developed that provide reagents for assays
in
dry stable form. U.S. Patent 5,413,732 describes certain dry reagent spheres
that are
capable of dissolving in a solution.
U.S. Patent 6,429,026 describes certain immunoassays using dry reagents and
time-resolved fluorescence detection. A catching antibody is immobilized on
the
surface of a microtitration well. An insulating layer containing carbohydrate
and/or
protein is dried on top of the catching antibody at the bottom of the well. A
labeled
antibody is added in a small volume and dried on top of the insulating layer.
The
antibody is labeled with a lanthanide chelate that can be detected using
dissociation
enhance lanthanide fluoroinununoassay (DELFIA) techniques. To start the
immunoassay, a sample and a common assay buffer is added. After allowing the
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antibody reactions to occur, the well is washed several times, a DELFIA
enhancement
buffer is added, and a fluorescence lifetime measurement is carried out.
U.S. Publication 2003/0108973 describes a sandwich immunoassay that
employed a test tube containing a lyophilized mixture comprising a capture
antibody
immobilized on 2.8 ptm magnetizable polystyrene beads and a detection antibody
labeled with an electrochemiluminescent label. The mixture could also include
blocking agents to reduce non-specific binding of the detection antibody to
the beads
during the lyophilization process. Addition of sample containing the analyte
of interest
resulted in the formation of sandwich complexes on the beads. A suspension of
beads
to was then aspirated into a reusable flow cell where they were collected
on an electrode
and analyzed using electrochemiluminescence (ECL) detection techniques.
U.S. Patent 6,673,533 of Wohlstadter et al. describes an ECL-based sandwich
immunoassay using dry reagents. A capture antibody was immobilized on a
composite
electrode. The other reagents used in assay were dried on the electrode
surface by
adding and lyophilizing a solution containing a detection antibody linked to
an ECL
label, phosphate, tripropylamine, bovine serum albumin, sucrose,
chloracetamide, and
TRITON X-100. Immunoassays were conducted by adding a sample to the dried
reagents on the electrodes, incubating the solutions, and applying a potential
to the
electrode to induce ECL. No washing step was required.
A variety of techniques have been developed for increasing assay throughput.
The use of multi-well assay plates (also known as microtiter plates or
microplates)
allows for the parallel processing and analysis of multiple samples
distributed in
multiple wells of a plate. Multi-well assay plates can take a variety of
forms, sizes, and
shapes. For convenience, some standards have appeared for instrumentation used
to
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process samples for high-throughput assays. Multi-well assay plates typically
are made
in standard izes and shapes, and have standard arrangements of wells.
Arrangements
of wells include those found in 96-well plates (12 x 8 array of w_911s), 384-
well plates
(24 x16 array of wells), and 1536-well plates (48 x 32 array of wells). The
Society for
Biomolecular Screening has published recommended microplate specifications for
a
variety of plate formats (see http://www.sbsonline.org).
U.S. Publications 2004/0022677 and 2005/0052646 of U.S. Applications
10/185,274 and 10/185,363, respectively, of Wohlstadter et al. describe
solutions that
are useful for carrying out singleplex and multiplex ECL assays in a multi-
well plate
to format. They include plates that comprise a plate top with through-holes
that form the
walls of the wells and a plate bottom sealed against the plate top to form the
bottom of
the wells. The plate bottom has patterned conductive layers that provide the
wells with
electrode surfaces. that act as both solid-phase supports for binding
reactions as well as
electrodes for inducing ECL. The conductive layers may also include electrical
contacts for applying electrical energy to the electrode surfaces.
Despite such known methods and systems for conducting assays, improved
assay modules for conducting chemical, biochemical, and/or biological assays
are
needed.
SUMMARY OF THE INVENTION
The invention relates to assay modules (e.g., assay plates, cartridges, or
multi-
well assay plates, reaction vessels, etc.), having assay reagents pre-loaded
in the wells,
chambers or assay regions of the assay module. In certain embodiments, these
assay
reagents are stored in a dry state. Furthermore, the assay modules may
comprise
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desiccant materials for maintaining these assay reagents in a stable dry
state. A method
is provided for making such assay modules and methods for using the assay
modules in
assays.
A multi-well plate is provided comprising at least one well having (1) a
binding
5 surface having a first binding reagent immobilized thereon and (2) at
least one
additional dry reagent, wherein at least one additional dry reagent does not
contact the
binding surface. The multi-well plate may have an electrode surface with a
binding
surface incorporated in at least one well of the multi-well plate.
A multi-well assay plate is provided comprising a plate body with a plurality
of
wells defined therein, the plurality of wells comprising a binding surface
having a
capture reagent immobilized thereon and a reconstitutable dry reagent.
Optionally, the
binding surface may be selected to be suitable for use as an electrode in an
electrochemical assay or electrochemiluminescence assay. Furthermore, the
binding
surfaces may be coated with a reconstitutable protective layer. The dry
reagent, which
may be a labeled detection reagent, is free standing or located on a surface
of the well
that does not overlap with the binding surface. In one specific example, the
binding
surface is located on the bottom of the well and the reconstitutable dry
reagent is
located on a wall of the well and, optionally, on a reagent storage helf
defined on the
wall. In another example, the binding surface and the reconstitutable dry
reagent are
both located on non-overlapping regions of the bottom surface of the well. In
another
specific example, the reconstitutable dry reagent is a free-standing pill.
The multi-well assay plate may further comprise a reconstitutable dry assay
control analyte which may have binding affinity for the immobilized capture
reagent
and/or, if present, the labeled detection reagent. In certain embodiments, the
control
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analyte has affinity for immobilized capture reagents and/or labeled detection
reagents
within the well, but is present in unbound form that is not in contact with
the binding
surface or labeled detection reagent.
The multi-well assay plate may further comprise one or more additional
immobilized capture reagents. The capture reagent and additional capture
reagents are
patterned on the binding surface to form an array of binding domains on the
binding
surface. These binding domains/capture reagents may differ in specificity or
affinity
for binding partners. In addition, the wells may contain a plurality of
different
reconstitutable dry labeled detection reagents that differ in specificity or
affinity for
to binding partners.
The multi-well plates, described above, may be used in methods of carrying out
assays comprising adding sample to one or more of the wells of a plate
comprising
immobilized capture reagents and reconstitutable dry labeled detection
reagents,
reconstituting reconstitutable dry materials in these wells to form a reaction
mixture(s),
incubating the reaction mixture(s) under conditions that promote binding of
said
capture and detection reagents to their corresponding binding partners, and
measuring
the formation of complexes comprising the immobilized capture reagents and
labeled
binding reagents. By appropriate choice of capture and detection reagents,
these
methods may include sandwich binding assay methods and competitive binding
assay
methods.
A method is provided of preparing multi-well assay plates for use in an assay
comprising carrying out the following on at least two weds of a plate:
immobilizing a
capture reagent on a surface of a well of said plate to form a binding
surface,
dispensing a liquid reagent comprising a labeled detection reagent to a
surface of the
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well that does not overlap the binding surface, and drying the liquid reagent
to form a
reconstitutable dry detection reagent. The method may also include dispensing
a
protecting reagent on the binding surface and drying the protecting reagent to
form a
reconstitutable dry protective layer on the binding surface. For example, the
protecting
feagent is dispensed and dried prior to dispensing the liquid reagent
comprising a
labeled detection reagent.
In certain specific embodiments, the binding surface is on a bottom surface of
the well and the liquid reagent is dispensed and dried on a non-overlapping
bottom
surface of the well or on a wall of the well. Optionally, the wall comprises a
liquid
storage shelf and the liquid reagent is (i) dispensed and dried on the shelf
or (ii)
dispensed on the wall at a location above the shelf such that liquid reagent
that runs
down the wall collects and is subsequently dried on the shelf.
The methods for preparing plates may further comprise immobilizing one or
more additional capture reagents so as to form an array of binding domains on
the
is binding surface that differ in their specificity or affinity for binding
partners. Similarly,
the liquid reagent may comprise one or more additional labeled detection
reagents that
differ in their specificity or affinity for binding partners. Furthermore, the
method may
include dispensing and drying an additional liquid reagent comprising an assay
control
analyte with binding affinity for the capture or labeled detection reagent,
the additional
liquid reagent being dispensed and dried such that it des not contact the
capture or
labeled detection reagents.
In certain alternate embodiments of the methods described above, dispensing
and drying a liquid reagent comprising a labeled detection reagent are omitted
and a
reconstitutable dry labeled detection reagent is added in free-standing form,
for
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example, as a free-standing pill. Preferably, prior to adding the detection
reagent, a
protecting reagent is dispensed and dried on the binding surface to form a
reconstitutable protective layer. The method may also include immobilizing one
or
more additional capture reagents so as to form an array of binding domains on
said
binding surface that differ in their specificity or affinity for binding
partners. Similarly,
the reconstitutable dry reagent may comprise one or more additional labeled
detection
reagents that differ in their specificity or affinity for binding partners.
Furthermore, the
method may include adding to the well an additional free standing dry reagent
comprising an assay control analyte that has binding affinity for the capture
and/or
detection reagents.
A multi-well plate is provided comprising a plate body with a plurality of
wells
defined therein including: a) a plurality of first reagent wells holding a
reconstitutable
first dry reagent and b) a plurality of second reagent wells holding a second
dry reagent,
wherein, the first and second reagents are matched reagents for conducting an
assay. A
method is provided for carrying out assays in these plates comprising: a)
adding a
sample to one of the first reagent wells, b) reconstituting reconstitutable
dry labeled
detection reagents in the first reagent well to form a reaction mixture, c)
transferring an
aliquot of the reaction mixture to one or more of the second reagent wells,
and d)
incubating the reaction mixture in the second reagent well(s) so as to carry
out said
assay on said sample. In one embodiment, the multi-well assay plate can be
divided
into a plurality of sets of wells consisting of one first reagent well and one
or more
second reagent wells and the method further comprises repeating the process of
(a)-(d)
for each set of wells.
