CA2221125A1 - Opposable-element assay device employing conductive barrier - Google Patents

Opposable-element assay device employing conductive barrier Download PDF

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Publication number
CA2221125A1
CA2221125A1 CA002221125A CA2221125A CA2221125A1 CA 2221125 A1 CA2221125 A1 CA 2221125A1 CA 002221125 A CA002221125 A CA 002221125A CA 2221125 A CA2221125 A CA 2221125A CA 2221125 A1 CA2221125 A1 CA 2221125A1
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Prior art keywords
chromatographic
sample
analyte
opposable
opposable component
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Abandoned
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CA002221125A
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French (fr)
Inventor
Howard M. Chandler
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SmithKline Diagnostics Inc
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Individual
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/505—Containers for the purpose of retaining a material to be analysed, e.g. test tubes flexible containers not provided for above
    • B01L3/5055—Hinged, e.g. opposable surfaces
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5023—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures with a sample being transported to, and subsequently stored in an absorbent for analysis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/90—Plate chromatography, e.g. thin layer or paper chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/5302—Apparatus specially adapted for immunological test procedures
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54386—Analytical elements
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54386—Analytical elements
    • G01N33/54387—Immunochromatographic test strips
    • G01N33/54388—Immunochromatographic test strips based on lateral flow
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0809—Geometry, shape and general structure rectangular shaped
    • B01L2300/0825—Test strips
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
    • B01L2400/0406—Moving fluids with specific forces or mechanical means specific forces capillary forces
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/28—Control of physical parameters of the fluid carrier
    • G01N30/30—Control of physical parameters of the fluid carrier of temperature
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/805—Test papers
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/81—Packaged device or kit
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/97—Test strip or test slide
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/973—Simultaneous determination of more than one analyte
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/975—Kit
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S436/00—Chemistry: analytical and immunological testing
    • Y10S436/805—Optical property
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S436/00—Chemistry: analytical and immunological testing
    • Y10S436/807—Apparatus included in process claim, e.g. physical support structures
    • Y10S436/808—Automated or kit
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S436/00—Chemistry: analytical and immunological testing
    • Y10S436/807—Apparatus included in process claim, e.g. physical support structures
    • Y10S436/81—Tube, bottle, or dipstick

Abstract

A chromatographic assay device for use with immunoassays which allows rapid and convenient assays of analytes of biological interest, and permits extractions to be carried out in situ, avoiding the use of separate extraction vessels. The device has a wide dynamic range and avoids interference from particulates or colored components. In one form, the device comprises: a first opposable component (12) comprising a sample preparation zone (18) adapted to receive a sample to be assayed; a second opposable component (14) comprising a chromatographic medium (20); and a conductive barrier (16) attached to the second opposable component. The first and second opposable components can be brought into opposition so as to cause the sample preparation zone to apply the sample to be tested to the chromatographic medium. Preferably, the analyte is detected with a visually detectable label. Other variations of the device vary the arangement of components to provide optimal chromatography for a variety of analytes. The devices can be incorporated in test kits, and assay methods, particularly sandwich immunoassays, using the devices are also disclosed.

Description

CA 0222112~ 1997-11-13 WO 96/38727 PCT/17S96~07663 OPPOSABLE-ELEM[ENT ASSAY DEVICE
EMPLOYING CONDUCTIVE BARRIER

BACKGROUND OF THE INVENTION

This invention is directed to test strips for determin~tion of characteristics of samples, lmiti7Pcl housings, and kits incorporating the test strips, and methods of d~ g the characteristics of samples using the test strips.

Among the many analytical systems used for detection and/or determination of analytes, particularly analytes of biological interest, are chromatographic assay systems.

Such chromatographic systems are frequently used by physicians and mP~ l technicians for rapid in-office diagnosis and theld~uLic monitoring of a variety of conditions and disorders. They are also increasingly used by patients themselves for at-home molliloling 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 mPtlillm.

Among the most important of tests that can be performed with such thin layer systems are immllnnassays, which depend on the specific 20 interaction between an antigen or hapten and a colle~ollding antibody. The use of i"-""~ s~ys 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 immllnP-based latex ag~ illA~ion test for detecting a factor associated with rhPum~tnid arthritis (Singer et al., Am. J.
25 Med. 22:8g8-892 (1956)).

CA 0222112~ 1997-11-13 Among the chromatographic techniques used in conjunction with immunoassays is a procedure known as immnnochromatography.

Tmmnnochromatographic assays fall into two principal categories:
5 "sandwich" and "competitive," according to the nature of the antigen-antibody complex to be ~letecte-l and the sequence of reactions required to produce that complex. In general, the sandwich immnnochromatographic 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 10 disclosing reagent, such as dyed latex, a colloidal metal sol, or a radioisotope.
This mixture is then applied to a chromatographic medium cont~ining a band or zone. This band or zone contains immobilized antibodies to the analyte of interest. The chromatographic m.orlillm often is in the form of a strip resembling a dipstick. When the complex of the molecule to be assayed and the labeled 15 antibody reaches the zone of the immobilized antibodies on the chromatographic m~rlinm, binding occurs and the bound labeled antibodies are localized at the zone. This in~lic~t~s the ~l~sellce of the molecule to be assayed. This technique can be used to obtain q~ re or semi-q -~ntit~tive results.

Examples of sandwich immnno~s~ys 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. both of which are incorporated herein by this lc~felcllce.

In addition to immlmnchromatographic assays, it is also known to use enzyme-based chromatographic assays. These techniques are roughly analogous to immllnt~chromatographic assays, but use an enzym~tir~lly catalyzed reaction instead of an antigen-antibody reaction. The enzym~ti~ally catalyzed reaction frequently generates a ~ietect~hle product. Other analogous chromatographic assays are known.
Although useful, ~;ull~lllly available chromatographic techniques using test strips have a number of drawbacks. Many samples, such as fecal CA 0222112~ 1997-11-13 samples, contain particulate matter that can clog the pores of the chromatographic medium, greatly hindering the immlln~chromatographic 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 hlLclrelcllce~ it is 5 frequently ~liffirnlt with existing chromatographic test devices to apply the sample to the chromatographic m~ m 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.

Sample preparation and waste generation are responsible for other problems with ~;ullcll~ly available devices and techniques for immlmnchromatography. 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 15 sampling device (such as a throat swab) directly to the chromatographic m~ m Several extraction and plcl,caLlllent 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 20 devices such as pipettes. Each of these devices is then co~.l;."-i"~lecl and must be disposed of using special l~lccauLions so that workers or people who may inadvcllcllLly come into contact with the waste do not become colllillllill~t~tl Accordingly, there is a need for an improved chromatographic 25 device for the pclrollllance of immlmochromatographic assays or other analogous assays. Such a device should be capable of receiving a possibly colll;~",i,~t~c~sample or a sample plel)Al~Lion device directly so as to elimin~t~ 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 immllnochromatographic assays30 on colored samples or samples cont~inin~ particulates without h~Lclrclcllce and should be able to de~iver the sample to the chromatographic medium uniformly and evenly to improve accuracy and precision of the tests. This aspect of an CA 0222112~ 1997-11-13 improved assay device is particularly important in avoiding false negatives and false positives.

I 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 col,l;.",i~ n. The device can perform 10 all types of immllnl-assays, including sandwich immllnl-assays, competitive immllnoassays, and assays 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. pylori. The device can pelrOll.l assays in which the antigen to be cletecte~l is detected indirectly by using a labeled second 15 antibody binding to the first antibody to the analyte. These assay devices all include a conductive barrier ~tt~rh~-~l to an opposable component of a device cont~inin~ at least two opposable components.

An assay device according to the present invention makes use of 20 pressure to transfer fluid from one opposable component to another opposable component, and also to drive fluid through the chromatographic m~ lm. The s~ul~ not only speeds up the operation of the device, but allows the performance of additional steps such as extraction steps to remove hlLelrelillg particulate components within a single device. The ~ ,S:jUl~ iS generated by 25 holding the opposable components together with engagers such as interlocking elementc on each of the opposable components. Preferably, a pred~ ed pressure is applied to ensure the o~lullulll pelrollllallce of 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 CA 0222112~ 1997-11-13 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 complement~ry nucleic acid segment according to the Watson-(~rick base pairing scheme.
In general, an assay device according to the present invention comprises:
(1) a first opposable component including a sample preparation means adapted to receive a liquid sample to be assayed;
(2) a second opposable component including a chromatographic m~ lm having at least one reagent binding specifically to an analyte to be detected, the reagent being bound at a detection zone on the chromatographic medium, the second opposable component being ~tt~h~hle to the first opposable component; and (3) a conductive barrier attached to the second opposable component.

The first and second opposable components can be brought into opposition from a position in which they are not in opposition so as to cause the 20 sample l~lc~alaLion means to apply the liquid sample to be tested to the cl~ullla~ographic mP-lillm through the conductive barrier and to flow through the chromatographic mP~ m The chromatographic assay is performed as a result of migration of the sample within the chromatographic me~ m so that an analyte is ~letecte~l within the chromatographic m.~ lm as the result of the migration by 25 binding of a labeled reagent that binds specifically to the analyte to be detected.
The analyte is detected at a position dirr~ than the position at which the sample is applied to the chromatographic medium. The analyte is det~ct~-l on thechromatographic medium after migration by binding of the labeled reagent to the analyte bound to the detection zone.
Typically, the sample p.~a.~Lion means includes at least one reagent for the tre~tment of the sample before the sample is applied to the CA 0222112~ 1997-11-13 chromatographic mt-~linm. The reagent for treatment of the sample can be an extraction reagent to extract analyte from the sample.

Generally, the detection zone is substantially smaller than the 5 chromatographic m~-linm In this arrangement, the detection zone can contain a first specific binding partner to the analyte immobilized thereto. When the analyte is an antigen or a hapten, the first specific binding partner can be an antibody to the antigen or hapten. In one particularly preferred alLellla~ive, the analyte is human hemoglobin and the first specific binding partner is an anti-10 human hemoglobin antibody.

Alternatively, the analyte can be an antibody and the first specific binding partner can be a hapten or antigen capable of being bound specifically by the antibody.
Typically, the chromatographic mP~lillm further includes a control zone substzlnti~lly smaller than the chromatographic m~-linm Typically, the control zone contains analyte immobilized thereto.

The chromatographic assay device can further comprise an absorbing means in operable contact with the second end of the chromatographic mP~ lm Typically, the sample pl~pdldLion means further contains a specific binding partner for the analyte labeled with a (l.-tect~hle label in a form that can be resolubilized by the addition of a liquid to the sample pl~pdld~ion means.
Generally, the liquid comprises an extraction reagent to extract analyte from the sample; however, other liquids can also be used.

Typically, in assay devices according to the present invention, the first and second opposable components can be brought into opposition by direct manual closure from a position in which they are not in opposition.

CA 0222112~ 1997-11-13 Another aspect of the present invention is a test kit for the detection and/or determination of an analyte. The test kit can comprise, st;~aldL~ly packaged:
. (1) the chromatographic assay device described above; and ~ S (2) a specific binding partner for the analyte labeled with a ~letrct~ble label, the specific binding partner to be used with the chromatographic assay device.

Other devices according to the present invention can also be incorporated into test kits, comprising, separately packaged, the device, along with either a specific binding partner for the analyte labeled with a ~letrct~hle label or a liquid for resolubilizing a resolubilizable labeled specific binding partner included in the device. In some cases, test kits can include a test card or an extraction reagent for extracting an analyte from a sample.
Yet another aspect of the present invention is a multiplex device capable of performing multiple assays on the same device ~imlllt~nrously. In general, this device comprises:
(1) a first opposable component including a plurality of laterally separated sample p,cpaldlion means, each adapted to receive a sample to be assayed;
(2) a second opposable component ~tt~rh~hle to the first opposable component and inrl~l~ling a chromatographic medium for each sample ~lel?ald~ion means on the first opposable component, the chromatographic media being laterally separated; and (3) a plurality of conductive barriers each ~tt~rhr-l to the second opposable component, one for each chromatographic medium.

