Note: Descriptions are shown in the official language in which they were submitted.
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OPPOSABLE-ELEMENT CF-~ROMATOGRAPHIC ASSAY DEVICE
BACKGROUND OF THE INVENTION
This invention is directed to test strips for
determination of characteristics of samples, unitized
housings, and kits incorporating the test strips and
housings, and methods of determining the characteristics
of samples using the test strips and housings.
Amcng the many analytical systems used for
detection and/or determination of analytes, particularly
analytes of biological interest, are chromatographic
assay systems. Among the analytes frequently assayed
with such systems are:
(1) hormones, such as human chorionic
gonadotropin (hCG), frequently assayed as a marker of
human pregnancy;
(2) antigens, particularly antigens specific
to bacterial, viral, and protozoan pathogens, such as
Streptococcus, hepatitis virus, and Giardia;
(3) antibodies, particularly antibodies
induced as a result of infection with pathogens, such as
antibody to the bacterium Helicobacter pylori and to
human immunodeficiency virus (HIV);
(4) other proteins, such as hemoglobin,
frequently assayed in determinations of fecal occult
blood, an early indicator of gastrointestinal disorders
such as colon cancer;
(S) enzymes, such as aspartate
aminotransferase, lactate dehydrogenase, alkaline
phosphatase, and glutamate dehydrogenase, frequently
assayed as indicators of physiological function and
tissue damage;
(6) drugs, both therapeutic drugs, such as
antibiotics, tranquilizers and anticonvulsants, and
AMEyOE~J ~u~ET
~~-CBS 7d
2
illegal drugs of abuse, such as cocaine, heroin, and
marijuana;
(7) environmental pollutants such as
pesticides and aromatic hydrocarbons and
(8) vitamins.
Such chromatographic systems are frequently
used by physicians and medical technicians for rapid in-
office diagnosis and therapeutic monitoring of a variety
of conditions and disorders. They are also increasingly
used by patients themselves for at-home monitoring of
such conditions and disorders.
Among the most important of such systems are
the "thin layer" systems in which a solvent moves across
a thin, flat absorbent medium.
Among the most important of tests that can be
performed with such thin layer systems are immunoassays,
which depend on the specific interaction between an
antigen or hapten and a corresponding antibody. The use
of immunoassays as a means of testing for the presence
and/or amount of clinically important molecules has been
known for some time. As early as 1956, J.M. Singer
reported the use of an immune-based latex agglutination
test for detecting a factor associated with rheumatoid
arthritis (Singer et al., Am. J. Med. 22:888-892
(1956) ) .
Among the chromatographic techniques used in
conjunction with immunoassays is a procedure known as
immunochromatography. In general, this technique uses a
disclosing reagent or particle that has been linked to
an antibody to the molecule to be assayed, forming a
conjugate. This conjugate is then mixed with a specimen
and, if the molecule to be assayed is present in the
specimen, the disclosing reagent-linked antibodies bind
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3
to the molecule to be assayed, thereby giving an
indication that the molecule to be assayed is present.
The disclosing reagent or particle can be identifiable
by color, magnetic properties, radioactivity, specific
reactivity with another molecule, or another physical or
chemical property. The specific reactions that are
employed vary with the nature of the molecule being
assayed and the sample to be tested.
Immunochromatographic assays fall into two
principal categories: "sandwich" and "competitive,"
according to the nature of the antigen-antibody complex
to be detected and the sequence of reactions required to
produce that complex. The antigen to be detected can
itself be an antibody, such as in serological assays for
H. pylori-specific antibody. In such cases, the
antibody to be detected can be bound to a specific
antigen. Alternatively, the antigen to be detected can
be detected indirectly by using a labeled second
antibody that binds to the first antibody to the analyte
to be detected.
In general, the sandwich immunochromatographic
procedures call for mixing the sample that may contain
the analyte to be assayed with antibodies to the
analyte. These antibodies are mobile and typically are
linked to a label or a disclosing reagent, such as dyed
latex, a colloidal metal sol, or a radioisotope. This
mixture is then applied to a chromatographic medium
containing a band or zone of immobilized antibodies to
the analyte of interest. The chromatographic medium
often is in the form of a strip resembling a dipstick.
When the comr~ex of the molecule to be assayed and the
labeled antibody reaches the zone of the immobilized
antibodies on the chromatographic medium, binding occurs
and the bound labeled antibodies are localized at the
zone. This indicates the presence of the molecule to be
Ar~E~!DF~ S''~~~T
CA 02158570 2003-12-30
4
assayed. This technique can be used to obtain
quantitative or semi-quantitative results.
Examples of sandwich immunoassays performed on test
strips are described by U.S. Patent No. 4,168,146 to
Grubb et al. and U.S. Patent No. 4,366,241 to Tom et al.
5 In competitive immunoassays, the label is typically
a labeled analyte or analyte analogue which competes for
binding of an antibody with any unlabeled analyte
present in the sample. Competitive immunoassays are
typically used for detection of analytes such as
10 haptens, each hapten being monovalent and capable of
binding only one antibody molecule. Examples of
competitive immunoassay devices are those disclosed by
U.S. Patent No. 4,235,601 to Deutsch et al., U.S. Patent
No. 4,442,204 to Liotta, and U.S. Patent No. 5,208,535
15 to Buechler et al.
Although useful, currently available
chromatographic techniques using teststrips have a
number of drawbacks. Many samples, such as fecal
samples, contain particulate matter that can clog the
20 pores of the chromatographic medium, greatly hindering
the immunochromatographic process. Other samples, such
as blood, contain cells and colored components that make
it difficult to read the test. Even if the sample does
not create interference, it is frequently difficult with
25 existing chromatographic test devices to apply the
sample to the chromatographic medium so that the sample
front moves uniformly through the chromatographic medium
to insure that the sample reaches the area where binding
is to occur in a uniform, straight-line manner.
2~~.~~ ~'~(~
Other problems exist with currently-available
test strips because of the nature of the sample to be
assayed or the assay to be carried out. With such
devices, it is impractical to perform washing steps
which are frequently desirable to improve sensitivity
and to reduce background. Also, it is difficult, and in
many cases impossible, to carry out preincubation steps
within the device.
Additionally, there is a need for an
immunochromatographic assay device that can carry out a
broad range of separations, such as the separation of
fat from milk or the separation of organic chemicals
such as the separation of benzene from toluene.
Sample preparation and waste generation are
responsible for other problems with currently available
devices and techniques for immunochromatography. The
increased prevalence of diseases spread by infected
blood and blood fractions, such as AIDS and hepatitis,
has exacerbated these problems. It is rarely possible
to apply a sample (such as feces) or a sampling device
(such as a throat swab) directly to the chromatographic
medium. Several extraction and pretreatment reactions
are usually required before the sample can be applied to
the chromatographic medium. These reactions are
typically carried out by the physician or technician
performing the test in several small vessels, such as
test tubes,.or microfuge tubes, requiring the use of
transfer devices, such as pipettes. Each of these
devices is then contaminated and must be disposed of
using special precautions so that workers or people who
may inadvertently come into contact with the waste do
not become contaminated.
Still another limitation on chromatographic
devices currently available for use by the clinician or
A~AENDED SHEET
215850
technician is their inability to perform two-directional
or two-dimensional chromatography. These techniques
have long been known to be powerful analytical tools,
but their complexity relative to simple unidirectional
chromatography has made it difficult to apply them to
test strip devices in the physician's office or a
clinical laboratory.
Accordingly, there is a need for an improved
assay device capable of handling a broad range of
chromatographic assays. Such a device should be able to
handle all types of immunoassays, including both
sandwich and competitive immunoassays as well as other
types of assays using chromatography. Such a device
should be capable of receiving a possibly contaminated
sample or a sample preparation device directly so as to
eliminate the need for extraction vessels and transfer
devices. Such a device, preferably in the form of a
test strip, should also be capable of performing
immunochromatographic assays on colored samples or
samples containing particulates without interference and
should be able to deliver the sample to the
chromatographic medium uniformly and evenly to improve
accuracy and precision of the tests. Additionally, such
an improved test strip should be capable of performing
two-directional or two-dimensional chromatography when
used in clinical laboratories or physicians' offices.
SUMMARY
We have developed an assay device that meets
these needs and provides improved assays for analytes of
biological interest, while simplifying the performance
of the assay and avoiding contamination. The device can
perform all types of immunoassays, including sandwich
immunoassays, competitive immunoassays, and assays
i - 'i r~T
I~~ItE~~U~~ ~t~CCI
7
employing combinations of these principles. The device
can perform serological assays in which the antigen to
be detected is itself an antibody, such as antibody to
H. Dylori. The device can perform assays in which the
antigen to be detected is detected indirectly by using a
labeled second antibody binding to the first antibody to
the analyte.
An assay device according to the present
invention makes use of pressure to transfer fluid from
one opposable component to another opposable component,
and also to drive fluid through the chromatographic
medium. The pressure not only speeds up the operation
of the device, but allows the performance of additional
steps such as extraction steps to remove interfering
particulate components within a single device. The
pressure is generated by holding the opposable
components together with engagers such as interlocking
elements on each of the opposable components.
Preferably, a predetermined pressure is applied to
ensure the optimum performance o~ each step of the assay
procedure.
Additionally, the device can perform other
types of specific binding assays, such as: (1) assays
based on the affinity of specific binding proteins such
as lectins, hormone receptors, or viral receptors for
their specific ligands; (2) assays based on the affinity
of enzymes for their corresponding substrates or
inhibitors; or (3) assays based on the affinity of a
nucleic acid (DNA or RNA) segment for a complementary
nucleic acid segment according to the Watson-Crick base
pairing scheme.
The device comprises:
(1) at least two substantially planar
opposable components, wherein one of the substantially
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~~~~~~o
8
planar components has on its surface a chromatographic
medium; and
(2) means for opposing the opposable
components and applying pressure thereto, the pressure
being sufficient to transfer fluid from one opposable
component to another opposable component in a direction
substantially normal to the opposable component so that
the sample is applied to the chromatographic medium for
detection and/or determination of the analyte thereon.
Devices according to the present invention can
also comprise:
(1) at least three substantially planar
opposable components, wherein one of the substantially
planar components has on its surface a chromatographic
medium, the chromatographic medium having first and
second ends;
(2) means for opposing the opposable
components pairwise in at least two different
combinations and applying pressure thereto, the pressure
being sufficient to transfer fluid from one opposable
component to another in a direction substantially normal
to the opposable components so that the sample is
applied to the chromatographic medium and flows through
the chromatographic medium from the first end to the
second end for detection and/or determination of the
analyte on the chromatographic medium; and
(3) at least one applicator and one absorber
located on one of the opposable components and
positioned in such manner that, when the opposable
component on which the applicator and the absorber is
located is brought into opposition to the opposable
component on which the chromatographic medium is
located, a second liquid is applied to the
chromatographic medium and flows through the
chromatographic medium from the second end to the first
end, thereby reversing the flow through the
AMENDED SHEET
d
chromatographic medium, with the detection and/or
determination of the analyte being made subsequent to
reversal of the flow through the chromatographic medium.
In one aspect, a chromatographic assay device
according to the present invention comprises:
(1) a first opposable component
including a sample preparation zone adapted to receive a
sample to be assayed; and
(2) a second opposable component
including a chromatographic medium.
An embodiment of the chromatographic assay
device suitable for sandwich immunoassays comprises:
(1) a first opposable component including:
(a) a chromatographic medium having
first and second ends;
(b) a detector application pad in
operable contact with the first end of the
chromatographic medium;
(c) a conductor in operable contact with
the detector application pad and in indirect contact
with the first end of the chromatographic medium; and
(d) an absorber in operable contact with
the second end of the chromatographic medium; and
(2) a second opposable component including a
sample preparation zone for receiving a sample to be
tested.
In this device, the first and second opposable
components are configured so that bringing the first and
second opposable components into opposition results in
the sample preparation zone being in contact with the
conductor to apply the sample to be tested to the
conductor and then to the first end of the
chromatographic medium through the detector application
pad.
AM~NC~D SHEET
10
Typically, the sample preparation zone
contains at least one reagent for treatment of the
sample. In some cases, such as when a urine or serum
sample is assayed, no extraction or other treatment of
the sample is required. Also typically, the detector
application pad contains a first specific binding
partner to the analyte in a form that can be
resolubilized by addition of an aqueous liquid to the
detector application pad, the first specific binding
partner being labeled with a detectable label, and the
chromatographic medium further comprises a detection
zone substantially smaller in area than the area of the
chromatographic medium. In this arrangement, the
detection zone contains a specific binding partner to
the analyte immobilized thereto, such that a ternary
complex comprising the first specific binding partner,
the analyte, and the second specific binding partner
forms at the detection zone if analyte is present in the
sample.
Preferably, the detectable label is a visually
detectable label.
Preferably, the first and second opposable
components are joined by a hinge. Typically, the hinge
is impermeable to an aqueous liquid. The opposable
elements preferably also include perimeter upstanding
walls which engage in abutting relationship when the
device is closed and locked.
A test kit can comprise, in separate
containers, the chromatographic assay device described
above and a specific binding partner for the analyte
labeled with a detectable label to be applied to the
detector application pad. Alternatively, if the
detector application pad contains a resolubilizable
labeled first specific binding partner, the kit can
A~~~~i~;DED SHIFT
comprise the assay device and an aqueous liquid for
resolubilizing the labeled specific binding partner.
Similar test kits can be constructed for other
embodiments of an assay device according to the present
S invention.
A method for detecting and/or determining an
analyte in a sample using this assay device to perform a
sandwich immunoassay can comprise the steps of:
(1) applying the aqueous sample to the sample
application pad of the chromatographic assay device
containing a resolubilizable labeled first specific
binding partner on the detector application pad;
(2) bringing the first and second opposable
components of the chromatographic assay device into
opposition, such that the sample comprises an aqueous
liquid resolubilizing the labeled specific binding
partner in the detector application pad, and such that
the sample and the resolubilized labeled specific
binding partner are applied to the conductor;
(3) allowing the sample and labeled specific
binding-partner to move through the conductor and then
through at least a portion of the chromatographic medium
so that the labeled specific binding partner gives a
detectable indication of the presence and/or quantity of
the analyte in the test sample; and
(4) observing and/or measuring the labeled
specific binding partner in at least a portion of the
chromatographic medium in order to detect and/or
determine the analyte.
In another version of this embodiment, the
second opposable component can also contain a second
detector application pad in operable contact with the
sample preparation zone. In this version, the second
detector application pad contains a second labeled
specific binding partner for the analyte in a form that
~~ ~~~5 ~~
12
can be resolubilized by the addition of a sample to the
sample preparation zone; the second labeled specific
binding partner is labeled with a detectable label. The
second labeled specific binding partner is positioned
such that application of the sample to the sample
preparation zone resolubilizes the second labeled
specific binding partner so that the sample preparation.
zone contains a mixture of the sample and the second
labeled specific binding partner. The first and second
labeled specific binding partners are preferably
identical and are labeled with an identical label.
Another embodiment of an assay device
according to the present invention comprises:
(1) a first opposable component including:
(a) a chromatographic medium having
first and second ends; and
(b) a conductor in operable contact with
the first end of the chromatographic medium; and
(2) a second opposable component including:
(a) a sample preparation zone for
receiving a sample to be assayed; and
(b) an absorber separated from the
sample preparation zone.
In this device, the first and second opposable
components are configured so that bringing the first and
second opposable components into opposition results in
the sample preparation zone coming into operable contact
with the conductor to apply the sample to be tested to
the conductor and then to the first end of the
chromatographic medium, and results in the absorber
coming into operable contact with the second end of the
chromatographic medium to withdraw fluid from the second
end of the chromatographic medium. Moving the absorber
to the second opposable component allows the use of a
A,~E~otc J'~~'~
i~~~
13
larger absorber, which can be advantageous if it is
desired to assay a sample of relatively large volume.
Another version of an assay device according
to the present invention with an absorber on the second
opposable component has a sample preparation zone on the
first opposable component, a chromatographic medium, and
a conductor in operable contact with the sample
preparation zone and the first end of the
chromatographic medium so that the conductor bridges the
sample preparation zone and the chromatographic medium.
The second opposable component has an applicator
containing a specific binding partner for an analyte in
a form that can be resolubilized by the addition of an
aqueous liquid to the applicator and an absorber
separated from the applicator. The specific binding
partner contained in the applicator is labeled with a
detectable label. In this device, bringing the first
and second opposable components into opposition results
in the applicator coming into operable contact with the
sample preparation zone such that, when a sample has
been added to the sample preparation zone, the labeled
specific binding partner for the analyte is
resolubilized, and results in the absorber coming into
operable contact with the second end of the
chromatographic medium to withdraw fluid from the
chromatographic medium.
In yet another version of an assay device
according to the present invention with an absorber on
the second component, the first opposable component
includes a chromatographic medium and a conductor. The
second opposable component includes a first applicator,
a second applicator, and an absorber. The absorber is
separated from both the first applicator and the second
applicator. The first and second applicator are
positioned on the second opposable component such that
AMENCEC SHE~T
14
they are not in operable contact when the first and
second opposable components are not in opposition. The
absorber is positioned such that it is in operable
contact with the second end of the chromatographic
medium when the first and second opposable components
are brought into opposition. The first and second
opposable components are configured so that bringing the
first and second opposable components into opposition
results in the conductor coming in operable contact with
the first applicator and results in the conductor coming
in operable contact with the second applicator, thereby
resulting in the first and second applicator coming into
operable contact with each other.
In this device, the second applicator can
include a detector application pad containing a labeled
first specific binding partner in resolubilizable form.
A method of use of this device according to
the present invention comprises applying the sample to
the first applicator, and bringing the first and second
opposable components into opposition so that the sample
resolubilizes the labeled specific binding partner in
the detector application pad. The chromatography and
detection steps are as described above.
Yet another assay device according to the
present invention including an absorber on the second
component comprises:
(1) a first opposable component including:
(a) a chromatographic medium having
first and second ends;
(b) a conductor in operable contact with
the first end of the chromatographic medium; and
(c) a detector application pad in direct
contact with the conductor and positioned such that it
r,,;,r._'.;yr~, ~. _~'
'~~~~~1~~
is in indirect contact with the first end of the
chromatographic medium; and
(2) a second opposable component including:
(a) a sample application pad; and
5 (b) an absorber separated from the
sample application pad.
In this device, the first and second opposable
components are configured so that bringing the first and
10 second opposable components into opposition results in:
(1) the sample application pad applying the
sample to the detector application pad and thus to the
first end of the chromatographic medium through the
conductor; and
15 (2) the absorber being in operable contact
with the second end of the chromatographic medium.
A detection method employing this version of
the assay device is similar to those described above.
Yet another version of an assay device
according to the present invention incorporating an
absorber on the second opposable component comprises:
(1) a first opposable component including:
(a) a chromatographic medium having
first and second ends; and
(b) a detector application pad in direct
contact with the first end of the chromatographic
medium; and
(2) a second opposable component including:
(a) a sample application pad; and
(b) an absorber separated from the
sample application pad.
In this device, the first and second opposable
components are configured that bringing the first and
second opposable components into opposition causes the
detector application pad and the sample application pad
AP~~IciVGcD SHEET
16
to come in contact except for the region of the detector
application pad directly adjacent to the first end of
the chromatographic medium. Bringing the first and
second opposable components of this device into
S opposition causes:
(1) the sample to be tested to be applied to
the detector application pad and then to the first end
of the chromatographic medium; and
(2) the absorber to be brought into operable
contact with the second end of the chromatographic
medium.
Yet another assay device according to the
present invention including an absorber on the second
opposable component comprises:
(1) a first opposable component including:
(a) a chromatographic medium having
first and second ends; and
(b) a conductor positioned such that it
is not in operable contact with the first end of the
chromatographic medium when the first opposable
component and the second opposable component are not in
opposition; and
(2) a second opposable component including:
(i) a first applicator;
(ii) a second applicator; and
(iii) an absorber separated from the
first and second applicators, the first and second
applicators being positioned on the second opposable
component such that they are not in operable contact
when the first and second opposable components are not
in opposition.
In this device, the first and second opposable
components are configured so that bringing the first and
second opposable components into opposition:
AP:1E?~;ncJ ~i~~T
21.~ ~ J'7 0
(1) results in the conductor coming in
operable contact with the first applicator, results in
the conductor coming in operable contact with the second
applicator, and results in the second applicator coming
in operable contact with the first end of the
chromatographic medium, thereby placing the first and
second applicator in operable contact with each other to
apply the contents of the first and second applicator of
the chromatographic medium; and
(2) results in the absorber coming in
operable contact with the second end of the
chromatographic medium.
Another embodiment of an assay device
according to the present invention suitable for the
performance of a sandwich immunoassay allows for the
washing of the chromatographic medium by a portion of
the sample to remove unbound labeled specific binding
partner and reduce the background. This embodiment
comprises:
(1) a first opposable component including:
(a) a chromatographic medium having
first and second ends;
(b) a conductor in operable contact with
the first end of the chromatographic medium; and
(c) an absorber in operable contact with
the second end of the chromatographic medium; and
(2) a second opposable component including an
applicator divided into two sectors:
(a) a first sector containing a first
specific binding partner for the analyte in a form that
can be resolubilized by addition of an aqueous liquid to
the applicator, the first specific binding partner being
labeled with a detectable label; and
(b) a second sector without the labeled
specific binding partner.
p,~;'~~;C~J SHEET
~~~~~~fl
In this device, the first and second opposable
components are configured so that bringing the first and
second opposable components into opposition places the
first sector, but not the second sector, of the
applicator into direct contact with the conductor, the
second sector being in indirect contact with the
conductor, to apply the contents of the first sector of
the applicator to the chromatographic medium.
Subsequent to the application of the contents of the
first sector of the applicator of the chromatographic
medium, the contents of the second sector of the
applicator are applied to the chromatographic medium to
provide a wash.
Another embodiment of the present invention
encompasses assay devices adapted for the performance of
a competitive immunoassay using a labeled analyte
analogue. One version of this embodiment comprises:
(1) a first opposable component including:
(a) a chromatographic medium having a
first end and a second end, and having immobilized
thereon analyte or an immunological analogue thereof in
a discrete area substantially smaller than the area of
the chromatographic medium;
(b) a first conductor in operable
contact with the first end of the chromatographic
medium; and
(c) a second conductor in operable
contact with the second end of the chromatographic
medium;
(2) a second opposable component including a
first applicator containing a first specific binding
partner to the analyte in a form that can be
resolubilized by addition of a first aqueous liquid to
the first applicator; and
(3) a third opposable component including:
SfIL'!
'IJ-J
~~~$~~.~ y~
19
(a) a second applicator containing a
second labeled specific binding partner to the analyte
in a form that can be resolubilized by the addition of a
second aqueous liquid to the second applicator, the
second specific binding partner being labeled with a
detectable label; and
(b) an absorber separated from the
second applicator.
In this de ice, the first and second opposable
components are configured so that bringing the first and
second opposable components into opposition places the
first conductor in operable contact with the first
applicator so that the contents of the first applicator
are applied to the chromatographic medium and are drawn
through at least a portion of the chromatographic
medium. The first and third opposable components are
configured so that bringing the first and third
opposable components into opposition places the absorber
in operable contact with the first conductor to withdraw
fluid from the chromatographic medium. Bringing the
first and third opposable components into opposition
causes the second applicator to come into operable
contact with the second conductor so that the contents
of the second applicator are applied to the
chromatographic medium and are drawn through at least a
portion of the chromatographic medium overlapping the
portion through which the contents of the first
applicator are drawn.