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In one specific embodiment of the multi-well plate having first and second
reagent wells, the first reagent wells are arranged in a regular two
dimensional pattern
and said first reagent wells have well floors and well walls, the well walls
having inner
wall surfaces and outer wall surfaces. Furthermore, the second reagent wells
have well
floors and well walls, the well walls being defined by outer wall surfaces of
the
detection wells and by rib elements connecting the outer wall surfaces of
adjacent
detection wells. Optionally, the first reagent wells have well opening
perimeters that
are round and/or the first reagent wells are arranged in an 8 x 12 square
array.
A multi-well assay plate is provided comprising a plate body with a plurality
of
to wells defined therein including: a) a plurality of detection wells, each
detection well
comprising a binding surface having a capture reagent immobilized thereon and
b) a
plurality of reagent reconstitution wells, each reagent reconstitution well
comprising a
reconstitutable labeled detection reagent, wherein, at least one detection
well and one
reagent reconstitution well comprise matched capture and detection reagents
for
measuring an analyte of interest. Optionally, the binding surface may be
selected to be
suitable for use as an electrode in an electrochemical or
electrochemiluminescence
assay. In one embodiment, the detection and reagent reconstitution wells are
grouped
into a plurality of assay sets consisting of one reagent reconstitution well
and one or
more detection wells, the reagent reconstitution well and detection wells
within a set
comprising matched capture and detection reagents for measuring an analyte of
interest.
These sets may consist of one reagent reconstitution well and one detection
well.
One specific embodiment of the multi-well plate with a detection wells and
reagent reconstitution wells includes:
a) a plurality of detection wells, wherein said detection wells,
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i) have well floors and well walls, said well walls having inner wall
surfaces and outer wall surfaces,
ii) are arranged in a regular two dimensional pattern, and
iii) comprise, on an inner surfaces of each of said detection wells, a binding
5 surface having a capture reagent immobilized thereon array;
b) a plurality of reagent reconstitution wells, wherein said reagent
reconstitution wells
i) have a well floors and well walls, said well walls being defined by
outer
wall surfaces of said detection wells and by rib elements connecting the
10 outer wall surfaces of adjacent detection wells, and
ii) comprise, in each reagent reconstitution well, a reconstitutable dry
labeled detection reagent.
Optionally, the detection wells have well opening perimeters that have no
reentrant angles or curves (e.g., round pierimeters) and the reagent
reconstitution wells
have well opening perimeters with reentrant angles or curves.
The detection or reagent reconstitution wells of the multi-well plates with
detection wells and reagent reconstitution wells may further comprise a
reconstitutable
dry assay control analyte. The detection wells may also comprise one or more
additional immobilized capture reagents. In this embodiment, the capture
reagents are
patterned to form a patterned array of binding domains on the binding surface
that =
differ in specificity or affinity for binding partners. Furthermore, the
reconstitutable
dry reagent may further comprise one or more additional labeled detection
reagents, the
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detection reagent and additional detection reagents differing in specificity
or affinity for
binding partners.
A method is provided fox carrying out assays in multi-well plates with
detection
wells and reagent reconstitution wells. One embodiment comprises a) adding a
sample
to one of the reagent reconstitution wells, b) reconstituting reconstitutable
dry labeled
detection reagents in the. reconstitution well to form a reaction mixture(s),
c)
transferring an aliquot of the reaction mixture to one or more detection
wells, c)
incubating the reaction mixture in the detection well(s) under conditions that
promote
binding of the capture and detection reagents to their corresponding binding
partners,
and d) measuring the formation of complexes comprising the immobilized capture
reagents and the labeled binding reagent. Optionally, the multi-well assay
plate can be
divided into a plurality of sets of wells consisting of one first reagent well
And one or
more second reagent wells and the method further comprises repeating the
process of
(a)-(d) for each of said set of wells.
A multi-well assay plate is provided comprising a plate body with a plurality
of
= wells defined therein having well floors and well walls that extend from
said floors to a
height hw above said floors, said walls being shaped so as to provide shelf
elements at a
height 1i, wherein 0< h <h. The wells may be arranged in standard multi-well
plate
formats including 4 x 6, 8 x 12, 16 x 24, and 32 x 48 arrays of wells arranged
in square
lattices. In certain embodiments, hs is greater than or equal to 0.02 hõ 0.05
h, or 0.1
hw but less than or equal to 0.1 h, 0.25h, or 0.5 hw. In other embodiments, h,
is
greater or equal to about 0.1 mm, 0.2 min 0.5 mm, or 1 mm but less than or
equal to
about 1 mm, 2 mm, or 5 mm. The shelf elements may be used to hold dry
reagents.
Thus, another embodiment is a plate with reconstitutable dry reagents on the
shelves.
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A method is provided for preparing plates for use in an assay that comprise
dispensing
a liquid reagent in a well of a multi-well plate that has a shelf element and
drying the
reagent to form a reconstitutable dry reagent, wherein the reagent is
dispensed and
dried on the shelf or dispensed on the wall above the shelf and dried such
that liquid
reagent that runs down the well wall collects on and is dried on the shelf.
In certain embodiments of the plate with wells with shelf elements, the plate
body is a one-piece injection-molded part. In other embodiments, the plate
body
comprises a plate top haying a plurality of through-holes that define the
walls of the
well and a plate bottom that is sealed against said plate top and defines the
well floors.
Optionally, the plate bottom provides conductive electrode surfaces that are
exposed to
the interior volume of the wells and may be used as electrodes in
electrochemical
assays or electrochemiluminescence assays.
A multi-well plate is provided comprising
a) a plate body with a plurality of wells defined therein including:
i) a plurality of assay wells comprising a dry assay reagent; and
ii) a plurality of desiccant wells comprising a desiccant, and
b) a plate seal sealed against said plate body thereby isolating said
plurality of
wells from the external environment.
The plate is optionally, arranged so that the wells are in a standard well
arrangement (e.g., 4x6, 8x12, 16x24 or 32x48 arrays of wells arranged in a
square
lattice). Suitable configurations of assay wells include, but are not limited
to, wells
with dry reagents (e.g., capture and/or detection reagents) as described in
the
embodiments described above. Advantageously, the desiccant wells may be
connected
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by drying conduits to the assay wells, the conduits permitting diffusion of
water vapor
from the assay wells to the desiccant wells but intersecting the wells at a
height in the
assay well above the location of the dry assay reagent. In one embodiment,
such
conduits may be provided by sealing the plate seal against recessed channels
in the top
surface of the plate body that connect assay wells to desiccant wells. In
certain
embodiments, the wells of said plate are divided into a plurality of assay
panels
comprising at least one assay well and at least one dessicant well. In these
embodiments, the wells in an assay panel are interconnected via drying
conduits but are
not connected to wells in other assay panels. In one specific embodiment, an
assay
panel comprises one assay well and one desiccant well.
In one embodiment of a multi-well plate with assay wells and desiccant wells,
the assay well comprises a binding surface having a capture reagent
immobilized
thereon and a reconstitutable dry labeled detection reagent. The assay well
may further
comprise one or more additional immobilized capture reagents, the capture
reagent and
additional capture reagents forming a patterned array of binding domains on
said
binding surface, the binding domains differing in specificity or affinity for
binding
partners. In addition, the reconstitutable dry reagent may further comprise
one or more
additional labeled detection reagents, the detection reagent and additional
detection
reagents differing in specificity or affinity for binding partners.
Optionally, the binding
surface is suitable for use as an electrode in an electrochemiluminescence
assay.
In certain embodiments of a multi-well plate with assay wells and desiccant
wells, the plate body is a one-piece injection-molded part. Alternatively, the
plate body
may comprise a plate top having a plurality of through holes that define well
walls and
plate bottom that is sealed against said plate top and defines well floors.
Said through
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holes and plate bottom may define all the wells or on only a portion of the
wells, e.g.,
only said Assay wells or only said desiccant wells. The plate bottom may,
optionally,
provide conductive electrode surfaces that are exposed to the interior volume
of the
wells.
A multi-well plate is also provided comprising
a) a plate body with a plurality of wells defined therein containing a dry
assay
reagent, said plate body comprising a plate top having a plurality of
through-holes that define well walls and a plate bottom that is sealed against
said plate top and defines well floors,
113 b) a plate seal sealed against said plate body, thereby isolating
said plurality of
wells from the external environment, and
c) a dessicant material.
The plate is optionally, arranged so that the wells are in a standard well
arrangement (e.g., 4 x 6, 8 x 12, 16 x 24, or 32 x 48 arrays of wells arranged
in a
square). The plate bottom may, optionally, provide conductive electrode
surfaces that
are exposed to the interior volume of the wells. Suitable configurations of
assay wells
include, but are not limited to, wells with dry reagents (e.g., capture and/or
detection
reagents) as described in the embodiments above. In certain embodiments, the
desiccant is comprised in the plate seal, a gasket layer between the plate
seal and the
plate top, the plate top, a gasket layer between the plate top and plate
bottom and/or the
plate bottom. For example, the desiccant may be impregnated in these
components or
in a coating on these components, etc. Alternatively, the plate body may
define one or
= more additional wells that hold the desiccant.
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In one embodiment, the assay well comprises a binding surface having a capture
reagent immobilized thereon and a reconstihitable dry labeled detection
reagent.
Optionally, the binding surface is suitable for use as an electrode in
electrochemical or
electrochemiluminescence assays. The assay well may further comprise one or
more
5 additional immobilized capture reagents, the capture reagent and
additional capture
reagents forming a patterned array of binding domains on said binding surface
that
differ in specificity or affinity for binding partners. In addition, the
reconstitutable dry
reagent may further comprise one or more additional labeled detection
reagents, the
detection reagent and additional detection reagents differing in specificity
or affinity for
10 binding partners.
BRIEF DESCRIPTION OF DRAWINGS
Figures la-le show non-scale schematic views of several embodiments of
multi-well plate wells that include dry reagents.
15 Figures 2a-2j show non-scale schematic top and cross-sectional views of
several
embodiments of wells having walls with shelf elements including ledges
(Figures 2a-
2f), bridges (Figures 2g-2h) and tables (Figures 2i-2j) that may be used to
support dry
reagents.