In this device, the first and second opposable components can be brought into opposition so as to cause each sample preparation means to apply each sample to be tested to the corresponding chromatographic medium through the corresponding conductive barrier.

CA 0222112~ 1997-11-13 In this multiplex device, at least one sample preparation means includes a collapsible well adapted for receiving a sample-cont~ining device. If a collapsible well is included, the first opposable component can further include hingedly foldable wings that fold over the second opposable component when the S first opposable component and second opposable component are brought into opposition.

An alternative embodiment of a multiplex assay device according to the present invention is adapted to receive a test card. This embodiment comprises:
(1) a first opposable component including a plurality of laterally separated reagent pads;
(2) a second opposable component adapted to receive a test card cont~ining a plurality of dried specimens, the second opposable component including:
(a) a chromatographic mt--linm for each sample ~-~ala~ion means on the first opposable component, the chromatographic media being laterally sepalat~d, each chromatographic m~-linm having a first and a second end, (b) a con~ cting means in operable contact with the first end of each chromatographic mPrlillm and in operable contact with each dried specimen of the test card when the test card is inserted into the second opposable component; and (c) an absorbing means in operable contact with the second end of each chromatographic medium; and (3) a plurality of conductive barriers each ~tt~Cl~ to the second opposable component, one for each chromatographic m~ lm In this embodiment of a multiplex assay device according to the present invention, the first and second opposable components can be brought intoopposition so as to cause each reagent pad to be applied to the corresponding dried specimen through the corresponding conductive barrier.

CA 0222112~ 1997-11-13 WO 96/38727 PCT/US96/(17663 In this embodiment, each reagent pad can include a specific binding partner for the analyte labeled with a (i~trct~hle label in a form that can be resolubilized by the addition of an aqueous reagent to the reagent pad. In one particularly !,lef~ d version of this embodiment, the analyte is human hemoglobin and the specific binding partner is an anti-human hemoglobin antibody.

Another embodiment of the present invention is an assay device in which the sample preparation means is located on the same opposable component as the chromatographic mr~ m. This device comprises:
(1) a first opposable component including:
(a) a sample preparation means; and (b) a chromatographic mr~lillm in operable contact with the sample preparation means;
(2) a second opposable component attachable to the first opposable component including an application means cont~ining a specific binding partner for the analyte labeled with a cletect~ble label in a form that can be resolubilized by the addition of a liquid to the application means; and (3) a conductive barrier att~rhpcl to the first opposable component.
In this device, bringing the first and second opposable components into opposition brings the application means into contact with the conductive barrier such that the labeled specific binding partner for the analyte is resolubilized by liquid passing through the conductive barrier. The chromatographic medium can have first and second ends with the con~lllr.ting means being in operable contact with the first end of the chromatographic medium; in this arrangement, the first opposable component can further include an absorbing means in operable contact with the second end of the chromatographic m.-flillm.

Another embodiment of the present invention has two application means on the opposable component not including the chromatographic mrtlillm.
This embodiment coll,~lises:

CA 0222112~ 1997-11-13 (1) a first opposable component including:
(a) a chromatographic medium having first and second ends;
(b) a con~ ctin3~ means in operable contact with the first 5 end of the chromatographic medium; and (c) an absorbing means in operable contact with the second end of the chromatographic mt~ m;
(2) a second opposable component comprising:
(a) a first application means; and (b) a second application means; and (3) a conductive barrier attached to the first opposable component.

The first and second application means are positioned on the second opposable component such that they are not in operable contact when the 15 first and second opposable components are not in opposition. Bringing the first and second opposable components into opposition places the con-lncting means in operable indirect contact with the first application means through the conductive barrier and places the con~lllcting means in operable contact with the second application means, thereby placing the first and second application means in 20 operable contact with each other.

Typically, in this embodiment, the first application means includes a sample application pad and the second application means comprises a detector application pad, to which cl~-tectin~ reagent can be applied, whereby, when the 25 first and second opposable components are brought into opposition, the contents of the sample application pad and the detector application pad are applied to the con-l~lcting means through the conductive barrier. Generally, the detector application pad contains a first specific binding partner to the analyte in a form that can be resolubilized by addition of a liquid to the detector application pad, 30 the first specific binding partner being labeled with a tlet~ct~ble label, and the cl~vlllaL~graphic m~lillm further comprises a detection zone substantially smaller in area than the chromatographic m~(lillm, the detection zone conf~ining a second CA 0222112~ 1997-11-13 specific binding partner to the analyte immobilized thereto, such that a ternarycomplex comprising the first specific binding partner, the analyte, and the second specific binding partner (i.e., a sandwich complex) forms at the detection zone if analyte is present in the sample.
S
Another embodiment of an assay device according to the present invention has a detector application pad on the opposable component also having the chromatographic metlillm and a sample application pad on the other opposable component. This embodiment comprises:
(1) a first opposable component including:
(a) a chromatographic me(lillm having first and second ends;
(b) a con~llcting means in operable contact with the first end of the chromatographic merlillm;
(c) an absorbing means in operable contact with the second end of the chromatographic mP(iillm; and (d) a detector application pad in direct contact with the con~ çting means and positioned such that it is in h~dile~;~ contact with the first end of the chromatographic me-linm;
(2) a second opposable component including a sample application pad; and (3) a conductive barrier ~ rh~cl to the first opposable component.

In this embodiment, bringing the first and second opposable 25 components into opposition causes the sample application pad to apply the sample to be tested to the detector application pad through the conductive barrier and thus to the first end of the chromatographic m~llinm though the conflllcting ~ means.

Yet another embodiment of the present invention comprises:
(1) a first opposable component including:

CA 0222112~ 1997-11-13 (a) a chromatographic mP~ m having first and second ends;
(b) an absorbing means in operable contact with the second end of the chromatographic mP-lium; and S(c) a detector application pad in direct contact with the first end of the chromatographic mP-lium; and (2) a second opposable component including a sample application pad; and (3) a conductive barrier ~tt~rllP-l to the first opposable component.
In this embodiment, when the first and second opposable components are brought into opposition, the detector application pad and the sample application pad are in indirect contact through the conductive barrier except for the region of the detector application pad directly adjacent to the first 15 end of the chromatographic mP.-lium. Bringing the first and second opposable components into opposition causes the sample application pad to apply the sampleto be tested to the detector application pad and thus to the first end of the chromatographic mPrlinm.

Yet another embodiment of an assay device according to the present invention comprises:
(1) a first opposable component including:
(a) a chromatographic mPrlium having first and second ends;
(b) a detector application pad in operable contact with the first end of the chromatographic mP(lillm, the detector application pad cont~ining at least one reagent for detection of the analyte;
(c) a conductor for allowing the passage of fluid in operable contact with the detector application pad and in indirect contact with the first end of the chromatographic mP~lium; and ~ (d) an absorber for absorbing fluid in operable contact with the second end of the chromatographic medium;

CA 0222112~ 1997-11-13 (2) a second opposable component including a sample preparation zone for receiving a sample to be tested, the second opposable component being ~tt~h~hle to the first opposable component so that the first andsecond opposable components are brought into opposition and fluid is transferred5 from the second opposable component to the first opposable component; and (3) a conductive barrier ~tt~rh~ot1 to the first opposable component.

In this embodiment, the first and second opposable components are configured so that a sample can be applied to the sample ~l~al~tion zone on the 10 second opposable component when the first and second opposable components are not in opposition and so that bringing the first and second opposable components into opposition results in the sample l~lepar~Lion zone being in indirect contact with the conductor to apply the sample to be tested through theconductive barrier to the conductor for flow through the conductor and then to 15 the first end of the chromatographic medium through the detector application pad to add the reagent for detection of the analyte to the sample. The flow from theconductor through the detector application pad to the first end of the chromatographic medium is aided by absorption of fluid by the absorber.

Preferably, the detector application pad contains a first specific binding partner to the analyte, as described above, and the chromatographic medium further comprises a detection zone, as described above.

Another embodirnent of an assay device according to the present invention includes two labeled specific binding partners. This embodiment compnses:
(1) a first opposable component including:
(a) a chromatographic mP~linm having f*st and second ends;
(b) a first detector application pad in operable contact with the fi*st end of the chromatographic medium, the first detector applicationpad cont~ining a first specific binding partner to the analyte in a form that can be CA 0222112~ 1997-11-13 resolubilized by the addition of a liquid to the first detector application pad, the first specific binding partner being labeled with a detectable label;
(c) a conductor for allowing the passage of fluid in operable contact with the first detector application pad so that the first detector S application pad bridges the conductor and the first end of the chromatographicmedium to allow fluid flow from the conductor through the first detector application pad and to the first end of the chromatographic medium; and (d) an absorber for absorbing fluid in operable contact with the second end of the chromatographic m-~inm;
(2) a second opposable component ~tt~rh~hle to the first opposable component so that the first and second opposable components are brought into opposition and fluid is transferred from the second opposable component to the first opposable component, the second opposable component including:
(a) a sample pl~l)dldLion zone for receiving a sample to be assayed; and (b) a second detector application pad in operable contact with the sample ~le~alaLion zone, the second detector application pad cont~inin~ a second specific binding partner for the analyte in a form that can be 20 resolubilized by the addition of a sample to the sample ~lcpaldlion zone, thesecond specific binding partner being labeled with a d~tect~hle label, the second detector application pad being located adjacent to the sample plcpaldlion zone on the second opposable component such that application of the sample to the sample ~lcpaldLion zone resolubilizes the second specific binding partner so that 25 the sample plc~aldtion zone contains a mixture of the sample and the second specific binding partner; and (3) a conductive barrier ~tt:~chf~-l to the first opposable component.

The first and second opposable components are configured so that 30 a sample can be applied to the sample plepaldLion zone on the second opposable component when the first and second opposable components are not in opposition. Bringing the first and second opposable components into opposition CA 0222112~ 1997-11-13 W~ 96/38727 PCT/US96107663 results in the sample preparation zone on the second opposable component being in operable indirect contact with the conductor on the first opposable componentthrough the conductive barrier to apply the sample to be tested and the second specific binding partner to the conductor for flow through the conductor and then 5 to the first end of the chromatographic medium through the first detector application pad to add the first specific binding partner to the sample and the second specific binding partner. The flow from the conductor through the first detector application pad to the first end of the chromatographic m~ lm is aided by absorption of fluid by the absorber.
Still another embodiment of an assay device according to the present invention has an applicator divided into two sectors. This embodiment comprises:
(1) a first opposable component including:
(a) a chromatographic medium having first and second ends;
(b) a conductor for allowing the passage of fluid in operable contact with the first end of the chromatographic m~ m; and (c) an absorber for absorbing fluid in operable contact with 20 the second end of the chromatographic m~ lm;
(2) a second opposable component ~tt~rh~hle to the first opposable component so that the first and second opposable components are brought into opposition and fluid is L.dl~rt:llc:d from the second opposable component to the first opposable component, the second opposable component 25 including an applicator for applying fluid to the conductor on the first opposable component when the first and second opposable components are brought into opposition, the applicator divided into two sectors:
(a) a first sector cont~ining a first specific binding partner for the analyte in a form that can be resolubilized by the addition of a30 liquid to the applicator when the first and second opposable components are not in opposition, the first specific binding partner being labeled with a detectable label; and CA 0222ll2~ l997-ll-l3 (b) a second sector lacking a first specific binding partner for the analyte; and (3) a conductive barrier ~tt~ch~-cl to the first opposable component.

S In this embodiment, the first and second opposable components areconfigured so that bringing the first and second opposable components into opposition places the first sector, but not the second sector of the applicator on the second opposable component into indirect contact with the conductor on the first opposable component through the conductive barrier, the second sector of the applicator being in indirect contact with the conductor through the first sector. This applies the contents of the first sector of the applicator to the clllulllatographic m~rlium, and, subsequent to the application of the contents of the first sector of the applicator to the chromatographic mP~ lm, applies the contents of the second sector of the applicator to the chromatographic medium.
The absorber withdraws fluid from the chromatographic m~o-lium to aid fluid flowfrom the applicator through the conductor and the chromatographic medium.