In this device, the first specific binding
partner and the second, labeled, specific binding
partner are preferably each antibody for the analyte.
Preferably, the immobilized analyte or analogue thereof
comprises analyte covalently linked to a protein lacking
specific binding activity for the analyte.
AME'VuED SHEET
20
A method for using this assay device for the
detection of an analyte by a competitive immunoassay
comprises:
(1) applying the sample to the first
applicator of the chromatographic assay device, the
sample comprising the first aqueous liquid;
(2) applying a reconstitution fluid to the
second applicator, the reconstitution fluid comprising
the second aqueous liquid;
(3) bringing the first and second opposable
components of the chromatographic assay device into
opposition, such that the sample and the resolubilized
first specific binding partner of the analyte are
applied to the first conductor and then to the first end
of the chromatographic medium;
(4) allowing a sample and a resolubilized
first specific binding partner to move through at least
a portion of the chromatographic medium blocking binding
sites on the immunological analogue immobilized in the
discrete area;
(5) separating the first and second opposable
components so that they are no longer in opposition;
(6) bringing the first and third opposable
components into opposition such that the resolubilized
labeled second specific binding partner is applied to
the second conductor and then to the second end of the
chromatographic medium;
(7) allowing the resolubilized labeled second
specific binding partner to move through at least a
portion of chromatographic medium overlapping the entire
chromatographic medium through which the sample and the
resolubilized first specific binding partner is drawn so
that, in the presence of analyte in the test sample, the
labeled second specific binding partner binds to the
analyte or immunological analogue thereof immobilized in
the discrete area due to the binding of sample and
analyte with the first specific binding partner; and
AME1VCEC SNEET
'~~.~8~7~
21
(8) observing and/or measuring the second
specific binding partner in the discrete area to detect
and/or determine the analyte.
Preferably, this method further comprises the
step of incubating the chromatographic assay device
following applying the sample of the first applicator in
order to promote the reaction between the analyte and
the first specific binding partner.
In an alternative assay method using this
device, a first reconstitution fluid can be applied to
the first applicator in addition to the sample.
Other versions of an assay device according to
the present invention suitable for competitive
immunoassays can be used similarly.
Another version of an assay device according
to the present invention for performing a competitive
immunoassay comprises:
(1) a first opposable component including:
(a) a chromatographic medium having a
first end and a second end and having immobilized
thereon, in separate discrete and non-overlapping areas,
each area being substantially smaller than the area of
the chromatographic medium:
(i) a specific binding partner for
the analyte; and
(ii) a secondary specific binding
partner, the secondary specific binding partner capable
of binding a member of a specific binding pair that
lacks affinity for the analyte, the secondary specific
binding partner being located closer to the first end of
the chromatographic medium than the first specific
binding partner;
~,i',!_'~~._~ Si-~~~T
~h
22
(b) a first conductor in operable
contact with the first end of the chromatographic
medium; and
(c) a second conductor in operable
S contact with the second end of the chromatographic
medium;
(2) a second opposable component including:
(a) an applicator containing an analyte
analogue, the analyte analogue comprising analyte
covalently linked to a member of a specific binding pair
lacking affinity for the analyte and bindable by the
secondary specific binding partner, the member of the
specific binding pair being labeled with a detectable
label, the analyte analogue being in a form that can be
resolubilized by the addition of an aqueous liquid to
the applicator; and
(b) an absorber separated from the
applicator.
In this device, the first and second opposable
components are configured so that bringing the first and
second opposable components into opposition places the
second conductor in operable contact with the applicator
so that the contents of the applicator are applied to
the chromatographic medium and are drawn through at
least a portion of the chromatographic medium, and
places the absorber in operable contact with the first
conductor to withdraw fluid from the chromatographic
medium.
This version of the device preferably further
comprises a cover hingedly attached to the first
opposable component so that it can be folded over the
first and second opposable components when they are
opposed. The cover has an aperture cut therein to
permit viewing of at least a portion of the
chromatographic medium when the first and second
AMENDED SHEET
.~ .~ ~ ~ ~,~'~ Q
23
opposable components are opposed and the cover is folded
over the first and second opposable components.
Preferably, in this version, the first
specific binding partner is an antibody specific for the
analyte and the secondary specific binding partner is a
second antibody capable of binding an immunoglobulin
lacking specificity for the analyte. Preferably, the
analyte analogue comprises analyte covalently linked to
an immunoglobulin from a species and the specific
binding partner is antibody specific for this species.
This version can further comprise a third
opposable component which contains an absorber
positioned such that when the first and third opposable
components are opposed, the absorber is brought into
contact with the entire chromatographic medium and both
conductors. In this version, an aperture is formed
behind the chromatographic medium which permits viewirLg
of at least a portion of the chromatographic medium from
the back when the second oppos.-ble component is folded
over the first opposable comps nt and the third
opposable component is folded over the second opposable
component.
In another variation, the area of the
secondary specific binding partner immobilized on the
chromatographic medium is divided into at least two
discrete and non-overlapping bands, with the quantity of
secondary specific binding partner in each band being
determined so that the quantity of analyte analogue
binding to the detection zone, and thus the
concentration of analyte in the test sample, is
indicated by the number of bands to which the analyte
analogue binds.
A~!~!~~~~ S'~ 1E~L
2.~,58~ ~~
24
Yet another version of an assay device
according to the present invention suitable for
competitive immunoassays employs a biotin-avidin link.
This version of the device comprises:
(1) a first opposable component comprising a
chromatographic medium having a first end and a second
end and having immobilized thereon, in separate discrete
non-overlapping areas, each area being substantially
smaller than the area of the chromatographic medium;
(a) a substance capable of specifically
binding biotin selected from the group consisting of
avidin, streptavidin, anti-biotin antibody, and
derivatives thereof; and
(b) a secondary specific binding partner
capable of specifically binding a three-component
complex, the three-component complex comprising:
(i) analyte;
(ii) a member of a specific binding
pair lacking specific binding affinity for the analyte,
the member covalently conjugated to the analyte; and
(iii) a detectable label bound to
the member of the specific binding pair;
(2) a second opposable component including a
first applicator containing a first specific binding
partner to the analyte in a form that can be
resolubilized by the addition of an aqueous sample to
the first applicator, the first specific binding partner
being covalently linked to biotin, the first specific
binding partner not capable of being bound by the
secondary specific binding partner;
(3) a third opposable component including:
(a) a second applicator containing the
three-component complex, the complex being in a form
that can be resolubilized by the addition of a second
aqueous liquid to the second applicator; and
(b) an absorber separated from the
second applicator.
~L~ I
file-.VL=j n
25
In this device, the first and second opposable
components are configured so that bringing the first and
second opposable components into opposition places the
first conductor in operable contact with the first
applicator so that the contents of the first applicator
are applied to the chromatographic medium and are drawn
through at least a portion of the chromatographic
medium. The first and third opposable components are
configured so that bringing the first and third
opposable components into oppc: Lion places the absorber
in contact with the first condu~cor to withdraw fluid
from the chromatographic medium, and causes the second
applicator to come into operable contact with the second
conductor so that the contents of the second applicator
are applied to the chromatographic medium and are drawn
through at least a portion of the chromatographic medium
overlapping the portion to which the contents of the
first applicator are drawn.
Preferably, the first specific binding partner
is an anti-analyte antibody. The member of the specific
binding pair in the three-component complex can be
rabbit immunoglobulin G, in which case the secondary
specific binding partner can be goat anti-rabbit IgG.
Preferably, the substance capable of specifically
binding biotin is streptavidin.
The area of the secondary specific binding
partner immobilized on the chromatographic medium can
divided into at least two discrete and non-overlapping
bands, with the quantity of secondary specific binding
partner in each band being determined so that the
quantity of three-component complex binding to the
detection zone, and thus the original analyte
concentration in the test sample, is indicated by the
number of bands to which the three-component complex
binds.
~~~8~70
26
Yet another version of a chromatographic assay
device according to the present invention suitable for
performing a competitive immunoassay comprises:
(1) a first opposable component including:
(a) a chromatographic medium having a
first end and a second end, and having immobilized
thereon in separate and non-overlapping discrete areas
each substantially smaller than the area of the
chromatographic medium:
10 (i) an analyte analogue capable of
binding a specific binding partner for the analyte; and
(ii) a secondary specific binding
partner that is capable of binding a specific binding
pair member that has affinity for the analyte, the
15 secondary specific binding partner itself lacking
binding affinity for the analyte; and
(b) a conductor in operable contact with
the first end of the chromatographic medium; .and
(2) a second opposable component including an
20 applicator containing a specific binding partner to the
analyte in a form that can be resolubilized by addition
of an aqueous liquid to the applicator.
In this device, the first and second opposable
25 components are configured so that bringing the first and
second opposable components into opposition places the
conductor in operable contact with the applicator so
that the contents of the applicator are applied to the
chromatographic medium and are drawn through at least a
30 portion of the chromatographic medium.
Preferably, the first specific binding partner
is antibody specific for the analyte and the analyte
analogue comprises analyte covalently linked to a
35 protein lacking specific binding activity for the
analyte or for the specific binding partner for the
analyte. Preferably, the secondary specific binding
AME~!DED SHEET
~~~~~~~o
27
partner binds the antibody specific for the analyte on
the basis of species-specific interactions not involving
the antigen-combining site of the antibody for the
analyte.
In this device, the area of the secondary
specific binding partner immobilized on the
chromatographic medium can be divided into at least two
discrete and non-overlapping bands, with the quantity of
secondary specific binding partner in each band being
determined so that the quantity of labeled specific
binding partner for the analyte binding to the detection
zone, and thus the quantity of analyte in the test
sample, is indicated by the number of bands to which the
labeled specific binding partner for the analyte binds.
Another version of an assay device according
to the present invention suitable for performing a
competitive immunoassay is a three-component device
involving two steps of reagent migration through the
chromatographic medium, both occurring in the same
direction. This device comprises:
(1) a first opposable component including:
(a) a chromatographic medium having a
first end and a second end and having immobilized
thereon, in separate discrete and non-overlapping areas,
each area being smaller than the area of the
chromatographic medium:
(i) a specific binding partner for
the analyte; and
(ii) a secondary specific binding
partner as described above, the specific binding partner
for the analyte being located closer to the first end of
the chromatographic medium;
(b) a first conductor in operable
contact with the first end of the chromatographic
., ,-,;..
28
medium, the first conductor capable of functioning as a
first applicator; and
(c) a second conductor in operable
contact with the second end of the chromatographic
S medium;
(2) a second opposable component including:
(a) a second applicator containing an
analyte analogue as described above, the analyte
analogue being in a form that can be resolubilized by
the addition of an aqueous liquid to the applicator; and
(b) a first absorber separated from the
second applicator; and
(3) a third opposable component including a
second absorber.
In this device, the first and second opposable
components are configured so that bringing the first and
second opposable components into opposition places the
second applicator into operable contact with the first
conductor, and places the first absorber into operable
contact with the second conductor. The first and third
opposable components are configured so that bringing the
first and third opposable components into opposition
places the second absorber into direct contact with the
first conductor and with the chromatographic medium to
withdraw fluid therefrom.
BRIEF DESCRIPTION OF THE DRAWINGS
These and other features, aspects, and
advantages of the present invention will become better
understood with reference to the following description,
appended claims, and accompanying drawings where:
.z~.~~~'~~
29
Figure lA is a drawing of one version of a
two-component chromatographic assay device according to
the present invention;
Figure 1B is a drawing of the two-component
chromatographic assay device of Figure lA shown with the
two components having been brought into opposition;
Figure 2 is a drawing of a version of a two-
component chromatographic assay device according to the
present inventicn in which the first opposable component
includes a samp~. preparation zone and a chromatographic
medium that is noc in communication with the sample
preparation zone, and the second opposable component
includes a conductive connecting member;
Figure 3 is a drawing of another version of a
two-component assay device according to the present
invention with a sample preparation zone incorporated
into the first opposable component;
Figure 4 is a drawing of an alternative of the
version of Figure 3 with an absorber on the second
opposable component rather than on the first opposable
component;
Figure 5 is a drawing of another version of a
two-component assay device according to the present
invention with a sample preparation zone and an absorber
located on the same opposable component;
Figure 6 is a drawing of yet another version
of a two-component assay device according to the present
invention with two detector application pads on
different opposable components;
Figure 7 is a drawing of yet another version
of a two-component assay device according to the present
invention incorporating two applicators on one of the
components;
Figure 8 is a drawing of an alternative of the
version of Figure 7 with an absorber on the second
opposable component rather than on the first opposable
component;
ANfENDE~ ~~~~T
30
Figure 9 is a drawing of yet another version
of a two-component assay device according to the present
invention incorporating a discontinuity between a
conductor and the chromatographic medium that is bridged
when the device is closed;
Figure l0A is a drawing of yet another version
of a two-component assay device according to the present
invention incorporating a detector application pad in
operable contact with the chromatographic medium;
Figure lOB is a sectional rear view of the
two-component assay device of Figure 10A, showing
details of the components in opposition;
Figure 11A is a drawing of yet another version
of a two-component assay device according to the present
invention, generally similar to the version of Figure
10, but with the detector application pad in direct
contact with the chromatographic medium;
Figure 11B is a sectional- rear view of the
two-component assay device of Figure 11A, showing
details of the components in opposition;
Figure l2A is a drawing of a version of a two-
component assay device according to the present
invention suitable for carrying out bidirectional
chromatography;
Figure 12B is a top view of the two-component
chromatographic assay device of Figure 12A shown with
the two components having been brought into opposition;
Figure 13A is a drawing of another version of
a two-component assay device suitable for carrying out
bidirectional chromatography with two applicators and a
conductor;
Figure 13B is a top view of the two-component
chromatographic assay device of Figure 13A shown with
the two components having been brought into opposition;
Figure 14 is a drawing of another two-
component assay device according to the present
invention with a detector application pad on the first
a~, ~ _ . .
=. ..
~~~g~'~ ~
31
opposable component and a sample preparation zone on the
second opposable component;
Figure 15 is a drawing of another two-
component assay device according to the present
invention with the applicator divided into sectors,
providing for a wash of the chromatographic medium with
sample free of label;
Figure 16A is a drawing of yet another two-
component assay device suitable for bidirectional
chromatography, incorporating a cover;
Figure 16B is a top view of the two-component
chromatographic assay device of Figure 16A shown with
the two components having been brought into opposition;
Figure 17A is a drawing of a three-component
assay device according to the present invention;
Figure 17B is a sectional rear view of the
three-component assay device of Figure 17A, showing
details of the components in opposition;
Figure 18 is a drawing of a three-component
assay desire according to the present invention, in
which the third component acts to absorb fluid from the
entire chromatographic medium ar_d both conductors;
Figure 19 is a drawing of a multiplex assay
device according to the present invention, suitable for
the simultaneous assay of one or more samples;
Figure 20 is a drawing of a version of a
multiplex assay device according to the present
invention, containing a collapsible well to accommodate
a sample;
Figure 21 is a drawing of a different version
of a multiplex assay device according =o the present
invention adapted to receive a test card;
Figure 22 is a drawing of yet another version
of a multiplex assay device according to the present
invention adapted to receive a test card;
Figure 23 is a drawing of a version of a
three-component assay device according to the present
AMEMDED SHEET
, 32
invention suitable for performing a competitive
immunoassay employing a labeled antibody binding to an
analyte analogue immobilized on the chromatographic
medium;
Figure 24 is a drawing of a two-component
assay device with a cover suitable for performing a
competitive immunoassay using a labeled analyte
analogue;
Figure 25 is a drawing of a three-component
assay device suitable for performing a unidirectional
competitive immunoassay using a labeled analyte
analogue, in which the third component acts to absorb
fluid from the entire chromatographic medium and both
conductors;
Figure 26 is a drawing of a three-component
assay device according to the present invention suitable
for performing a competitive immunoassay, the device
using a biotin-avidin link;
Figure 27 is a drawing of a two-component
assay device suitable for performing a competitive
immunoassay using a labeled anti-analyte antibody; and
Figure 28 is a depiction of an assay device
according to the present invention suitable for
receiving a swab or similar sampling device and designed
for detection of Streptococcus A antigen.
DESCRIPTION
Definitions
In the context of this disclosure, the
following terms are defined as follows unless otherwise
indicated:
Specific Binding Partner: A member of a pair
of molecules that interact by means of specific non-
~.,_ .~-, c~=_T
f'SI~iL:'~r-J VIILr)
33
covalent interactions that depend on the t'_~_ee-
dimensional structures of the molecules involved.
Typical pairs of specific binding partners include
antigen-antibody, hapten-antibody, hormone-receptor,
nucleic acid strand-complementary nucleic acid strand,
substrate-enzyme, inhibitor-enzyme, carbohydrate-lectin,
biotin-avidin, and virus-cellular receptor.
Operable Contact: Two solid components are in
operable contact when they are in contact, either
directly or indirectly, in such a manner that an aqueous
liquid can flow from one of the two components to the
other substantially uninterruptedly, by capillarity or
otherwise. "Direct contact" means that the two elements
are in physical contact, such as edge-to-edge or front-
to-back. Typically, when two components are in direct
contact, they are overlapped with an overlap of about
0.5 to about 3 mm. However, the components can be
placed with abutting edges. "Indirect contact" means
that the two elements are not in physical contact, but
are bridged by one or more conductors.
Finite Capacity: An absorber has finite
capacity when it becomes saturated by liquid received
during the normal performance of an assay in the device
in which the absorber is located. At that point, the
absorber can release additional liquid absorbed and
become at least partially conductive.
Analyte: The term "analyte" includes both the
actual molecule to be assayed and analogues and
derivatives thereof when such analogues and derivatives
bind another molec~~~e used in the assay in a manner
substantially equivalent to that of the analyte itself.
Antibody: The term "antibody" includes both
intact antibody molecules of the appropriate specificity
aMFr!o~~ ~H~ET
34
and antibody fragments (including Fab, F(ab'), and
F(ab')2 fragments) as well as chemically modified intact
antibody molecules and antibody fragments, including
hybrid antibodies assembled by in vitro reassociation of
subunits.
Secondary Specific Binding Partner: An
additional specific binding partner that binds to a
member of a pair of specific binding partners when the
pair of specific binding partners is interacting is
designated a secondary specific binding partner. For
example, a pair of specific binding partners can
comprise Giardia antigen and rabbit anti-Giardia
antibody. In that case, the secondary specific binding
partner can be goat anti-rabbit IgG antibody. The
secondary specific binding partner can be specific for
the species, class, or subclass of an antibody specific
binding partner to which it binds. Alternatively, when
one of the specific binding partners is labeled with
biotin, the secondary specific binding partner can
comprise a molecule conjugated to avidin.
Outline of the Disclosure
For the convenience of the reader, and as an
aid to understanding the organization of the disclosure,
the following outline of the disclosure is presented,
setting out the organization of the disclosure in terms
of the section headings.
I. CHROMATOGRAPHIC ASSAY DEVICES
A. Two-Component Devices
1. General Arrangement
2. Particular Embodiments of Two-Component
Device
a. Device with Conductive Connecting
Member on Second Component
~~"~~,~'1 n~)IVCT
hIJILI .L 'J
35
b. Device wit~:_ Sample Preparation Zone
on First Opposable Component
c. Device with Absorber on Second
Component
d. Device with Sample Preparation Zone
and Absorber on Same Opposable
Component
e. Device with Two Detector Application
Pads on Different Opposable
i0 Components
f. Device Including Two Separate
Applicators on Same Opposable
Component
g. Device with Two Applicators and
Absorber on Second Opposable
Component
h. Device with Gap or Discontinuity
Between Conductor and
Chromatographic Medium
i. Device with Pad for Labeled Specific
Binding Partner on Same Opposable
Component as Chromatographic Medium
j. Device with Detector Application
Pad
in Direct Contact with First End
of
Chromatographic Medium
k. Bidirectional Device Including
Second Applicator and Absorber on
Second Opposable Component
1. Hidirectional Device Including Two
Applicators and Conductor
m. Device with Detector Application
Pad
on First Component
n. Device with Two-Sector Applicator
to
Provide Wash
B. Two-Compo nent Device with Cover
C. Three-Component
Device
D. Multiplex Devices
hiJl~'i~U~v Ci"i~
36
1. Basic Multiplex Device
2. Multiplex Device with Collapsible Well
3. Multiplex Devices Adapted to Receive Test
Card
II. CHROMATOGRAPHIC ASSAY DEVICES FOR COMPETITIVE
ASSAYS
A. Three-Component Bidirectional Flow Device
B. Two-Component Bidirectional Flow Device with
Cover
C. Three-Component Unidirectional Flow Device
with Absorber
D. Three-Component Bidirectional Flow Device
Using Specificity of Biotin
E. Two-Component Device for Competitive
Inhibition Immunoassay
III. ANALYTES AND ANTIBODIES FOR USE WITH ASSAY DEVICES
IV. TEST KITS
I. CHROMATOGRAPHIC ASSAY DEVICES
One aspect of the present invention comprises
chromatographic assay devices particularly useful for
the assay of analytes in biological samples. These
devices are suitable for the direct application of
biological samples, without preliminary extraction
steps, and are constructed so as to minimize
interference with assay results caused by particulates
or colored samples.
The device has at least two substantially
planar opposable components. One of the substantially
planar components has on its surface a chromatographic
medium.
The device also has means for opposing the
opposable component and applying pressure thereto. The
pressure applied is sufficient to transfer fluid from
.Sq~,mr,i_r ~'.~C=.
H~u_.m__~ .: __
~o
. 37
one opposable component to another opposable component
in a direction substantially normal to the opposable
components so that the sample is applied to the
chromatographic medium for detection and/or
determination of the analyte thereon. The pressure also
drives fluid through the chromatographic medium to
accelerate the process of chromatography, giving a
detectable result in less time. Additionally, the
pressure makes possible the performance of steps, such
as extraction steps, in the device, and can be used to
remove excess fluid from the chromatographic medium by
absorbers to reduce the background of the assays. The
pressure is generated by placing the opposable
components into opposition and maintained by holding the
components into opposition by engagers such as locks or
clasps.
Devices according to the present invention can
be constructed for the performance of either a sandwich
or a competitive assay.
When the device is constructed for the
performance of a sandwich immunoassay, typically at
least one of the opposable components of the device has
incorporated thereon a first, labeled, specific binding
partner to the analyte. This first, labeled, specific
binding partner to the analyte is in a form that can be
resolubilized by an aqueous liquid. The aqueous liquid
used for resolubilization can be the sample. The first,
labeled, specific binding partner is positioned so that
it can react with the analyte in the sample.
In a device suitable for the performance of a
sandwich immunoassay, the chromatographic medium has
incorporated thereon a detection zone of a second,
unlabeled, specific binding partner for the analyte
immobilized thereto. The detection zone is
a,.:-.",~
2~~~5'~0
38
substantially smaller than the chromatographic medium.
These components are arranged so that a ternary complex
comprising: (1) the first, labeled, specific binding
partner; (2) the analyte; and (3) the second, unlabeled,
specific binding partner binds at the detection zone if
analyte is present in the sample.