Figures 3a-3c show schematic illustrations of multi-well plates having
detection
wells and reagent reconstitution wells.
Figures 4a-4b show top and cross-sectional schematic views of one embodiment
of a plate having detection wells and reagent reconstitution wells, the
reagent
reconstitution wells being located in interstitial spaces between the
detection wells.
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Figures 5a-5f show schematic views of multi-well plates 500 (Figures 5a-5b),
520 (Figures 5c-5d) and 540 (Figures 5e-50 having assay wells and desiccant
wells.
Figure 6 is a schematic exploded view of one einbodiment of a multi-well assay
plate.
Figures 7a-7c show three schematic views of a multi-well plate that is
configured to carry out array-based multiplexed electrochemiluminescence
assays.
Figure 8 shows one embodiment of a square well plate with dry reagent ledges
and seven spots per well.
Figure 9 shows one embodiment of a square well plate with dry reagent ledges,
lo seven spots per well, and drying conduits between pairs of adjacent
wells.
Figure 10 shows the effect of incorporating desiccant wells into assay plates
on
the stability of dry reagents stored in the plates.
DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION
We describe assay modules (for example, assay plates, cartridges, multi-well
assay plates, reaction vessels, etc.) having assay reagents pre-loaded in the
wells,
chambers, or assay regions of the assay module. In certain embodiments, these
assay
reagents are stored in a dry state. Furthermore, the assay modules may
comprise
desiccant materials for maintaining the assay reagents in a dry state. The
assay =
modules preloaded with the assay reagents can greatly improve the speed and
reduce
the complexity of assay measurements while maintaining excellent stability
during
storage. We also describe methods for making such assay modules and methods
for
using the assay modules in assays.
=
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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 ways, 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. In certain
embodiments, a
dry reagent (e.g., a reconstitutable dry reagent) is included that comprises
ammonium
phosphate as a buffering component, comprises other ammonium salts, and/or
comprises less than about 1% (w/w) or less than about 0.1% (w/w) of sodium or
potassium ions.
Many of the embodiments will be described in the context of multi-well plates
holding dry capture and detection reagents for binding assays where the
capture and
detection reagents are stored on the plate in a manner that prevents them from
contacting each other. But it will be clear to the skilled artisan that such
embodiments
can be more generally applied to the storage of any number of different dry
assay
reagents (whether they are binding reagents, immobilized or not, labeled or
not, etc.) in
a manner that prevents them from contacting each other prior to use. Likewise,
while
many of the drawings use a "Y" symbol to represent reagents or binding
reagents, the
use of this symbol should not be interpreted as limiting these reagents to
antibodies
unless specifically stated. It will also be clear that the embodiments can be
more
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generally applied to assay reagents stored in other types of assay modules in
compartments other than wells (e.g., chambers, channels, flow cells, etc.).
The descriptor "reconstitutable dry" may be used to refer to dry reagents as
in
reconstitutable dry reagents with labeled detection reagents or dry
reconstitutable
protective layers, etc. This terminology is used to refer to dry reagents that
are
reconstituted by the addition of a sample or solvent to form a solution or
suspension.
Preferably, they are water-soluble or otherwise reconstitutable by addition of
an
aqueous sample. By comparison, an "immobilized" reagent, as the term is used
herein,
refers to the reagent that will normally remain on a surface after addition of
a sample
during the conduct of an assay, although there may be specific conditions that
can be
used to actively dissociate it from the surface.
Reconstitutable dry reagents may be prepared in situ in a compartment of an
assay module (e.g., in the well of a multi-well assay plate). By way of
example, a
volume of a liquid reagent may be dispensed into the well or other compartment
and
dried (e.g., by air drying, vacuum drying, freeze drying, etc.) to form the
reconstitutable
dry reagent. By adding a small volume that remains confined on a discrete
surface of
the compartment (e.g., a discrete location on the bottom or wall of a well),
the resulting
dry reagent may remain fixedly confined to that location. Alternatively, a
volume may
be added that is sufficient to spread across the bottom surface or to fill the
compartment/well so as to form a dry reagent layer over the contacted
surfaces.
Reconstitutable dry reagents may be prepared outside the assay module and
added to a
compartment of the module (e.g., a well of a multi-well plate) in dry fonn
(e.g., as a dry
powder or as a free-standing dry pill). Pill refers herein to a contiguous dry
object such
as a pressed dry tablet or a lyophilized dry bead (as in U.S. Patent
5,413,732).
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19
Some embodiments include or employ dry binding reagents that are useful in
carrying out binding assays. Binding reagents that can be used in the assay
modules
and methods include, but are not limited to, antibodies, receptors, ligands,
haptens,
antigens, epitopes, mimitopes, aptamers, hybridization partners, and
intercalaters.
Suitable binding reagent compositions include, but are not limited to,
proteins, nucleic
acids, drugs, steroids, hormones, lipids, polysaccharides, and combinations
thereof.
Nucleic acids and proteins (in particular, antibodies) have proven especially
useful in
binding assays. The skilled artisan will be able to identify appropriate
binding reagents
for a specific application. As used herein, 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 & Weir,
J. Cell.
Physiol., 67 (Suppl. 1):51-64, 1966 and Hochman et al. Biochemistry 12:1130-
1135,
1973). The term also includes intact antibody molecules, antibody fragments
and
antibody constructs that have been chemically modified, e.g., by the
introduction of a
label. As used herein, the term nucleic acid will be generally applied to
include not
only DNA and RNA but also analogs (such as peptide nucleic acids or
phosphorothioate linked nucleic acids) that can participate in specific Watson-
Crick or
Hoogstein hybridization reactions with DNA or RNA sequences and also includes
nucleic acids and analogs that have been chemically modified, e.g., by the
introduction
of a label.
The term "capture reagent" is used herein to refer to binding reagents that
are
immobilized on surface to form a binding surface for use in a solid phase
binding assay.
The assay modules and methods may also employ or include another binding
reagent,
CA 02893383 2015-06-01
"the detection reagent" whose participation in binding reactions on the
binding surface
can be measured. The detection reagents may be measured by measuring an
intrinsic
characteristic of the reagent such as color, luminescence, radioactivity,
magnetic field,
charge, refractive index, mass, chemical activity, etc. Alternatively, the
detection
5 reagent may be labeled with a detectable label and measured by measuring
a
characteristic of the label. Suitable labels include, but are not limited to,
labels selected
from the group consisting of electrochemiluminescence labels, luminescent
labels,
fluorescent labels, phosphorescent labels, radioactive labels, enzyme labels,
electroactive labels, magnetic labels and light scattering labels.
10 Assays that may be carried out include "sandwich assays" that employ
an
immobilized capture reagent and a detection reagent that can bind
simultaneously to an
analyte of interest so as to have the effect of sequestering the detection
reagent on the
binding surface. Thus, the presence of the analyte can be measured by
measuring the
accumulation of the detection reagent on the surface. Assays may also include
15 "competitive assays" that i) employ an immobilized capture reagent that
competes with
an analyte for binding to a detection reagent or ii) a detection reagent that
competes
with an analyte for binding to an immobilized capture reagent. In the case of
the
competitive assay, the presence of analyte .leads to a measurable decrease in
the amount
of detection reagent on the binding surface.
20 Capture or detection reagents may directly bind to (or compete with)
an analyte
of interest or may interact indirectly through one or more bridging ligands.
=
Accordingly, the dry assay reagents may include such bridging ligands. By way
of
example, streptavidin or avidin may be used as capture or detection reagents
by
employing biotin-labeled bridging reagents that bind or compete with the
analyte of
CA 02893383 2015-06-01
21
interest. Similarly, anti-hapten antibodies may be used as capture or
detection reagents
by employing hapten labeled binding reagents that bind or compete with the
analyte of
interest. In another example, anti-species antibodies or Fc receptors (e.g.,
Protein A, G
or L) are used as capture or detection reagents through their ability to bind
to analyte
specific antibodies. Such techniques are well established in the art of
binding assays
and one of average skill in the art will be able to readily identify suitable
bridging
ligands for a specific application.
Certain embodiments of the assay modules/plates include a capture reagent
immobilized on a surface of the module/plate so as to form a binding surface.
Immobilization may be carried out using well established immobilization
techniques in
the art of solid phase binding assays such as the techniques that have been
established
for carrying out ELISA assays or array-based binding assays. in one example,
binding
reagents may be non-specifically adsorbed to a surface of a well of a multi-
well plate.
The surface may be untreated or may have undergone treatment (e.g., treatment
with a
plasma or a charged polymer) to enhance the adsorbance properties of the
surface. In
another example, the surface may have active chemical functionality that
allows for
covalent coupling of binding reagents. After immobilizing the reagent, the
surface
may, optionally, be contacted with a reagent comprising a blocking agent to
block
uncoated sites on the surface. For conducting multiplexed measurements,
binding
surfaces with arrays of different capture reagents may be used. A variety of
techniques
for forming arrays of capture reagents are now well established in the art of
array based
assays.
The binding surfaces are, optionally, coated with a reconstitutable dry
protective
layer. The protective layer may be used to stabilize a binding surface, to
prevent a
CA 02893383 2015-06-01
22
binding surface from contacting detection reagents during manufacture or
storage, or
simply as a location to store assay reagents such as bridging reagents,
blocking
reagents, pH buffers, salts, detergents, electrochempurninescence coreactants,
etc.