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 accoll~al,yillg drawings where:
Figure lA is a drawing of one version of a two-component chromatographic assay device employing a conductive barrier according to the present invention;
Figure lB is a drawing of the two-component chromatographic assay device of Figure lA shown with the two components having been brought into opposition;
Figure lC is a side view of the device of Figures lA and lB
showing the relationships of the sample preparation zone, the conductive barrier, and the conductor;

CA 0222112~ 1997-11-13 WO 96/38727 PCT/lJS96107663 Figure 2 is a drawing of a version of a two-component chromatographic assay device with a conductive barrier according to the present invention in which the first opposable component includes a sample preparation zone;
S Figure 3 is a drawing of another version of a two-component assay device with a conductive barrier according to the present invention with two applicators on the same opposable component;
Figure 4A is a drawing of another version of a two-component assay device with a conductive barrier according to the present invention incorporating a pad for a labeled specific binding partner on the same opposablecomponent as the chromatographic medium and a sample preparation zone on the other opposable component;
Figure 4B is a sectional rear view of the two-component assay device of Figure 4A, showing details of the chromatographic mPtlillm, the first and second opposable components, and the conductive barrier;
Figure S is a drawing of a variation of the two-component assay device shown in Figures 4A and 4B with the absorber on the second opposable component;
Figure 6A is a drawing of yet another version of a two-component assay device according to the present invention, generally similar to the version of Figures 4A and 4B, but with the detector application pad in direct contact with the chromatographic medium;
Figure 6B is a sectional rear view of the two-component assay device of Figure 6A, showing details of the components in opposition;
Figure 7 is a drawing of yet another version of a two-component assay device employing a conductive barrier according to the present invention, in which a detector application pad is on the first opposable component and is located between the chromatographic m.--lillm and a conductor;
Figure 8 is a drawing of yet another version of a two-component assay device employing a conductive barrier according to the present invention, employing a two-sector applicator to provide a wash;

CA 0222112~ 1997-11-13 Figure 9 is a drawing of yet another version of a two-component assay device employing a conductive barrier according to the present invention, with two detector application pads, one on each opposable component;
Figure 10 is a drawing of a multiplex assay device according to 5 the present invention, suitable for the simlllt~n~--ous assay of one or more samples;
Figure llA is a drawing of another version of a multiplex assay device according to the present invention, cont~ining a collapsible well to accom~nodate a sample;
Figure 11B is a drawing of a version of a multiplex assay device similar to that of Figure 11A, except substituting hingedly foldable wings for the gasket;
Figure 12 is a drawing of a dirrer~ version of a multiplex assay device according to the present invention adapted to receive a test card; and Figure 13 is a drawing 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 as constructed in Example 1.

Definitions In the context of this disclosure, the following terms are defined as 25 follows unless otherwise in~ t.o~1 Specific Binding Partner: A member of a pair of molecules that interact by means of specific non-covalent interactions that depend on the three-dimensional structures of the molecules involved. Typical pairs of specific 30 binding ~alLll~l~ include antigen-antibody, hapten-antibody, hormone-receptor, nucleic acid strand-complementary nucleic acid strand, substrate-enzyme, inhibitor-enzyme, carbohydrate-iectin, biotin-avidin, and virus-cellular receptor.

CA 0222112~ 1997-11-13 WC> 96S3g727 PCTtUS96/07663 Operable Contact: Two solid components are in operable contact when they are in contact, either directly or indirectly, in such a manner that aliquid can flow from one of the two components to the other subst~nti~lly uni~ Lecl!y, by capillarity or otherwise. "Direct contact" means that the two ~ 5 elements are in physical contact, such as edge-to-edge or front-to-back. "Indirect contact" means that the two elements are not in physical contact, but are bridged by one or more concl--rting means. This bridging by one or more con-lllc.ting means can be either edge-to-edge or front-to-back, such as by the opposition of planar elements.
Conductive Barrier: The term "conductive barrier" is used herein to describe an element that is capable of con-lllcting liquids and solutes contained in the liquids while serving as a barrier to partir~ tes and preventing liquid flow from one element to another except through the conductive barrier. As described lS below, such barriers can be constructed of cellulose, such as paper.

Finite Capacity: An absorbing means has finite capacity when it becomes sdLuldl~d by liquid received during the normal performance of an assay in the device in which the absorbing means is located. At that point, the absorbing means can release additional liquid absorbed and become at least partially conductive.

Analyte: The term "analyte" inrllllles both the actual molecule to be assayed and analogues and derivatives thereof when such analogues and derivatives bind another molecule used in the assay in a marmer substantially equivalent to that of the analyte itself.

Antibody: The term "antibody" includes both intact antibody molecules of the ~ypl'oyliate specificity and antibody fr~gment~ (including Fab,F(ab'), and F(ab')2 fragments) as well as ch.-mie~lly modified intact antibody molecules and antibody fragments, including hybrid antibodies assembled by in CA 0222112~ 1997-11-13 vitro reassociation of subunits. Both polyclonal and monoclonal antibodies are included unless otherwise specified.

. Secondary Specific Binding Partner: An additional specific 5 binding partner that binds to a member of a pair of specific binding partners when the pair of specific binding palLllcl~ 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 10 secondary specific binding partner can be specific for the species, class, orsubclass of an antibody specific binding partner to which it binds. AlL~ ati~ely, when one of the specific binding partners is labeled with biotin, the secondary specific binding partner can comprise a molecule conjugated to avidin.

I. CHROMATOGRAPHIC ASSAY DEVICES

One aspect of the present invention comprises chromatographic assay devices particularly useful for the assay of analytes in biological samples.
20 These devices are suitable for the direct application of biological samples, without prelilllindly extraction steps, and are constructed so as to minimi hlLclrc;lcllce with assay results caused by particulates or colored samples.

These devices include a conductive barrier ~tf~Ch~l to one of the 25 opposable components that serves to provide a more even flow of material Lldn~fcllcd from one opposable component to another opposable component.
This provides a more reproducible assay and reduces the likelihood of false positive or false negative results.

The device has at least two substantially planar opposable components. One of the substantially planar components has on its surface a chromatographic medium.

CA 0222112~ 1997-11-13 When there are two opposable components, one of the opposable components is ~lesign~t~l the first opposable component and the other is ~lesi~n~tecl the second opposable component. This designation is albill~ly and for convenience in description; the role of each of the opposable components is S determined by the element or elements located on it.

The device also has means for opposing the opposable components and applying pressure thereto. The opposable components can be brought into opposition from a position in which they are not in opposition by direct manual 10 closure, i.e., by manipulation by the operator. The plCS~iUlC applied is sufficient to transfer fluid from 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 dcle~ alion of the analyte thereon. The pressure also drives fluid through the 15 chromatographic mP~ lm to accelerate the process of chlolllatography, giving a ~letect~ble result in less time. Additionally, the plCS~ulc makes possible the performance of steps, such as extraction steps, in the device, and can be used to remove excess fluid from the chromatographic m~inm by absorbers to reduce the background of the assays. The ~lCS~u-c is generated by placing the 20 opposable components into opposition and m~int~into-l by holding the components into opposition by engagers such as locks or clasps.

Devices according to the present invention can be constructed for the pclrollllance of either a sandwich or a culll~cliLi~le assay.
A type of assays for which devices according to the present invention are particularly useful is sandwich immlmoassays. As used herein, the term "immllno~s~y" is used generally to include specific binding assays and need not n~cess~rily be restricted to assays in which the specific binding partner 30 is an antibody, unless so specified.

CA 0222112~ 1997-11-13 A. Principles of Devices and Methods Accordin~ to the Present Invention All of the devices according to the present invention have a S chromatographic medillm and have application of liquid to the chromatographic m~ m controlled by a conductive barrier. The barrier provides for smoother flow and prevents high local concentrations of analyte or, in some cases, a labeled specific binding partner, at a particular location on the chromatographic medium at the start of chromatography. This ensures even progress of 10 chromatography and prevents retardation of the rate of chromatography as a result of analyte concentration.

Although the basic principle of the invention can be used in a single-component assay device, it is generally plcr~lled to construct assay 15 devices cont~ining two or more opposable components connPcted by a hinge or other connection and fastenable by engagers such as locks. This allows plCS~ C
to be placed on the component to drive fluid from one component to another and to accelerate the rate of flow. The degree of pressure employed can be regulatedso that it is O1JIU11U111 for the characteristics of the chromatographic m~illm,20 analyte, and label.

B. Elements Common to Devices Accordin~ to the Present Invention A number of elements are common to assay devices according to 25 the present invention and are discussed here for convenience.

1. The Chromato~raphic Medium The chromatographic medium is a strip. Typically, the strip is 30 substantially planar, although this is not required in all applications. It is typically rectangular; having first and second ends and first and second surfaces.
Throughout this description, the term "first end" refers to the end at or near CA 0222112~ 1997-11-13 W<~ 96138727 PCTIUS96/~17663 which liquid is first applied to the chromatographic medium and the term "second end" applies to the opposite end of the chromatographic medium. The liquid applied at or near the first end of the chromatographic medium can be, but is not nPce~rily, a sample or a treated sample. The chromatographic mP~ lm S is composed of material suitable as a mPriillm for thin layer chromatography of analyte and analyte-antibody conjugates, such as nitrocellulose, nylon, rayon, cellulose, paper, or silica. The chromatographic medium can be ~.~Lleat~d or modified as needed. Typically, the chromatographic medium is translucent, so that colored zones appearing on it can be viewed from either side.
2. The Conductive Barrier The conductive barrier is typically a thin layer of fibrous, porous material such as cellulose (i.e., paper) or nitrocellulose that is permeable to 15 fluids and does not substantially absorb the fluids applied. Typically, the conductive barrier is rectangular or square. The conductive barrier is positioned in the assay device so that one side of the conductive barrier is in direct contact with one of the opposable components of the device and the other side is in direct contact with the second opposable component of the device. The 20 conductive barrier has the characteristic that liquid applied to one side of the conductive barrier pellc;Lld~s to the other side.

3. Absorbers In a number of devices according to the present invention, absorbers are in operable contact with one or both ends of the chromatographic medium. The absorbers can be made of any bibulous material that will hold a liquid sufficiently so liquid can be drawn through the chromatographic medium and ~rcllmlll~tl-~l in the absorber. Typical materials include, but are not limited to, filter paper.

CA 0222112~ 1997-11-13 4. Other Fluid-Carryin~ Elements As described below, in particular devices according to the present invention, other fluid-carrying elements can be employed as sample preparation 5 zones, applicators, distribution membranes, and/or conductors. These elements are lJlc;palcd of hydrophilic media that pass liquids without subst~nti~lly absorbing them. Such materials are well-known in the art. In some cases, these elements can have incorporated therein a component in dry form that can be resolubilized by addition of a liquid to the element.
5. Opposable Components Many of the embodiments of the assay device according to the present invention comprise two opposable components. The bodies of the 15 opposable components are preferably made of l~min~te~l cardboard that is sufficiently ~ iOUS to moisture to contain the liquids involved in the performance of the assay carried out by the device. Other cellulose-based materials, such as paperboard or solid bleached sulfite (SBS) can also be used.
Alternatively, the bodies of the opposable components can be made of plastic that 20 is impervious to moisture. A suitable plastic is a polycarbonate plastic such as Lexan~.

The opposable components are joined by a hinge, preferably made of a material impermeable to liquids, such as a plastic that can be compatibly 25 joined with or is the same as the material used for the first and second opposable components.
6. Labeled Components For assay devices intended to perform a sandwich immunoassay, the labeled component is typically a labeled specific binding partner to the analyte. This labeled component is typically mobile, in that it can migrate CA 0222112~ 1997-11-13 through the chromatographic medium, whether free or bound to analyte. The label is preferably a visually detectible label, such as a colloidal metal label.
Preferably, the colloidal metal label is gold, silver, bronze, iron, or tin; most preferably, jt is gold. The preparation of gold-labeled antibodies and antigens is - 5 described in J. DeMey, "The Preparation and Use of Gold Probes," in Tmmllnncytochemistry: Modern Methods and Applications (J. M. Polak and S.
VanNoorden, eds., Wright, Bristol, Fngl~n-l, 1986), ch. 8, pp. 115-145, incorporated herein by this rcfclcllce. Antibodies labeled with colloidal gold are commercially available, such as from Sigma Chemical CU1111)a11Y~ St. Louis, Missouri.