In a device suitable for the performance of a
competitive immunoassay, the chromatographic medium has
incorporated thereon in at least one zone substantially
smaller than the chromatographic medium a member of a
specific binding pair selected from the group consisting
of the analyte or an analogue thereof and a specific
binding partner for the analyte. Several different
arrangements are possible for devices according to the
present invention that are suitable for the performance
of competitive immunoassays. These different
arrangements are described below.
Typically, detection and/or determination of
the analyte after the sample is applied in a
chromatographic medium occurs by use of a visually
detectable labeled component. The labeled component is
preferably either the analyte or an analogue thereof
linked to a visually detectable label, or a specific
binding partner for the analyte linked to a visually
detectable label.
Assay devices according to the present
invention can be constructed for the performance of more
than one assay simultaneously. In such devices, one of
the substantially planar components has at least two
separate and non-contacting chromatographic media
thereon.
A variation of the device is particularly
suitable for bidirectional chromatography, although not
AME~ICE~ ~;'=~T
y
39
restricted to that use. This device has at least three
substantially planar opposable components. One of the
substantially planar components has on its surface a
chromatographic medium having first and second ends.
The three-component device also has means for opposing
the opposable components pairwise in at least two
different combinations and applying pressure thereto, by
holding the opposed components together by engagers such
as locks or clasps. The pressure is sufficient to
transfer fluid from one opposable component to another
in a direction substantially normal to the opposable
components and to drive the fluid through the
chromatographic medium. This causes the sample to be
applied to the chromatographic medium and flow through
the chromatographic medium from the first end to the
second end.
The device also has at least one applicator
and one absorber located on one of the opposable
components and positioned in such manner that, when the
opposable component on which the applicator and the
absorber are located is brought into opposition to the
opposable component on which the chromatographic medium
is located, a second liquid is applied to the
chromatographic medium. The second liquid flows through
the chromatographic medium from the second end to the
first end. This reverses the flow through the
chromatographic medium. Detection and/or determination
of the analyte is made subsequent to reversal of the
flow.
A. Two-Component Devices
One embodiment of the assay device of the
present invention is a two-component chromatographic
assay device operating in one dimension with one-
directional flow.
. 40
1. General Arrangement of Two-Component
Device
In general, a two-component chromatographic
S assay device according to the present invention
comprises:
(1) A first opposable component including a
sample preparation zone adapted to receive a sample to
be assayed; and
(2) A second opposable component including a
chromatographic medium.
In this device, the first and second opposable
components can be brought into opposition when the
device is closed so as to cause a sample preparation
zone to apply the sample to be assayed to the
chromatographic medium. In use, the first and second
opposable components are typically brought into
opposition after a detection reagent is applied to the
sample preparation zone. When the first and second
opposable components are brought into opposition, the
sample preparation zone applies the sample and detection
reagent to the chromatographic medium. After the sample
and detection reagent is allowed to traverse at least a
portion of the chromatographic medium, so that the
detection reagent gives a detectable indication of the
presence and/or quantity of the analyte; the detection
reagent is then observed and/or measured in at least a
portion of the chromatographic medium. This results in
detection and/or determination of the analyte.
The description of the details of construction
of this basic device also applies, as far as possible,
to other two-component and three-component assay devices
according to the present invention.
AME!~'~~~ ~w =~T
. 41
o-,~
The detection reagent comprises the first
yes' specific binding partner for the analyte as described
above; it may comprise additional components.
This process can give a qualitative and/or
quantitative indication of the analyte, depending upon
the density of the second specific binding partner in
the detection zone and the size of the detection zone.
Typically, to achieve results, the assay
requires from 30 seconds to 10 minutes, more typically,
from 1 to 5 minutes, including any period of incubation
of the sample on the sample preparation zone, as well as
the time required for chromatography itself. Typically,
the assay is performed at room temperature, although it
can be performed at 4°C or up to 37°C or higher in some
cases, depending upon the nature of the analyte and
specific binding partners. In some cases, performing
the assay at a lower temperature may be desirable to
limit degradation, while in other cases, performing the
assay at a higher temperature with suitable analytes and
specific binding partners may speed up the assay.
This general arrangement of the
chromatographic assay device is shown in Figure lA. The
chromatographic assay device 10 has a first opposable
component 11 and a second opposable component 12. The
first opposable component 11 includes a sample
preparation zone 13. The second opposable component 12
contains a chromatographic medium 14. The
chromatographic medium 14 has a first end 15 and a
second end 16; the chromatographic medium 14 contains a
detection zone 17 and a control zone 18. The first
opposable component 11 and the second opposable
component 12 are joined by a hinge 19. The first and
second opposable components 11 and 12 preferably further
comprise engagers that secure the first and second
A~~E's~~=S S. ;SST
~~_585~~
. 42
opposable components in opposition. The engagers can
comprise locks, such as locks 20 and 21 that are engaged
when the first opposable component 11 and the second
opposable component 12 are brought into opposition. The
S construction and dimensions of the locks 20 and 21 can
be varied to exert the optimal degree of pressure on the
opposable components 11 and 12. The degree of pressure
that is optimal may depend on the thickness and
construction of the chromatographic medium 14, the
intended sample volume, and other factors. To guard
against leakage of samples or reagents, a sealing ridge
or gasket 22 is positioned around the perimeter of the
first and second opposable components 11 and 12.
Although the use of the engagers, such as the locks 20
and 21, and of the sealing ridge or gasket 22, is
generally preferred, these components are not necessary
to construct a basic device according to the present
invention. The second opposable component 12 has a
first window 23; optionally, the first opposable
component 11 can have a second window 24 to permit
viewing of the chromatographic medium 14 from either
side. The second window 24 permits viewing of the
chromatographic medium 14 from the surface opposite the
surface to which the reagents are applied. As another
option, the first window 23 can be absent and the second
window 24 used for viewing of the chromatographic medium
14. In general, two-component devices according to the
present invention can have either one or two windows,
also known as apertures, to allow viewing of the
chromatographic medium either through the opposable
component not carrying the chromatographic medium, as
window 23 in Figure 1, or through the opposable
component carrying the chromatographic medium, as window
24 in Figure 1. Alternatively, the first and/or second
opposable components 11 and 12 can be made of
transparent or translucent materials, so that the
A~IIE~D~D S. ;EET
y
43
chromatographic medium 14 can be viewed without a
separate aperture or window.
Figure 1B shows the device 10 after the
opposable components 11 and 12 have been brought into
opposition. The chromatographic medium 14, including
the detection zone 17 and the control zone 18, is
visible through window 24. The sample preparation zone
13 contacts the chromatographic medium 14 at or near the
first end 1. ~o that the contents of the sample
preparation zone 13 can flow through the chromatographic
medium 14, including the detection zone 17 and the
control zone 18.
The device 10 can, optionally, further
comprise a conductor 25 in operable contact with the
first end 15 of the chromatographic medium 14, as shown
in Figure lA. The conductor 25 can be a material such
as cellulose or other material that can conduct an
aqueous liquid without substantially absorbing it. The
conductor 25 can be treated with a surfactant so that
the reagents can be applied more evenly to the
chromatographic medium 14. When the conductor 25 is
present, the sample preparation zone 13 preferably
contacts the conductor 25 when the first and second
opposable components 11 and 12 are brought into
opposition.
The device l0 can further comprise an absorber
26 in operable contact with the second end 16 of the
chromatographic medium 14 to aid in drawing fluid
through the chromatographic medium 14 from the first end
15 toward the second end 16, as shown in Figure 1A.
The sample preparation zone 13 can be made of
any suitable material, such as, but not limited to,
cellulose, paper, nylon, rayon, glass fiber, fleeces, or
2I5~5'~0
44
non-woven synthetic fabrics. The porosity of the sample
preparation zone 13 can be chosen to filter out cellular
or particulate matter in samples such as whole blood or
fecal samples. The sample preparation zone 13 can
S contain at least one reagent for treatment of the sample
before the sample is applied to the chromatographic
medium 14.
The reagents that can be present in the sample
preparation zone 13 vary with the sample to be applied
to the sample. preparation zone 13 and with the analyte
to be assayed. They can include, but are not limited
to, acids or alkalis to adjust the pH, buffers to
stabilize the pH, chelating agents such as EDTA or EGTA
to chelate metals, hydrolytic enzymes to lyse the cell
membrane of animal cells or the cell wall of bacteria to
liberate analytes, substrates or coenzymes for enzymes,
and the like. One particularly useful extraction
reagent is a mixture of sodium nitrite and acetic acid
to generate nitrous acid. The sodium nitrite can be
present in dried form on the sample preparation zone 13,
and the acetic acid can be added to the sample
preparation zone 13 after the addition of the sample.
The sample, or optionally, a sampling device
such as a throat swab or a microporous filter, can be
placed by the operator on the sample preparation zone
13; if needed, other reagents can be added.
The bodies of the first and second opposable
components 11 and 12 are preferably made of laminated
cardboard that is sufficiently impervious to moisture to
contain the liquids involved in the performance of the
assay. Other cellulose-based materials, such as
paperboard or solid bleached sulfite (SBS) can also be
used. Alternatively, the bodies can be made of plastic
AMENDED SHEET
45
that is impervious to moisture. A suitable plastic is a
polycarbonate plastic such as Lexan".
The hinge 19 is preferably made of material
that is impermeable to an aqueous liquid, such as a
plastic that can be compatibly joined with or is the
same as the material used for the bodies of the first
and second opposable components 11 and 12.
Typically, the chromatographic medium 14,
absorber 26, conductor 25, and other liquid-receiving
components are secured to the bodies of the first and
second opposable components 11 and 12 by adhesive.
Suitable adhesives are well known in the art. Other
joining methods, such as stapling or tacking, can also
be used.
The analyte is detected either by means of a
labeled specific binding partner to the analyte or by
the use of a labeled secondary specific binding partner
for a specific binding partner to the analyte. In most
cases, the use of a labeled specific binding partner to
the analyte is preferred. The label of the labeled
specific binding partner is preferably a visually
detectable label, such as a colloidal metal label.
Preferably, the colloidal metal label is gold, silver,
bronze, iron, or tin; most preferably, it is gold. The
preparation of gold-labeled antibodies is described in
J. DeMey, "The Preparation and Use of Gold Probes," in
Immunocvtochemistry: Modern Methods and Applications
(J. M. Poiak & S. VanNoorden, eds., Wright, Bristol,
England, 1986), Ch. 8, pp. 115-145. Antibodies labeled
with colloidal gold are commercially available, such as
from Sigma Chemical Company, St. Louis, Missouri.
Alternatively, other colloidal labels, such as
a colloidal sulfur label or a dye-silica label, can also
46
be used. In a less preferred alternative, the visually
detectable label can be a colored latex label. It is
also possible to use other labels, such as radioactive
labels.
Although Applicants do not necessarily intend
to be bound by this theory, when an aqueous liquid
containing a sample is applied to a resolubilizable
specific binding partner labeled with a colloidal metal
label, such as colloidal gold, the kinetics of the
reaction between the analyte and the labeled specific
binding partner are extremely rapid. These rapid
kinetics result in the substantially complete labeling
of analyte before the combination of the analyte and the
labeled specific binding partner is applied to the
chromatographic medium. Thus, in a one-directional
chromatographic procedure performed with an assay device
according to the present invention, what is
chromatographed is predominantly the binary complex of
the analyte and the corresponding labeled specific
binding partner. This allows separation of this complex
from contaminants not binding the specifis binding
partner and improves accuracy of the assay.
In this embodiment, the labeled specific
binding partner preferably is present on the sample
preparation zone 13 in a form that can be resolubilized
by the addition of an aqueous liquid to the sample
preparation zone. Typically, the aqueous liquid is the
sample itself. In some cases, particularly where small
sample volumes are used, it may be desirable to add
additional buffer or other aqueous liquid to the sample
preparation zone.
In other embodiments discussed below, the
labeled specific binding partner can be present on an
element of the chromatographic assay device that is
47
~~y separate from the sample preparation zone but comes into
contact with it during the performance of the assay. In
these embodiments, the labeled specific binding partner
is preferably present in a resolubilizable form on this
element, and is resolubilized when the sample comes into
contact with the element. In some cases, the labeled
specific binding partner can be resolubilized by the
addition of a separate aqueous liquid, distinct from the
sample, to the element.
The chromatographic medium 14 on the second
opposable component 12 is a flat strip. It is typically
rectangular, having first and second ends 15 and 16.
Throughout this Description, the term "first end" 15
refers to the end of the chromatographic medium 14 at
which the sample is applied, and the term "second end"
16 refers to the opposite end. The original direction
of flow of the sample is from the first end 15 toward
the second end 16 of the chromatographic medium 14. The
chromatographic medium 14 is composed of a material
suitable as a medium for thin-layer chromatography of
analytes and analyte-antibody conjugates, such as
nitrocellulose, nylon, rayon, cellulose, paper, or
silica. The chromatographic medium 14 can be pretreated
or modified as needed. Typically, the chromatographic
medium 14 is translucent, so that colored zones
appearing on it as a result of the assay can be viewed
from either side.
In some applications, it is preferable to
place a second flexible transparent support on the top
of the chromatographic medium 14 to regulate the flow of
the sample through the chromatographic medium 14 and
prevent migration over the top of the chromatographic
medium. Suitable flexible transparent supports include
polyethylene, vinyl, Mylar~, and cellophane.
~~~$5r~0
48
When the chromatographic assay device 10 is to
be used for an assay such as a sandwich immunoassay, the
chromatographic medium can further comprise a detection
17 zone substantially smaller than the chromatographic
medium 14. This detection zone can contain a second
specific binding partner to the analyte immobilized
thereto against diffusion. The second specific binding
partner can be bound to the chromatographic medium by
either covalent or non-covalent means. If the analyte
to be assayed is an antigen or hapten, the second
specific binding partner can be an antibody to the
antigen or the hapten. Alternatively, the analyte can
be an antibody and the second specific binding partner
can be a hapten or an antigen capable of being bound
specifically by the antibody.
The chromatographic medium 14 can further
comprise a control zone 18 substantially smaller than
the chromatographic medium 14, and separate from the
detection zone 17. The control zone 18 can comprise
analyte immobilized thereto non-diffusibly in order to
bind labeled antibody that is not bound at the detection
zone 17 by the formation of a ternary "sandwich"
complex. Any such antibody is bound by the immobilized
analyte and forms a detectable zone or band. This
provides a check on the operation of the assay and the
correct binding of the reagents, as described below.
The methods used to bind the second specific binding
partner in the detection zone 17 and the analyte in the
control zone 18 are well known in the art and need not
be described further.
Alternatively, for some analytes, such as
carbohydrates, it may be difficult or impossible to fix
the analyte stably to the chromatographic medium 14. In
such cases, the control zone 18 can comprise an
immobilized zone of antibody specific for the labeled
AR",~~0=D SHLET
c'~~ . 4 9
~
anti-analyte antibody. For example, if the analyte is
Ei ~v
~Y the Streptococcus A-specific carbohydrate, and the
labeled antibody is rabbit IgG specific for
Streptococcus A antigen, the control zone 18 can
comprise goat antibody to rabbit IgG. In such cases, to
prevent complete capture of the labeled anti-analyte
antibody in the detection zone 17 at high analyte
concentration and consequent disappearance of the
labeled anti-analyte antibody from the control zone 18,
it can be desirable to add labeled antibody not specific
for the analyte and of a different species than the
labeled anti-analyte antibody. Such antibody can
constitute immunologically indifferent immunoglobulin
or
an antibody to an analyte not found in the test sample.
The control zone 18 would then comprise anti-species
antibody or analyte not found in the test sample.
Several variations of this device are
possible. In one variation, as discussed above, the
sample preparation zone 13 can further contain a
specific binding p._, tner for the analyte labeled with a
detectable label i:. a form that can be resolubilized by
the addition of an aqueous liquid to the sample
preparation zone 13. The aqueous liquid can be the
sample itself. The labeled specific binding partner can
be freeze-dried or reversibly precipitated so that it is
resolubilized and mobilized by the addition of the
sample to the sample preparation zone. In this
variation, it is not necessary to add a detection
reagent to the sample preparation zone 13, as the
detection reagent is automatically generated by the
additi- of the sample to the sample preparation zone
13.
In another variation, the conductor 25 in
operable contact with the first end 15 of the
chromatographic medium 14 on the second operable
Aj~,%~~''vnC~ ~t'~='
. 50
component can be replaced by an absorber 25a of finite
capacity in operable contact. The absorber 25a is
located so that it comes into contact with the sample
preparation zone 13 when the first 11 and second 12
opposable components are placed into opposition, to
apply the sample to the absorber. This may be useful in
controlling the flow of sample into the chromatographic
medium 14 so that the chromatographic medium 14 is not
overloaded.
In this version., the absorber 25a can contain
a labeled specific binding partner for the analyte in a
form that can be resolubilized, as described above. In
this arrangement, the labeled specific binding partner
is resolubilized when the first and second opposable
components 11 and 12 are brought into opposition,
applying the sample to the absorber 25a. The
combination of the sample and the resolubilized labeled
specific binding partner then enters the chromatographic
medium 14 at its first end 15.
~",'''..r~ c'-:
i~~
51
2. Particular Embodiments of Two-Component
Device
a. Device with Conductive Connecting
Member on Second Component
In one embodiment of a two-component device
according to the present invention, the first opposable
component comprises:
(1) A sample preparation zone; and
(2) A chromatographic medium that is not in
communication with the sample preparation zone.
The second opposable component comprises a
conductive connecting member. The first and second
opposable components can be brought into opposition so
as to cause the connecting member to establish a
communication between the sample preparation zone and
the chromatographic medium so as to result in the
application of the sa~ple to the chromatographic medium.
This embodiment is depicted in Figure 2. The
chromatographic assay device 40 comprises a first
opposable component 41 and a second opposable component
42. The first opposable component includes a sample
preparation zone 43 and a chromatographic medium 44,
with a first end 50 and a second end 51. The
chromatographic medium 44 has a detection zone 45 and a
control zone 46. The second opposable component 42
includes a conductive connecting member 47. The first
and second opposable components 41 and 42 are connected
by a hinge 48. The first and second opposable
components 41 and 42 also include engagers such as locks
52 and 53, with a gasket 54 surrounding the first and
second opposable components 41 and 42. The second
opposable component 42 has an aperture 49 to permit
viewing of the chromatographic medium 44.
~~ ~~(~ ,1 .. -r_T
~~~L~~F'J ',.
~~.5$~'~~
52
When the first and second opposable components
41 and 42 are brought into opposition, the sample
preparation zone 43 on the first opposable component 41
is brought into contact with the conductive connecting
member 47 on the second opposable component 42, which in
turn is brought into contact with the first end 50 of
the chromatographic medium 44 to apply the sample to the
chromatographic medium 44.
In variations of the device of Figure 2, the
conductive connecting member 47 can contain a specific
binding partner for the analyte labeled with a
detectable label in a form that can be resolubilized by
the addition of an aqueous liquid. In this variation,
the sample is added to the sample preparation zone 43.
Alternatively, the sample preparation zone 43 can be
used for addition of labeled specific binding partner
for the analyte in liquid form, with the sample itself
being added to the conductive connecting member 47. In
yet another alternative, the sample preparation zone 43
can contain resolubilizable specific binding partner for
the analyte, with the sample again being added to the
conductive connecting member 47.
b. Device with Sample Preparation Zone
on First Opposable Component
Another embodiment of a chromatographic assay
device according to the present invention is a device
that incorporates a sample preparation zone on the first
opposable component, i.e., the component on which the
chromatographic medium is located. Typically, in this
embodiment, the second opposable component comprises an
applicator incorporating a labeled specific binding
partner for the analyte in a form that can be
resolubilized.
.4.,~__
fi~:LIVL~u .,f~L_
~,,
C~
53
I: this embodiment, bringing the first and
second opposable components into opposition brings the
applicator into contact with the sample preparation zone
so that the labeled specific binding partner for the
analyte is resolubilized.
Preferably, the first opposable component
further comprises a conductor, and operable contact
between the sample preparation zone and the
chromatographic medium is achieved by having the sample
preparation zone and the chromatographic medium both in
operable contact with the conductor.
Preferably, the first opposable component
further comprises an absorber in operable contact with
the second end of the chromatography medium.
The chromatographic medium is preferably
constructed as described above, with detection and
control zones.
This embodiment of the assay device is shown
in Figure 3. The chromatographic assay device 60 has a
first opposable component 61 and a second opposable
component 62. The first opposable component 61 includes
a sample preparation zone 63, a conductor 64 in operable
contact with the sample preparation zone 63, a
chromatographic medium 65 having a first end 66 and a
second end 67, and an absorber 68 in operable contact
with the second end 67 of the chromatographic medium 65.
The chromatographic medium 65 contains a detection zone
69 and a control zone 70. The second opposable
component 62 contains an applicator 71, preferably
incorporating a labeled specific binding partner in a
form that can be resolubilized. The first opposable
component 51 and the second opposable component 62 are
joined by a hinge 72. The second opposable component 62
~I1»~~r,rl~ J
IW n'.L IJ~
~~~85,~0
54
contains a window 73 to allow viewing of at least a
portion of the chromatographic medium 65. The first and
second opposable components 61 and 62 have engagers such
as locks 74 and 75, with a gasket 76 surrounding the
first and second opposable components 61 and 62.
In operation, a sample is applied to the
sample preparation zone 63. The first and second
opposable components 61 and 62 are then brought into
opposition so that the sample in the sample preparation
zone 63 resolubilizes the contents of the applicator 71,
including the labeled specific binding partner. The
contents of the sample preparation zone 63 and the
applicator 71 are then applied to the chromatographic
medium 65 through the sample preparation zone 63 and the
conductor 64.
c. Device with Absorber on Second
Component
In an alternative of this embodiment, the
absorber can be placed on the second opposable component
instead of the first opposable component. In this
alternative, the absorber comes into operable contact
with the second end of the chromatographic medium when
the first and second opposable components are placed in
opposition. This allows for a larger absorber, which
can be desirable when it is necessary to use a larger
sample, such as in the detection of an analyte present
only in low concentrations.
This alternative of the embodiment is shown in
Figure 4. The chromatographic assay device 80
comprises the first opposable component 81 and the
second opposable component 82. The first opposable
component 81 includes the sample preparation zone 83,
the conductor 84 in operable contact with the sample
AP'~~:(~~r ~;~==T
. _ ~ , ; ~ ~~58~v~w
. .
preparation zone 83, and the chromatographic medium 85
having the first end 86 and the second end 87, with the
detection zone 88 and the control zone 89. The second
opposable component 82 contains the applicator 90 and
S the absorber 91; the applicator 90 is separated from the
absorber 91 on the second opposable component 82. The
first opposable component 81 and the second opposable
component 82 are joined by a hinge 92. The second
opposable component 82 also contains an aperture 93 to
l0 permit viewing of at least a part of the chromatographic
medium 85. The first and second opposable components 81
and 82 also include engagers such as locks 94 and 95 and
a gasket 96, as described above.