Stabilizers that may be found in the protective layer include, but are not
limited to,
sugars (sucrose, trehalose, marmitol, sorbitol, etc.), polysaccharides and
sugar polymers
(dextran, FICOLL, etc.), polymers (polyethylene glycol, polyvinylpyrrolidone,
etc.),
zwitterionic osmolytes (glycine, betaine, etc.) and other stabilizing
osmolytes
(trimethylamine-N-oxide, etc.). Blocking agents are materials that prevent non-
specific
binding of assay components, especially detection reagents, to binding
surfaces and
include proteins (such as serum albumins, ganuna globulins, immunoglobulins,
dry
milk or purified casein, gelatin, etc.), polymers (such as polyethylene oxide
and
polypropylene oxide) and detergents (e.g., classes of non-ionic detergents or
surfactants
are known by the trade names of BRIJ, TRITON, TWEEN, THESIT, LUBROL,
GENAPOL, PLURONIC, TETRONIC, and SPAN). In certain embodiments, a
protective layer is included that comprises ammonium phosphate as a buffering
component, comprises other ammonium salts, and/or comprises less than 1% or
0.1%
(w/w) sodium or potassium ions.
One embodiment is a multi-well plate comprising at least one well having (1) a
first dry assay reagent and (2) a second dry assay reagent wherein one or both
of said
first and second dry reagents is a reconstitutable thy reagent and wherein
said first and
second dry reagents do not contact each other. The well may further include
one or
more additional dry reagents. These may include one or more additional
reconstitutable dry reagents that do not contact the first and/or second dry
reagents.
The embodiment also includes methods for conducting assays in these plates for
an
CA 02893383 2015-06-01
23
analyte of interest comprising adding' liquid samples to one or more wells of
a plate,
reconstituting reconstitutable dry reagents in the wells and measuring an
analyte-
dependent assay signaLso as to measure analyte in the sample. The skilled
artisan will
be able to readily select reagents and detection methodology for measuring a
wide
variety of analytes based on knowledge in the assay art. Detectable signals
that may be
measured include, but are limited to, optical absorbance, photoluminescence
(e.g.,
fluorescence), chemiluminescence, electrical current or potential, catalytic
activity,
chemical activity, light scattering, agglutination, radioactivity,
electrochemiluminescence, magnetism, changes in refractive index, and other
signals
that have been used in assay measurements.
Another embodiment is a multi-well plate comprising at least one well having
(1) a binding surface having a first binding reagent immobilized thereon and
(2) at least
one additional dry reagent, wherein at least one additional dry reagent is a
reconstitutable dry reagent that does not contact the binding surface. The
multi-well
plate may have an electrode surface with a binding surface incorporated in at
least one
well of the multi-well plate.
Figures la-le show non-scale schematic views of several embodiments of well
100 of a multi-well plate. The well is defined by well floor 120 and well
walls 110.
Floor 120 and walls 110 may be formed of a single contiguous material or may
be
separate components (e.g., a plate top and plate bottom) that are mated
together. Well
100 also contains a first dry reagent 130 located on floor 120 that, as shown,
may be
one or more capture reagents that are immobilized on floor 120 to form a
binding
surface. First dry reagent 130 may include a plurality of immobilized capture
reagents
(e.g., reagents 130a, 130b, and 130c) that are patterned into a plurality of
discrete
CA 02893383 2015-06-01
24
binding domains (e.g., an array). Advantageously, the binding reagents/domains
may
have different affinity or specificity for binding partners; such binding
domains may be
used to carry out multiplexed array-based measurements. A reconstitutable
protective
layer 140 covers dry reagent 130. Protective layer 140 may be omitted, e.g.,
when it is
not required to physically separate reagents 130 and 150. Well 100 also
comprises a
second dry reagent 150 that is a reconstitutable dry reagent. Second dry
reagent 150
may comprise a detection reagent such as labeled detection reagent 160.
Optionally,
second dry reagent 150 comprises a plurality of detection reagents that differ
in affinity
or specificity for binding partners. Well 100 may also include an, optional,
additional
reconstitutable dry reagent 170 that comprises an assay control analyte 180
(as shown
in Figures Ic-le). Also shown is plate seal 190. Plate seal 190, which may be
omitted,
is sealed against the top surface of walls 110 to protect the dry reagents
from the
environment.
Figure la shows an embodiment in which first dry reagent 130 is coated with
= 15 reconstitutable protective layer 140. Second dry reagent 150 is
layered onto of
protective layer 140 which prevents second dry reagent 150 from contacting
first dry
= reagent layer 130. In one example of this embodiment, second dry reagent
150 is
deposited by dispensing it in liquid form on protective layer 140; protective
layer 140 is
chosen to have enough thickness or mass such that it can adsorb this liquid
without
allowing it to=contact dry reagent 130. The liquid is then dried to form
second dry
reagent 150. In an alternate example, protective layer 140 is introduced in
liquid form
and frozen in the well to form a first frozen layer. Reagent 150 is then
introduced in
liquid form and frozen as a second frozen layer over the first frozen layer.
Lyophilization of the two frozen layers provides the layered dry reagent
structure.
CA 02893383 2015-06-01
Figure lb shows an embodiment where reagents 130 and 150 are both fixedly
located on non-overlapping regions of floor 120. Additional dry reagents, such
as
assay control reagents (not shown), could be located on other non-overlapping
regions
of floor 120. The localization of reagents on selected regions of floor 120
may be
5 carried out using standard techniques in patterned reagent deposition or
dispensing.
Optionally, floor 120 has relatively hydrophilic domains surrounded by
relatively
hydrophobic areas such that appropriate volumes of feagents dispensed on the
hydrophilic domains will spread to defined boundaries determined by the
hydrophobic
areas. In this and other embodiments where reconstitutable dry reagents are
located on
10 a surface, one may pre-treat the surface with blocking agents to prevent
adsorption of
the reagents to the surfaces and/or include blocking agents in the reagent
composition.
Figure lc shows an embodiment where second dry reagent 150 is fixedly
located, as one or more dry reagent pills, on walls 110. The pills may be
formed, e.g.,
by dispensing one or more droplets of the reagent (in liquid form) on walls
110 and
15 drying them to form the dry reagent pills. Figure lc also shows optional
additional dry
reagent 170 with control analyte 180 fixedly located on another non-
overlapping region
of walls 110. Figure ld shows an embodiment that is like that shown in Figure
lc
except that reagents 150 and 170 are located on shelves 115 on walls 110. Dry
reagents
150 and 170 may be formed from liquid reagents by dispensing and drying them
on
20 shelves 115 or dispensing them above shelves 115 so that they run down
walls 110 onto
shelves 115 where they are dried. Alternatively, free-standing dry reagent
pills may be
placed on shelves 115.
Finally, Figure le shows an embodiment where reagent 150 and optional
reagent 170 are free standing dry reagent pills. Also included are embodiments
of well
CA 02893383 2015-06-01
26
100 in which there is some combination of reconstitutable dry reagents on the
well
floor, well walls, well shelves, and/or in free-standing form. In alternate
embodiments,
some combination of fixedly located and free standing reconstitutable dry
reagents is
employed.
As shown in the embodiments in Figure I, the multi-well plates include those
having wells with multiple, physically-distinct, dry reagents. Similarly, for
carrying
out different assays in different wells, there may be different dry reagents
in different
wells. It may be desirable, for example for QC purposes, to be sure that the
correct dry
reagents are present in the wells of a plate. Accordingly, the dry reagents
may include
indicators (such as dyes or fluorophores) that can be used in optical
inspection of the
plates. By using different distinguishable indicators in different dry
reagents, it is
possible to optically inspect a plate to ensure that the correct reagents are
in the
appropriate locations in the appropriate wells of a plate.
Figure 2 shows non-scale schematic views of several embodiments of wells that
have shelf elements on which liquid reagents can be held and dried and/or on
which
free-standing dry reagents may be supported above the well bottom. The shelf
elements may include ledges, bridges or tables as described below. Figure 2a
is a
cross-section of a well 200 showing well bottom 220 and well wall 210, the
well wall
having ledges such as ledges 230 and 235 that can support dry reagents. Ledge
230 has
an angle that is substantially 90 or less than 90 relative to the wall
directly above the
ledge such that an appropriate volume of reagent can be dispensed on ledge 230
and
accumulate on ledge 230 without overflowing onto well bottom 220. The ledges
may
also have additional features to help contain reagents such as lip 240 on
ledge 235.
CA 02893383 2015-06-01
27
Shelf elements such as ledge 235 may be located at any height (he) above well
bottom 240 (hi, =0) and below the height of the well (hw). In some
embodiments, hs is
greater than or equal to 0.02 Kff, 0.05 hw or 0.1 hw but less than or equal to
0.1 hw, 0.25
hw or 0.5 hw. In other embodiments, he is greater or equal to about 0.1 mm,
0.2 mm, 0.5
mm, or 1 mm but less than or equal to about 1 mm, 2 mm, or 5 mm. Through
proper
selection of shelf height and volumes of sample/reagent added during the
course of an
assay, it may be possible to control the order or timing of assay reactions.
In one
example, the shelf height and sample volume are chosen such that addition of
sample to
the well provides a height of liquid that contacts reagents on the bottom of
the well and
to also reconstitutes reagents on one or more shelves. Alternatively, shelf
height may be
chosen so that addition of defined volume of a first liquid contacts dry
reagents on the
bottom of the well (reconstituting reconstitutable reagents on the bottom
and/or
allowing reactions to proceed involving reagents stored on the bottom) but
does not
reach the height of one or more shelves. Reactions involving reagents on the
shelves
can be commenced at a later time by adding sufficient volume of a second
liquid so that
the liquid level reaches the height of the shelves so as to reconstitute dry
reagent on the
shelves. In conducting an assay, the sample to be measured may be the first
liquid,
second liquid or both.
Figures 2b-2f show top views of several embodiments of well 200 and show
that the well openings may have a variety of shapes including, but not limited
to, square
(Figures 2b-2d) and round (Figures 2e-2f). Furthermore, the shelf elements may
extend
around the perimeter of the well as in Figures 2b and 2e or there may be one
or more
isolated shelf elements that only extend partially around the well as in
Figures 2c-2d
and 2f. A well may also include a plurality of shelf elements at different
heights within
CA 02893383 2015-06-01
28
a well. Figures 2g-2h show cross-section and top views, respectively, of a
well 290 in
which a shelf element is provided by bridge 250 that extends across the well.
Figures
2i-2j show cross-section and top views, respectively of a well 295 in which a
shelf
element I provided by a table 260 that extends vertically from an area of well
bottom
220.