Alternatively, other colloidal labels, such as a colloidal sulfur label or a dye-silica label, can also be used. In a less l~lc~cllcd alternative, the visually detectible label can be a colored latex label. It is also possible to use other labels, such as a radioactive label or an enzyme label.

C. General Alldl~elllent of Two-Component Device In general, a two-component chromatographic assay device according to the present invention comprises:

(1) A first opposable component including a sample plepal~lion zone adapted to receive a sample to be assayed; and (2) A second opposable component including a chromatographic mPrlillm; and (3) a conductive barrier attached to the second opposable component.

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 CA 0222112~ 1997-11-13 brought into opposition after a detection reagent is applied to the sample yl~paldtion 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 meflillm through the conductive barrier so that the 5 combination of sample and detection reagent is dispersed within the conductivebarrier before being applied to the chromatographic me-lium. After the sample and detection reagent are allowed to traverse at least a portion of the chromatographic medium, the detection reagent gives a detectable indication of the presence and/or quantity of the analyte; the detection reagent is then observed 10 and/or measured in at least a portion of the chromatographic medium to obtainthis indication. 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 assay devices according15 to the present invention.

The detection reagent comprises the first specific binding partner for the analyte as described above; it may comprise additional components.

This process can give a qualitative and/or q~ e indication of the analyte, depending upon the concentration 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 25 10 mimltes, more typically, from 1 to 5 minutes, including any period of incubation of the sample on the sample yl~yaldtion zone, as well as the time required for cl~o~l~atography 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 30 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 CA 0222112~ 1997-11-13 WO 96)38727 PCT/IJS96/07663 higher temperature with suitable analytes and specific binding partners may speed up the assay.

In devices according to the present invention, detection of the - 5 analyte occurs without contact of the sample with any additional liquid once the sample is applied to the chromatographic m~-lium This is true even if a wash of sample is being used. In other words, the device is self-cont~inf d, and there is no need to dilute the sample or label with additional liquid once the sample is applied to the chromatographic medium. This provides ~ i,-,u", concentrations of sample and labeled specific binding partner for m~ximllm sellsiliviLy.

In devices according to the present invention, a chromatographic assay is performed as a result of migration of the sample within the chromatographic m~lillm The analyte is cletecte(l at a position dirr~lc"~ than the position at which the sample is applied to the chlull,atographic medium.

This general arrangement of the chroll,-dtographic assay device is shown in Figure lA. The chromatographic assay device 10 has a first opposable component 12, a second opposable component 14, and a conductive barrier 16 attached to the second opposable component 14. Typically, the conductive barrier 16 is flexibly ~tt:~rhto~l to the second opposable component 14, such as by an adhesive that permits relative motion of the conductive barrier 16 and the second opposable co",~o,.ellL 14. In Figure lA and other figures, the conductivebarrier 16 is shown, for convenience in depiction, separated from the second opposable component 14; however, during the operation of the device 10, the conductive barrier 16 is directly in contact with the second opposable component14. The conductive barrier 16 can be ~tt~chPcl to the second opposable component 14 by an edge of the conductive barrier 16.

The first opposable component 12 includes a sample plepaldtion zone 18. The second opposable component 14 contains a chromatographic me-lillm 20. The chromatographic m~ m 20 has a first end 22 and a second CA 0222ll2~ l997-ll-l3 W096/38727 PCTrUS96/07663 end 24; the chromatographic medium 20 contains a detection zone 26 and a control zone 28. The first opposable component 12 and the second opposable component 14 are joined by a hinge 30. The first and second opposable components 12 and 14 preferably further comprise engagers that secure the first S and second opposable components in opposition. The engagers can comprise locks, such as locks 32 and 34 that are engaged when the first opposable component 12 and the second opposable component 14 are brought into opposition. The construction and dimensions of the locks 32 and 34 can be varied to exert the optimal degree of pressure on the opposable components 12 10 and 14. The degree of pressure that is optimal may depend on the thickn~s.c and construction of the chromatographic m~ lm 20, the intended sample volume, and other factors. To guard against leakage of samples or reagents, a sealing ridge or gasket 36 iS positioned around the perimeter of the first and second opposable components 12 and 14. Although the use of the engagers, such as the 15 locks 32 and 34, and the use of the sealing ridge or gasket 36, iS generally ~cr~llcd, these components are not nt~cess~ry to construct a basic device according to the present invention. The second opposable component 14 has a first window 38; optionally, the first opposable component 12 can have a second window 40 to permit viewing of the chromatographic meAinm 20 from either 20 side. The second window 40 perrnits viewing of the chromatographic me~ m 20 from the surface opposite the surface to which the reagents are applied. As another option, the first window 38 can be absent and the second window 40 used for viewing of the chromatographic medium 20. Alternatively, the first and/or second opposable components 12 and 14 can be made of L-dl~l.a.c-l~ or 25 translucent materials, so that the chromatographic m~l1illm 20 can be viewed without a separate aperture or window.

Figure lB shows the device 10 after the opposable components 12 and 14 have been brought into opposition. The chromatographic met1ium 20, 30 including the detection zone 26 and the control zone 28, is visible through window 38. The flow from the sample preparation zone 18 through the conductive barrier 16 contacts the chromatographic me-1illm 20 at or near the CA 0222ll2~ l997-ll-l3 W096/38727 PCTrUS96/07663 first end 22 SO that the contents of the sample ~lcpald~ion zone 18 can flow through the chromatographic m~ m 20, including the detection zone 26 and the control zone 28.

The device 10 can, optionally, further comprise a conductor 42 in operable contact with the first end 22 of the chromatographic m~ m 20, as shown in Figure lA. The conductor 42 can be a material such as cellulose or other material that can conduct a liquid without substantially absorbing it. Theconductor 42 can be treated with a surfactant so that the reagents can be applied more evenly to the chromatographic m~ lm 20. When the conductor 42 is present, the conductive barrier 16 preferably contacts the conductor 42 when thefirst and second opposable components 12 and 14 are brought into opposition.

Figure lC shows, in greater detail, a side view of the device 10 with a conductor 42 after the first and second opposable components 12 and 14 have been brought into opposition, showing the approximate dimensional relationships of the sample ~ aldlion zone 18, the conductive barrier 16, and the conductor 42. Typically, the edge of the conductive barrier 16 extends past the edge of the sample ~l~aldLion zone 18, and, in turn, the edge of the conductor 42 extends past the edge of the conductive barrier 16. This is to minimi7e or elimin~te ullwdll~d flow of reagents past these edges. The arrow in Figure lC in~ .t~s the flow path of fluid through the sample pl~dlation zone 18, the conductive barrier 16, and the conductor 42.

The device 10 can further comprise an absorber 44 in operable contact with the second end 24 of the chromatographic mf~ m 20 to aid in drawing fluid through the chromatographic mt~-lillm 20 from the first end 22 - toward the second end 24, as shown in Figure lA.

The sample preparation zone 18 can be made of any suitable material, such as, but not limited to, cellulose, paper, nylon, rayon, glass fiber, fleeces, or non-woven synthetic fabrics. The porosity of the sample ~le~aldLion CA 0222112~ 1997-11-13 zone 18 can be chosen to filter out cellular or particulate matter in samples such as whole blood or fecal samples. The sample ~l~aldLion zone 18 can contain at least one reagent for treatment of the sample before the sample is applied to the chromatographic m~itlm 20 through the conductive barrier 16. The sample 5 preparation zone 18 is adapted to receive a liquid sample. As used herein, theterm "liquid sample" is defined to mean a sample having sufficient liquid so that chromatography can be performed, and includes semisolid samples or samples cont~ining particulate matter.

The reagents that can be present in the sample preparation zone 18 vary with the sample to be applied to the sample preparation zone 18 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 15 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 ~ Lul~ of sodium nitrite and acetic acid to generate nitrous acid. The sodium nitrite can be present in dried form on the sample preparation zone 18, and the acetic acid can be added to the sample pl~paldlion zone 18 after the 20 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 ~elator on the sample ~l~aldLion zone 18; if nPe-le(1, other reagents can be added.
The bodies of the first and second opposable components 12 and 14 are preferably made of l~ l 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 30 also be used. AlLelll-dLively, the bodies can be made of plastic that is impervious to moisture. A suitable plastic is a polycarbonate plastic such as Lexan~.

CA 0222112~ 1997-11-13 The hinge 30 is preferably made of material that is impermeable to a liquid, such as a plastic that can be compatibly joined with or is the same asthe material used for the bodies of the first and second opposable components 12and 14.
Typically, the chromatographic m~ m 20, absorber 44, conductor 42, conductive barrier 16, and other liquid-receiving components are secured to the bodies of the first and second opposable components 12 and 14 by adhesive. Suitable adhesives are well known in the art. Other joining methods, 10 such as stapling or tacking, can also be used.

The analyte is ~lPtecte(l 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 that itself is 15 unlabeled. In most cases, the use of a labeled specific binding partner to the analyte is ~l~r~llc~d. The label of the labeled specific binding partner is preferably a visually clet~ct~ble label, such as a colloidal metal label, as described above.

AlL~ aLiv~:ly, other colloidal labels, such as a colloidal sulfur label or a dye-silica label, can also be used. In a less ~l~r~ d all~".~ e, the visually detect~ble label can be a colored latex label. It is also possible to use other labels, such as radioactive labels.

Although Applicant does not nPcess~rily intend to be bound by this theory, when a liquid cont~ining 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 subst~3nti~lly complete labeling of analyte before the combination of the analyte and the labeled specific binding partner is applied to the chromatographic medium 20 through theconductive barrier 16. Thus, in a one-directional chromatographic procedure CA 0222ll2~ l997-ll-l3 W 096/38727 PCTrUS961n7663 performed with an assay device according to the present invention, what is chromatographed is predomin~ntly the binary complex of the analyte and the corresponding labeled specific binding partner. This allows separation of this complex frorn cont~min~nt~ not binding the specific binding partner and improves5 accuracy of the assay.

In this embodiment, the labeled specific binding partner preferably is present on the sample plc~ala~ion zone 18 in a form that can be resolubilizedby the addition of a liquid to the sample ~lc~aldLion zone 18. Typically, the 10 liquid is an aqueous liquid. Typically, the 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 liquid to the sample preparation zone.

In other emboAimf nt~ Ai~c~l~seA below, the labeled specific binding 15 partner can be present on an element of the chromatographic assay device that is separate from the sample plcl)al~lion zone but comes into contact with it duringthe performance of the assay. In these embo~lim~nt~, 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 liquid, distinct from the sample, to the element.

The chromatographic m.-Aillm 20 on the second opposable component 14 iS a flat strip. It is typically rectangular, having first and second 25 ends 22 and 24. Throughout this Description, the term "first end" 22 refers to the end of the chromatographic m~Aillm 20 at which the sample is applied, and the term "second end" 24 refers to the opposite end. The direction of flow of the sample during the performance of the assay is from the first end 22 toward the second end 24 of the chromatographic m~Aillm 20. The chromatographic mt?(lillm 20 iS composed of a material suitable as a medium for thin-layer chromatography of analytes and analyte-antibody conjugates as described above.

CA 0222112~ 1997-11-13 W~ 96138727 PCTIUS96/07663 In some applications, it is preferable to place a second flexible Lld~ls~a-c~L support on the top of the chromatographic medium 20 to regulate theflow of the sample through the membrane and prevent migration over the top of the membrane. Suitable flexible transparent supports include polyethylene, vinyl, 5 Mylar~9, and cellophane.