15 In use, the sample is applied to the sample
preparation zone 83, where extraction or other treatment
of the sample can occur. The sample then enters the
first end 86 of the chromatographic medium 85 by flowing
through the conductor 84, as described above. When the
20 first opposable component 81 and the second opposable
component 82 are brought into opposition, the applicator
90 is brought into operable contact with the conductor
84 to resolubilize the labeled specific binding partner
in the applicator 90 and apply the resolubilized labeled
25 specific binding partner to the conductor 84 and then to
the first end 86 of the chromatographic medium 85, which
causes the resolubilized labeled specific binding
partner to enter the chromatographic medium 85. The
absorber 91 is simultaneously brought into operable
30 contact with the second end 87 of the chromatographic
medium 85 to withdraw fluid from the chromatographic
medium 85.
d. Device with Samgle Preparation Zone
35 and Absorber on Same Opposable
Component
AMENDED SHEET
CA 02158570 2003-12-30
56
Another embodiment of a chromatographic assay
device according to the present invention is a two-
component device in which the sample preparation zone
and the absorber are located on the same opposable
5 component. This has the advantage in certain
applications of being able to use a larger absorber to
withdraw fluid more rapidly and efficiently from the
second end of the chromatographic medium.
10 This embodiment of the two-component
chromatographic assay device is shown in Figure 5. The
chromatographic device 100 has a first opposable
component 101 and a second opposable component 102
connected by a hinge 103. The first opposable component
15 101 includes a chromatographic medium 104 having a first
end 105 and a second end 106, and a conductor 107 in
operable contact with the first end 105 of the
chromatographic medium 104. The chromatographic medium
104 contains a detection zone 108 and, optionally, a
20 control zone 109.
The second opposable component 102 includes a
sample preparation zone 110 for receiving a sample to be
assayed and an absorber 111 separated from the sample
25 preparation zone 110. The sample preparation zone 110
can contain a labeled specific binding partner for the
analyte in resolubilizable form. The second opposable
component 102 has an aperture 112 therein to permit
viewing of the chromatographic medium. The first and
30 second opposable components 101 and 102 have engagers
such as locks 113 and 114, and a gasket 115, as
described above.
When the first 101 and second 102 opposable
35 components are brought into opposition, the sample
preparation zone 110 comes into operable contact with
the conductor 107 to apply the sample to the conductor
2~. ~~5'~Q
57
107 and then to the first end 105 of the chromatographic
medium 104. The absorber 111 comes into operable
contact with the second end 106 of the chromatographic
medium 104 to withdraw fluid from the second end 106 of
the chromatographic medium 104. In other respects, the
design and operation of this device is similar to that
depicted in Figure 4, above.
e. Device with Two Detector Application
Pads on Different Op~~osable
Components
Yet another embodiment of a two-component
assay device according to the present invention
incorporates two separate detector application pads on
different opposable components. This arrangement is
particularly useful when it is desired to use a large
volume of a labeled specific binding partner, as when a
labeled antibody is only available in dilute form and
attempts to concentrate the antibody would denature or
inactivate it.
This embodiment of the two-component
chromatographic assay device is depicted in Figure 6.
The chromatographic assay device 120 has a first
opposable component 121 and a second opposable component
122 connected by a hinge 123. The first opposable
component 121 includes a chromatographic medium 124
having a first end 125 and a second end 126. The
chromatographic medium 124 includes a detection zone
127, and, optionally, a control zone 128.
The first opposable component 121 also has a
first detector application pad 129 in operable contact
with the first end 125 of the chromatographic medium
124. The first detector application pad 129 contains a
first specific binding partner to the analyte in a form
AMENO=p ~~;EET
58
that can be resolubilized by the addition of an aqueous
liquid to the first detector application pad 129. The
first specific binding partner is typically labeled with
a detectable label. The first opposable component 121
S also has a conductor 130 in operable contact with the
first detector application pad 129 so that the first
detector application pad 129 bridges the conductor 130
and the first end 125 of the chromatographic medium 124.
The first opposable component 121 also has an absorber
131 in operable contact with the second end 126 of the
chromatographic medium 124.
The second opposable component 122 includes a
sample preparation zone 132 for receiving a sample to be
assayed. The second opposable component 122 also
contains a second detector application pad 133 in
operable contact with the sample preparation zone 132,
with the sample preparation zone 132 being placed over
the second detector application pad 133. The sample
preparation zone 132 and the second detector application
pad 133 can be held together by a fastener or adhesive.
The second detector application pad 133 and the sample
preparation zone 132 are positioned so that a sample
applied to the sample preparation zone 132 must pass
through the sample preparation zone 132 before entering
the second detector application pad 133. The second
detector application pad 133 contains a second specific
binding partner for the analyte in a form that can be
resolubilized by the addition of a sample to the sample
preparation zone 132. The second detector application
pad 133 is positioned such that application of the
sample to the sample preparation zone 132 resolubilizes
the second specific binding partner so that the sample
preparation zone 132 contains a mixture of the sample
and the second specific binding partner.
A~~ENDED SWEET
59
The second specific binding partner is labeled
with a detectable label. Preferably, the first and
second specific binding partners are identical and the
detectable labels labeling the first and the second
specific binding partners are identical.
The second opposable component 122 also
contains an aperture 134 therein to allow viewing of at
least a portion of the chromatographic medium 124,
including the detection zone 127 and, if present, the
control zone 128. The first and second opposable
components 121 and 122 also have engagers such as locks
135 and 136, and a gasket 137 as described above for the
basic two-component device.
When the first and second 121 and 122
opposable components are brought into opposition, the
sample preparation zone 132 is brought into contact with
the conductor 130 to apply the sample and the second
specific binding partner to the conductor 130 and then
to the first end 125 of the chromatographic medium 124
through the first detector application pad 129. Thus,
the sample sequentially contacts the second specific
binding partner and then the first specific binding
partner before being applied to the first end 125 of the
chromatographic medium 124 for chromatography. This
results in a greater volume of labeled specific binding
partner being in contact with the sample to increase the
sensitivity of the assay.
f. Device Includina Two Separate
Applicators on Same Opposable
Component
Yet another embodiment of a chromatographic
assay device according to the present invention
including two separate applicators on the same opposable
An,aG.,,~~,r!~ c~,~'~
.~~~~~5r~
component. These two applicators are not in operable
contact until they are bridged by a conductor on the
opposing element when the elements are brought into
opposition.
5
This embodiment of the chromatographic assay
device is shown in Figure 7. The chromatographic assay
device 140 has a first opposable component 141 and a
second opposable component 142. The first opposable
10 component 141 includes a chromatographic medium 143
having a first end 144 and a second end 145, a conductor
146 in operable contact with the first end 144, and an
absorber 147 in operable contact with the second end 145
of the chromatographic medium 143. The chromatographic
15 medium 143 contains a detection zone 148 and a control
zone 149. The second opposable component 142 contains a
first applicator (sample application pad) 150 and a
second applicator (detector application pad) 151. The
first applicator 150 and the second applicator 151 are
20 not in operable contact until the first opposable
component 141 and the second opposable component 142 are
brought into opposition. When the first opposable
component 141 and the second opposable component 142 are
brought into opposition, the first applicator 150 is
25 brought into contact with the conductor 146 and the
second applicator 151 is brought into contact with both
the conductor 146 and the first end 144 of the
chromatographic medium 143. The overlap is typically
several millimeters; i.e., enough to ensure transfer of
30 fluid. This results in the first applicator 150 and the
second applicator 151 being bridged by the conductor.146
so that the contents of the first applicator 150 and th
second applicator 151 are applied to the chromatograph
medium 143. The first opposable component 141 and the
35 second opposable component 142 are joined by a hinge
152. The second opposable component 142 contains a
window 153 to allow viewing of the chromatographic
~~r,~r~c~~ ~;;~~i
61
medium 143. The first and second opposable components
141 and 142 also include engagers such as locks 154 and
15S and a gasket 156.
The first applicator 150 can comprise a sample
application pad and the second applicator 151 can
comprise a detector application pad, to which detecting
reagent can be applied. When the first and second
opposable components 141 and 142 are brought into
opposition, the contents of the sample application pad
and the detector application pad are applied to the
chromatographic medium 143 via the conductor 146.
Preferably, the second applicator 151
(detector application pad) contains a specific binding
partner for the analyte labeled with a detectable label
in a form that can be resolubilized by the addition of
an aqueous liquid to the second applicator 151. The
aqueous liquid is typically the sample itself, which
resolubilizes the labeled specific binding partner when
the first 141 and second 142 opposable components are
brought into opposition. In some assays, it may be
desirable to add a separate reconstituting aqueous
liquid to the detector application pad. Alternatively,
the labeled specific binding partner can be applied in
liquid form to the second applicator 151.
g. Device with Two Applicators and
Absorber on Second Opposable
Component
In another variation of this embodiment, the
absorber can be relocated from the first opposable
component (i.e., the opposable component containing the
chromatographic medium) to the second opposable
component (i.e., the opposable component containing the
a~,f~;:J__ _ -._
CA 02158570 2003-12-30
62
first and second applicators). This allows the use of a
larger sample and can be desirable for detection of
analytes present in low concentration.
5 This variation is shown in Figure 8. The
chromatographic assay device 160 has a first opposable
component 162 and a second opposable component 164. The
first opposable component 162 includes a chromatographic
medium 166 having a first end 168 and a second end 170
10 and a conductor 172 in operable contact with the first
end 168 of the chromatographic medium 166. The
chromatographic medium 166 contains a detection zone 174
and a control zone 176. The second opposable component
164 contains a first applicator (sample application pad)
15 178 and a second applicator (detector application pad)
180. The first applicator 178 and the second applicator
180 are not in operable contact until the first
opposable component 162 and the second opposable
component 164 are brought into opposition, and are then
20 bridged as described above for the assay device depicted
in Figure 7. The second opposable component 164 also
contains an absorber 182 that is separated from the
first applicator 178 and the second applicator 180.
When the first opposable component 162 and the second
25 opposable component 164 are brought into opposition, the
first applicator 178 and the second applicator 180 are
bridged by the conductor 172 so that the contents of the
first applicator 178 and the second applicator 180 are
applied to the chromatographic medium 166.
30 Simultaneously, the absorber 182 is brought into
operable contact with the second end 170 of the
chromatographic medium 166 to absorb fluid from the
chromatographic medium 166. The first opposable
component 162 and the second opposable component 164 are
35 joined by a hinge 184. The second opposable component
164 contains a window 186 to allow viewing of the
chromatographic medium 166. The first and second
~-~~~~7~
63
opposable components 162 and 164 also include engagers
such as locks 185 and 187, and a gasket 188.
h. Device with Gap or Discontinuitv
Between Conductor and
Chromatographic Medium
A further variation of this device
incorporates a gap or discontinuity between the
conductor and the chromatographic medium so that the
path of fluid flow is from the first applicator through
the conductor, then through the second applicator, and
finally through the chromatographic medium.
This variation of the device is shown in
Figure 9. The chromatographic assay device 190 has a
first opposable component 191 and a second opposable
component 192. The first opposable component 191
includes a chromatographic medium 193 having a first end
194 and a second end 195, a conductor 196 not in
operable contact with the first end 194 of the
chromatographic medium 193 when the device 190 is in
open position, and an absorber 197 in operable contact
with the second end 195 of the chromatographic medium
193. The chromatographic medium 193 contains a
detection zone 198 and a control zone 199. The second
opposable component 192 contains a first applicator
(sample application pad) 200 and a second applicator
(detector application pad) 201. The first applicator
200 and the second applicator 201 are not in operable
contact until the first opposable component 191 and the
second opposable component 192 are brought into
opposition. When the first opposable component 191 and
the second opposable component 192 are brought into
opposition, by closing a hinge 203 connecting the first
opposable component 191 and the second opposable
component 192, the first applicator 200 and the second
. . J .W
~~ "~'~_T
~Z~ ~~~~ ~
64
applicator 201 are bridged by the conductor 196 and the
second applicator 201 contacts the chromatographic
medium 193. Thus, the path of fluid flow is from the
first applicator 200 through the conductor 196, then
through the second applicator 201, and then into the
chromatographic medium 193. The second opposable
component 192 contains a window 202 to allow viewing of
the chromatographic medium 193. The first and second
opposable components 191 and 192 also include engagers
such as locks 204 and 205, and a gasket 206, as
described above.
i. Device with Pad for Labeled Specific
Binding Partner on Same Opposable
Component as Chromatoaraohic Medium
Yet another embodiment of a chromatographic
assay device according to the present invention is a
two-component device incorporating a pad for a labeled
specific binding partner on the same opposable component
as the chromatographic medium. In this device, the
sample applicator is located on the other opposable
component.
This embodiment of a chromatographic assay
device according to the present invention is depicted in
Figure 10A. The chromatographic assay device 210 has a
first opposable component 212 and a second opposable
component 214. The first opposable component 212 has a
chromatographic medium 216 having a first end 218 and a
second end 220. The chromatographic medium has a
detection zone 222 and, optionally, a control zone 224,
as described above for other variations of assay devices
suitable for sandwich immunoassays. The first opposable
component 212 also has a conductor 226 in operable
contact with the first end 218 of the chromatographic
medium 216, and an absorber 228 in operable contact with
A~r'~'~C~;~ _ _-.
~~~~~ l
r~
the second end 220 of the chromatographic medium 216.
The first opposable component 212 also has a detector
application pad 230 in direct contact with the conductor
226 and positioned such that it is in indirect contact
5 with the first end 218 of the chromatographic medium
216. The second opposable component 214 has a sample
application pad 232. The first opposable component 212
and the second opposable component 214 are joined by a
hinge 234. When the first opposable component 212 and
10 the second opposable component 214 are brought into
opposition, the sample application pad 232 is brought
into contact with the detector application pad 230. The
second opposable component 214 contains a window 236 to
allow viewing of the chromatographic medium 216. The
15 first and second opposable components 212 and 214 have
engagers such as locks 233 and 235, and a gasket 237, as
described above.
A sectional rear view of the device 210 is
20 depicted in Figure lOB. Figure lOB shows the first
opposable component 212 and second opposable component
214 in opposition. The sample application pad 232 is
shown in contact with the detector application pad 230.
The detector application pad 230 is in contact with the
25 conductor 226, which is in turn in contact with the
first end 218 of the chromatographic medium 216. The
detection zone 222 and control zone 224 of the
chromatographic medium 216 are shown. The second end
220 of the chromatographic medium 216, nearer the
30 control zone 224, is in contact with the absorber 228.
Bringing the first and second opposable
components 212 and 214 into opposition causes the sample
application pad 232 to apply the sample to be tested to
35 the detector application pad 230 and thus to the first
end 218 of the chromatographic medium 216 though the
conductor 226.
:r~~n-~ ''
Aw:~,.~;=~ Si~S~T
~~~~r~
::..~~~ 1; ~ 66
Preferably, the detector application pad 230
contains a first specific binding partner to the analyte
in a form that can be resolubilized by addition of an
aqueous liquid to the detector application pad 230, and
the first specific binding partner is labeled with a
detectable label.
Preferably, the contents of the sample
application pad 232 after a sample is applied thereto
comprises an aqueous liquid, and the aqueous liquid
applied to the detector application pad 230 comprises
the contents of the sample application pad. In this
arrangement, there is no additional liquid needed to
resolubilize the labeled specific binding partner.
In a variation of this device, the absorber is
located on the second opposable component instead of
being located on the first opposable component. The
absorber is separated from the sample application pad
also located on the second opposable component and is
placed in operable contact with the second end of the
chromatographic medium when the first opposable
component and the second opposable component are brought
into opposition, as shown above in Figure 5.
j. Device with Detector Application Pad
in Direct Contact with First End of
Chromatographic Medium
A further variation of this device omits the
conductor between the detector application pad and the
chromatographic medium, so that the detector application
pad is in direct contact with the first end of the
chromatographic medium. In this variation, when the
first and second opposable components are brought into
opposition, the detector application pad and the sample
applicaticn pad are in contact except for the region of
,h;u~.,_:_~ ___ .
67
the detector application pad directly adjacent to the
first end of the chromatographic medium. There is a
slight gap or offset at that region of the detector
application pad, so that sample cannot flow directly
from the sample application pad to the chromatographic
medium. This gap or offset is typically from about 0.5
mm to about 2 mm, more typically from about 0.5 mm to
about 1 mm.
This variation is particularly suitable for
the detection of fecal occult blood by use of a labeled
anti-hemoglobin antibody, and can detect concentrations
of hemoglobin corresponding to as much as 17 ml of blood
per 100 g feces (13 mg hemoglobin per gram of feces),
without the occurrence of false negatives due to a high
dose "hook" effect.
This variation is depicted in Figure 11A. The
chromatographic assay device 250 has a first opposable
component 252 and a second opposable component 254. The
first opposable component 252 has a chromatographic
medium 256 having a first end 258 and a second end 260.
The chromatographic medium 256 has a detection zone 262
and a control zone 264. The first opposable component
252 also has an absorber 266 in operable contact with
the second end 260 of the chromatographic medium 256.
The first opposable component 252 also has a detector
application pad 268 in direct contact with the first end
258 of the chromatographic medium 256. The second
opposable component 254 has a sample application pad
270. The first opposable component 252 and the second
opposable component 254 are joined by a hinge 272. When
the first opposable component 252 and the second
opposable component 254 are brought into opposition, the
sample application pad 270 is brought into contact with
the detector application pad 268, except for a narrow
gap or offset 274 at the end of the detector application
APJ1ENCEC~ EuEET
CA 02158570 2003-12-30
68
pad 268 in contact with the first end 258 of the
chromatographic medium 256. This gap 274 prevents
sample applied to the sample application pad 270 from
flowing directly into the chromatographic medium 266.
5 The second opposable component 254 has a window 276 to
allow viewing of the chromatographic medium 256. The
first and second opposable components 252 and 254 have
engagers such as locks 278 and 280, and a gasket 282 as
described above.
10 A sectional rear view of the device 250 of Figure
11A is depicted in Figure 11B. Figure 11B shows the
first opposable component 252 and second opposable
component 254 in opposition, with the hinge 272 in
closed position. The sample application pad 270 is
15 shown in contact with the detector application pad 268,
except for the small gap 274 at the end of the detector
application pad 268 nearest the chromatographic medium
256. The detector application pad 268 is in direct
contact with the first end 258 of the chromatographic
20 medium 256. The detection zone 262 and control zone 264
of the chromatographic medium 256 are shown. The second
end 260 of the chromatographic medium 256, nearer the
control zone 264, is in contact with the absorber 266.
k. Bidirectional Device Including
25 Second Applicator and Absorberon
,Second Opposable Component
Another embodiment of a chromatographic assay
device according to the present invention comprises a
30 device capable of carrying out bidirectional
chromatography.
This embodiment of the chromatographic assay device
is shown in Figure 12A. The assay device 300 has
CA 02158570 2003-12-30
69
a first opposable component 302 and a second opposable
component 304. The first opposable component 302 has a
chromatographic medium 306 having a first end 308 and a
second end 310. The first opposable component 302 also
5 has a first applicator 312 in operable contact with the
first end 308 of the chromatographic medium 306. The
chromatographic medium 306 also comprises a detection
zone 314 and, optionally, a control zone 316 located
between the detection zone 314 and the second end 310 of
10 the chromatographic medium 306. The detection zone 314
and control zone 316 are constituted as discussed above
for other embodiments of the device suitable for
sandwich immunoassays. The second opposable component
304 includes an absorber 318, which can be an absorbent
15 pad, and a second applicator 320. The first302 and
second 304 opposable components are joined by a hinge
322. The second opposable component 304 includes a
window 324 to permit viewing of the chromatographic
medium 306. The first and second opposable components
20 302 and 304 include engagers 323 and 325, and a gasket
326, as described above.
When the hinge 322 is closed, the device 300
appears as shown in a top view in Figure 12B. The
25 chromatographic medium 306, including the detection zone
314, and if present, the control zone 316, is visible
through the window 324.
In this embodiment of the device, addition of
30 a first liquid to the first applicator 312 causes the
first liquid to be applied to the first end 308 of the
chromatographic medium 306. Bringing the first and
second opposable components 302 and 304 into opposition
then causes the second applicator 320 to come into
35 operable contact with the second end 310 of the
chromatographic medium 306 so as to apply a second
liquid to the second end 310 of the chromatographic
~0
medium 306, and causes the absorber 318 to come into
operable contact with the first applicator 312 so as to
withdraw fluid from the chromatographic medium 306
through the first applicator 312, thus reversing the
flow. In the operation of this device, the sample is
applied to the first applicator 312 and a solution of a
labeled specific binding partner is applied to the
second applicator 320. The sample then moves through
the chromatographic medium 306 from the first end 308
toward the s~:~or_ =nd 310 so that any analyte present in
the sample is bo.__.d to the immobilized antibody at the
detection zone 314. When the first and second opposable
components 302 and 304 are brought into opposition, the
labeled specific binding partner is applied to the
chromatographic medium 306 and moves through the
chromatographic medium 306 from the second end 310
toward the first end 308. The labeled specific binding
partner then binds to any analyte bound to the
immobilized antibody at the detection zone 314,
generating a detectable ternary complex. The labeled
specific binding partner also binds to the immobilized
analyte or analogue at the control zone 316, giving an
indication of correct performance of the assay.
1. Bidirectional Device Including Two
Applicators and Conductor
Another embodiment of a chromatographic assay
device according to the present invention is a device
suitable for bidirectional chromatography that includes
an absorber to reverse the flow and a reagent pad in
operable contact with the chromatographic medium.
This embodiment of the chromatographic assay
device is shown in Figure 13A. The assay device 340 has
a first opposable component 342 and a second opposable
component 344. The first opposable component 342 has a
~~fr~~r,rn C~L-=i
71
chromatographic medium 346 having a first end 348 and a
second end 350. Adjacent to and in operable contact
with the first end 348 of the chromatographic medium 346
is a first applicator 352. Adjacent to and in operable
contact with the second end 350 of the chromatographic
medium 346 is a conductor 354. The chromatographic
medium 346 contains a detection zone 356, and
optionally, a control zone 358. The second opposable
component 344 comprises an absorber 360 and a second
applicator 362. The first and second opposable
components 342 and 344 are.joined by a hinge 364. The
second opposable component 344 contains a window 366 to
permit viewing of the chromatographic medium 346. The
first and second opposable components 342 and 344
include engagers 365 and 367, and a gasket 368 as
described above.
When the first and second opposable components
342 and 344 are brought into opposition, the absorber
360 is brought into operable contact with the first
applicator 352, and the second applicator 362 is brought
into operable contact with the conductor 354, thereby
reversing the flow. The portion of the chromatographic
medium 346, including the detection zone 356 and, if
present, the control zone 358, can be viewed through the
window 366 in the second opposable component 344 when
the first 342 and second 344 opposable components are
placed into opposition.
Figure 13B shows a top view of the device 340
when the first and second opposable components 342 and
344 are placed into opposition; the detection zone 356
and the control zone 358 are visible through the window
366 in the second opposable component 344.
In this device, the first applicator 352 can
comprise a sample application pad, to which the sample
AP~~E~DED SHEET
y~~~~ t~
72
to be tested is applied, and the second applicator 362
can comprise a buffer application pad to which buffer is
added. The first applicator 352 (sample application
pad) can contain at least one reagent for treatment of
S the sample before it is applied to the chromatographic
medium, as described above. The second applicator 362
(buffer application pad) typically contains a specific
binding partner for the analyte in a form that can be
resolubilized by the addition of an aqueous liquid, as
described above.
The first applicator 352 (sample application
pad) preferably further comprises an inert dye so that
the flow of the sample through the chromatographic
medium 346 can be monitored visually. Preferably, the
inert dye is of a contrasting color to that of the
detectable label. For example, when the detectable
label is pink colloidal gold, the inert dye can be blue.