A multi-well plate is provided comprising a plate body with a plurality of
wells
defined therein including: a) a plurality of first reagent wells holding a
reconstitutable
first dry reagent and b) a plurality of second reagent wells holding a second
dry reagent
(which may be a reconstitutable dry reagent or an immobilized reagent),
wherein, the
first and second reagents are matched reagents for conducting an assay (i.e.,
they are
both used in conducting an assay of interest). The reagents may be located in
a variety
of locations with the wells such as well bottom, well walls, on shelf
elements, as free-
standing pills or powders, etc. A method is provided for carrying out assays
in these
plates comprising: a) adding a sample to one of the first reagent wells, b)
reconstituting
reconstitutable dry labeled detection reagents in the first reagent well to
form a reaction
mixture, c) transferring an aliquot of the reaction mixture to one or more of
the second
reagent wells, and d) incubating die reaction mixture in the second reagent
well(s) so as
to carry out said assay on said sample. In one embodiment, the multi-well
assay plate
can be divided into a plurality of sets of wells consisting of one first
reagent well and
one or more second reagent wells and the -method further comprises repeating
the
process of (a)-(d) for each set of wells.
Figure 3a is a (not to scale) schematic illustration of one embodiment showing
cross-sectional views of two wells of a multi-well plate 300. Well 302 is a
reagent
reconstitution well comprising one or more reconstitutable dry reagents which
may
CA 02893383 2015-06-01
29
include a labeled detection reagent (such as dry reagent 350 comprising
labeled
detection reagent 360) or a an assay control analyte (such as dry reagent 370
comprising assay control analyte 380). These dry reagents may include
additional
reagent components such as blocking agents, stabilizers, preservatives, salts,
pH
buffers, detergents, bridging reagents, ECL coreactants and the like. The
reagents may
be located on well bottoms, specific locations on well bottoms, on well walls,
shelf
elements or may be free-standing (as per the discussion of Figures 1 and 2).
Well 301
is a detection well comprising one or more dry reagents which. may include
reconstitutable dry reagents or an immobilized dry reagent. As shown, well 301
comprises immobilized capture reagents 330 that are patterned into three
binding
domains 330a, 330b, and 330c to form a binding surface. Well 301 also
comprises a
reconstitutable protective layer 340 which may be omitted. In one embodiment
of an
assay, sample is added to the reagent reconstitution well where
reconstitutable dry
reagents are reconstituted. The sample is then transferred to the detection
well where
the assay measurement is carried out. Alternatively, a reconstitution buffer
may be
used to reconstitute reagents in the reagent reconstitution well; the
reconstitution buffer
is then combined with sample in the detection well. Figure 3a also shows plate
seal
390 which seals against the openings of wells 301 and 302 to protect the
contents of the
wells from the environment.
The detection and reagent reconstitution wells in a multi-well plate may be
grouped into a plurality of assay sets consisting of one reagent
reconstitution well and
one or more detection wells, the reagent reconstitution well and detection
wells within a
set comprising matched capture and detection reagents for measuring an analyte
of
interest. Figure 3b shows an arrangement where a set has one reagent
reconstitution
CA 02893383 2015-06-01
wells 302 and three detection wells 301. During an assay, a sample added to
well 302
may then be distributed among the three associated detection wells 301 so as
to conduct
multiple replicates or, Where the detection wells hold different reagents,
multiple
different assays. Figure 3c shows an arrangement where a set has one reagent
5 reconstitution well 302 and one detection well 301.
Reagent reconstitution wells and detection wells may be similar in size and
shape or may have different sizes and/or shapes. In some embodiment, the wells
in a
standard multi-well plate are divided between the two types of wells. Figure 4
shows a
non-scale schematic views of an alternative arrangement of wells. Figure 4a
shows a
to top view of multi-well plate 400 having detection wells 440 that are
arranged in a
regular two dimensional pattern and that have detection wells walls 430 with
inner wall
surfaces and outer wall surfaces. Multi-well plate also has reagent
reconstitution wells
460 in interstitial spaces between detection wells. Reagent reconstitution
wells 460
have well walls that are defined by the outer well surfaces of detection well
walls 430
15 and rib elements 450 that connect the outer surfaces of well walls 430
of adjacent
detection wells (and, in the outermost of the wells, by the inner surface of
plate frame
wall 410). As shown, the detection wells may be shaped to have no reentrant
(i.e.,
inward pointing) curves or angles while the interstitial wells may have
reentrant curves
and/or angles. Figure 4b shows a cross-sectional view along the dotted line in
Figure
20 4a and shows the bottom surfaces of the two types of wells (which may be
at different
heights in the plate body). Plate 400 may be formed from a single contiguous
material.
In an alternate embodiment, plate 400 is formed from a plate top 405 and a
plate
bottom 420 that are mated along the dotted line shown in Figure 4b.
Advantageously,
the basic arrangement of arrays of round wells with interstitial wells defined
by the well
CA 02893383 2015-06-01
31
walls and rib elements is a common feature of many injection-molded 96-well
plates
and plate tops and allows these components to be used to form dry reagent
plates as
shown in Figure 4.
A multi-well plate is provided comprising a) a plate body with a plurality of
wells defined therein including: i) a plurality of assay wells comprising a
dry assay
reagent; and ii) a plurality of desiccant wells comprising a desiccant, and b)
a plate seal
sealed against said plate body thereby isolating said plurality of wells from
the external
environment. In some embodiments; the assay wells comprise the necessary
reagents
for conducting an assay in the assay well. Also included are embodiments in
which the
desiccant wells are connected by drying conduits to the assay wells, the
conduits
permitting diffusion of water vapor from tha assay wells to the desiccant
wells but
intersecting the wells at a height in the assay well above the location of the
dry assay
reagent. In addition to multi-well plates containing dry reagents and
desiccants, the
plates themselves (i.e., without dry reagents and desiccants), in particular,
plates having
conduit or channel elements (e.g., as shown in Figure 5 described below) that
are
suitable for connecting sets of desiccant and assay wells with dry reagents
are provided.
Figure 5 shows non-scale schematic views of a multi-well plate 500 having
assay wells 501 and desiccant wells 502 (desiccant and dry reagents are not
shown).
Figure 5a is a top view showing well walls 510 and conduits 508 connecting
dessicant
wells with one (e.g., as in row A) or more assay wells (e.g., as in row B).
Figure 5b
shows a cross-sectional view along the dotted line in Figure 5a and together
with Figure
5a shows how conduits 508 may be formed by sealing plate seal 515 against
channels
in the top surface of the plate body. Plate seal 515 seals against these
channels and the
tops of the wells to form sets of assay and dessicant wells that are
interconnected by
CA 02893383 2015-06-01
32
conduits but are isolated from the environment and from other sets of wells.
Accordingly, one or more sets of wells may be unsealed and used in an assay
and the
remaining sets of wells will be maintained in a dry environmentally protected
environment. Plate 500 may be formed from a single contiguous material. In an
alternate embodiment, plate 500 is formed from a plate top 505 and a plate
bottom 512
that are mated along the dotted line shown in Figure 5b, plate bottom 512
defining the
floor of at least some of the wells.
The assay wells or sets of wells in plate 500 may include one or more of any
of
the dry reagent-containing wells described above, for example, in the
descriptions of
Figures 1-4 and may include both detection wells and reagent reconstitution
wells. The
desiccants used in the desiccant well may be selected from known desiccant
materials
including, but not limited to, silica, activated alumina, activated clays,
molecular sieves
and other zeolites, hydroscopic salts (e.g., anhydrous 'calcium sulfate,
magnesium
sulfate, sodium sulfate, sodium hydroxide and lithium chloride), hydroscopic
solutions
(e.g., concentrated solutions of lithium chloride) and water reactive
materials such as
phosphorous pentoxide. In some embodiments, the desiccant is present as a free
dry
powder or granular material. In other embodiments, the desiccant is present as
a dry
pill, for example a pressed tablet or a desiccant impregnated polymeric
material. In
other embodiments, the desiccant is contained in a water vapor permeable bag
or
container (e.g., as in commercial silica pouches). Advantageously, desiccant
in pill
form or pre-packaged containers may be "press fit" into desiccant wells to
prevent
movement in the well during shipping or use.
Figures 5c-5d show top and cross-sectional views of one embodiment of a
multi-well plate 520 with assay and desiccant wells. Plate 520 has assay wells
521
CA 02893383 2015-06-01
33
(which may contain dry assay reagents) that are arranged in a regular two
dimensional
pattern and that have assay well walls 523 with inner wall surfaces and outer
wall
surfaces. It also has desiccant wells 522 in interstitial spaces betweerk
detection wells
(alternatively, wells 521 are used as desiccant wells and wells 522 are used
as assay
wells). Desiccant wells 522 have well walls that are defined by the outer well
surfaces
of detection well walls 523 and rib elements 525 that connect the outer
surfaces of well
walls 523 of adjacent assay wells (and, in the outermost of the wells, by the
inner
surface of plate frame wall 526). Channels 524 notched into the top of well
walls 523
provide, when mated to a plate seal, paths for water vapor to travel from
assay wells to
desiccant wells. As shown, the assay wells may be shaped to have no reentrant
(i.e.,
inward pointing) curves or angles while the interstitial wells may have
reentrant curves
and/or angles. Figure Sd shows a cross-sectional view along the dotted line in
Figure
5c and shows plate seal 527 which is mated to the top of the plate to form
sets of assay
and desiccant wells that are connected via conduits 524 but isolated from
other wells
and from the environment. Plate 520 may be formed from a single contiguous
material.
In an alternate embodiment, plate 520 is formed from a plate top 528 and a
plate
bottom 529 that are mated along the dotted line shown in Figure 5d.