When the chromatographic assay device 10 is to be used for an assay such as a sandwich immllnoassay, the chuomatographic medium 20 can further comprise a detection zone 26 subst~nti~lly smaller than the 10 chromatographic medium 20. This detection zone 26 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; covalent means are generally ~ler~ d.
If the analyte to be assayed is an antigen or hapten, the second specific binding 15 partner can be an antibody to the antigen or the hapten. AlL~lllalively, 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 20 can further comprise a control 20 zone 28 substantially smaller than the chromatographic m~ m 20, and separate from the detection zone 20. The control zone 28 can comprise analyte immobilized thereto non-diffusibly in order to bind labeled antibody that is notbound at the detection zone 26 by the formation of a ternary "sandwich"
complex. Any such antibody is bound by the immobilized analyte in the control 25 zone 28 and forms a tletect~hle zone or band at the control zone 28. 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 26 and the analyte in the control zone 28 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 mP~ lm CA 0222112~ 1997-11-13 20. In such cases, the control zone 28 can comprise an immobilized zone of antibody specific for the labeled anti-analyte antibody. For example, if the analyte is the Streptococcus A-specific carbohydrate, and the labeled antibody is rabbit IgG specific for Streptococcus A antigen, the control zone 28 can comprise 5 goat antibody to rabbit IgG. In such cases, to prevent complete capture of thelabeled anti-analyte antibody in the detection zone 26 at high analyte concentration and consequent disappearance of the labeled anti-analyte antibody from the control zone 28, it can be desirable to add labeled antibody not specific for the analyte and of a different species than the labeled anti-analyte antibody.
10 Such antibody can con~LiLuL~ immllnl logically indirrclelll immunoglobulin or an antibody to an analyte not found in the test sample. The control zone 28 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 15 discussed above, the sample ~ aldlion zone 18 can further contain a specific binding partner for the analyte labeled with a detectable label in a form that can be resolubilized by the addition of a liquid to the sample plcpaldLion zone 18.
The liquid can be the sample itself. The labeled specific binding partner can befreeze-dried or reversibly precipitated so that it is resolubilized and mobilized by 20 the addition of the sample to the sample pl~a.dLion zone. In this variation, it is not n--cess~ry to add a detection reagent to the sample p~cl)aldLion zone 18, as the detection reagent is autom~tir~lly generated by the addition of the sample to the sample pl~aldlion zone 18.

In another variation, the conductor 42 in operable contact with the first end 22 of the chromatographic m~ m 20 on the second operable component 14 can be replaced by an absorber 42a of finite capacity in operable contact. The absorber 42a is located so that it comes into contact with the conductive barrier 16 when the first 12 and second 14 opposable components are placed into opposition, to apply the sample to the absorber 42a. This may be useful in controlling the flow of sample into the chromatographic medium 20 so that the chromatographic m~-linm 20 is not overloaded.

CA 0222112~ 1997-11-13 In this version, the absorber 42a 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 12 and 14 are brought into ~ 5 opposition, applying the sample to the absorber 42a through the conductive barrier 16. The combination of the sample and the resolubilized labeled specificbinding partner then enters the chromatographic medium 20 at its first end 22.

The use of the conductive barrier in assay devices according to the present invention provides a more regular and reproducible process of chromatography, by preventing excessively high local concentrations of reagents and assuring that the flow of reagents into and Ihrough the chromatographic me~ m is substantially synch~ol~iG~d.

D. ~ltern~tive Arrangements of Two-Component Device The general principles of construction of the device shown in Figures lA, lB, and lC, above, can also be used to construct other devices according to the present invention. Details of these devices are given below.
1. Device with Sample Preparation Zone on First OPposable Component Another embodiment of a chromatographic assay device according 25 to the present invention is a device that incorporates a sample ~l~al~.Lion zone on the first opposable component, i.e., the component on which the chromatographic m.-(lillm 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. In this 30 emborliment~ a conductive barrier is ~tt~rhe~l to the first opposable component so that operable contact between the applicator and the sample preparation zone occurs through the conductive barrier.

CA 0222112~ 1997-11-13 W096/38727 PCTrUS96/07663 In this embodiment, bringing the first and second opposable components into opposition brings the applicator into contact with the sample preparation zone through the conductive barrier 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 ~lc~ald~ion zone and the chromatographic mt~ lm is achieved by having the sample plcpaldtion zone and the chromatographic m~rlillm 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 mt?~lillm.

The chromatographic medium is preferably constructed as 15 described above, with detection and control zones.

This embodiment of the assay device is shown in Figure 2. The chromatographic assay device 60 has a first opposable component 62 and a second opposable component 64. The first opposable component 62 includes a 20 sample pl~cpdldLion zone 66, a conductor 68 in operable contact with the sample c~ald~ion zone 66, a chromatographic m~lillm 70 having a first end 72 and a second end 74, and an absorber 76 in operable contact with the second end 74 of the chromatographic mt?tlillm 70. The chrvlllaLographic m~rlillm 70 contains a detection zone 78 and a control zone 80. A conductive barrier 82 iS z~ ht~l to 25 the first opposable component 62. The second opposable component 64 contains an applicator 84, preferably incorporating a labeled specific binding partner in a form that can be resolubilized. The first opposable component 62 and the second opposable component 64 are joined by a hinge 86. The second opposable component 62 contains a window 88 to allow viewing of at least a portion of the 30 chromatographic m~ lm 70. The first and second opposable components 62 and 64 have engagers such as locks 90 and 92, with a gasket 94 surrounding the firstand second opposable components 62 and 64.

CA 0222112~ 1997-11-13 In operation, a sample is applied to the sample pr~ald~ion zone 66, allowing operation of the device as described above.

2. Device Includin~ Two Separate Applicators on Same ~ S Opposable Component Yet another embodiment of a chromatographic assay device according to the present invention including two separate applicators on the same opposable component. These two applicators are not in operable contact until 10 they are bridged by a conductor on the opposing element when the elements arebrought into opposition, operable contact again being made through the conductive barrier.

This embodiment of the chromatographic assay device is shown in 15 Figure 3. The chromatographic assay device 100 has a first opposable component 102 and a second opposable component 104. The first opposable component 102 includes a ch.u-llaL~graphic m~ m 106 having a first end 108 and a second end 110, a conductor 112 in operable contact with the first end 108, and an absorber 114 in operable contact with the second end 110 of the chromatographic m~-lillm 106. The cl~lunl~lugraphic m~ lm 106 contains a detection zone 116 and a control zone 118. A conductive barrier 120 iS ~tt~f.hP-l to the first opposable component 102. The second opposable component 104 contains a first applicator (sample application pad) 122 and a second applicator(detector application pad) 124. The first applicator 122 and the second applicator 124 are not in operable contact until the first opposable component 102 and the second opposable component 104 are brought into opposition. When the first opposable component 102 and the second opposable component 104 are brought into opposition, the first applicator 122 is brought into operable contact with the conductor 112 through the conductive barrier 120 and the second applicator 124 30 is brought into operable contact with both the conductor 112 and the first end 108 of the ch.ulllatographic m~lillm 106 through the conductive barrier 120.
The overlap is typically several millimt-t~rs; i.e., enough to ensure transfer of CA 0222112~ 1997-11-13 fluid. This results in the first applicator 122 and the second applicator 124 being bridged by the conductor 112 so that the contents of the first applicator 122 and the second applicator 124 are applied to the chromatographic m~ lm 106 via the conductive barrier 120 and then via the conductor 112. The first opposable 5 component 102 and the second opposable component 104 are joined by a hinge 126. The second opposable component 104 contains a window 128 to allow viewing of the chromatographic medium 106. The first and second opposable components 102 and 104 also include engagers such as locks 130 and 132 and a gasket 134.
The first applicator 122 can comprise a sample application pad and the second applicator 124 can comprise a detector application pad, to which cletecting reagent can be applied. The term "detector application pad", as used herein, refers to a component that contains a detection reagent, typically a 15 labeled specific binding partner for the analyte. The use of the term "detector application pad" is not intem1e(1 to imply that detection occurs in this component;
typically, detection occurs in the detection zone of the chromatographic m~flinm Preferably, the second applicator 124 (detector application pad) 20 contains a specific binding partner for the analyte labeled with a lietect~ble label in a form that can be resolubilized by the addition of a liquid to the second applicator 124. The liquid is typically the sample itself, which resolubilizes the labeled specific binding partner when the first 102 and second 104 opposable components are brought into opposition. In some assays, it may be desirable to 25 add a separate reco~ liquid to the detector application pad. Alternatively, the labeled specific binding partner can be applied in liquid form to the secondapplicator 124.

CA 0222112~ 1997-11-13 WC) 96138?27 PCT/US96/07663 3. Device with Pad for Labeled Specific Bindin~ Partner on Same Opposable Component as Chromato~raphic Medium Yet another embodiment of a chromatographic assay device ~ 5 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 m~ m. 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 4A. The chromatographic assay device 140 has a first opposable component 142 and a second opposable component 144. The first opposable component 142 has a chromatographic m~-lillm 146 having a first end 148 and a second end 150. The chromatographic m~ lm 146 has a detection zone 152 and, optionally, a control zone 154, as described above for other variations of assay devices suitable for sandwich i~...,~...,o~.c~ys. The first opposable component 142 also has a conductor 156 in operable contact with the first end 148 of the chromatographic mrflillm 146, andan absorber 158 in operable contact with the second end 150 of the 20 chromatographic m~lillm 146. The first opposable component 142 also has a detector application pad 160 in direct contact with the conductor 156 and positioned such that it is in indirect contact with the first end 148 of the chromatographic mrtlillm 146. ~ r~ l to the first opposable component 142 is a conductive barrier 162. The second opposable component 144 has a sample 25 application pad 164. The first opposable c~,lllpol~llL 142 and the second opposable component 144 are joined by a hinge 166. When the first opposable component 142 and the second opposable component 144 are brought into opposition, the sample application pad 164 is brought into operable contact withthe detector application pad 160 through the conductive barrier 162. The second 30 opposable component 144 contains a window 168 to allow viewing of the chromatographic mr~ lm 146. The first and second opposable components 142 CA 0222112~ 1997-11-13 and 144 have engagers such as locks 170 and 172, and a gasket 174, as described above.

A sectional rear view of the device 140 is depicted in Figure 4B.
5 The section shown in Figure 4B is taken from the view of Figure 4A, between the chromatographic mP~ m 146 and the hinge 166 looking toward the edge opposite the hinge 166. Figure 4B shows the first opposable component 142 and second opposable component 144 in opposition, with the conductive barrier 162 between them. The sample application pad 164 is shown in indirect contact with 10 the detector application pad 160 through the conductive barrier 162. The detector application pad 160 is in contact with the conductor 156, which is in turn in contact with the first end 148 of the chromatographic mPriillm 146. The detection zone 152 and control zone 154 of the chromatographic mPtlillm 146 are shown. The second end 150 of the chromatographic m~ lm 146, nearer the lS control zone 154, is in contact with the absorber 158. The arrow in Figure 4Bin(li~tes the flow path of fluid through the sample application pad 164, the conductive barrier 162, and the detector application pad 160.

Bringing the first and second opposable components 142 and 144 20 into opposition causes the sample application pad 164 to apply the sample to be tested to the detector application pad 160 through the conductive barrier 162 and thus to the first end 148 of the clll;ollla~graphic medium 146 though the conductor 156.

Preferably, the detector application pad 160 contains a first specific binding partner to the analyte in a form that can be resolubilized by addition of a liquid to the detector application pad 160, and the first specificbinding partner is labeled with a detectable label.

Preferably, the contents of the sample application pad 164 after a sample is applied thereto comprises a liquid, and the liquid applied to the detector application pad 160 comprises the contents of the sample application CA 0222ll2~ l997-ll-l3 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 - 5 opposable component instead of being located on the first opposable component.
The absorber is sepal~ted 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 m~ lm when the first opposable component and the second opposable component are brought into opposition. This variation of the 10 device is shown in Figure 5. In Figure 5, all element.c of the device are thesame as in Figure 4 except that the absorber 158 iS relocated to the second opposable component 144 and comes into operable contact with the second end 150 of the chromatographic m~ lm 146 when the first and second opposable components 142 and 144 are brought into opposition. This allows for the use of 15 a larger absorber. The advantages of a larger absorber include the ability to use a larger sample volume, so that dilute samples can be assayed. This can provide a greater dynamic range for the assay.