The migration of sample is monitored by observing the
inert dye. After the sample has migrated a sufficient
distance, for example, two-thirds or three-fourths of
the length of the chromatographic medium 346, the first
and second opposable components 342 and 344 are brought
into opposition and the absorber 360 is brought into
contact with the first applicator 352. This reverses
the flow of the sample through the chromatographic
medium 346, allowing additional capture of the analyte
at the detection zone 356. It also brings the second
applicator 362 into contact with the conductor 354 and
causes the buffer solution containing resolubilized
labeled specific binding partner to the analyte to be
applied to the chromatographic medium 346. The buffer
solution migrates through the chromatographic medium 346
from the second end 350 toward the first end 348. When
it reaches the detection zone 356, labeled specific
binding partner to the analyte binds to the analyte
already bound to the detection zone 356. Detection
..r~~ m:-
Ai~~t,;~ ~~ ~~ ,~~
CA 02158570 2003-12-30
73
and/or determination of the analyte is then performed as
described above.
Because the first and second opposable components
342 and 344 are not in contact when the sample is
applied as the first liquid to the first applicator 352,
5 it is possible to apply either the sample to the first
applicator 352 first or to apply the buffer to the
second applicator 362 first to reconstitute the labeled
specific binding partner.
A variation of this bidirectional device replaces
10 the conductor 354 on the first opposable component 342
with a first absorber of finite capacity. The absorber
360 on the second opposable component 344 then is a
second absorber. The absorber of finite capacity on the
first opposable component 344 has the property that
15 liquid can be drawn back through it when the second
applicator 362, containing a resolubilizable labeled
specific binding partner, is placed in operable contact
with it and the second absorber 360 is placed in
operable contact with the first applicator 352.
20 m. Device with Detector application Pad
on First Component
Another embodiment of a two-component
chromatographic assay device according to the present
invention has a detector application pad located on the
25 first opposable component and a sample preparation zone
located on the second opposable component. In this
device, the detector application pad is located so that
it is in operable contact with the first end of the
chromatographic medium. The detector application pad
30 preferably contains a labeled specific binding partner
to the analyte in a form that can be resolubilized by
74
the addiCion of an aqueous liquid to the detector
application pad. The device further comprises a
conductor in operable contact with the detector
application pad and in indirect contact with the first
end of the chromatographic medium, as well as an
absorber in operable contact with the second end of the
chromatographic medium.
In the operation of this embodim~~~ of the
device, the sample is applied to the sampl reparation
zone on the second opposable component, af~ ~ which the
first and second opposable components are brought into
opposition. This applies the contents of the sample
preparation zone to the conductor, and then to the first
end of the chromatographic medium through the detector
application pad. When the sample reaches the detector
application pad, the contents of the detector
application pad are resolubilized. When the contents of
the detector application pad include a specific binding
partner for the analyte, the passage of the sample
through the detector application pad results in the
specific binding partner binding to any analyte present
in the sample.
This embodiment of the device is shown in
Figure 14. The assay device 380 has a first opposable
component 382 and a second opposable component 384. The
first opposable component 382 includes a chromatographic
medium 386 having a first end 388 and a second end 390.
The first opposable component 382 also includes a
detector application pad 392 in operable contact with
the first end 388 of the chromatographic medium 386, a
conductor 394 in operable contact with the detector
application pad 392 and in indirect contact with the
first end 388 of the chromatographic medium 386, and an
absorber 396 in operable contact with the second end 390
of the chromatographic medium 386. The chromatographic
AME»rE~ ~I~cET
75
medium 386 includes a detection zone 398 and a control
zone 400. The second opposable component 384 includes a
sample preparation zone 402. The first and second
opposable components 382 and 384 are joined by a hinge
404. The second opposable component 384 has an aperture
406 to permit viewing of at least a portion of the
chromatographic medium 386. The first and second
opposable components include engagers 405 and 407, and a
gasket 408 as described above.
A variation of this embodiment incorporates a
specific binding partner for the analyte in a form that
can be resolubilized on the second opposable component
384 as well as on the first opposable component 382.
When the resolubilizable specific binding partner is
located on the second opposable component 384, it is
preferably not located directly in the sample
preparation zone 402 itself. Rather, it preferably
surrounds the sample preparation zone 402 in an area 403
such that the sample first passes through the sample
preparation zone 402 and then moves into the area 403
surrounding the sample preparation zone 402,
resolubilizing the specific binding partner. For
example, the sample preparation zone 402 can comprise a
piece of suitably treated filter paper placed on the
surface of the second opposable component 384, adhered
by an adhesive or a fastener. This allows for treatment
of the sample, e.g., to adjust the pH, lyse intact
cells, and/or remove particulates, before the sample
contacts the resolubilizable specific binding partner.
This variation of this embodiment can provide a wider
dynamic range and can be useful when the available
antibody has a low affinity or low concentrations of
analytes are to be detected.
n. Device with Two-Sector Applicator to
Provide wash
a~',!''..~~ '~~'C
76
Another embodiment of a two-component assay
device according to the present invention has a two-
sector applicator to provide a wash of sample unreacted
with the labeled specific binding partner after the
S mixture of the sample and the labeled specific binding
partner has passed through the chromatographic medium.
This embodiment has the advantage of providing a clearer
background and making it easier to read a weakly
positive result.
In this embodiment, the first opposable
component includes a chromatographic medium having first
and second ends, a conductor in operable contact with
the first end of the chromatographic medium, and an
absorber in operable contact with the second end of the
chromatographic medium. The second opposable component
includes an applicator divided into two sectors: a first
sector containing a labeled specific binding partner for
the analyte in resolubilizable form, and a second sector
without the labeled specific binding partner. Bringing
the first and second opposable components into
opposition places the first sector, but not the second.
sector, of the applicator into direct contact with the
conductor, to apply the contents of the first sector of
the applicator to the conductor and then to the first
end of the chromatographic medium. The second sector is
placed in indirect contact with the conductor, as the
contents of the second sector flow through t~-:e first
sector and then to the conductor. Thus, subsequent to
the application of the contents of the first sector of
the applicator to the conductor, the contents of the
second sector are applied to the conductor. The
contents of the second sector, which includes sample but
no labeled specific binding partner, serve to wash out
unbound labeled specific binding partner from the
chromatographic medium, thereby reducing the background
of visible label seen in the chromatographic medium and
77
improving the reading of the assay device. This is
particularly advantageous for weakly positive assays.
This embodiment of the assay device is shown
in Figure 15. The assay device 420 has a first
opposable component 422 and a second opposable component
424. The first opposable component 422 includes a
chromatographic medium 426 with a first end 428 and a
second end 430, a conductor 432 in operable contact with
the first end 428 of the chromatographic medium 426, and
an absorber 434 in operable contact with the second end
430 of the chromatographic medium 426. The second
opposable component 424 has an applicator 436 divided
into two sectors: a first sector 438 in direct contact
with the conductor 432 when the first and second
opposable components 422 and 424 are brought into
opposition, and a second sector 440 in indirect contact
with the conductor 432 when the first and second
opposable components 422 and 424 are brought into
opposition. The chromatographic medium 426 has a
detection zone 442 and a control zone 444. The first
and second opposable components 422 and 424 are joined
by a hinge 446. The second opposable component 424 has
a window 448 to permit viewing of at least a portion of
the chromatographic medium 426. The first and second
opposable components 422 and 424 also include engagers
423 and 425 and a gasket 427, as described above.
B. Two-Component Device with Cover
Another embodiment of a chromatographic assay
device according to the present invention is a two-
component device with a cover.
This embodiment of the assay device is shown
in Figure 16A. The assay device 460 has a first
opposable component 462, a second opposable component
.~ .,. . r
~~T
Afv~ti~~~E~ .:~,;.
78
464, and a cover 466. The second opposable component
464 is hingedly attached to one side of the first
opposable component 462 by a first hinge 468. The cover
466 is hingedly attached to the opposite side of the
first opposable component 462 by a second hinge 470.
The first opposable component 462 has a chromatographic
medium 472 having a first end 474 and a second end 476.
Adjacent to and in operable contact with the first end
474 of the chromatographic medium 472 is a first
applicator or sample pad 478 which is located in a
recess 480. The first applicator 478 can include a
sample preparation zone that can contain at least one
reagent for treatment of the sample before it is applied
to the chromatographic medium, as described above. The
first applicator 478 can also contain an inert dye to
indicate the progress of chromatography as described
above. A first absorber 482 is adjacent to and in
operable contact with the second end 476 of the
chromatographic medium 472. The chromatographic medium
472 contains a detection zone 484 and a control zone 486
and is optionally marked with a limit line 488. The
limit line 488 can be optionally marked on the first
opposable component 462 as well. The second opposable
component 464 comprises a second applicator 490,
typically containing a labeled specific binding partner
for the analyte, and a second absorber 492. The cover
466 contains a first aperture 494. Preferably, the
second opposable component 462 contains a second
aperture 496. In this device, the second opposable
component 464 and the cover 466 also include engagers
491 and 493. The device 460 is surrounded by a gasket
495 as described above.
Figure 16B depicts a top view of this device
460 when the second opposable component 464 is folded
over the first opposable component 462 and the cover 466
is folded over the second opposable component 464. A
_ . -, c"_
79
portion of the chromatographic medium 472 is visible
through the first aperture 494 of the cover 466 and
through the second aperture 496 of the second opposable
component 464, including the detection zone 484 and the
control zone 486.
In the operation of this device, the addition
of a first liquid to the first applicator 478 causes the
first liquid to be applied to the first end 474 of the
chromatographic medium 472. Typically, the first liquid
is a sample that may contain an analyte.
Ir. this device, the function of the recess 480
in the first opposable component 462 is to position the
second absorber 492 so that it can be placed at least
partially in direct contact with the chromatographic
medium 472 in order to remove excess sample, while
keeping the first applicator 478 from blocking this
contact.
In use, the sample is added to the first
applicator 478 so that chromatography can proceed in the
chromatographic medium 472 from the first end 474 toward
the second end 476. When the dye reaches the limit line
488, the first and second opposable components 462 and
464 are brought into opposition, which causes the second
applicator 490 to come into operable contact with the
first absorber 482 so as to apply the second liquid to
the second end 476 of the chromatographic medium 472 and
causes the second absorber 492 to come into operable
contact with the first applicator 478 so as to withdraw
fluid from the chromatographic medium 472 via the first
applicator 478. This reverses chromatographic flow and
draws the labeled specific binding partner back through
the chromatographic medium 472 from the second end 476
toward the first end 474. This device is therefore
useful for bidirectional chromatography.
.. ,_, t-, ., _
., _ .
a0
The cover 466 can be folded over the second
opposable component 464 to more securely lock and hold
the second opposable component 464 against the first
opposable component 462. This allows more convenient
storage of the device, after use, as in a medical
record.
C. Three-Component Device
Another embodiment of a chromatographic assay
device according to the present invention is a three-
component assay device utilizing bidirectional
chromatography.
This embodiment of the three-component assay
device is shown in Figure 17A. The assay device 520 has
a first opposable component 522, a second opposable
component 524, and a third opposable component 526. The
second opposable component 524 is hingedly attached to
one side of the first opposable component 522 by a first
hinge 528; the third opposable component 526 is hingedly
attached to the opposite side of the first opposable
component 522 by a second hinge 530. The first
opposable component 522 has a chromatographic medium 532
having a first end 534 and a second end 536. The
chromatographic medium 532 has a detection zone 538 and
a control zone 540. In operable contact with the first
end 534 of the chromatographic medium 532 is a first
conductor 542 and in operable contact with the second
end 536 of the chromatographic medium 532 is a second
conductor 544. The second opposable component 524
comprises a 'first absorber 546 and a first applicator
548, intended for application of the sample. Typically,
the first applicator 54~ contains a first specific
binding partner for the analyte in a form that can be
resolubilized by the application of an aqueous sample to
the first applicator 548. A first aperture 550 is cut
AME!~ncJ ~!~~rT
81
in the second opposable component 524 to allow viewing
of at least a portion. of the chromatographic medium 532.
The first aperture S50 is between the first absorber 546
and the first applicator 548. The third opposable
S component 526 comprises a second applicator 552,
intended for a labeled secondary specific binding
partner, and a second absorber 554. A second aperture
556 is cut in the third opposable component 526 to allow
viewing of at least a portion of the chromatographic
medium 532. The second aperture 556 is between the
second applicator 552 and the second absorber 554. When
the device 520 is closed, with the second opposable
component 524 folded over the first opposable component
522 and the third opposable component 526 folded over
the first opposable component 522, at least a portion of
the chromatographic medium 532 is visible through the
first aperture 550 and the second aperture 556 (Fig.
17B). The second and third opposable components 524 and
526 includes engagers 551 and 555 to hold the components
together. A flexible ridge or gasket 557 surrounds the
edges of the components to retain reagents or samples
within the device 520. A similar arrangement of
engagers and a ridge or gasket is used for other three-
component devices, as described below.
In this device, bringing the first and second
opposable components 522 and 524 into opposition causes
the first absorber 546 to come into operable contact
with the second conductor 544 and the first applicator
548 to come into operable contact with the first
conductor 542 to apply fluid to the chromatographic
medium 532, so that a first liquid applied to the first
applicator 548 is drawn through at least a portion of
the chromatographic medium 532, including the detection
zone 538 and control zone 540.
82
The first and second opposable components 522
and 524 are then withdrawn from opposition and the first
and third opposable components 522 and 526 are brought
into opposition. This causes the second absorber 554 to
come into operable contact with the first conductor 542
to withdraw fluid from the chromatographic medium 532
and causes the second applicator 552 to come into
operable contact with the second conductor 544 to apply
fluid to the chromatographic medium 532. This causes a
reversal of flow so that a second liquid applied to the
second applicator 552 is drawn through at least a
portion of the chromatographic medium through which the
first liquid has been drawn in the direction opposite to
the direction in which the first liquid was drawn
through the chromatographic medium 532. The first 522,
second 524, and third 526 opposable components are in
such a configuration that, when the third opposable
component 526 is brought into opposition with the first
opposable component 522, the second opposable component
524 can be folded over the first and third opposable
components 522 and 526 to form a cover.
In the performance of a serological assay
using this device, firm pressure between the second
absorber 554 and the first conductor 542 is important to
assure that non-specific immunoglobulin is withdrawn
from the chromatographic medium 532 before the advancing
front of labeled secondary specific binding partner, or
to assure the withdrawal of free analyte before advance
of labeled specific anti-analyte. If mixing between
anti-immunoglobulin label and non-specific
immunoglobulin occurs, the non-specific immunoglobulin
neutralizes the anti-immunoglobulin label (conjugate)
leaving less or none available for labeling the
specifically captured immunoglobulin. The third
opposable component 526 of the device helps keep
consistent pressure applied to reliably effect this
A. y_. r.1 r,~.~
83
function. This is one of the advantages of assay
devices according to the present invention.
The first specific binding partner can be
directly labeled with a detectable label.
Alternatively, indirect labeling of the first specific
binding partner can be used. In indirect labeling, the
first specific binding partner is not labeled, but is
detected by application of a labeled secondary specific
binding partner that specifically binds the first
specific binding partner by means of the second
applicator. Indirect labeling is particularly useful
for testing for Giardia or other antigens for which
commercially available antibodies are directly labeled
only with difficulty. When the first specific binding
partner is not directly labeled, the second applicator
552 preferably contains a labeled secondary specific
binding partner for the first specific binding partner,
as discussed below in Section III.
Another embodiment of a three-component device
employs an absorber on the third component that is
positioned to remove fluid from the entire
chromatographic medium and components in operable
contact with it. This serves the purpose of removing
excess sample that may create a background color, thus
reducing sensitivity.
This embodiment is shown in Figure 18. The
assay device 560 has a first opposable component 561, a
second opposable component 562, and a third opposable
component 563. The second opposable component 562 is
hingedly attached to one side of the first opposable
component 561 by a first hinge 564. The third opposable
component 563 is hingedly attached to the other side of
the first opposable component 561 by a second hinge 565.
The first opposable component 561 has a chromatographic
Ar"~~~~rE~ ~NE~i
84
medium 566 having a first end 567 and a second end 568.
Adjacent to and in operable contact with the first end
567 of the chromatographic medium S66 is a first
applicator 569. Adjacent to and in operable contact
with the second end 568 of the chromatographic medium
566 is a conductor 570. The chromatographic medium
contains a detection zone 571, and, optionally, a
control zone 572. The second opposable component 562
comprises a first absorber 573 and a second applicator
574, typically incorporating a labeled specific binding
partner to the analyte in resolubilizable form. The
third opposable component 563 comprises a second
absorber 575 that extends substantially along the length
of the chromatographic medium 566 in order to absorb
fluid from the chromatographic medium 566, the first
applicator 569, and the conductor S70 when the third
opposable component 563 is brought into opposition with
the first opposable component 561. The first opposable
component 561 has an aperture S76 behind the
chromatographic medium 566 for viewing at least a
portion of the chromatographic medium 566 at the back of
the first opposable component 561. The second and third
opposable components 562 and 563 also include engagers
577 and 579, and a gasket 580 as described above.
In use, the sample to be tested is applied to
the first applicator 569 and a buffer solution is
applied to the second applicator 574. The sample is
then allowed to flow through the chromatographic medium
566, including the detection zone 571 and control zone
572. The first and second opposable components 561 and
562 are then brought into opposition, so that the second
applicator S74 contacts the first applicator 569 and the
first absorber 573 contacts the conductor 570 at the
second end 568 of the chromatographic medium 566. This
causes the resolubilized labeled specific binding
partner originally in the second applicator 574 to flow
. - ~ - _ _-.
85
through the chromatographic medium. After completion of
flow of the sample and the resolubilized specific
binding partner through the chromatographic medium 566,
the third opposable component 563 is brought into
opposition with the first opposable component 561 so
that the second absorber 575 can remove fluid from the
chromatographic medium 566, the first applicator 569,
and the conductor 570. The second opposable component
562 is then folded back over the third opposable
component 563, and the chromatographic medium 566,
including the detection zone 567 and the control zone
568, is viewed through the apertures 576 located behind
the chromatographic medium 566.
D. Multiplex Devices
Another embodiment of a chromatographic assay
device according to the present invention is a multiplex
assay device that can perform multiple assays
simultaneously. The assays can be performed on the same
analyte or different analytes. In general, all versions
of the device described above are suitable for multiplex
use by providing first and second opposable components,
and third opposable components if necessary, with
multiple chromatographic media, sample preparation
zones, applicators, conductors, absorbers, and other
required elements.
1. Basic Multiplex Device
One version of a multiplex assay device
according to the present invention is shown in
Figure 19. The assay device 590 has a first opposable
component 592 and a second opposable component 594. The
second opposable component 594 is hingedly attached to
the first opposable component 592 by a hinge 596. The
first opposable component 592 comprises a plurality of
1 ~
86
chromatographic media 598. Each of the chromatographic
media 598 has a first end 600 and a second end 602, and
comprises a detection zone 604 and a control zone 606.
The second end 602 of each chromatographic medium 598 is
S in operable contact with an absorber 608 to drive flow
through the chromatographic medium 598. There is a
separate absorber 608 for each chromatographic medium
598. The second opposable component 594 comprises a
plurality of sample preparation zones 610, one for each
chromatographic medium 598. Typically, each sample
preparation zone 610 contains labeled specific binding
partner for the analyte to be tested in a form that can
be resolubilized by the addition of a liquid sample to
the sample preparation zone 610. Alternatively, the
labeled specific binding partner in a liquid form can be
added to the sample preparation zone 610 before or after
the addition of the sample thereto. Bringing the first
and second opposable components 592 and 594 into
opposition causes each of the sample preparation zones
610 to be applied to the corresponding chromatographic
medium 598 at the first end 600. The second opposable
component 594 contains a plurality of apertures 612, one
for each chromatographic medium 598. The first
opposable component 592 and second opposable component
594 include engagers 614 and 616 and a gasket 618, as
described above.
This multiplex device can contain from 2 to 12
or more sample preparation zones and chromatographic
media, depending upon the assay for which the device is
to be employed. Typically, the device contains from 2
to 5 separate.sample preparation zones and
chromatographic media.
This embodiment of the device can be used to
assay a number of different analytes in different
aliquots of the same sample, or can be used to assay the
~w;rrl~~J ~f'!r~T
~~~8
same analyte in a number of different samples. This
latter mode is particularly useful in assaying for a
condition for which samples taken at different times
from the same patient must be assayed for the analyte of
interest, such as fecal occult blood. The presence of
fecal occult blood is frequently determined by means of
a series of stool samples taken once a day or at other
intervals for a prescribed period. Alternatively, one
or more of the assays can be used for controls or
l0 reference standards.
2. Multiplex Device with Collapsible Well
In yet another variation of the multiplex
device, at least one sample preparation zone could
comprise a collapsible well, to which an extraction swab
or other sample-containing device can be added. In this
variation, the first opposable component can further
comprise hingedly foldable wings that fold over the
second opposable component when the first opposable
component and second opposable component are brought
into opposition.
This variation of the multiplex device is
shown in Figure 20. The device 620 has a first
opposable component 622 and a second opposable component
624. The second opposable component 624 is hingedly
attached to the first opposable component 622 by a hinge
626. Hingedly attached to the first opposable component
622 are two foldable wings 628 and 630. The first
opposable component 622 has a control well 632 and a
collapsible sample well 634, i.e., made of a sponge-like
material. The second opposable component 624 has a
plurality of chromatographic media 636, in this example,
two, each with a detection zone 638 and a control zone
640. The second opposable component has an aperture 642
for viewing of a portion of each of the
a'.'_ '_ "~ ~__ _
'z~.~~~'~0
88
chromatographicmedia 636, including the detection zone
638 and the control zone 640. The first and second
opposable components 622 and 624 include engagers 641
and 643. When the first opposable component 622 and the
second opposable component 624 are opposed, samples in
the control well 632 and the collapsible sample well 634
are applied to the corresponding chromatographic media
636 for chromatography.
3. Multiplex Devices Adagted to Receive Test
Card
Yet another variation of the multiplex device
is particularly useful for determination of hemoglobin
1S in fecal occult blood. This device is adapted to
receive a test card that includes several dried fecal
samples, typically taken on consecutive days.
This device is shown in Figure 21. The assay
device 660 comprises a first opposable component 662, a
second opposable component 664 hingedly attached to the
first opposable component 662 by a first hinge 666, and
a third opposable component 668 hingedly attached to the
first opposable component 662 by a second hinge 670.
The first opposable component 662 is adapted to receive
a test card 672 that has a plurality of dried specimens
674 mounted thereon. The second opposable component 664
has reagent pads 676 incorporated therein. The third
opposable component 668 has a plurality of
chromatographic media 678 each with a detection zone 680
and a control zone 682 as described above. There is a
separate chromatographic medium 678 for each sample to
be tested. The third opposable component 668 has an
aperture 684 to permit viewing of at least a portion cf
the chromatographic media 678, including each detection
zone 680 and control zone 682. The second and third
opposable components include engagers 681 and 685, with
. _ ,, _ _ __
CA 02158570 2003-12-30
89
the device 660 including a gasket 686 as described above
for three-component devices.