Figure Se shows a schematic view of another embodiment of a multi-well plate
with assay wells (which may contain dry reagents) and desiccant wells and
shows a
plate 540 with assay wells 541 and desiccant wells 543 that are connected into
sets of
wells via channels 542 in the plate body. Multi-well plate 540 is largely
analogous to
the embodiment of plate 500 pictured in Figures 5a-5b except that in plate
540,
desiccant wells 542 are much shallower and smaller in area than the assay
wells
allowing a larger portion of the plate footprint to be dedicated to wells used
in assay
CA 02893383 2015-06-01
34 ,
measurements. Figure 5f shows a cross-sectional view along the dotted line in
Figure
5e and also shows plate seal 544 that is sealed against the top of the plate
to form
connected sets of assay and desiccant weUs. Plate 540 may be formed from a
single
contiguous material. In an alternate embodiment, plate 540 is formed from a
plate top
545 and a plate bottom 546 that are mated along the dotted line shown in
Figure 5f,
plate bottom 546 also defining the floor of assay wells 541.
Figure 6 is a schematic exploded view of one embodiment of a multi-well assay
plate. Multi-well assay plate 600 comprises a plate top 610 with through-holes
615 that
define the walls of wells. Plate top 610 is sealed against plate bottom 620
through
gasket 625 such that plate bottom 620 defines the bottom surface of the wells.
=
Optionally, plate top 610 is sealed directly to plate bottom 620 and gasket
625 is
omitted. Sealing may be accomplished through traditional sealing techniques
such as
adhesives, solvent welding, heat sealing, sonic welding and the like. In
another
optional embodiment, plate top 610 fully defines the sides and bottom of the
wells and
plate bottom 620 and gasket 625 may be omitted. The contents of the wells,
which may
include wells configured to contain dry reagent and/or desiccant as described
above,
may be protected from the outside environment by sealing (e.g., via
traditional sealing
techniques) plate seal 630 to plate top 610 directly or via optional gasket
635.
The components of plate 600 may be made from a variety of different materials
=
including, but not limited to, plastics, metals, ceramics, rubbers, glasses or
combinations thereof. In accordance with the requirements of the particular
detection
technology used with the plates, the components some or all of the components
may be
selected to be transparent, colored, opaque, or highly light scattering. In
one
embodiment, plate top 610 is an injection-molded plastic such as injection-
molded
=
CA 02893383 2015-06-01
polystyrene, polypropylene, or cyclic olefin copolymer (COC). Optionally, one
or
more of the components may be made of or comprise (for example in the form of
a
coating) a maierial that has a low water vapor transmission rate, e.g., a
water vapor
transmission rate less than 1 g/m2 per day through a 100 um thickness. Low
water
5 vapor transmission materials include, but are not limited to, glass,
metals or metal films
(e.g., aluminum films), COC, polyvinylidene chloride (PvDC), polypropylene,
polychlorotrifluoroethylene (PCTFE), and liquid crystal polymers (LCP).
Plate 600 may include desiccant wells as described above. Alternatively, or in
addition, desiccant may be incorporated directly into plate top 610, plate
bottom 620,
to plate seal 630, gasket 625 and or gasket 635. For example, U.S. Patent
6,174,952 to
Hekal et al. describes desiccant containing polymer blends that may be molded,
cast
into liners, or formed into films, sheets, beads or pellets.
In some embodiments, plate bottom 620 has features to facilitate the
patterning
of reagents on the bottom of wells (e.g., patterned hydrophilic features
surrounded by
15 hydrophobic areas) and/or conductive layers that provide electrodes that
are exposed to
the interior volumes of the wells of plate 600 so that electrochemical or
electrode
induced luminescence assays (e.g., electrochemiluminescence assays) may be
carried
out. Plate bottom 620 may also include electrode contacts to allow an external
instrument to apply electrical potential/current to the electrodes. Suitable
approaches,
20 configurations and compositions for such features, conductive layers and
electrode =
contacts include those described in 'U.S. Publications 2004/0022677 and 2005/
0052646
to Wohlstadter et al. Suitable instrumentation and methods that can be used to
conduct
ECL measurements using assay modules include those described in U.S.
Publications
2004/0022677 and 2005/0052646 of U.S. Applications 10/185,274 and 10/185,363,
CA 02893383 2015-06-01
36
respectively; U.S. Publication 2003/0113713 of U.S. Application 10/238,391;
U.S.
Publication 2005/0142033 of U.S. Application 10/980,198; and the concurrently
filed
U.S. Application 11/ of Clinton et al. entitled "Assay Apparatuses,
Methods
and Reagents."
Figure 7 provides schematic illustrations of one specific embodiment that
includes some of the inventive concepts disclosed above in the context of a
multi-well
plate configured to carry out array-based multiplexed electrochemiluminescence
assays.
Figure 7a shows a section of multi-well plate 700 that has a plurality of
assay wells 710
which may comprise dry reagents and a plurality of desiccant wells 720 which
may
comprise a desiccant. Channels 725 on the top surface of plate 700 link each
desiccant
well to an assay well. Optionally, desiccant wells 720 and channels 725 are
omitted.
Assay wells 710 have ledges 712 which may be used to support a reconstitutable
dry
reagent (e.g., dry reagents comprising assay controls and/or ECL labeled
detection
reagents). Assay wells also have working electrode surfaces 714 which are
covered by
patterned dielectric layer 716 so as to expose a plurality of exposed
electrode surfaces
or "spots" (shown as circles within the wells). In addition, counter
electrodes 718 are
provided to provide for a complete electrochemical circuit. Optionally, the
surface of
dielectric layer 716 is hydrophobic relative to electrode surface 714 so that
small
volumes of reagents patterned onto the spots may be kept confined to the
spots. The
different spots may have different capture reagents immobilized thereon to
form a
binding surface with an array of binding domains differing in specificity or
affinity for
binding partners (e.g., analytes of interest). Alternatively, some of the
spots may have
reconstitutable dry reagents confined thereon which, e.g., may contain assay
controls
CA 02893383 2015-06-01
37
and/or ECL labeled detection reagents. The assay well may further comprise a
reconstitutable protective layer over the binding surface.
Figure 7b provides an exploded cross-sectional view along the dotted line in
Figure 7a and illustrates one approach to forming the electrode/dielectric
layers in assay =
wells 710. The multi-well plate comprises a plate top 730 that defines
desiccant wells
720 and has through-holes that define the walls of assay wells 710 and ledges
712.
Plate top 730 also has channels 725 that form conduits between assay wells 710
and
desiccant wells 720 when plate seal 750 is sealed against the top surface of
plate top
730. In one non-limiting example, plate top 730 is an injection-molded part
molded
from a plastic with low water vapor transmission. In another non-limiting
example,
plate seal 750 is a heat sealable film comprising a low water vapor
transmission plastic
or a metal (e.g., aluminum) foil.
Figure 7b also shows plate bottom 740 which seals against plate top 730 and
defines the bottom of assay wells 710. Plate bottom 740 comprises substrate
715 which
supports patterned conductive layers that provide for electrodes 714 and 718.
Patterned
dielectric layer 716 on the electrodes defines the exposed electrode spots. A
variety of
materials may be used to provide for the substrate and the conductive and
dielectric
layers (see, e.g., U.S. Publications 2004/0022677 and 2005/0052646). In one
non-
limiting example, the substrate is a plastic film (made, e.g., of a polyester
such as
MYLAR, polyvinylchloride, or a low water vapor transmissive material such as
COC),
the conductive layers are screen printed conducting inks (e.g., screen printed
carbon
inks) and the dielectric layer is a screen printed insulating ink. Also shown
in Figure 7b
are electrode contacts 780 and 785 which are conductive layers on the bottom
of
substrate 715 that provide connectivity (e.g., via conductive through holes in
substrate
CA 02893383 2015-06-01
38
715 to electrodes 714 and 718. The electrode contacts may also be provided by
screen
printed conductive inks which during printing can be caused to fill holes in
substrate
715 to also provide the conductive through-holes. Advantageously, the
conductive
through-holes may be located directly below well walls to limit water vapor
transmission through the holes. In addition, an optional bottom sealing layer
790 may
be sealed to the bottom of substrate 715. Bottom sealing layer 790 is made of
a low
water vapor transmissive material and covers most of the bottom surface of
substrate
715 except for defined openings in sealing layer 790 that are located so as to
allow a
plate reading instrument to contact electrode contacts 780 and 785.
Figure 7c shows a more detailed angled view of one embodiment of plate 700
and shows desiccant pills 722 that are press-at into desiccant wells 720.
A variety of samples which may contain an analyte or activity of interest may
be
assayed. In one example, a sample is introduced to an assay plate or one or
more wells
of an assay plate having reconstitutable dry reagents pre-loaded thereon, thus
reconstituting these assay reagents and an assay signal is measured so as to
measure
(quantitatively or qualitatively) the amount of analyte in the sample. The
reagents may
include a luminescent, electrochemiluminescent, chemiluminescent, and/or redox-
active
substance. Accordingly, the assay signal is preferably a luminescent or
electrochemical
signal. Assays formats that may be carried out include homogeneous and
heterogeneous methods.
Assays that may be carried out include formats that employ solid-phase
supports so as to couple the measurement of an analyte or activity to the
separation of
labeled reagents into solution-phase and solid phase supported portions.
Examples
include solid-phase binding assays that measure the formation of a complex of
a
CA 02893383 2015-06-01
39
material and its specific binding partner (one of the pair being immobilized,
or capable
of being immobilized, on the solid phase support), the formation of sandwich
complexes (including a capture reagent that is immobilized, or capable of
being
immobilized, on the solid phase support), the competition of two competitors
for a
binding partner (the binding partner or one of the competitors being
immobilized, or
capable of being immobilized, on the solid phase support), the enzymatic or
chemical
cleavage of a label (or labeled material) from a reagent that is immobilized,
or capable
of being immobilized on a solid phase support and the enzymatic or chemical
attachment of a label (or labeled material) to a reagent that is immobilized
or capable of
to being immobilized on a solid-phase support. The term "capable of being
immobilized"
is used herein to refer to bridging reagents that may participate in reactions
in solution
and subsequently be captured on a solid phase during or prior to detection.