4. Device with Detector Application Pad in Direct Contact with First End of Chromato~raphic Medium A further variation of this device omits the conductor between the detector application pad and the chlulllatographic m~-lillm, so that the detector application pad is in direct contact with the first end of the chromatographic me-lillm In this variation, when the first and second opposable components are brought into opposition, the detector application pad and the sample applicationpad are in operable contact through the conductive barrier except for the regionof 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 30 detector application pad, so that sample carmot flow directly from the sampleapplication pad or conductive barrier to the chromatographic m~-1illm This gap CA 0222ll2~ lgg7-ll-l3 wo 96/38727 PCT/USg6/07663 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 5 occult blood by use of a labeled anti-hemoglobin antibody, without the occurrence of false negatives due to a high dose "hook" effect. It can also be used for other analytes.

This variation is depicted in Figure 6A. The chromatographic 10 assay device 200 has a first opposable component 202 and a second opposable component 204. The first opposable component 202 has a chromatographic medium 206 having a first end 208 and a second end 210. The chromatographic m~ lm 206 has a detection zone 212 and a control zone 214. The first opposable component 202 also has an absorber 216 in operable contact with the 15 second end 210 of the cl.l~,.l.aL~Jgraphic m~ lm 206. The first opposable component 202 also has a detector application pad 218 in direct contact with thefirst end 208 of the chromatographic m.Q-lillm 206. ~tt~h~cl to the first opposable component 202 is a conductive barrier 220.

The second opposable component 204 has a sample applir~ti~ n pad 222. The first opposable component 202 and the second opposable component 204 are joined by a hinge 224. When the first opposable component 202 and the second opposable component 206 are brought into opposition, the sample application pad 222 is brought into contact with the detector application pad 218 through the conductive barrier 220, except for a narrow gap or offset 226 at theend of the detector application pad 218 in contact with the first end 208 of thechromatographic medium 206. This gap 226 pl~vellL~ sample applied to the sample application pad 222 from flowing directly into the chromatographic mf~ m 206. The second opposable component 204 has a window 228 to allow viewing of the chromatographic m~ lm 206. The first and second opposable components 202 and 204 have engagers such as locks 230 and 232, and a gasket 234 as described above.

CA 0222112~ 1997-11-13 A sectional rear view of the device 200 of Figure 6A is depicted in Figure 6B. The section shown in Figure 6B is taken from the view of Figure 6A
between the chlolllalographic m~ lm 206 and the hinge 224 looking toward the edge opposite the hinge 224. Figure 6B shows the first opposable component ~ 5 202 and second opposable component 204 in opposition, with the conductive barrier 220 between them and with the hinge 224 in closed position. The sample application pad 222 is shown in indirect contact with the detector application pad 218, except for the small gap 226 at the end of the detector application pad 218nearest the clllulllatugraphic ,..~-lil.... 206. The detector application pad 218 is in 10 direct contact with the first end 208 of the chromatographic medium 206. The detection zone 212 and control zone 214 of the chromatographic medium 206 are shown. The second end 210 of the chromatographic medium 206, nearer the control zone 214, is in contact with the absorber 216.

5. 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 locatedon the first opposable component and a sample p,cpa,alion zone located on the 20 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 clllu",at~graphic m~ m. The detector application pad preferably contains a labeled specific binding partner to the analyte in a form that can be resolubilized by the addition of a liquid to the detector application pad. The device further comprises a 2~ conductor in operable contact with the detector application pad and in indirect contact with the first end of the chromatographic m~ m, as well as an absorber in operable contact with the second end of the chromatographic m~ lm.

In the operation of this embodiment of the device, the sample is 30 applied to the sample ~lcpaldlion zone on the second opposable component, after which the first and second opposable components are brought into opposition.
This applies the contents of the sample prcpaldLion zone to the conductor through CA 0222112~ 1997-11-13 the conductive barrier, 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 5 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 7. The assay 10 device 240 has a first opposable component 242 and a second opposable component 244. The first opposable component 242 includes a chromatographic mPrlillm 246 having a first end 248 and a second end 250. The first opposable component 242 also includes a detector application pad 252 in operable contact with the first end 248 of the chromatographic mPdillm 246, a conductor 254 in 15 operable contact with the detector application pad 252 and in indirect contact with the first end 248 of the chromatographic m~-lillm 246, and an absorber 256 in operable contact with the second end 250 of the chromatographic m~rlillm 246.The chlolllatographic mPrlillm 246 includes a detection zone 258 and a control zone 260. ~tt~hf~d to the first opposable component 242 is a conductive barrier 20 262.

The second opposable component 244 includes a sample c~dLion zone 264. The first and second opposable components 242 and 244 are joined by a hinge 266. The second opposable component 244 has an ape.L~llc 25 268 to permit viewing of at least a portion of the chromatographic m~-lillm 246.
The first and second opposable components 242 and 244 include engagers 270 and 272, and a gasket 274 as described above.

A variation of this embodiment incorporates a specific binding 30 partner for the analyte in a form that can be resolubilized on the second opposable component 244 as well as on the first opposable component 242.
When the resolubilizable specific binding partner is located on the second CA 0222112~ 1997-11-13 opposable component 244, it is preferably not located directly in the sample preparation zone 264 itself. Rather, it preferably surrounds the sample ~-~aldLion zone 264 in an area 276 such that the sample first passes through thesample plepa~dLion zone 264 and then moves into the area 276 ~ulloullding the 5 sample preparation zone 264, resolubilizing the specific binding partner. For example, the sample plepal~Lion zone 264 can comprise a piece of suitably treated filter paper placed on the surface of the second opposable component 244, adhered by an adhesive or a fastener. This allows for tre~tment of the sample, e.g., to adjust the pH, lyse intact cells, and/or remove partir,nl~tes, before the 10 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 ,t~ct~

6. Device with Two-Sector Applicator to Provide Wash Another embodiment of a two-component assay device according to the present invention has a two-sector applicator to provide a wash of sampleunreacted with the labeled specific binding partner after the llli~LUle of the 20 sample and the labeled specific binding partner has passed through the c'~ulllaLugraphic mr(lillm This embodiment has the advantage of providing a clearer background and making it easier to read a weakly positive result.

In this embo~1im~nt, the first opposable component includes a 25 chromatographic mP(1ium having first and second ends, a conductor in operablecontact with the first end of the chromatographic m~rlillm, and an absorber in operable contact with the second end of the cnromatographic me~ lm The second opposable component includes an applicator divided into two sectors: a first sector cont~ining a labeled specific binding partner for the analyte in 30 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 indirect CA 0222ll2~ l997-ll-l3 W096/38727 PCTrUS96/07663 contact with the conductor through the conductive barrier, to apply the contentsof the first sector of the applicator to the conductor and then to the first end of the chromatographic m~ m The second sector is placed in indirect contact with the conductor, as the contents of the second sector flow through the first S sector and then to the conductor via the conductive barrier. 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 in~ lcles sample but no labeled specific binding partner, serve to wash out unbound labeled specific binding partner from10 the chromatographic medium, thereby reducing the background of visible label seen in the chromatographic medium and 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 8. The 15 assay device 300 has a first opposable component 302 and a second opposable co~ ol~llL 304. The first opposable component 302 includes a chromatographic mP~illm 306 with a first end 308 and a second end 310, a conductor 312 in operable contact with the first end 308 of the chromatographic mPdillm 306, and an absorber 314 in operable contact with the second end 310 of the 20 cl~olllatographic medium 306. ~tt~hPd to the first opposable component 302 iS a conductive barrier 316.

The second opposable component 304 has an applicator 318 divided into two' sectors: a first sector 320 in direct contact with the conductor 25 312 when the first and second opposable components 302 and 304 are brought into opposition, and a second sector 322 in indirect contact with the conductor 312 when the first and second opposable components 302 and 304 are brought into opposition.

30 - The chromatographic mP~ lm 306 has a detection zone 324 and a control zone 326. The first and second opposable components 302 and 304 are joined by a hinge 328. The second opposable component 304 has a window 330 CA 0222112~ 1997-11-13 WO 96138727 PCTtUS9610'7663 to permit viewing of at least a portion of the chromatographic m~ m 306. The first and second opposable components 302 and 304 also include engagers 332 and 334 and a gasket 336, as described above.

~ 5 7. Device with Two Detector Application Pads on Dirre Opposable Components Yet another embodiment of a two-component assay device according to the present invention incorporates two separate detector application pads on dirr~l~n~ opposable components. This arrangement is particularly useful when it is desired to use a relatively large volume of a labeled specific binding partner, as when a labeled antibody is only available in dilute form and ~LL~ L~to concentrate the antibody would denature or inactivate it.

This embodiment of the two-component chromatographic assay device is depicted in Figure 9. The chromatographic assay device 350 has a firstopposable component 352 and a second opposable component 354 conn~c~e~l by a hinge 356. The first opposable component 352 includes a cl.,~,ll,atographic medium 358 having a first end 360 and a second end 362. The chromatographic medium 358 includes a detection zone 364, and, optionally, a control zone 366.

The first opposable component 352 also has a first detector application pad 368 in operable contact with the first end 360 of the chromatographic m~ lm 358. The first detector application pad 368 contains a first specific binding partner to the analyte in a form that can be resolubilized by the addition of a liquid to the first detector application pad 368. The first specific binding partner is typically labeled with a detectable label. The firstopposable component 352 also has a conductor 370 in operable contact with the first detector application pad 368 so that the first detector application pad 368 bridges the conductor 370 and the first end 360 of the chromatographic m~ m 358. The first opposable component 352 also has an absorber 372 in operable CA 0222112~ 1997-11-13 W096/38727 PCTrUS96107663 contact with the second end 362 of the chromatographic medium 358. ~tt~rh~d to the first opposable component 352 is a conductive barrier 374.

The second opposable component 354 includes a sample S prepaldtion zone 376 for receiving a sample to be assayed. The second opposable component 354 also contains a second detector application pad 378 in operable contact with the sample preparation zone 376, with the sample preparation zone 376 being placed over the second detector application pad 378.
The sample preparation zone 376 and the second detector application pad 378 can 10 be held together by a fastener or adhesive. The second detector application pad 378 and the sample ~lc~aldlion zone 376 are positioned so that a sample applied to the sample preparation zone 376 must pass through the sample preparation zone 376 before entering the second detector application pad 378. The second detector application pad 378 contains a second specific binding partner for the 15 analyte in a form that can be resolubilized by the addition of a sample to the sample l!lc~alalion zone 376. The second detector application pad 378 iS
positioned such that application of the sample to the sample ~r~dldLion zone 376resolubilizes the second specific binding partner so that the sample ~lc~aldlionzone 376 contains a mixture of the sample and the second specific binding 20 partner.

The second specific binding partner is labeled with a detect~hle label. Preferably, the first and second specific binding partners are iriPnti~l and the ~l~t~ct~hle labels labeling the first and the second specific binding partners are 25 identical.

The second opposable component 352 also contains an aperture 380 therein to allow viewing of at least a portion of the chromatographic m~
358, including the detection zone 364 and, if present, the control zone 366. The30 first and second opposable components 352 and 354 also have engagers such as locks 382 and 384, and a gasket 386 as described above for the basic two-component device including a conductive barrier.

CA 0222ll2~ l997-ll-l3 WO 96/38727 PCT~US96/07663 When the first and second opposable components 352 and 354 are brought into opposition, the sample pl~dldLion zone 376 iS brought into contact with the conductive barrier 374 to apply the sample and the second specific binding partner to the conductor 370 and then to the first end 360 of the ~ 5 chromatographic medium 358 through the first detector application pad 368, via the conductive barrier 374. Thus, the sample sequentially contacts the second specific binding partner and then the first specific binding partner before being applied to the first end 360 of the chromatographic medium 358 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.