In use, the test card 672 containing the plurality of
dried specimens 674 is inserted into the first opposable
5 component 662. The second opposable component 664 is
folded over the first opposable component 662, so that
the reagent pads 676 contact the plurality of dried
specimens 674 in the test card 672 and extract analyte
from the dried specimens 674. The second opposable
10 component 664 is then unfolded from the first opposable
component 662. Finally, the third opposable component
668 is folded over the first opposable component 662 to
apply the contents of the reagent pads 676 and the
analyte extracted from the dried specimens 674 to the
15 chromatographic media 678 so that chromatography can
occur.
In this device, the reagent pads 676 comprise a
specific binding partner for the analyte labeled with a
detectable label in a form that can be resolubilized by
20 the addition of an aqueous reagent to the reagent pads
676. The reagent added is an extration reagent for the
analyte to be assayed, such as hemoglobin.
When the second opposable component 664 is opposed
to the first opposable component 662, the analyte is
25 extracted from the samples on the test card 672 and
binds to the labeled specific binding partner. When the
third opposable component 668 is subsequently opposed to
the second opposable component, any analyte bound to the
labeled specific binding partner migrates through the
30 chromatographic media 668, and binds to the detection
zone 680 in each of the chromatographic media 678.
90
Yet another variation of a multiplex device
according to the present invention is a multiplex device
similar to that shown in Figure 19, but adapted to
receive a test card. The test card can contain a
plurality of samples, such as dried fecal samples when a
fecal occult blood test is performed.
This variation is shown in Figure 22. The
assay device 700 has a first opposable component 702 and
a second opposable component 704. The second opposable
component 704 is hingedly attached to the first
opposable component 702 by a hinge 706. The first
opposable component 702 comprises a plurality of
chromatographic media 708. Each of the chromatographic
media 708 has a first end 710 and a second end 712, and
comprises a detection zone 718 and a control zone 720.
The first end 710 of each chromatographic medium 708 is
in operable contact with a conductor 714, and the second
end 712 of each chromatographic medium 708 is in
operable contact with an absorber 716. There is a
separate conductor 714 and absorber 716 for each
chromatographic medium 708. The first opposable
component 702 is adapted to receive a test card 722
containing a plurality of dried specimens 724 positioned
so that they are in operable contact with each conductor
714. The second opposable component 704 comprises a
plurality of applicators 726, one for each
chromatographic medium 708. Preferably, each applicator
726 contains labeled specific binding partner for the
analyte in resolubilizable form.
In use, a buffer or other aqueous liquid is
applied to each applicator 726 to reconstitute the
labeled specific binding partner. Bringing the first
and second opposable components 702 and 704 into
opposition causes each of the applicators 726 to be
applied to the corresponding dried specimen 724 so that
a,~,1_ . _ =~ ._
91
~~~c~
the contents of each dried specimen 724 and each
applicator 726 are applied to each conductor 714, and
thus to each chromatographic medium 708. The test card
722 holds each of the specimens 724 in position so that
they can receive the contents of the applicators 726,
and so that analyte in the specimens 724 is extracted,
reacts with the labeled specific binding partner, is
applied to the conductors 714. The second opposable
component 704 contains a plurality of apertures 728, one
for each chromatographic medium 708, for viewing of each
chromatographic medium 708. The first and second
opposable components 702 and 704 each includes engagers
725 and 727 and a gasket 730 to retain samples and
reagents.
II. CHROMATOGRAPHIC ASSAY DEVICES FOR COMPETITIVE
ASSAYS
Assay devices similar in construction to those
disclosed above can be used for competitive assays for
monovalent analytes. The monovalent analytes are
typically haptens, but the same principles could be used
to assay any analyte that is monovalent, such as a
normally multivalent antigen on which the additional
antibody-binding sites are blocked or modified.
Assayable analytes include the following: theophylline,
digoxin, disopyramide, lidocaine, procainamide,
propranolol, quinidine, amikacin, penicillin and other
g-lactam antibiotics, chloramphenicol, gentamicin,
kanamycin, netilmycin, tobramycin, tricyclic
antidepressants, ethosuximide, phenobarbital, diazepam,
phenytoin, primidone, valproic acid, acetaminophen,
acetylsalicylic acid, ibuprofen, methotrexate, drugs of
abuse such as morphine, codeine, cocaine, fentanyl, 3-
methylfentanyl, amphetamines, lysergic acid
diethylamide, phencyclidine, and heroin and their
metabolites, DNP, 1-substituted-4-hydroxy-2-
AM~i'IDED S'~'LC i
,z,~r~~r~ ~
92
nitrobenzenes, 4-substituted-2-vitro-trialkylanilinium
salts, and environmental contaminants such as benzene,
toluene, xylene, ethylbenzene, chlordane, DDT and its
metabolites, 2,4-D, 2,4,5-T, and atrazine.
Specific binding partners suitable for the
performance of these assays include, but are not limited
to, antibodies and specific binding proteins. An
example of the latter is the penicillin binding protein
(PBP) isolated from Bacillus stearothermobhilus.
Unlike previous assay devices adapted for
carrying out competitive immunoassays, devices according
to the present invention have the advantage that the
presence of analyte in the sample gives a positive or
detectable result, each as a colored line in the
detection zone of the device. Typically, in competitive
immunoassays, the development of a detectable signal
indicates a negative result (no analyte present). Thus,
an inverse relationship exists between the observed
result and detection. The devices of the present
invention, which yield a direct relationship between the
analyte concentration and the detectable signal, are
less likely to yield false negative results. These
devices also possess an expanded dynamic range.
A. Three-Component Bidirectional Flow Device
One embodiment of a chromatographic assay
device for competitive assays according to the present
invention is a three-component bidirectional flow
device.
The device has three opposable components.
The first opposable component includes a chromatographic
medium having a first end and a second end; the flow in
the first direction is from the first end to the second
end and in the second direction, from the second end to
the first end. The chromatographic medium has
A.MS'~DSD SNSET
93
immobilized thereon in a discrete area substantially
smaller than the area of the chromatographic medium
analyte or an immunological analogue thereof.
An immunological analogue is, preferably, an
analyte covalently linked to a protein lacking specific
binding activity for the analyte, such as normal IgG or
other non-immune IgG. Such IgG is preferably is
absorbed against the analyte of interest to remove any
immunoglobulin fractions with specific binding activity,
either natural antibodies for the analyte or antibodies
capable of cross-reacting with the analyte.
The conjugation of haptens to proteins,
including non-immune IgG, is well-known in the art and
is described, for example, in P. Tijssen, "Practice and
Theory of Enzyme Immunoassays" (Elsevier, Amsterdam,
1985) pp.~279-296. Briefly, haptens containing carboxyl
groups or that can be carboxylated, can be coupled by
the mixed anhydride reaction, by reaction with a water-
soluble carbodiimide; or esterification with N-
hydroxysuccinimide. Carboxylation can be performed by
reactions such as alkylation of oxygen or nitrogen
substituents with haloesters, followed by hydrolysis of
the ester, or the formation of hemisuccinate esters or
carboxymethyloximes on hydroxyl or ketone groups of
steroids.
Haptens with amino groups or nitro groups
reducible to amino groups can be converted to diazonium
salts and reacted with proteins at mildly alkaline pH,
for aromatic amines. Haptens with aliphatic amines can
be conjugated to proteins by various methods, including
reaction with carbodiimides, reaction with the
homobifunctional reagent tolylene-2,4-diisocyanate, or
reaction with maleimide compounds. Aliphatic amines can
also be converted to aromatic amines by reaction with g-
hi.~~:". ~.l
~z~.~~~~~
94
nitrobenzoylchloride and subsequent reduction to a g-
aminobenzoylamide, which can then be coupled to proteins
after diazotization. Also, bifunctional imidate esters
such as dimethylpimelimidate, dimethyladipimidate, or
dimethylsuberimidate, can be used to conjugate amino
group-containing haptens to proteins.
Thiol-containing haptens can be conjugated to
proteins with malemides, such as 4-(N-maleimidomethyl)-
cyclohexane-1-carboxylic acid N-hydroxysuc~inimide
ester.
For haptens with hydroxyl groups, an alcohol
function can be converted to the hemisuccinate, which
introduces a carboxyl group available for conjugation.
Alternatively, the bifunctional reagent
sebacoyldichloride converts an alcohol to an acid
chloride, which then reacts with proteins.
Phenols can be activated with diazotized g-
aminobenzoic acid, which introduces a carboxyl group,
and can then be reacted with the proteins by the mixed
anhydride reaction. Sugars can be activated by forming
a p-nitrophenyl glycoside, followed by reduction of the
nitro group to an amino group and conjugation by
diazotization. Other methods include the cleavage of
vicinal glycols of sugars to aldehydes by reaction with
periodate, followed by coupling to amines by reductive
alkylation with sodium borohydride. Alternatively,
hydroxyl containing haptens can be conjugated after
conversiur~ to chlorocarbonates by reaction with
phosgenE
For haptens with aldehyde or ketone groups,
carboxyl groups can be introduced through the formation
of O-carboxymethyloximes. Ketone groups can also be
derivatized with g-hydrazinobenzoic acid. to produce
.,, ,.
s
95
carboxyl groups. Haptens containing aldehydes can be
directly conjugated through the formation of Schiff
bases that are stabilized by reaction with a reducing
agent such as sodium borohydride.
The chromatographic medium is preferably
nitrocellulose, to which the analyte analogue is bound
noncovalently by a hydrophobic interaction. Other
chromatographic media can be used, and the analyte
analogue can alternatively be coupled covalently to the
chromatographic medium.
The first opposable component further
comprises a first conductor. in operable contact with the
first end of the chromatographic medium, and a second
conductor in operable contact with the second end of the
chromatographic medium.
The second opposable component includes a
first applicator containing a first specific binding
partner to the analyte in a form that can be
resolubilized by addition of a first aqueous liquid to
the first applicator. The first application liquid can
be the sample; preferably, however, it is a combination
of the sample and a first reconstitution fluid, with the
first reconstitution fluid being applied first.
The third opposable component includes a
second applicator containing a second specific binding
partner to the analyte in a form that can be
resolubilized by addition of a second reconstitution
fluid to the second applicator. The second specific
binding partner is labeled with a detectable label,
preferably a visually detectable label such as a gold
sol label as disclosed above. The third opposable
component also includes an absorber separated from the
second applicator on the third opposable component.
~"~~1~'JDEu S~'~'
~~~a~~~ 9
96
The first and second specific binding partners
are typically antibodies to the analyte. They can be
identical, but need not be. In place of intact bivalent
antibodies, monovalent antibody fragments such as Fab or
Fab' fragments can be used. This may be preferable for
some applications, inasmuch as there is no requirement
in such competitive assays for any antibody molecule to
react with more than one corresponding analyte.
In use, the first specific binding partner on
the first applicator is resolubilized, preferably by the
application of the sample alone, or, alternatively, by
the application of a first reconstitution fluid followed
by the addition of the sample. The second specific
binding partner on the second applicator is also
reconstituted by addition of a second reconstitution
fluid. Typically, the first and second reconstitution
fluids are the same. The reconstitution fluid is
preferably a buffer solution that can contain additional
components such as chelating agents, detergents,
antibacterials, and preservatives. A suitable
reconstitution fluid is prepared by mixing equal volumes
of phosphate buffered saline, 0.1 M, pH 7.2, containing
0.8% Tween-20TM and 2.5 mM HEPES buffer, pH 7.5,
containing 0.005% Triton X-100"", 0.003% tetrasodium
EDTA, and 0.05% sodium azide. Preferably, the volume of
the reconstitution fluid applied to each of the first
and second applicator is from about 5 ~1 to about 200
~,1, more preferably from about 10 ~1 to about 100 ~.1,
most preferably about 20 ul. The volume of sample
ap~lied is preferably from about 5 ~,l to about 40 ~.1,
me preferably about 30 ~.1.
After application of the sample to the first
applicator, the device is incubated to allow
resolubilization of the resolubilizable specific binding
partner and to allow reaction of the first specific
,..,-
.. ~~~~~°'r .
97
binding partner with the analyte. Preferably, this
incubation is from about 5 minutes to about 30 minutes;
most preferably, about 15 minutes. The incubation is
typically performed at room temperature, but can be at
lower or higher temperatures. Incubation at higher
temperatures can speed the reaction; incubation at lower
temperatures may be desirable to prevent oxidation or
other breakdown of sensitive samples.
After the incubation, the first opposable
component and the second opposable component are brought
into opposition, applying the sample and the
resolubilized first specific binding partner to the
analyte to the first conductor and then into the first
end of the chromatographic medium. The sample and the
resolubilized specific binding partner are then allowed
to move through at least a portion of the
chromatographic medium and past the analyte or
immunological analogue immobilized in the discrete area
on the chromatographic medium. This typically requires
about 10 to about 20 seconds.
At this point, the first and second opposable
components are separated by opening, and the first and
third opposable components are brought into opposition
to bring the absorber into contact with the first
conductor and to bring the second applicator into
contact with the second conductor. This causes the
absorber to withdraw fluid from the first end of the
chromatographic medium and causes the second applicator
to apply fluid, including the resolubilized labeled
specific binding partner, to the second end of the
chromatographic medium. Preferably, the second
opposable component is then folded back over the first
and third opposable components to ensure that sufficient
liquid is forced out of the second applicator and
absorbed by the absorber so that reversal of flow is
" -. _..
i. _. . .
98
efficient. The pressure exerted on the chromatographic
medium by the folding back of the second component
enhances the flow reversal.
The labeled resolubilized specific binding
partner is then allowed to move through at least a
portion of the chromatographic medium overlapping the
portion of the chromatographic medium through which the
sample and the first specific binding partner migrated,
and including the discrete area containing the
immobilized analyte or immunological analog. If analyte
is present in the sample, the labeled specific binding
partner binds in the discrete area and is detected. If
the detectable label is a visually detectable label, a
line is visible in the discrete area.
The assay can be performed as a qualitative
assay, i.e., an on/off assay. In such an assay, the
first specific binding partner is present at a
concentration that binds virtually all immobilized
analyte or immunological analogue thereof in the
discrete area. The sites in the discrete area that are
bound to the first specific binding partner are blocked
to further binding. If the analyte is present in the
sample, the analyte binds the first specific binding
partner, which prevents the first specific binding
partner from binding to the immobilized analyte or
analyte analogue in the discrete area. Thus, the
second, labeled, specific binding partner can bind to.
the analyte or immunological analogue thereof in the
discrete area, giving a detectable signal.
A quantitative version can employ two or more
chromatographic media within one housing, at least one
being designed for a control standard to be used as a
reference.
AME~~CcL~ S.-S
99
A device suitable for this version of a
competitive assay is shown in Figure 23. The
chromatographic assay device 740 has a first opposable
component 742, a second opposable component 744, and a
third opposable component 746. The second opposable
component 744 is hingedly attached to one side of the
first opposable component 742 by a first hinge 748. The
third opposable component 746 is hingedly attached to
the other side of the first opposable component 742 by a
second hinge 750. The first opposable component 742 has
a chromatographic medium 752 with a first end 754 and a
second end 756, with a first conductor 758 in operable
contact with the first end 754 of the chromatographic
medium 752 and a second conductor 760 in operable
contact with the second end of the chromatographic
medium 756. The chromatographic medium 752 also has a
discrete area 762 of analyte or immunological analogue
thereof immobilized thereon, as discussed above, which
is read as the signal.
The second opposable component 744 has a first
applicator 764 containing a first specific binding
partner for the analyte in resolubilizable form, to
which the sample preferably is applied.
The third opposable component 746 has a second
applicator 766 that contains a second specific binding
partner for the analyte that is labeled with a
detectable label and is present in a form which can be
resolubilized by addition of a second aqueous liquid to
the second applicator 766. The third opposable
component 746 has an absorber 768 separated from the
second applicator 766. The third opposable component
746 also has an aperture 770 for viewing of at least a
portion of the chromatographic medium 752, including the
discrete area 762. Alternatively, an aperture 772 is
located behind the discrete area 762 of the
,a,_, _.
.. .. .. . .. ....
.. .. . .. . . ~ . ..
. ... . . ~ . . . ...
, . . . . . . . .... . ...
, . . . .
.. .. .. . .. ...
100
chromatographic medium 752 in the first opposable
component 742 for viewing from the rear side of the
device 740 when it is closed. The second and third
opposable components 744 and 746 have engagers 769 and
773, and the device 740 includes a gasket 774 to contain
reagents.
When the first opposable component 742 and the
second opposable component 744 are brought into
opposition, the first applicator 764 is brought into
operable contact with the first conductor 758 to apply
the contents of the first applicator 764 to the first
conductor 758 and then to the first end 754 of the
chromatographic medium 752. When the third opposable
component 746 and the first opposable component 742 are
brought into opposition, the absorber 768 is brought
into operable contact with the first conductor 758
adjacent to the first end 754 of the chromatographic
medium 752 and the second applicator 766 is brought into
operable contact with the second conductor 760 adjacent
to the second end 756 of the chromatographic medium 752,
reversing the direction of fluid flow within the
chromatographic medium 752.
B. Two-Com onent Bidirectional Flow Device with
Cover
A second embodiment of a chromatographic assay
device for competitive assays according to the present
invention is a two-component bidirectional flow device
with cover. In this embodiment, the sample to be tested
is not preincubated with antibody but is applied
directly to a conductor in direct contact with a
chromatographic medium.
The second embodiment of the chromatographic
assay device for competitive immunoassays can comprise a
~:;';~~:~ ": .;,-~_e
~~ ~' ~~ ~D
first opposable component and a second opposable
component. The first opposable component has a
chromatographic medium having a first end and second
end. The chromatographic medium has immobilized
thereon: (1) a first specific binding partner for the
analyte; and (2) a secondary specific binding partner
that is capable of binding a specific binding pair
member that lacks affinity for the analyte. These
components are placed on the chromatographic medium in
separate discrete and non-overlapping areas. The
secondary specific binding partner is located closer to
the first end of the chromatographic medium than the
first specific binding partner for the analyte.
Preferably, the first specific binding partner is
located about 0.25 inch (6.35 mm) from the secondary
specific binding partner in the chromatographic medium
to increase efficiency.
The first specific binding partner for the
analyte is typically an antibody for the analyte or an
antibody fragment, as disclosed above.
The secondary specific binding partner is
typically an antibody that is capable of binding another
immunoglobulin on the basis of a species-specific
determinant and not on the basis of the antibody
specificity, if any, of the immunoglobulin to be bound.
For example, the secondary specific binding partner can
be goat anti-rabbit IgG antibody, which binds all rabbit
immunoglobulin G molecules, regardless of their
immunological specificity.
The first opposable component also includes a
first conductor in operable contact with the first end
of the chromatographic medium and a second conductor in
operable contact with the second end of the
chromatographic medium. Preferably, the first opposable
4Pf EI~~CED SNcE i
102
component is marked with a limit line indicating the
point at which flow reversal is to occur in the
bidirectional chromatographic process.
The second opposable component includes: (1)
an applicator containing an analyte analogue in a form
that can be resolubilized by the addition of a
reconstitution fluid to the applicator; and (2) an
absorber separated from the applicator.
The analyte analogue is analyte covalently
linked to a member of a specific binding pair bindable
by the secondary specific binding partner. The member
of the specific binding pair in the analyte analogue is
labeled with a detectable label. Preferably, the
detectable label is a visually detectable label. Most
preferably, the visually detectable label is a metal
sol, such as gold, silver, or copper sol.
For example, when the secondary specific
binding partner is goat anti-rabbit IgG, the member of
the specific binding pair conjugated to the analyte is
normal rabbit IgG without antibody activity for the
analyte.
An example of an analyte analogue suitable for
this version of the assay, when penicillin is the
analyte, is 7-aminocephelosporanic acid covalently
linked to rabbit IgG, with the rabbit IgG being labeled
with colloidal gold.
Bringing the first and second opposable
components into opposition places a second conductor
into operable contact with the applicator so that the
contents of the applicator are applied to the second
conductor and are drawn through at least a portion of
the chromatographic medium. It also places the absorber
,,1 ._ _.
=.; _ _ _
103
in operable contact with the first conductor to withdraw
fluid from the first end of the chromatographic medium
to reverse the flow in the chromatographic medium.
During the first phase of downward flow, the
analyte present in a positive specimen passes the area
of the secondary specific binding partner on the
chromatographic medium unhindered and then binds the
area of the first specific binding partner for the
analyte. For a positive sample, during the second phase
of upward flow, the labeled analyte analogue is blocked
from binding to the first specific binding partner for
the analyte and then binds the secondary specific
binding partner. If the sample lacks analyte, there is
no binding of analyte to the first specific binding
partner during the downward flow phase, and the analyte
analogue is bound to the first specific binding partner
during the second, upward flow phase and does not reach
the secondary specific binding partner, so that no
signal is developed at the secondary specific binding
partner.
Preferably, the device further comprises a
cover hingedly attached to the first opposable component
so that it can be folded over the first and second
opposable components when they are opposed. The cover
can have a aperture cut therein to permit viewing of at
least a portion of the chromatographic medium in the
first and second opposable components are opposed.
Alternatively, an aperture is located behind the
chromatographic medium and viewing occurs on the back
side of the closed device.
This device can also contain two or more
chromatographic media in one housing, at least one being
designed for a control standard to be used as a
reference to give a semi-quantitative or quantitative
_. '~iv~ I
C'
104
indication of the concentration of analyte present in
the sample.
In another version of this embodiment, the
secondary specific binding partner can be immobilized in
the detection zone in multiple bands so that the labeled
analyte analogue available for binding to the secondary
specific binding partner is titrated. The quantity of
secondary specific binding partner in each band is
determined so that the quantity of analyte analogue
binding to the detection zone, and thus the
concentration of analyte in the test sample, is
indicated by the number of bands to which the analyte
analogue binds. This gives a semiquantitative estimate
of analyte concentration. Typically, in this version,
the band of secondary specific binding partner closest
on the chromatographic medium to the first specific
binding partner to the analyte has the lowest
concentration of secondary specific binding partner,
with each successive band having a greater concentration
of secondary specific binding partner than the
concentration in the lowest band.
If the concentration of analyte were
sufficiently high that enough unbound labeled analyte
analogue were present to saturate the first band of
secondary specific binding partner, some of the labeled
analyte analogue would bind to the second band of
secondary specific binding partner. Thus, two lines
would be visible at the detection zone of the
chromatographic medium. A range of analyte
concentrations is accordingly indicated by the
appearance of one, two, or three lines in the detection
zone.
The expected range of concentrations of the
analyte to be assayed determines the concentrations that
AME~JDE~ SHEET
2~~$5'~~
105
trigger the appearance of one or more lines in the
detection zone. For example, using the nicotine
metabolite cotinine, a 10 ppb concentration in a body
fluid can indicate significant passive exposure to
cigarette smoke, a 500 ppb concentration can indicate an
active smoker, and a 10,000 ppb concentration can
indicate a very heavy smoker. The assay device can be.
arranged so that one line appears at the detection zone
at 10 ppb of cotinine, two lines at 500 ppb, and three
lines at 10,000 ppb.
In operation, reconstitution fluid, as
described above, is applied to the applicator to
resolubilize the analyte analogue. The sample is then
applied to the first conductor and allowed to migrate to
the limit line. Typically, the chromatography in the
first direction takes about 5 to about 25 seconds, more
typically about 10 seconds. The second opposable
component is then folded over the first opposable
component so that they are in opposition. The cover is
then folded over the first and second opposable
components. After a period of incubation from about 30
seconds to about 10 minutes, typically about 2 minutes,
a visible line is seen at the secondary specific binding
partner if analyte is present in the sample. Preferred
volumes of samples and reconstitution fluids are
described above for the first embodiment of the
competitive immunoassay device.