For
example, the reagent may be captured using a specific binding partner of the
reagent
that is immobilized on the solid phase. Alternatively, the reagent is linked
to a capture
moiety and a specific binding partner of the capture moiety is immobilized on
the solid
phase. Examples of useful capture moiety-binding partner pairs include biotin-
streptavidin (or avidin), antibody-hapten, receptor-ligand, nucleic acid ¨
complementary nucleic acid, etc.
In assays carried out on solid-phase supports, the amount of analyte or
activity
may be determined by measuring the amount of label on the solid phase support
and/or
in solution using i) a surface selective technique, ii) a solution selective
technique
and/or iii) after separation of the two phases. In electrochemiluminescence
methods,
the solid phase support may also be the working electrode used to induce
electrochemiluminescence from labels bound to the solid phase. The
CA 02893383 2015-06-01
electrochemiluminescence methods may include washing to remove unbound
electrochemiluminescence labeled reagents prior to addition of an ECL
coreactant (e.g.,
tertiary amines such as tripropylamine or piperazine-1,4-bis(2-ethanesulfonic
acid)) and
application of a potential to induce ECL from bound labels. Alternatively,
because of
5 the surface selectivity of electrochemiluminescence measurements, the
method may be
run without washing. Advantageously, in unwashed assays, the ECL coreactant
maybe
pre-added to assay wells in the form of a reconstitutable dry reagent or
protective layer.
Another embodiment relates to kits for use in conducting assays that comprise
the assay modules/multi-well plates. The kit may include one or more
additional
w reagents in one or more containers including, but not limited to, assay
calibrators, assay
controls, assay diluents, ECL coreactants, and wash buffers.
According to one embodiment, the kit comprises one or more of the assay
components in one or more plate wells, preferably in dry form. In one
preferred
embodiment, the kit comprises an assay plate having a binding immobilized on
one or
15 more working electrodes within an assay module and one or more
additional assay
reagents deposited in the form of a dry bead, pellet or a pill directly into
the well,
preferably at a position spacially separated from a working electrode, or
alternatively
deposited into one or more interstitial wells. Preferably, the kits do not
contain any
liquids in the wells.
= EXAMPLES
The following examples are illustrative of some of the methods and
instrumentation falling within the scope of the present invention. They are,
of course,
not to be considered in any way limiting of the invention. Numerous changes
and
CA 02893383 2015-06-01
41
modifications can be made with respect to the invention by one of ordinary
skill in the
art without undue experimentation.
Materials & Methods
Labeled Detection Antibodies
Labeled detection antibodies were labeled with SULFO-TAG NHS ester (Meso
Scale Discovery, Gaithersburg, MD), an electrochemiluminescent label based on
a
sulfonated derivative of ruthenium-tris-bipyridine (compound 1 pictured
below).
Labeled antibodies were purified by size exclusion chromatography on SEPHADEX
G-
50 (Pharmacia).
frO
0 S03-
N-0
I
0
N -.. SO3Na
.., N... N
1. N NI
S03"
=
Na03S
Lyophilized Detection Antibody Pills
Pills comprising one or more labeled detection antibodies were formed from a
solution containing 1 pg/mL of each of the labeled antibodies, 2% bovine serum
albumin and 20% sucrose in a phosphate buffered saline. Frozen droplets of
this
solution were formed by dispensing 20 p,L droplets into liquid nitrogen. The
frozen
droplets were transferred onto chilled (.....-78 C) aluminum trays which were
placed on
the shelves of an ADVANTAGE XL. lyophilizer (Virtis). The shelves of the
lyophilizer
CA 02893383 2015-06-01
42
were pre-cooled to 5-45 C prior to introduction of the aluminum trays and a
conductive paste was used to improve the heat transfer between the shelves and
the
trays containing beads. In a typical lyophilization protocol, the lyophilizer
chamber
was evacuated and the shelf temperature was slowly increased to -30 C, -20 C, -
15 C
and finally to +20 C (ambient conditions) over the course of about 24 hours.
The
temperature was held at each of these levels for sufficient time to allow for
equilibration while controlling the chamber pressure 0.01 torr. Karl Fisher
titrations of
lyophilized beads typically showed water contents of less than 4% by weight.
The
water content could be reduced to under 2% by extended storage in the presence
of a
dessicant.
Multi-Well Plates for Electrochemiluminescence Measurements
Electrochemiluminescence measurements were carried out using specially
designed multi-well plates having integrated screen printed carbon ink
electrodes for
carrying out eIectrochemiltuninescence measurements (MULTI-ARRAY or MULTI-
SPOT plates, Meso Scale Discovery, a division of Mean Scale Diagnostics, LLC,
Gaithersburg, MD). A patterned dielectric layer patterned over the working
electrode
on the bottom of each well exposes one or more regions or "spots" on the
working
electrode. In some experiments, the electrode surfaces were treated with an
oxygen
plasma prior to immobilizing antibodies on them. Different capture antibodies
were
immobilized on the different spots by patterned micro-dispensing of solutions
of the
antibodies on the spots using a nanoliter dispenser (Bio-Dot, Inc.). The
volumes
dispensed on the spots were selected so that they spread to the boundaries
defined by
the dielectric layers but remained confined on the spots, thus allowing for
the
=
CA 02893383 2015-06-01
, 43
immobilization of each antibody (via passive adsorption) on a defined region
of the
working electrode; if the electrode surfaces were not plasma treated, a small
amount of
TRITON X-100 detergent was added to the spotting solutions to enhance
spreading.
Adsorption was allowed to proceed for at least 2 hours after which the plates
were
washed with a. stabilizing wash buffer (2% sucrose, 185 m.M dibasic ammonium
phosphate, 13 m.M monobasic ammonium phosphate, 0.1% TWEEN 20, and KATHON
CG/ICP II preservative), dried, and stored in the presence of a desiccant. By
controlling the amount of wash buffer left in the wells before drying
(typically between
5-20 AL), sucrose films of different thicknesses could be left over the
working electrode
surfaces.
Electrochemiluminescence Measurement Instrument =
Electrochemiluminescence was induced and measured in the MULTI-SPOT
plates using a Sector Imager 6000 reader or a Sector PR 400 reader (both
from
Meso Scale Discovery, a division of Meso Scale Diagnostics, LLC, Gaithersburg,
MD).
The Sector Imager 6000 instrument applies electrical potentials to the
working
electrodes in the plate and images the resultant ECL. Image analysis
algorithms are
employed to distinguish and quantitated the light emitted from each spot in a
well. The
=
Sector PR 400 instrument applies electrical potential to the working
electrodes in one
column of the plate at a time. An array of photodiodes is used to measure the
ECL
emitted from the wells in the column.
CA 02893383 2015-06-01
44
EXAMPLE 1. Multiplexed Cytokine Detection Using Labeled Detection Antibodies
in
Lyophilized Beads
High binding MULTI-SPOT plates having a 7 spot array of capture antibodies
against seven different human cytokines (TNF-a, IL1-A 112, ILS, 116, 118,1112,
and
= s GM-CSF) and lyophilized beads containing labeled detection
antibodies against the
same seven cytokines were prepared as described above. One bead was placed in
each
well and the plates were stored in the presence of dessicant until used.
Multiplexed
cytokine assays were carried out by introducing cytokine solutions (40 Al per
well
prepared in RPMI cell culture media supplemented with 10% fetal calf serum) of
pre-
defined concentrations into the wells of the plate and incubating for 2 hours
at room
temperature on a plate shaker. MSDa0 READ BUFFER P (Meso Scale Discovery), a
solution containing a tertiary amine ECL coreactant, was added at 2x
concentration to
the wells (110 Al/well) and the plate was analyzed on a Sector t Imager 6000
instrument. The resultant signals on each spot showed good linearity for all
seven
cytokines between 10 and 10,000 pg/rnl. The standard deviations of the signals
were
typically less than 10% of the average signals. Background signals and
calculated
sensitivities were similar to those obtained when the antibodies were added to
the wells
as liquid solutions.
EXAMPLE 2. Cytokine Measurements Using a Labeled Detection Antibody that is
Dried on a Protective Layer Covering a Capture Swface
This assay used a small spot MULTI-ARRAY plate with a single spot per well.
The spot treated, as described in the Materials and Methods section with a
solution
containing an anti-human TNF-a capture antibody to immobilize the antibody on
the
CA 02893383 2015-06-01
spot surface. The well was then filled with 75 L of 4x MSDO READ BUFFER P that
was supplemented with 7% FICOLL (a highly branched hydrophilic polymer of
sucrose), and the plate was cooled to freezing and lyophilizegl overnight to
provide a
protective "cake" layer over the bottom of the well. A small droplet (35 nL)
of a
5 concentrated solution of a labeled anti-human TNF-a detection antibody
was dispensed
on the surface of the cake. The plate was then vacuum dried for 5 minutes and
stored
in the presence of dessicant until used. Assays were carried out by adding to
the wells
150 itL of solutions containing pre-determined concentrations of human TNF-a
in
RPM' cell Culture media supplemented with 10% fetal calf serum and shaking for
two
10 hours. The plate was then analyzed on a Sector Imager 6000 instrument.
The
calculated detection limits of 5-6 pg/mL are comparable to those observed in
non-
washed assays using liquid detection antibody solutions.
EXAMPLE 3. Cytokine Measurements Using a Labeled Detection Antibody that is
15 Dried on the Sides of the Wells of a Multi-Well Plate
This assay used a small spot MULTI-ARRAY plate with a single spot per well.
The spot was coated, as described in the Materials and Methods section with an
anti-
human TNF-a capture antibody. Droplets (1 AL) of a 24 ptg/mL solution of the
detection antibody in 4.8% sucrose were dispensed on the inside walls of the
wells and
20 allowed to dry. The plates were stored in the presence of dessicant
until used in an
assay. The assay protocol involved adding 80 a of a TNF-ci solution to each
well,
shaking the plate for 30 minutes at room temperature, washing the plate,
adding 150 AL
of lx MSD READ BUFFER T (Meso Scale Discovery) and analyzing the plate on a
Sector Imager 6000 instrument. Plates stored for 18 days at room temperature
or 4 C
CA 02893383 2015-06-01
46
gave detection limits that were less than 1 pg/mL and comparable to those
observed in
washed assays employing liquid detection antibody solutions.