E. Multiplex AssaY Devices The description of the devices above is directed to assay devices that perform one assay at a time. However, assay devices according to the present invention can also be constructed that can perform multiple assays at the same time. The assays can be yclro~ ed for the same analyte or di~ferent analytes. In general, all versions of the device described above are suitable for multiplex use by providing first and second opposable components with multiple chromatographic media, sample ~lcpa.dlion zones, conductive barriers, applicators, conductors, absorbers, and other required elements. The following descriptions of multiplex devices according to the present invention are therefore inten~ rl to be exemplary and not exclusive.

1. Basic Multiplex Device One version of a multiplex assay device according to the present invention is shown in Figure 10. The assay device 400 has a first opposable component 402 and a second opposable component 404. The second opposable 30 component 404 iS hingedly ~tt~ch~ll to the first opposable component 402 by ahinge 406. The first opposable component 402 comprises a plurality of chromatographic media 408. Each of the chromatographic media 408 has a first CA 0222112~ 1997-11-13 end 410 and a second end 412, and includes a detection zone 414 and a control zone 416. The second end 412 of each chromatographic medium 408 is in operable contact with an absorber 418 to drive flow through the chromatographic m~ lm 408. There is a separate absorber 418 for each chromatographic 5 medium 408. Attached to the first opposable component 402 is a plurality of conductive barriers 420. The conductive barriers 420 can be physically separate;alternatively, they can be combined in one single structure and functionally isolated by substantially impermeable barriers.

The second opposable component 404 comprises a plurality of sample pl'cl)aldlion zones 422, one for each chromatographic medium 408.
Typically, each sample preparation zone 422 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 ~lcpaldLion zone 422. Alternatively, 15 the labeled specific binding partner in a liquid form can be added to the sample preparation zone 422 before or after the addition of the sample thereto. Bringing the first and second opposable components 402 and 404 into opposition causes each of the sample plc~aldlion zones 422 to be applied to the corresponding chromatographic m~-1illm 408 at the first end 410. The second opposable 20 component 404 contains a plurality of a~clLulcs 424, one for each chromatographic m~rlillm 408. The first opposable component 402 and second opposable component 404 include engagers 426 and 428 and a gasket 430, as described above.

This multiplex device can contain from 2 to 12 or more sample c~dl~llion 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 ~lirrelcnl analytes iri dirrelcl~l aliquots of the same sample, or can be used to assay the same analyte in a number of dirrelellL samples. This latter mode is CA 0222112~ 1997-11-13 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 deL~ ed by means of a series of stool samples taken once a day or at other ~ 5 intervals for a prescribed period. Alternatively, one or more of the assays can be used for controls or reference standards.

2. Multiplex Device with Collapsible Well In yet another variation of the multiplex device, at least one sarnple preparation zone can comprise a collapsible well, to which an extractionswab or other sample-conf~ining device can be added.

This variation of the multiplex device is shown in Figure llA.
The device 440 has a first opposable component 442 and a second opposable component 444. The second opposable component 444 is hingedly attached to the first opposable component 442 by a hinge 446. The first opposable component 442 has a control well 448 and a collapsible sample well 450, i.e., made of a sponge-like material. The second opposable component 14'1 has a plurality of laterally s~alaL~d chromatographic media 452, in this example, two,each with a first end 454 and a second end 456. Each of the chromatographic media 452 has a detection zone 458 and a control zone 460. Attached to the second opposable component 444 is a plurality of conductive barriers 462, one for each chromatographic m~r~ m 452.
The first opposable component has an aperture 464 for viewing of a portion of each of the chromatographic media 452, including the detection zone458 and the control zone 460. The first and second opposable components 442 and 144 include engagers 466 and 468 and a gasket 470. When the first opposable component 442 and the second opposable component 444 are opposed, samples in the control well 448 and the collapsible sample well 450 are applied CA 0222112~ 1997-11-13 to the corresponding chromatographic media 452 through the conductive barriers 462 for chromatography.

If a collapsible well is included, the first opposable component can 5 include, in place of the gasket 470, hingedly foldable wings 472 that fold over the second opposable component when the first opposable component and second opposable component are brought into opposition.
This version of a multiplex assay device according to the present invention is shown in Figure llB.
In this and other embodiments of the present invention that are adapted to the use of a swab, the device typically can accommodate a wide variation in the quantity of liquid applied to the swab, so that oversaturation of the swab is not deleterious. The device is (lesign~ so that liquid that migrates15 into the perimeter area surrounding the swab does not hllelr~le with the operation of the device.

3. Multiplex Device Adapted to Receive Test Card Yet another variation of the multiplex device is particularly useful for cletPrmin~tion of hemoglobin 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 variation is shown in Figure 12. The assay device 480 has a first opposable component 482 and a second opposable component 484. The second opposable component 484 is hingedly ~tt~chP~l to the first opposable component 482 by a hinge 486. The first opposable component 482 includes a plurality of laterally separated reagent pads 488, i.e., applicators. Preferably, each applicator 488 contains labeled specific binding partner for the analyte inresolubilizable form.

CA 0222112~ 1997-11-13 The second opposable component 484 includes a chromatographic medium 490 for each reagent pad 488 on the first opposable component 482.
The chromatographic media 490 are laterally separated. Each of the chromatogr~phic media 490 has a first end 492 and a second end 494, and ~ S comprises a detection zone 496 and a control zone 498. The first end 492 of each chromatographic medium 490 is in operable contact with a conductor 500, and the second end 494 of each chromatographic medium 490 is in operable contact with an absorber 502. There is a separate conductor 500 and absorber 502 for each chromatographic mP~ lm 490. The second opposable component 484 is adapted to receive a test card 504 cont~inin~ a plurality of dried specimens 506 positioned so that they are in operable contact with each conductor 500, such as by a recess 508 in the second opposable component 484.

~tt~ Prl to the second opposable component 484 is a plurality of conductive barriers 510, one for each chromatographic mPtlinm 490.

The second opposable component 482 contains a plurality of apertures 512, one for each cllrol,latographic mP~ m 488, for viewing of each chromatographic mP~ m 488. The first and second opposable components 482 and 484 each includes engagers 514 and 516 and a gasket 518 to retain samples and reagents.

In use, a buffer or other liquid is applied to each applicator 488 to reconstitute the labeled specific binding partner. Bringing the first and secondopposable components 482 and 484 into opposition causes each of the applicators 488 to be applied to the corresponding dried specimen 506 so that the contents of each dried specimen 506 and each applicator 488 are applied to each conductive barrier 510, and then to the conductors S00, and thus to the first end 492 of each chromatographic mP~ lm 490. The test card 504 holds each of the specimens 506 in posltion so that they can receive the contents of the applicators 488, and so that analyte in the specimens 506 is extracted, reacts with the labeled specific CA 0222112~ 1997-11-13 binding partner, and is applied to the conductive barriers 510 and then to the conductors 500.

Still other variations of test devices according to the present 5 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.

II. ANALYTES AND ANTIBODIES FOR USE WITH ASSAY DEVICES

The analytes suitable for detection with an assay device according to the present invention include antigens, haptens, and antibodies. Antigens 15 ~let(~ct~ble 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 ~E~ and to viruses, including HIV. Haptens ~letect~hle include haptens to 20 which antibodies of sufficient specificity can be prepared.

Two antigens for which devices according to the present invention are particularly suitable are human hemoglobin and Streptococcus A antigen.
The detection of human hemoglobin is clinir~lly ~ignifir~nt, because the presence 25 of human hemoglobin in fecal material is a marker of int-?stin~l or rectal bleeding, which is indicative of the presence of cancer in the gastroint~stin~l system or other pathogenic conditions. The detection of Streptococcus A antigen is also clinically significant, because streptococcal infections are fast-moving and can be life-Ll.lc:atemllg.
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.

CA 0222112~ 1997-11-13 WO 96/38727 PCT~IJS96~07663 In many applications, it is preferable that the first and second specific binding partners are antibodies to dirr~lcllt 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 plerellcd, as their 5 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 ~lcfcll~;;d that the first and second specific binding partners be 10 antibodies to dirrelell~ epitopes; otherwise, there may be an undesirable competition reaction set up that may h~clrclc with binding of the complex of thelabeled specific binding partner and the analyte to the immobilized second specific binding partner. It is recognized that not all haptens are large enough to accommodate more than one epitope; however, some haptens, though not large 15 enough to induce antigen formation efficiently when injected by themselves, are nevertheless large enough that they possess more than one epitope. In cases where antibodies to more than one epitope of a hapten cannot be obt ined, col"l,eLi~ e assay procedures are generally plcr~llcd.

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 plcrt;llcd that the first specific binding partner to an antibody analyte binds to the constant region of the antibody analyte, in order to prevent hlLclrt;lcllce. 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 ~lrst specific binding partner can be labeled.
Typically, the labeled secondary specific binding partner binds to the antibody CA 0222112~ 1997-11-13 that is the first specific binding partner on the basis of species, class, or subclass specificity.

As an alLelllaliv~ to the use of a secondary specific binding 5 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 immnnoassays are ~ullllllaliG~d in Table I below.

W~ 96~38727 PCT/~JS96/07663 TABLE I
SCHEMES OF BINDING FOR SANDWICH IMMUNOASSAYS

lST SBP 2ND SBP SECONDAR
ANALYT (MOBIL(FIXED) Y COMPLEX FORMED
E E) SBP
Ag Abl* Ab2 --- Ab2-Ag-Ab,*
H Abl* Ab2 --- Ab2-H-Ab,*
Ab Abc* Ag Ag-Ab-AbC*
Ag Abl Ab2 Abc* Ab2-Ag-Abl-Abc*
Ab Abcl Ag Abc2* Ag-Ab-Abcl-Abc2*
Ag Ab,-Bi Ab2 Av-L Ab2-Ag-Ab,-Bi-Av-L
Ag = Antigen H = Hapten Ab = Antibody Abl = 1st Antibody Ab2 = 2nd Antibody AbC, AbCl, AbC2 = Antibody specific for another antibody Bi = Biotin Av = Avidin L = Label *Indicates labeled component (1) Ab2 and Abl* preferred to bind to different epitopes;' - not all haptens possess such different epitopes.

CA 0222112~ 1997-11-13 III. 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 nPcess~ry reagents required to treat or extract the sample; and (3) optionally, if the assay device does not incorporate a 10 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, cryst~lli7Pcl, precipitated, or 15 aggregated). If the latter, they are resolubilized by the user, typically with distilled or purified water, with physiological saline, or a buffer solution.

The reagents required to treat or extract the sample are those described above. In some cases, such reagents can interact with a reagent 20 incorporated in the device in resolubilizable form. An example is the generation of nitrous acid by the reaction of sodium nitrite with acetic acid or another weak acid.

In some cases, test kits can also include a recolls~i~u~ion fluid for a 25 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.

The invention is illustrated by the following Examples. The 30 Examples are for illustrative purposes only and are not to be construed as limiting the scope of the invention in any manner.

CA 0222112~ 1997-11-13 EXAMPLES

Example 1 Construction of Device for Detectin~
Streptococcal Anti~en A device was constructed for detecting Streptococcus A antigen using labeled antibody to Streptococcus A antigen. The device was constructed 10 essentially as depicted in Figure 13.

Figure 13 shows a chromatographic assay device 540 according to the present invention with a first opposable cu,l,~ollc;"~ 542, and a second opposable component 544 hingedly :~tt~ let1 to the first opposable component 54215 by a hinge 545. The first opposable component 542 inrllldes a chlo",atographic me~ lm 546 with first and second ends 548 and 550, a detection zone 552, and a control zone 554. The first opposable component 542 also includes a zone of resolubilizable anti-Streptococcus A antibody 556 (detector application pad) adjacent to the first end 458 of the chromatographic m~rlillm 546. The first 20 opposable component also includes an absorber 558 adjacent to the second end 550 of the chromatographic mP~ lm 546. ~tt~C~ to the first opposable component 542 is a conductive barrier 560. The second opposable component 544 includes a well 562 that can receive a throat swab. The well 562 is covered with an impermeable barrier 564 that has two apertures, one at each end of the 25 well, a first aperture 566 for insertion of the swab and a second aperture 568 for application of the extracted sample to the conductive barrier 560. The first opposable component 542 contains a window 570.