A device suitable for this embodiment of the
competitive immunoassay is shown in Figure 24. The
chromatographic assay device 780 has a first cpposable
component 782, a second opposable component 784, and a
cover 786. The second opposable component 784 is
hingedly attached to one side of the first opposable
component 782 by a first hinge 788. The cover 786 is
hingedly attached to the ocher side of the first
~I~l.~ni~=J ,:v__.
:1,.,
C<~,~ 10 6
opposable component 782 by a second hinge 790. The
first opposable component 782 has a chromatographic
medium 792 with a first end 794 and a second end 796.
The chromatographic medium 792 has an area of a first
specific binding partner for the analyte 798 immobilized
thereon, and a non-overlapping area of a secondary
specific binding partner 800 immobilized thereon. The
first opposable component 782 also includes a first
conductor 802 in operable contact with the first end 794
of the chromatographic medium 792 and a second conductor
804 in operable contact with the second end 796 of the
chromatographic medium 792. The first opposable
component 782 also includes a limit line 806 to indicate
the point at which the first 782 and second 784
opposable components are to be brought into opposition
to reverse the direction of fluid flow during the
performance of the assay.
The second opposable component 784 includes an
applicator 808 containing the analyte analogue in a form
that can be resolubilized by the addition of the
reconstitution fluid. The second opposable component
784 also includes an absorber 810 separated from the
applicator 808, and an aperture 814. When the first 782
and the second 784 opposable components are brought into
opposition, the applicator 808 is placed in operable
contact with the second conductor 804 and the absorber
810 is placed in operable contact with the first
conductor 802 to reverse the direction of fluid flow.
The cover 786 has an aperture 812 cut therein to allow
viewing of a portion of the chromatographic medium 792.
Preferably, the aperture 812 allows viewing of the area
of the secondary specific binding partner 800 and not of
the area of the first specific binding partner to the
analyte 798. The second opposable component 784 and
the cover 786 also include engagers 811 and 813. The
device is surrounded by a gasket 815.
AP~Ic~~'~J '"= .T
107
C. Three-Component Unidirectional Flow Device
with Absorber
Another device suitable for this embodiment of
the competitive immunoassay is shown in Figure 25. This
device has a third component that contains an absorber
or blotter that withdraws fluid from the chromatographic
medium and both conductors. The absorber extends across
a substantial portion of the chromatographic medium when
it is in operable contact with it. The chromatographic
assay device 820 has a first opposable component 822, a
second opposable component 824, and a third opposable
component 826. The second opposable component 824 is
hingedly attached to one side of the first opposable
component 822 by a first hinge 828. The third opposable
component 826 is hingedly attached to the other side of
the first opposable component 822 by a second hinge 830.
The first opposable component 822 has a chromatographic
medium 832 with a first end 834 and a second end 836.
The chromatographic medium has an area of a first
specific binding partner for the analyte 840 and a non-
overlapping area of a secondary specific binding partner
838 immobilized thereon. The first opposable component
822 also includes a first conductor 842, which serves as
an applicator, in operable contact with the first end of
the chromatographic medium 832 and a second conductor
844 in operable contact with the second end 836 of the
chromatographic medium 832. The first opposable
component 822 also includes a limit line to indicate the
point at which the first 822 and second 824 opposable
components are to be brought into opposition.
The second opposable component 824 includes a
second applicator 850 containing the analyte analogue.
Typically, an aqueous liquid is added to the second
applicator 850 to resolubilize the analyte analogue on
the second applicator 850. The second opposable
~:,,'= _ _ _ ~'- _~T
t
108
component 824 also includes a first absorber 848
separated from the second applicator 850. When the
first 822 and second 824 opposable components are
brought into opposition, the second applicator 850 is
placed in operable contact with the first conductor 842
and the first absorber 848 is placed in operable contact
with the second conductor 844 to withdraw fluid. An
aperture 852 is located behind the chromatographic
medium allowing viewing of a portion of the
l0 chromatographic medium 832 from behind. The second and
third opposable components 824 and 826 include engagers
849 and 853. The device 820 also includes a gasket 858.
The third opposable component 826 includes a
second absorber 854 positioned such that it is in
operable contact with a substantial area of the
chromatographic medium 832 when the third 826 and first
822 opposable components are brought into opposition.
This removes excess fluid from the first conductor 842
and from the second conductor 844 as well as from the
chromatographic medium 832.
In use, sample is applied to the first
conductor or applicator 842 and allowed to travel
through the chromatographic medium 832. The third
opposable component 826 is brought into opposition with
the first opposable component 822 to bring the second
absorber 854 into direct contact with the first
conductor or applicator 842 and the chromatographic
medium 832 drawing excess liquid into the second
absorber 854. The second opposable component 824 and
the first opposable component 822 are then opposed
bringing the second applicator 850 containing
resolubilized labeled analyte analogue into contact with
the conductor or applicator 842 at the first end 834 of
the chromatographic medium 832. Also, the first
S , a ~ _ i.. . _ __
z~.~~~~o
109
absorber 848 is brought into contact with the second
conductor 844 on the second end of the chromatographic
medium 832. The third opposable component 826 is then
closed over the second opposable component 824 as a
cover. A portion of the chromatographic medium is
visible from the aperture 856 located behind the
chromatographic medium 832.
Detection and/or determination of the analyte
occurs in the same manner as discussed in Section II(B),
above, for the bidirectional flow device.
D. Three-Component Bidirectional Flow Device
Usincr Specificity Of Biotin
Another embodiment of a chromatographic device
according to the present invention is a three-component
bidirectional flow device using the specificity of the
biotin-avidin link.
The first opposable component has a
chromatographic medium having a first end and a second
end, as described above. The chromatographic medium has
immobilized thereon: (1) a substance capable of
specifically binding biotin, selected from the group
consisting of avidin, streptavidin, anti-biotin
antibody, and derivatives thereof; and (2) a secondary
specific binding partner. The secondary specific
binding partner is capable of specifically binding a
three-component complex, where the three-component
complex comprises: (a) analyte; (b) a member of a
specific binding pair lacking specific binding affinity
for the analyte covalently conjugated to the analyte;
and (c) a detectable label bound to the member of the
specific binding pair. This three-component complex is
disposed on a third opposable component as described
below.
4~'ES! ~'~-, :,~,--=i
. 110
The binding components are immobilized on the
r~,y chromatographic medium in separate discrete non-
overlapping areas, with each area being substantially
smaller than the area of the chromatographic medium.
The secondary specific binding partner is immobilized in
the first discrete area and is located closer to the
first end of the chromatographic medium than is the
substance capable of binding biotin (referred to
generically herein as "avidin"), which is immobilized in
the second discrete area.
The first opposable component also includes a
first conductor in operable contact with the first end
of the chromatographic medium and a second conductor in
operable contact with the second end of the
chromatographic medium.
The second opposable component includes a
first applicator containing a first specific binding
partner to the analyte in a form that can be
resolubilized by the addition of aqueous liquid to the
applicator. As disclosed above, the aqueous liquid can
be, preferably, the sample alone, or a first
reconstitution fluid, followed by the sample. The first
specific binding partner is not capable of being bound
by the secondary specific binding partner. The first
specific binding partner is conjugated to biotin.
Methods for linking biotin to globular
proteins, including antibodies, are well-known in the
art and are described, for example, in P. Tijssen,
suQra, at pp 25-31. Biotin can be conjugated to prot,in
as a reactive ester, either directly or by introduct~._:
of a spacer such as e-aminocaproic acid. Typical
reactive esters are biotinyl-g-nitrophenyl ester,
biotinyl-N-hydroxysuccinimide ester, and
caproylamidobiotinyl-N-hydroxysuccinimide ester, the
__~ .. -._
1:.'. _ . _.. . m
~'~.58~~~
last of which provides a spacer between the biotin
moiety and the protein. Biotin can also be activated by
other groups.
The third opposable component includes: (1) a
second applicator containing the three-component complex
as described above comprising the analyte, the member pf
the specific binding pair lacking affinity for the
analyte, and the detectable label and (2) an absorber
separated from the first applicator.
In the device, bringing the first and second
opposable components into opposition places the first
conductor in operable contact with the first applicator
so that the contents of the first applicator are applied
to the first conductor and into the first end of the
chromatographic medium. Thus, the contents of the first
applicator migrate through at least a portion of the
chromatographic medium. Bringing the first and third
opposable components into opposition causes the second
applicator to come into operable contact with the second
conductor to apply the contents of the second applicator
to the second end of the chromatographic medium, and
causes the absorber to come into operable contact with
the first conductor to withdraw fluid from the first end
of the chromatographic medium and reverse the flow.
An example of a suitable combination of
antibodies for use in this embodiment of the
chromatographic assay device, with theophylline as the
analyte, is biotinylated murine monoclonal anti-
theophylline IgG antibody as the biotinylated first
specific binding partner, goat anti-rabbit IgG antibody
as the immobilized secondary specific binding partner,
and rabbit IgG lacking anti-theophylline activity as the
member of the specific binding pair in the three-
component complex.
A~~~~~~~ ~;~E~r
.
c~
. 112
Another example of an analyte that can be
assayed by a device according to this embodiment is
penicillin, using penicillin binding protein (PBP) as
the specific binding partner, and a three-component
complex comprising 7-aminocephelosporanic acid
covalently linked to rabbit IgG, with the rabbit IgG
being labeled with colloidal gold. Other 8-lactam
antibiotics can be assayed similarly.
In operation, the biotiny° wed first specific
binding partner on the first applic~ .r is
resolubilized, either, preferably by the application of
the sample alone or by the application of a first
reconstitution fluid followed by the application of the
sample as discussed above. The three-component complex
on the second applicator is resolubilized by a second
reconstitution fluid, as described above in Section
II(A). After a period of incubation sufficient to allow
any analyte in the sample to bind to the first specific
binding partner, the first and second opposable
components are brought into opposition, to apply the
sample and the resolubilized first specific binding
partner to the first conductor a:~d then to the first end
of the chromatographic medium. The sample and the first
specific binding partner migrate through the
chromatographic medium and past the two discrete non-
overlapping areas of immobilized reagents in the
chromatographic medium. The first biotinylated specific
binding partner binds the component capable of binding
biotin in the second discrete area. If the analyte is
present in the sample, this biotinylated specific
binding partner bound to the solid support via the
biotin-avidin link is itself bound to analyte. If
analyte is absent from the sample, analyte is not bound
to the bound biotinylated specific binding partner.
Al:jr',~~_~ ,L,yT
113
After the sample and the resolubilized first
specific binding partner pass the second discrete area,
the first and second opposable components are separated
and the first and third opposable components are brought
into opposition, reversing the flow and applying the
three-component complex to the second conductor. The
three-component complex then migrates through the
chromatographic medium beginning at the second end
toward the first end. If analyte was present in the
test sample, the biotinylated specific binding partner
bound to the avidin in the second discrete area cannot
bind the analyte in the three-component complex, so the
three-component complex reaches the secondary specific
binding partner. The secondary specific binding partner
then binds the member of the specific binding pair in
the three-component complex, where it is detected.
Typically, the third opposable component of the device
has an aperture so that only the first discrete area of
the secondary specific binding partner, and not the
second discrete area of the avidin, is visible on the
chromatographic medium when the third opposable
component is in opposition with the first opposable
component. However, when no analyte is in the sample,
the three-component complex is bound by the first
specific binding partner which is bound to the avidin,
whose antigen-binding site is unoccupied. The three-
component complex then does not reach the first discrete
area containing the secondary specific binding partner
for detection.
A chromatographic assay device suitable for
performing this version of the competitive immunoassay
is shown in Figure 26. The chromatographic assay device
860 has a first opposable component 862, a second
opposable component 864, and a third opposable component
866. The second opposable component 864 is hingedly
attached to one side of the first opposable component
AN1E''~~1
114
862 by a first hinge 868, and the third opposable
component 866 is hingedly attached to the other side of
the first opposable component 862 by a second hinge 870.
The first opposable component 862 has a chromatographic
medium 872 with a first end 874 and a second end 876,
with a first conductor 878 in operable contact with the
first end 874 of the chromatographic medium 872 and a
second conductor 880 in operable contact with the second
end 876 of the chromatographic medium 872. The
chromatographic medium 872 has a first discrete area of
a secondary specific binding partner 882 and a second
discrete area of avidin 884. The first 882 and second
884 discrete areas are non-overlapping, with the first
discrete area 882 being located closer to the first end
1S 874 of the chromatographic medium 872. The second
opposable component 864 has a first applicator 886
containing a resolubilizable first specific binding
partner for the analyte. The third opposable component
866 has a second applicator 888 containing a
resolubilizable three-component complex and an absorber
890 separated from the second applicator 888. The
second opposable component 864 and the third opposable
component 866 also have apertures 892 and 894 therein
for viewing a portion of the chromatographic medium 872.
Preferably, the apertures 892 and 894 permit viewing of
the first discrete area 882 containing the secondary
specific binding partner but not of the second discrete
area 884 containing the avidin. The second and third
opposable components 864 and 866 also include engagers
891 and 895. the device 860 also includes a gasket 896.
Bringing the first 862 and s :ond 864
opposable components into opposition causes the first
applicator 886 to come into operable contact with the
first conductor 878 to begin flow through the
chromatographic medium 872. Subsequently, bringing the
ae''= ~'~-~ ''~;~cT
.._~'.~~~J .:~
115
first 862 and third 864 opposable components into
opposition causes the second applicator 888 to come into
operable contact with the second conductor 880 and the
absorber 890 to come into operable contact with the
first conductor 878, reversing direction of the flow
through the chromatographic medium 872.
In a variation of this embodiment of the
device, the detection zone can include two or more bands
of secondary specific binding partner, as described
above in Section II(B). This can titrate the quantity
of labeled three-component complex available for binding
to give an indication of the quantity of analyte present
in the test sample by the number of visible bands in the
detection zone.
E. Two-Component Device for Competitive
Inhibition Immunoassavs
Another embodiment of a chromatographic assay
device according to the present invention is suitable
for performing competitive immunoassays. This device
gives a positive indication of analyte concentration by
providing a detectable signal when analyte is present in
the test sample.
This embodiment is a two-component device and
flow is unidirectional throughout the device. The first
opposable component includes a chromatographic medium
having a first end and a second end. The
chromatographic medium has immobilized thereon: (1) an
analyte analogue capable of binding a specific binding
partner for the analyte; and (2) a secondary specific
binding partner that is capable of binding a specific
binding pair member that has affinity for the analyte,
the secondary specific binding partner itself lacking
affinity for the analyze. The components are placed on
'. _ ~ J
116
the chromatographic medium in separate discrete and non-
overlapping areas. The analyte analogue is located
closer to the first end of the chromatographic medium
than is the secondary specific binding partner.
The analyte analogue is analyte covalently
linked to a protein molecule, such as a nonspecific
immunoglobulin, that can be bound to the chromatographic
medium. The protein molecule should not have binding
affinity for analyte or for a specific binding partner
to the analyte.
The secondary specific binding partner
specifically binds a specific binding partner for the
analyte on the basis of a species-specific determinant,
as described above. The interaction between the
secondary specific binding partner and the specific
binding partner for the analyte does not involve the
antigen-combining sites of the specific binding partner
for the analyte.
The first opposable component also includes a
conductor in operable contact with the first end of the
chromatographic medium. Preferably, the first opposable
component also includes an absorber in operable contact
with the second end of the chromatographic medium.
The second opposable component includes an
applicator containing a specific binding partner for the
analyte labeled with a detectable label. Preferably,
the detectable label is a visually detectable label such
as a metal sol. In one alternative, the second
opposable component can further include an absorber
separated from the applicator if the first opposable
component lacks an absorber. Preferably, the second
opposable component also includes an aperture to allow
viewing of a portion of the chromatographic medium.
~, r
A,M«1~~~ ._~~~T
117
Preferably, the aperture allows viewing of the area of
the secondary specific binding partner and not of the
area of the analyte analogue.
In use, the sample to be tested is applied to
the applicator to resolubilize the labeled specific
binding partner. Typically, the sample is incubated
with the specific binding partner for about 1 minute to
about 3 minutes, although this time period can be
increased if greater sensitivity is desired.
The first and second opposable components are
then brought into opposition, applying the contents of
the applicator to the conductor and then to the first
end of the chromatographic medium. Analyte present in a
positive test sample occupies the analyte-binding sites
on the labeled specific binding partner, so the labeled
specific binding partner does not bind to the analyte
analogue on the chromatographic medium. Instead, the
labeled specific binding partner proceeds to the region
of the secondary specific binding partner, where it is
bound, forming a detectable line that gives a positive
indication of the presence of analyte.
In the absence of analyte in the test sample,
all of the labeled specific binding partner for the
analyte is bound by the analyte analogue and does not
reach the secondary specific binding partner. The
quantities of reagents used can be titrated so that the
concentration of analyte required to give a positive
response can be adjusted to suit clinical or
pharmacological requirements.
A device suitable for this embodiment of the
competitive immunoassay is shown in Figure 27. The
device 900 has a first opposable component 902 and a
second opposable component 904 hingedly attached by a
P.'''~'~'~:C~~ '''-,~~i
<r~, 118
~~
v.,~~,'' hinge 906. The first opposable component 902 has a
chromatographic medium 908 with a first end 910 and a
second end 912. The chromatographic medium 908 has an
area of an analyte analogue 914 immobilized thereon, and
a non-overlapping area of a secondary specific binding
partner 916 immobilized thereon. The first opposable
component 902 also has a conductor 918 in operable
contact with the first end 910 of the chromatographic
medium 908 and an absorber 920 in operable contact with
'_0 the second end 912 of the chromatographic medium 908.
The second opposable component 904 includes an
applicator 922 containing the labeled specific binding
partner for the analyte in a form that can be
resolubilized by the addition of the sample. The second
opposable component 904 also includes an aperture 924 to
allow viewing of at least a portion of the
chromatographic medium 908. Preferably the aperture 924
allows viewing of the area of the secondary specific
binding partner 916 and not of the area of the analyte
analogue 914. The first and second opposable components
also include engagers 923 and 925, and a gasket 926 as
described above for two-component devices.
In another version of this embodiment of the
device, the detection zone can include two or more bands
of secondary specific binding partner to give a
semiquantitative indication of analyte concentration in
the test sample, as described above in Section II(B).
This titrates the quantity of labeled specific binding
partner available for binding to give an indication of
the quantity of analyte present in the test sample by
the number of bands visible in the detection zone.
III. ANALYTES AND ANTIBODIES FOR USE WITH ASSAY DEVICES
AMEfdDE~ S~;~ET
119
The analytes suitable for detection with an
assay device according to the present invention include
antigens, haptens, and antibodies. Antigens detectable
with the device include hemoglobin, Streptococcus A and
B antigens, antigens specific for the protozoan parasite
Giardia, and viral antigens, including antigens specific
for HIV and the Australia antigen specific for
hepatitis. Antibodies that can be assayed include
antibodies to bacteria such as Helicobacter pylori and
to viruses, including HIV. Haptens detectable include
the haptens enumerated above in Section III, su ra, as
well as other haptens to which antibodies of sufficient
specificity can be prepared.
If the analyte is an antigen or a hapten and a
sandwich procedure is used, the first and second
specific binding partners are preferably antibodies. In
many applications, it is preferable that the first and
second specific binding partners are antibodies to
different epitopes on the analyte, but this is not
required. The antibodies can be polyclonal or
monoclonal, and can be IgG, IgM or IgA. In many
applications, polyclonal antibodies are preferred, as
their natural variability may allow more accurate
detection in systems where antigenic polymorphisms exist
or may exist.
When the analyte is a hapten and a sandwich
assay procedure is used, it is strongly preferred that
the first and second specific binding partners be
antibodies to different epitopes; otherwise, there may
be an undesirable competition reaction set up that may
interfere with binding of the complex of the labeled
specific binding partner and the analyte to the
immobilized second specific binding partner. It is
recognized that not all hapLens are large enough to
accommodate more than or_e epitope; however, some
A(~~~IVC r ~ ~r, ~..~'T
120
haptens, though not large enough to induceantigen
formation when injected themselves, are nevertheless
by
large enough that they pos sess more than
one epitope.
In cases where antibodies to more than epitope of
one a
hapten cannot be obtained, competitive assay
procedures
are generally preferred.
Where the analyte is an antibody and a
sandwich assay procedure is used, the first specific
binding partner is typically a labeled antibody that
binds to the analyte on the basis of species, class, or
subclass (isotype) specificity. It is highly preferred
that the first specific binding partner to an antibody
analyte binds to the constant region of the antibody
analyte, in order to prevent interference. When the
analyte is antibody, the second specific binding partner
is preferably an antigen or hapten for which the
antibody analyte is specific.
In some applications, it is desirable to
employ indirect labeling. For example, in testing for
Giardia antigen, an IgM antibody can be used that may be
difficult to label directly. In that case, a secondary
specific binding partner specific for the mobile first
specific binding partner can be labeled. Typically, the
labeled secondary specific binding partner binds to the
antibody that is the first specific binding partner on
the basis of species, class, or subclass specificity.
As an alternative to the use of a secondary
specific binding partner, the first specific binding
partner can be conjugated to biotin and an avidin-
conjugated label can be used.
These relationships between analytes, specific
binding partners, and labels for sandwich immunoassays
are summarized in Table 1: below.
Airl~!vl.~~ ~ __
~~5~5"~~
121
TABLE I
SCHEMES OF BINDING FOR SANDWICH IMMUNOASSAYS
1ST SBP 2ND SBP SECONDARY
ANALYTE (MOBILE)(FIXED) SBP COMPLEX FORMED
Ag Ab,. Ab2 --- Ab;-Ag-Ab,*
H Ab,* Abz --- Abz H-Ab,*~
Ab Ab~* Ag --- Ag-Ab-Abp
Ag Ab, Abi Ab~* Abz Ag-Ab,-Ab
Ab Ab~, Ag AbG* Ag-Ab-Ab~,-Ab~z*
Ag Ab,-Bi Ab~ Av-L Ab2-Ag-Ab,-Bi-Av-L
Ag - Antigen
H - Hapten
Ab - Antibody
Abl - 1st Antibody
Ab2 - 2nd Antibody
Ab~~ Ab~l~ Ab~2 - Antibody specific for another
antibody
Bi - Biotin
Av - Avidin
L - Label
*Indicates labeled component
Ab2 and Abl* preferred to bind to different
epitopes; not all haptens possess such different
epitopes.
w '. ?~' li ~~ !~!;"
122
,.,~ The relationships between analytes, specific
binding partners, labels, and other participants in the
reaction schemes for competitive immunoassays are shown
in Table II below.
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A~::_. _ ~~ ~~,-~T
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rn
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H
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w a a a
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c ~n cn ~n
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H
W
H O H z W
H
M fx ~ W W U7 I
W
OW En ~ U7
W ~
H
z w N ~
H H
Ca g E1
W W
z H
H
x x H x
U (~ ~
W
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U~ H U I ~ tT C71 I
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II II
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124
H . Hapten
i ~."