EXAMPLE 4. Cytokine Measurements Using a Multi-Well Plate with Wells Having a
Capture Layer Coated with a Protein-Containing Protective Layer and Dried
Labeled
Detection Antibody on the Well Walls
This assay used a MULTI-ARRAY plate with a single spot per well. The
working electrode in each well was pre-coated with streptavidin (streptavidin
MULTI-
ARRAY plate, Meso Scale Discovery). Anti-IL1-3 monoclonal antibody was
immobilized on the working electrode according to the following protocol. The
wells
were washed three times with 1,)13S and then treated with 20 AL of a 3
I.I.g/mL solution
of biotin-labeled anti-Ill-O. The immobilization was allowed to proceed over 2
hours
under agitation on a plate shaker. The wells were then washed three times with
PBS.
A 20 ptL volume of a buffered solution of BSA and sucrose was added to the
wells and
then dried in the wells under vacuum to form a dry film on the bottom of the
plates.
A dry pill of SULFO-TAG labeled anti-11,1-fl polyclonal antibody was formed
on the well wall according to the following protocol. A 100 nL microdroplet of
a 482
fig/mL solution of the labeled antibody was dispensed on each well wall using
a BIO-
DOT microdispensor (Bio-Dot, Inc.) with an angled tip. The droplet remained on
the
well wall where it was allowed to dry for 30 minutes in a dessicator chamber.
The
wells were then sealed with a plate heat seal. In some experiments, a low
concentration
of fluorescein was added to the detection antibody solution. The fluorescein
fluorescence could be used to provide a quality control check by identifying
any well in
CA 02893383 2015-06-01
47
which the detection antibody ran down the well wall or splattered on the well
bottom.
The fluorescein did not affect assay performance.
Assays for IL1-0 were carried out by adding 125 AL of solutions containing
known amounts of 11,143 to the wells and incubating for 37 minutes while
shaking the
plate. The plate was then washed with PBS, MSD READ BUFFER T was added and
the plate was analyzed on a Sector PR 400 instrument. Assays using plates
with dry
detection antibodies performed in a comparable fashion to assays that used
liquid
detection antibody solutions.
EXAMPLE 5. Assays in Multi-Well Plates Using Dry Reagents: Storage of Dried
Labeled Antibodies on Ledges in the Wells
This assay used a Multi-Spot plate configured as shown in Figure 8. The plate
was similar to that shown in Figure 7 except for the use of a 7-spot pattern
and the
omission of desiccant wells 720, channels 725, desiccant pills 722, and bottom
sealing
Is layer 790. The plate top was injection-molded polypropylene. Capture
antibodies
against were immobilized by dispensing, on the individual spots, antibodies
against
botulinum toxin A (BotA), dinitrophenyl (DNP), ricin, staphylococcal
enterotoxin B
(SEB), Venezuelen equine encephalitis (VEE), and Yersinia peals (YP). Non-
immune
mouse IgG was immobilized on the remaining spot for use as a negative control.
Immobilization was carried out by dispensing 75 nL of solutions comprising
between
100-500 Ag/mL of an antibody, 750 Ag/mL of BSA, and 0.03% TRITON X-100. One
exception was the BotA capture antibody which was biotinylated and immobilized
after
pre-binding it to 1200 Ag/mL avidin and which was immobilized in the absence
of
BSA.
CA 02893383 2015-06-01
48
The non-immune IgG should not participate in a sandwich complex and should
give a low signal for all samples. Elevation of this signal outside of a
selected range
can be used as an indication that a measurement artifact is producing elevated
non-
specific binding of detection antibodies and that there is a risk of false
positive results.
More generally, any binding reagent that is not paired with a corresponding
detection
reagent may be used. Optionally, the binding reagent may be selected to share
structural properties with the test capture reagents, for example, in an
iimmunoassay it
may include immunoglobulins from one or more of the species from which the
other
capture antibodies were derived. The anti-DNP spot will be used as a positive.
control.
The well will also include a dry SULFO-TAG labeled anti-fluorescein (FL)
antibody
and a defined quantity of dry BSA labeled with both DNP and FL (DNP-FL-BSA).
The positive control signal should, therefore give a constant positive signal
indicative
of the defined quantity of DNP-FL-BSA. Reduction of this signal below a
selected
range can be used as an indication that a sample interferes with binding
reactions or
signal generation and that there is a risk of false negative results. More
generally, the
positive control may be an assay for any analyte that can be spiked into the
reaction
mixture. Prefefably, there is a low likelihood of finding the analyte in the
samples of
interest.
The capture antibody solutions were allowed to dry for 30 minutes in a
desiccated environment and then dried for 30-60 minutes under vacuum. The
wells
were washed with a the stabilizing wash buffer containing sucrose described in
the
Materials and Methods section, blocked with 5% BSA for 45 minutes and washed
once
more with the stabilizing wash buffer. A stabilizing/blocking solution (20 AL
of 305
mM ammonium phosphate, 100 mM ammonium chloride, 0.02% TRITON X-100, 2%
CA 02893383 2015-06-01
49
sucrose, 2%BSA, and 0.02% KATHON preservative, pH 7.4) was added and the
solution was dried in the well under vacuum to form a dry reagent cake on the
well
bottom.
A mixture of STAG-labeled detection antibodies (0.5 AL of a mixture of
antibodies against BotA, FL, ricin, SEB, VEE, and YP at between 40-240 )hg/mL
in the
stabilizing/blocking solution) was dispensed (using a BIO-DOT dispenser with
an
angled dispense tip) on the walls of the wells just above the dry reagent
ledge and
allowed to flow down onto the ledge. A solution containing 80 ng/mL of the
positive
control analyte (DNP-FL-BSA) was dispensed on the opposite wall. The detection
io antibody and control solutions were allowed to dry for 30-60 minutes
under vacuum.
The plates were then packaged with desiccant until used.
The protocol used to conduct assays with these plates was: add 80 iL of sample
(defined amounts of one or more of the target analytes in 0.1% TRITON X-100 in
phosphate buffered saline (PBS)), incubate 1 hour with shaking, wash with PBS,
add
150 AL lx MSD READ BUFFER T (Meso Scale Diagnostics, LLC) and analyze plate
using an MSD Sector Imager 6000 instrument. VEE and YP used in this assay
were
inactivated by irradiation. BotA was used in the assay was inactivated with
formalin.
The table below shows that the signals observed at each spot for samples
= containing no analyte (-) or for samples containing 10 ng/mL BotA, 1
ng/mL ricin, 50
ng/mL SEB, 1000 ng/mL VEE, or 10,000 CFU/mL YP. The table shows sensitive and
specific detection of the target analytes and proper performance of the
positive and
negative control spots.
CA 02893383 2015-06-01
Capture Spot
Analyte BotA Ricin SEB VEE YP Neg Pos
BotA 28893 178 97 182 159 134 9456
Ricin 243 15502 106 222 142 129 8776
SEB 276 165 9518 230 177 162 8288
VEE 1516 233 188 11821 204 237 8506
YP 243 129 107 211 4280 152 8656
= 249 81 75 212 115 121 8923
The next table compares the signals observed with these plates to assays
carried
out under comparable conditions except for the use of liquid detection
reagents. The
table provides only the signal on the specific spot for a given analyte and
provides both
5 signal in the presence of analyte (10 ng/mL BotA, 1 ng/mL ricin, 50 ng/mL
SEB, 1000
ng/mL VEE, or 10,000 CFU/mL YP) and background signal in the absence of
analyte.
The table shows that the dry and wet assays perform comparably.
Signal on S micific Spot
Dry Wet
Analyte Backrd = Signal Backrd Signal
BotA 249 12098 156 4809
Ricin 81 15502 94 12531
SEB 75 9518 68 6299
VEE 212 11821 260 7497
YP 115 4280 145 3876
10 EXAMPLE 6. Assays in Multi-Well Plates With Assay Wells and Desiccant
Wells
This assay used a MULTI-SPOT plate configured as shown in Figure 9. The
plate was similar to that shown in Figure 8 except for the inclusion of
conduits 910
connecting pairs of adjacent wells. The conduits were provided by shallow
notches that
were cut into the walls separating adjacent wells. In this example, one well
of each pair
15 of wells was used to carry out a multiplexed immunoassay and, the other
was used to
hold desiccant for maintaining the assay well in a dry state during storage.
The assay
used a multiplexed sandwich immunoassay with dry capture and detection
reagents
prepared as in Example 5. The capture antibodies were anti-Bacillus subtilis
var. Niger
CA 02893383 2015-06-01
51
(I3G), anti-MS2 phage, anti-FL, anti-DNP, and mouse IgG as a negative control.
The
dry detection antibody pill included labeled anti-BG, anti-MS2, and anti-
ovalbumin
(Ova) (for detecting FL-Ova and DNP-Ova).
After the plates were prepared, the desiccant wells were filled with roughly
50
mg to 200 mg of silica gel or DRIERITE (calcium sulfate) desiccants and the
plates
were sealed with an aluminum foil heat seal. After sealing the foil seal to
the plate top,
the notches in the wells provided conduits between the sets of assay and
desiccant
wells. Some plates were prepared with no desiccant in the desiccant wells for
comparison. The plates were kept at 4 C under dry conditions for several days
to allow
the dry reagents to fully dry. The plates were then exposed to elevated
temperature and
humidity for several days prior to using them to carry out measurements of the
target
organisms (using the assay protocol of Example 5).
Figure 10 provides signals for samples containing defined amounts of the
target
analytes and compares the signals from plates with silica desiccant, calcium
sulfate
desiccant and no desiccant after exposure to 60% humidity at 30 C for 7 days.
Signals
are provided as a percentage of the signal obtained from a plate that was
prepared at the
same time as the others but that had been kept dry a 4 C for the 7 day period.
The
results indicate that the desiccant wells were very effective at improving the
stability of
the dry reagents to heat and humidity.