Abl, Abz . First and second antibodies specific for
hapten
'~°~t Ig . Immunoglobulin lacking specific binding
activity
Abs . Secondary antibody binding Abl or Abz
gi . Biotin
Av . Avidin
Label
Noncovalent bond
_ . Covalent bond
~1~~~7~
125
IV. TEST KITS
Another aspect of the present invention is
test kits that can be used to detect particular
analytes. A test kit comprises, in separate containers:
(1) a chromatographic assay device according.
to the present invention;
(2) any necessary reagents required to treat
or extract the sample; and
(3) optionally, if the assay device does not
incorporate a labeled specific binding partner to the
analyte in a form that can be resolubilized, the
required specific binding partner.
The components required in (2) and (3) are
packaged separately and can be in liquid or solid form
(freeze-dried, crystallized, precipitated, or
aggregated). If the latter, they are resolubilized by
the user, typically with distilled or purified water,
with physiological saline, or a buffer solution.
In some cases, test kits can also include a
reconstitution fluid for a reagent present on the device
in resolubilizable form, either a specific binding
partner or an analyte analogue. Specific examples are
disclosed above, with the disclosure of the operation of
each type of device.
Still other variations of test devices
according to the present invention are possible. For
example, any of the two-component devices described can
have a cover hingedly attached to one of the opposable
components. This cover can have an aperture cut therein
to allow viewing of at least a portion of the
chromatographic medium.
An~Ft~!~ED SHEET
L-°~
a0 12 6
The invention is illustrated by the following
Examples. The Examples are for illustrative purposes
only and are not to be construed as limiting the scope
of the invention in any manner.
EXAMPLES
Example 1
Construction of Device for Detecting
Streptococcal Antigen
A device was constructed for detecting
Streptococcus A antigen using labeled antibody to
Streptococcus A antigen. The device was constructed
essentially as depicted in Figure 28.
Figure 28 shows a chromatographic assay device
940 according to the present invention with a first
opposable component 942, a second opposable component
944 hingedly attached to the first opposable component
942, and a cover 946 hingedly attached to the second
opposable component 944. The first opposable component
942 includes a chromatographic medium 948. The second
opposable component 944 includes a ribbon 952 that holds
in place a teardrop-shaped receptacle 950 by the tension
of the ribbon 952 stretched across the second opposable
Component 944. The teardrop-shaped receptacle 950 forms
a well when held in place by the tension of the ribbon
952. The first opposable component 942 contains a first
window 954 and the cover 946 contains a second window
956.
The opposable components were made of a hard,
impervious plastic such as polycarbonate. The first and
second opposable components, as well as the cover, each
were about 7.62 cm (3 inches)in length; the first
P.M~(dDED S'~'~ET
127
opposable component was about 5.71 cm (2.25 inches) in
width, while the second opposable component and the
cover was each about 6.04 cm (2.375 inches) in width.
The second opposable component was lined with a foam
rubber receptacle, into which a teardrop-shaped well was
cut to accept a swab or other sampling device. The swab
was held in place with a ribbon separately inserted into
the second opposable component across the well.
The chromatographic medium was a
nitrocellulose strip of S um pore size and 1.27 cm (0.5
inch) in length, (MSI, Westborough, Mass.), affixed to
the plastic backing by means of double-sided tape (3M,
Minneapolis, Minnesota). The conductor and absorber
were cellulose strips (Ahlstrom Filtration, Holly
Springs, Pennsylvania), 1.35 cm (17/32 inch) in length
for the absorber, which was Ahlstrom Grade 939, and
0.635 cm (0.25 inch) in length for the conductor, which
was Ahlstrom Grade 1281. The detector application pad
was also Ahlstrom Grade 1281, and was 0.95 cm (0.375
inch) wide. The detector application pad overlapped
slightly with the conductor, which in turn overlapped
slightly with the chromatographic medium at its first
end. The chromatographic medium overlapped slightly at
its second end with the absorber.
The required reagents were first incorporated
in the chromatographic medium and the detector
application pad, after which the device was assembled
using double-sided tape to hold the components to the
backing.
The detection zone comprised rabbit anti-
Streptococcus A antibody at 2 mg/ml in 0.001 mole/1
phosphate buffered saline, pH 7.2. The control zone
comprised goat anti-rabbit IgG at a similar
concentration in the same buffer. The antibody
Q
z~..
128
solutions were applied to the appropriate regions of the
chromatographic medium and dried at 37.8°C (100°F) in a
low humidity environment. The chromatographic medium
was wet in excess blocking solution (Blocking Reagent
for ELISA, Boehringer Mannheim, Mannheim, Germany,
diluted 1:10 with distilled water containing 0.2% Tween
20T"") and again dried at 37.8°C (100°F) .
The detector application pad contained rabbit
anti-Streptococcus antibody labeled with 40-nm colloidal
gold particles. To apply the labeled antibody to the
detector application pad, the labeled antibody was
diluted 1:1.5 with DBN (1.5 mole/1 Tris-HCl, pH 7.4, 1%
(v/v) Tween 20r"", 0 . 4 % (v/v) Brij 35~", 0 . 02 % (w/v) sodium
azide, 3 mg/ml rabbit IgG). Per test, 15 ~.l of diluted
labeled antibody was added to the detector application
pad. The detector application pad was dried for 30
minutes at 37.8°C (100°F).
Example 2
Detection of StreP.rtococcal Antigen
Using Device or Example 1
The device of Example 1 was used to detect
Streptococcus A antigen. A woven dacron swab to which
varying quantities of Streptococcus type A bacteria had
been added was inserted into the sample well. Three
drops of Extraction Reagent A (0.25% acetic acid, 5%
Tween 20~'), and three drops of Extraction Reagent B (2
mole/1 sodium nitrite, 5% Tween 20~") were added to the
swab, mixed by gently rotating the swab, and incubated
for one minute. The device was then closed, so that the
first and second opposable components were brought into
contact, and the cover was then folded over the first
opposable component. The result was read after an
incubation period of from 2 minutes to 5 minutes. The
AMENDED ~~~~
.. .. .. . .. ...
.. .. . . . . . . . . .
. . . . . ... . . . . . ...
.
1 . 1 1
t
1
. t 1 1
129
development of a pink-red band in the detection zone of
the chromatographic medium indicated the detection of
Streptococcus A antigen.
The device of Example 1 could detect 1 x 105
Streptococcus A organisms after a 2-minute incubation,
and could detect 5 x 104 Streptococcus A organisms after
a 5-minute incubation. For a comparison, the Concise"
immunoassay of Hybritech (La Jolla, California) could
detect 1 x 105 Streptococcus A organisms only after a 5-
minute incubation, and could not detect 5 x 104
Streptococcus A organisms even after a 20-minute
incubation. Similarly, the Smart" immunoassay of New
Horizons could detect 1 x 105 Streptococcus A organisms
only after a 7-minute incubation, and gave an equivocal
result with 5 x 104 Streptococcus A organisms after a 7-
minute incubation.
Example 3
Competitive Immunoassay for Theophylline
Using Three-Component Assay Device
A competitive immunoassay device was
constructed to detect the bronchodilator theophylline.
A 12-micron nitrocellulose membrane from Schleicher and
Schuell (Keene, New Hampshire) was secured on a double-
backed adhesive in 6.35 cm (2.5 inch) by 1.75 cm (11/16
inch) blocks. A theophylline analogue capable of
hydrophobically binding to nitrocellulose was prepared
by covalently coupling theophylline to normal rabbit
immunoglobulin G by the following procedure: A solution
of 8-(3-carboxypropyl)-1,3-dimethylxanthine (18 mg,
0.068 mM) in 2 ml tetrahydrofuran and 1 ml
dimethylformamide was treated with N-hydroxysuccinimide
(17 mg, 0.15 mM), and dicyclohexylcarbodiimide (27 mg,
APB~E.\C~D S;~,.=ET
.. .. .. . .. ....
.. .. . . . . . . . . .
. . ... . . . . . . ...
. . . . . . .... . ...
. . . . s . . . . . .
. w .. .. .. . .. ...
130
L.~
0.13 mM). After standing at room temperature overnight,
the rE2ttion rilixture was refrigerated for two hours and
then fil~.ered through glass wool. The crystals from the
reactit~n mixture were washed with tetrahydrofuran and
the solutions were combined. The solvents were removed
under vacuum .and the residue was washed with 3-4 ml
diethyl ether ~o remove excess carbodiimide. The washed
residuewas ~.ashed in 1 ml dimethylformamide. Normal
rabbit ~gG (3 .75 ml of 13.7 mg/ml solution) was then
dilute to 1C ml with water. The active N-
hydroxysuccimirinide ester was slowly added to the
immuno globulin G solution, followed by 10 ~.1 of
triethylamine . Thus the reaction mixture contained
about 19p ec~ui~valents of active ester per mole of
proteit~. The reaction mixture was allowed to stand in
the refrigeraE.cr for 3 days.
Ten ~l of a solution of the theophylline-
rabbit~IgG co n7ugate (1 mg/ml in 0.05 M phosphate
buffer was uniformly applied via a Hamilton syringe
across the up her region of the 6.35 cm (2.5 inch) length
of the membra n es. The membranes were dried in a
dehydrator fort' 15 minutes, then dipped in a protein
blocking solu'~ion (0.2°s nonfat dried milk, 0.2% Tween-
20~') and redried for another 15 minutes. The line of
theophylline rabbit immunoglobulin G was designated the
top of the membrane. A 0.635 cm (1/4 inch) strip of
Ahlstrpm Cytosep 799-13 (Ahlstrom Filtration) was
adhered to the top of the membrane to act as a
conducEing region. A second conductant band of Ahlstrom
992 mernbrane was secured to the lower region of the
membrane. The membrane blocks were then cut into strips
of 0.6;5 cm (1/4 inch), and stored desiccated.
For reagent pads, squares of 0.635 cm (1/4
inch) x 0.635 cm (1/4 inch) of Lipore (Grade 9254 glass
fiber filter, Lydall Technical Papers, Rochester, New
r,:.'.~'~~~=~ '~;,c~T
.. .. .. . .. ....
.. .. . .. . .. . , .
. ... . . . . . . ...
. . . . . . .... . ...
.. . . ~ .
.. .. .. . .. ..c~~ CQC"i~
131
Hampshire) were cut. For the first reagent pad, 40 ul
of antitheophylline antibody (murine monoclonal, O.E.M.
Concepts, Toms River, New Jersey) in a concentration of
180 ~.g/ml of drying/stabilizing buffer (0.5 M Tris-HC1,
pH 7.2, 0.1% Tween-20T"", 0.1% Brij-35"", 1.0% bovine serum
albumin) was applied to the pad and dried for 2 hours.
For the second reagent pad, 20 ~,1 of a working dilution
of anti-theophylline gold conjugate (E-Y Laboratories)
using monoclonal anti-theophylline antibody was applied
to the pad; the second reagent pad was also dried for
two hours.
The membrane strips were secured in a
cardboard housing such that the theophylline-rabbit IgG
line was visible through the housing windows, as shown
in Figure 23. The first reagent pad containing anti-
theophylline antibody was placed in the upper region of
the left third of the housing so that when closed it
would contact the upper conductant region on the
membrane, like applicator 764 in Figure 23. The second
reagent pad containing the anti-theophylline-gold
conjugate was placed in the lower region of the right
third of the housing so that it would contact the lower
conductant region of the membrane, like applicator 768
in Figure a~. A 2 cm x 2 cm square of Ahlstrom 270 was
used as an absorbent pad and was placed in the upper
region of the right third of the housing such that when
the right t bird was closed over the center, it made
contact with the upper conductant region.
ArJIE~~~'~'' '~~' "-;
CA 02158570 2003-12-30
132
Example 4
Con~uaate of Atrazine W
Rabbit And Goat Immunoalobulin G
To demonstrate the feasibility of the conjugation
of haptens to immunoglobulin G, conjugates of atrazine
5 with rabbit and goat immunoglobulin G were prepared.
For the conjugate with rabbit immunoglobulin G, rabbit
IgG (34.1 mg in 5 ml of phosphate buffered saline) was
treated with 12 mg thiolacetylsuccinic anhydride and 20
/~l triethylamine (approximately 300 equivalents of
10 anhydride per mole of protein). The solution was
allowed to stand in the refrigerator overnight and was
then dialyzed once against 2 liters of water and 4 times
against 2 liters of phosphate buffered saline. A
solution of hydroxylamine hydrochloride was prepared at
15 a concentration of 63 mg/ml. To the rabbit IgG as
prepared above in 8 ml PBS, was added 52 ~.cl of the
hydroxylamine solution (3.3 mg, approximately 200
equivalents). The solution was allowed to stand at room
temperature for 45 minutes.
20 An atrazine derivative, in which the terminal
methyl group of the substituent on the nitrogen is
activated, was dissolved in acetonitrile at a
concentration of 19.7 mg/ml. A portion of this solution
(200 eq, 16.6 mg, 844E,c1) was added to the rabbit IgG-
25 thiol solution. The solution was allowed to stand
overnight at room temperature.
CA 02158570 2003-12-30
133
A similar procedure was used to couple the
atrazine to goat immunoglobulin G. Goat IgG (7.8 mg
in 1.5 ml PBS) was treated with 8 mg of
thiolacetylsuccinic anhydride and 10 ,ul of
triethylamine. The solution was allowed to stand in
5 the refrigerator overnight and then dialyzed as for
the rabbit IgG. The dialyzed goat IgG solution was
treated with 10 ,ul hydroxylamine solution (0.75 mg,
approximately 200 eq). The solution was allowed to
stand at room temperature for 45 minutes. A second
10 portion of the atrazine derivative (200 eq, 3.8 mg,
190 ~cl) was added to the goat IgG-thiol solution and
reacted overnight. An ELISA, in which the goat IgG
atrazine conjugate was immobilized on polystyrene
microtiter plates, showed that the reaction was
15 successful.
Example 5
Competitive Immunoassay Device
Usincr Labeled Three-Component ConZugate
20
A competitive immunoassay device according to
the present invention for the detection of
theophylline using a labeled three-component
conjugate was constructed. The device was a two-
25 component device with a cover similar to the device
shown in Figure 24, above. The orientations
described here are with respect to the device
oriented laterally, as shown in that figure.
Nitrocellulose membrane (12 ,um pore size)
30 (Schleicher and Schuell) was secured on double back
- __ ~.._ ~ ._.._.... _~_
~-.,."°~
.. .. .. . .. ....
.. .. . .. . ~ ~ ' ~'
. . . . . ... . . . . . . ...
. . . . . . .... . ... .
.. . .
.. .. .. . .. ...
134
adhesive (3 M) in 6.35 cm (2.5 inch) x 1.75 cm (11/16
inch) blocks. A block of clear plastic had already been
positioned to the back side of the adhesive. Monoclonal
antitheophylline antibody (see Example 4) in phosphate
buffered saline (10 ~.1) was uniformly applied with a
Hamilton syringe across the upper region of the 6.35 cm
(2.5 inch) length of the nitrocellulose membrane, 0.79
cm (5/16 inch) from the designated top. Goat anti-
rabbit IgG was obtained from Pel-Freez Biologicals
=0 (Rogers, Arkansas). Pure IgG was obtained from the
serum by precipitation with caprylic acid and ammonium
sulfate. Ten ~,1 of purified goat anti-rabbit IgG (3.5
mg/ml in phosphate buffered saline) was applied above
the first line of antitheophylline antibody, 0.32 cm
_5 (1/8 inch) from the top. The membranes were dried in a
circulating dehydrator for 10 minutes, then dipped in
blocking solution (see Example 4) and redried for 15
minutes. A 0.635 cm (1/4 inch) band of Ahlstrom 1281
(Ahlstrom Filtration) was adhered to the top of the
20 membrane to act as a conductant region. A second
conductant band, Ahlstrom 992, was secured to the lower
region. The membrane blocks were then cut into 0.635 cm
(1/4 inch) strips.
25 For the reagent pads, 0.635 cm x 0.635 cm (1/4
x 1/4 inch) squares of Ahlstrom 1281 were cut.
Prediluted theophylline-rabbit IgG-gold (E. Y.
Laboratories) was applied to each square. The squares
were dried for 2 hours. Membrane strips were secured in
30 the housing such that the top goat anti-rabbit IgG line
only was visible through the windows. The reagent pad
was placed in the lower region of the left third of the
housing such that when closed over the center panel it
would make contact with the lower conductant region on
35 the membrane. A 2 cm x 2 cm square of Ahlstrom 270 was
placed in the upper region of the left third of the
housing, just above the window, where it would serve as
AMEP;~~D ~-~:.ET
135
an absorbent pad. Tt was positioned to contact the
upper conductant region of the membrane when closed.
Example 6
Two Component Device for Competitive
Inhibition Immunoassav
A two-component device was constructed using
the nitrocellulose membrane strips, adhesive, and
reagent pads of Example 4, su ra. The device was
similar to that depicted in Figure 27, su ra. The
orientations described here are with respect to the
device oriented laterally, as shown in that figure. Two
lines were immobilized on the nitrocellulose
chromatographic membrane. The first line contained 10
~,l of affinity-purified goat anti-mouse IgG (O.E.M.
Concepts) diluted in drying buffer (0.01 M phosphate,
3.0% sucrose, 0.5% bovine serum albumin, 0.5% Tween-20TM,
0.05% sodium azide (pH 7.4)). The second line contained
10 ul of the theophylline-rabbit IgG conjugate of
Example 4 in 0.05 M phosphate buffer at 1 ~g/ml. The
membranes were dried in a dehydrator and dipped in
protein blocking solution (0.1 M Tris-HC1, 0.1% BSA, pH
7.4), then redried. For the applicator on the second
opposable component, 20 ~.1 of a working dilution of
murine monoclonal anti-theophylline antibody (O.E.M.
Concepts) conjugated with colloidal gold (E-Y
Laboratories) was applied in drying buffer and dried for
2 hours.
For the assay, theophylline (Aldrich Chemical,
Milwaukee, Wisconsin) was dissolved in methanol to form
a stock solution and subsequent dilations were made in
pgS or serum. The sample (30 ~.l) was added to the
"'.'._ _ _~
136
'"'~r applicator on e second opposable component to
th
resolubilize the anti-theophylline-gold conjugate.
After a 1-minute incubation, the two opposable
components were brought into opposition so that the
applicator came into contact with the lower conductor
on
the test strip. Within 2 to 5 minutes a visually
detectable line appeared at the zone of goat anti-mouse
IgG antibody,
the only line
visible through
the aperture
of the housing. Concentrations as low as 1 ppb of
theophylline could
be detected.
Example 7
Two-Component Device for Competitive Immunoassay
Yielding Semiguantitative Results
A two-component device for competitive
immunoassay of atrazine was constructed according to
Example 9, except that the detection zone contained
three lines of affinity-purified goat anti-mouse IgG
(GAM) (O.E.M. Concepts), in addition to a line of
atrazine-goat IgG conjugate (Example 6). The line
closest on the chromatographic medium to the goat IgG-
atrazine conjugate contained the most dilute antibody,
at a concentration of 0.5 mg/ml; the other two lines
contained more concentrated antibody, each at a
concentration of 1.0 mg/ml. Murine monoclonal anti-
atrazine antibody was purchased from Agri-Diagnostics
(Cinnaminson, N.J.). The drying/stabilizing buffer for
the anti-atrazine-gold conjugate applied to the second
opposable component was 4% sucrose, 10 mM phosphate,
0.5% bovine serum albumin, 0.25% Tween-20~", 0.05% sodium
azide, pH 7.4.
For the assay, atrazine (Supelco, Inc.,
Bellefonte, Pennsylvania) was dissolved as a standard in
~~~~5"~~
137
methanol, and subsequent dilutions were prepared in
deionized water for use as working standards. Thirty ~l
of sample was added to the applicator on the second
opposable component. After a 1 minute incubation, the
two opposable components were brought together to apply
the sample and the resolubilized anti-atrazine-gold to
the chromatographic medium. Within three minutes, an
atrazine concentration in the sample of 1 to 10 ppb gave
one line on the chromatographic medium; a concentration
of 11 to 100 ppb, two lines, and a concentration of 101
to 1000 ppb, three lines.
ADVANTAGES OF THE INVENTION
Chromatographic assay devices according to the
present invention provide an advantage in being
constructed of opposable elements. The use of opposable
elements provides great versatility, as it permits the
performance of reactions in a number of different
sequences. This is possible because the use of such
opposable elements allows the delivery of reagents to
precisely defined regions of a test strip or other
reaction component. The use of opposable elements also
provides optimum performance with minimum consumption of
reagents by ensuring that reagents are not wasted by
being sequestered in dead volumes of apparatus.
Finally, the use of opposable components provides
optimum containment of possibly contaminated blood
samples, each as those containing HIV or hepatitis
virus.
Another advantage of assay devices according
to the present invention lies in the ability of the
devices to use pressure to drive fluid from one
opposable component to another and through the
chromatographic medium and to control the pressure
AM~,,rc~ ym?
. _.
138
''~''~' applied so that it is optimum for each assay to be
carried out. This accelerates the assay process and
allows the performance of operations such as extraction
within the assay device. It also reduces the dead
volumes of reagents remaining in components, allowing
the use of smaller samples and smaller quantities of
expensive or hard-to-purify reagents such as labeled
antibodies.
Additionally, chromatographic assay devices
according to the present invention allow the rapid. and
accurate detection of clinically important analytes,
such as Streptococcus A and B antigen, hemoglobin for
the determination of fecal occult blood, and antibody to
Helicobacter pylori, as well as clinically important
haptens. The construction of the devices allows more
even application of the samples to the chromatographic
medium, and reduces interference that might otherwise be
introduced by particulates or colored samples. The use
of colloidal metal labels in a resolubilizable form
provides extremely rapid kinetics of labeling and allows
substantially complete formation of binary analyte-label
complexes before the sample is applied to the
chromatographic medium. This aids in the separation of
contaminants and improves the performance of the assay.
Additionally, the construction and arrangement of the
housing of the device aids in the performance of the
assay by assuring the withdrawal of excess
immunoglobulin-containing sample that could otherwise
create interference.
Extraction of biological samples such as
blood, sputum, or feces can be performed directly in the
devices, reducing the quantity of contaminated material
that must be disposed and reducing the likelihood of
accidental infection of physicians, technicians, or the
public by such contaminated material. Additionally, the
~.~_ ~:JWW
, 139
devices are capable of performing bidirectional
chromatography to further increase accuracy and reduce
interference. Test methods using devices according to
the present invention have a wide dynamic range and are
substantially free from false negatives that may occur
in other test methods at high concentrations of analyte.
Although the present invention has been
described with considerable detail, with reference to
certain preferred versions thereof, other versions and
embodiments are possible. These versions include other
arrangements of two- or three-component devices that
operate by the basic principles described herein and
utilize any of: (a) in situ extraction of samples; (b)
resolubilization of a labeled specific binding partner
and rapid binding to analyte; and (c) arrangement of the
chromatographic medium and absorber to remove excess
sample that could otherwise create interference. These
versions include assay devices adapted for competitive
immunoassays as well as sandwich immunoassays, in
various arrangements. In particular, devices according
to the present invention can be adapted to make use of
radial or circumferential flow through a chromatographic
medium rather than linear flow. The present invention
further encompasses variations in which the two or three
components of the device are not held in a permanently
fixed arrangement, but can be separated and brought
together to perform the assay, such as by electrical or
magnetic forces or by using a separable fastener such as
a hook-and-eye fabric, for example Velcro"". Therefore,
the scope of the invention is determined by the
following claims.
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