The opposable componerlts were made of a hard, impervious 30 plastic such as polycarbonate. The first and second opposable components eachwere about 3" in length, the first opposable component was about 2.25" in width,while the second opposable component was each about 2.375" in width. The CA 0222112~ 1997-11-13 second opposable component was lined with a foarn rubber receptacle, into which a ~aldlop-shaped well was cut to accept a swab or other sampling device.

The chromatographic mt~ lm was a nitrocellulose strip of 8 ,~4m S pore size and 0.5" in length, (MSI, Westborough, Mass.), affixed to the plastic backing by means of-double-sided tape (3M, Minneapolis, Minnesota). The conductive barrier and absorber were cellulose strips (Ahlstrom Filtration, Holly Springs, Pennsylvania), 17/32" in length for the absorber, which was Ahlstrom Grade 939, and 0.25" in length for the conductive barrier, which was Ahlstrom 10 Grade 1281. The detector application pad was also Ahlstrom Grade 1281, and was 0.375" wide. The chromatographic m~ lm overlapped slightly at its second end with the absorber.

The required reagents were first incorporated in the 15 chromatographic m~ lm and the zone of resolubilizable antibody, 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 20 at 2 mg/ml in 0.001 mole/l phosphate buffered saline, pH 7.2. The control zone comprised goat anti-rabbit IgG at a similar concentration in the same buffer. The antibody solutions were applied to the ~lu~liate regions of the chromatographic m~rlillm and dried at 100~F in a low hllmi(iity environment. The chromatographic m~llillm was wet in excess blocking solution (Blocking Reagent 25 for ELISA, Boehringer Mannheim, Mannheim, Germany, diluted 1:10 with distilled water cont~ining 0.2% Tween 20) and again dried at 100~F.

The detector application pad contained rabbit anti-Streptococcus antibody labeled with 40-nm colloidal gold particles. To apply the labeled 30 antibody to the detector application pad, the labeled antibody was diluted 1:1.5 with DBN (1.5 mole/l Tris-HCl, pH 7.4, 1% (v/v) Tween 20, 0.4% (v/v) Brij 35, 0.02% (w/v) sodium azide, 3 mg/ml rabbit IgG). Per test, 15 ,ul of diluted CA 0222ll2~ l997-ll-l3 labeled antibody was added to the detector application pad. The detector application pad was dried for 30 minutes at 100~F.

Example 2 Detection of Streptococcal Anti~en Usin~ Device of Example 1 The device of Example 1 was used to detect Streptococcus A
10 antigen. A woven dacron swab to which varying ql-~ntities 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/l sodium nitrite, 5 % Tween 20) were added to the swab, mixed by gently rotating the swab, and inrllbatPd for one minute. The 15 device was then closed, so that the first and second opposable components were brought into contact. The result was read after an incubation period of from 2 mimltec to 5 ,.,i"~ s. The development of a pink-red band in the detection zone of the chromatographic mP~linm in-lir~ted the detection of StrePtOCOCCUS A
antigen.
The device of Example 1 could detect 1 x 105 Streptococcus A
organi~m~ after a 2-minute inrllbation, and could detect 5 x 104 Streptococcus Aolg~l-i~lll~ after a 5-minute inrllb~tion. For a comr~ri~Qn, the ConciseTU
llulloassay of Hybritech (La Jolla, California) could detect 1 x 105 25 Streptococcus A organisms only after a S-minute inrllb~tion, and could not detect S X 104 Streptococcus A org~ni~mc even after a 20-minute inrl1b~tion. Similarly,the SmartsM immlln~assay of New Horizons could detect 1 x 105 Streptococcus A
org~ni~m~ only after a 7-minute incubation, and gave an equivocal result with 5 x 104 Streptococcus A org~ni~m~ after a 7-minute incubation.

CA 0222112~ 1997-11-13 Example 3 Device for Detectin~ Hemo~lobin in Fecal Occult Blood An assay device for the dçtçctiQn of hemoglobin in fecal occult blood was constructed according to Figures 4A and 4B, with details of materials and construction as in Example 1. A labeled specific binding partner was applied to the sample application pad in resolubilizable form. The labeled specific binding partner was goat anti-human antibody labeled with colloidal 10 gold. A fecal sample of 60 ,ul was applied to the sample application pad and allowed to mix with conjugate. The device was closed and the combination of the fecal sample and reco~ (i antibody contacted the conductor and moves through the chromatographic medium. Chromatography was allowed to proceed for a period of about 1 minute to about 5 mimltçc. The chromatographic 15 m~tlillm contained a detection zone of immobilized anti-human Hb antibody, and a control zone of immobilized rabbit anti-goat IgG antibody.

Color appearing at both the detection zone and the control zone intljr~t~s a positive result, i.e., the presence of occult blood in the fecal sample.
20 Color appearing at the control zone, but not at the detection zone, inrlir~tPc the absence of occult blood and the correct performance of the test.

This device is capable of dçtçcting hemoglobin in fecal occult blood in a collcellllalion range of from about 0.2 ml blood/100 g feces to about25 17 ml blood/100 g feces. This device is free from hllelr~,ellce caused by peroxidase and dietary (non-human) hemoglobin.

ADVANTAGES OF THE INVENTION
Chromatographic assay devices according to the present invention provide an advantage in being constructed of opposable elements. The use of CA 0222112~ 1997-11-13 opposable elements provides great versatility, as it permits the perform~nre 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 ~ S elements also provides Op~ ulll performance with minimnm consumption of reagents by ensuring that reagents are not wasted by being sequestered in dead volumes of al~dl~LuS. Finally, the use of opposable components provides opLilllulll cont~inment of possibly cont~min~t.o~l blood samples, each as those CC~ 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 plC~i:!iUlC: applied so that it is ~JIJLhllulll for each assay to be carried 15 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 rem~inin~ in components, allowing the use of smaller samp!es and smaller qn~ntities of expensive or hard-to-purify reagents such as labeled antibodies.
Yet another advantage of assay devices according to the present invention results from the use of the conductive barrier to control flow and prevent irregular flow or high local concentrations of a reactant in a specific binding reaction. This provides greater reproducibility and reliability in the 25 pelr~,llllallce of assays using assay devices according to the present invention.

Additionally, chromatographic assay devices according to the present invention allow the rapid and accurate detection of clinically importantanalytes, such as Streptococcus A and B antigen, hemoglobin for the 30 det~rmin~t-on 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 CA 0222112~ 1997-11-13 WO 96/38727 PCT~US96/07663 hl~elrelellce that might otherwise be introduced by particulates or colored samples. The use of colloidal metal labels in a resolubilizable form provides extremely rapid kineties of labeling and allows substantially complete formationof binary analyte-label complexes before the sample is applied to the 5 chromatographic medium. This aids in the separation of cont~min~nf~ 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 byassuring the withdrawal of excess immlln~globulin-cont~ining sample that could otherwise create hlL~lrt;lellce.
Extraction of biological samples such as blood, sputum, or feces can be performed directly in the devices, reducing the quantity of cont~min~tPclmaterial that must be disposed and reducing the likelihood of accidental infection of physicians, technicians, or the public by such co~ r~1 material.
15 Additionally, the devices are capable of performing bidirectional chromatography to further increase accuracy and reduce illL~lrelc;llce. 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 rerel~llce to certain ~l~r~lled versions thereof, other versions and embo-liment~ are possible. These versions include other arrangements of two- or three-component devices that operate by the basic 25 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 hlLelr~ ce. These versions include assay devices adapted for competitive immunoassays as well as sandwich 30 immlmo~ ys, 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 metlinm rather than linear flow. The present invention further encomp~ses 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 m~yn~ti~. forces or by using a separable fastener such as a hook-and-eye fabric,- S for example Velcros~. Therefore, the scope of the invention is determined by the following claims.

Claims (39)

We claim:
1. A chromatographic assay device comprising:
(a) a first opposable component including a sample preparation means adapted to receive a liquid sample to be assayed;
(b) a second opposable component including a chromatographic medium having at least one reagent binding specifically to an analyte to be detected, the reagent being bound at a detection zone on the chromatographic medium, the second opposable component being attachable to the first opposable component; and (c) a conductive barrier attached to the second opposable component;
wherein the first and second opposable components can be brought into opposition from a position in which they are not in opposition so as to cause the sample preparation means to apply the liquid sample to be tested to the chromatographic medium through the conductive barrier and to flow through the chromatographic medium, the chromatographic assay being performed as a result of migration of the sample within the chromatographic medium so that an analyte is detected within the chromatographic medium as the result of the migration by binding of a labeled reagent that binds specifically to the analyte to be detected, the analyte being detected at a position different than the position at which the sample is applied to the chromatographic medium, the analyte being detected on the chromatographic medium after migration by binding of the labeled reagent to the analyte bound to the detection zone.
2. The device of claim 1 wherein the sample preparation means includes at least one reagent for the treatment of the sample before the sample is applied to the chromatographic medium and wherein the reagent for treatment of the sample is an extraction reagent to extract analyte from the sample.
3. The chromatographic assay device of claim 1 wherein the detection zone is substantially smaller than the chromatographic medium and wherein the detection zone contains a first specific binding partner to the analyte immobilized thereto.
4. The chromatographic assay device of claim 3 wherein the analyte is an antigen or hapten and the first specific binding partner is an antibody to the antigen or hapten.
5. The chromatographic assay device of claim 4 wherein the analyte is human hemoglobin and the first specific binding partner is an anti-human hemoglobin antibody.
6. The chromatographic assay device of claim 3 wherein the analyte is an antibody and the first specific binding partner is a hapten or antigen capable of being bound specifically by the antibody.
7. The chromatographic assay device of claim 1 wherein the chromatographic medium further includes a control zone substantially smaller than the chromatographic medium.
8. The chromatographic assay device of claim 7 wherein the control zone contains analyte immobilized thereto.
9. The chromatographic assay device of claim 1 further comprising an absorbing means in operable contact with the second end of the chromatographic medium.
10. The chromatographic assay device of claim 1 wherein the sample preparation means further contains a specific binding partner for the analyte labeled with a detectable label in a form that can be resolubilized by the addition of a liquid to the sample preparation means.
11. The chromatographic assay device of claim 1 wherein the first and second opposable components can be brought into opposition by direct manual closure from a position in which they are not in opposition.
12. A test kit for the detection and/or determination of an analyte comprising, separately packaged:
(a) the chromatographic assay device of claim 1; and (b) a specific binding partner for the analyte labeled with a detectable label, the specific binding partner to be used with the chromatographic assay device.
13. A chromatographic assay device for detection and/or determination of at least one analyte comprising:
(a) a first opposable component including a plurality of laterally separated sample preparation means, each adapted to receive a sample to be assayed;
(b) a second opposable component attachable to the first opposable component and including a chromatographic medium for each sample preparation means on the first opposable component, the chromatographic media being laterally separated; and (c) a plurality of conductive barriers each attached to the second opposable component, one for each chromatographic medium;
wherein the first and second opposable components can be brought into opposition so as to cause each sample preparation means to apply each sample to be tested to the corresponding chromatographic medium through the corresponding conductive barrier.
14. The chromatographic assay device of claim 13 wherein each chromatographic medium further comprises a detection zone substantially smaller than the chromatographic medium and wherein each detection zone contains a first specific binding partner to the analyte to be assayed on that chromatographic medium.
15. The chromatographic assay device of claim 13 wherein at least one sample preparation means includes a collapsible well adapted for receiving a sample-containing device.
16. The chromatographic assay device of claim 15 wherein the first opposable component further includes hingedly foldable wings that fold over the second opposable component when the first opposable component and second opposable component are brought into opposition.
17. A test kit for the detection and/or determination of at least one analyte comprising, separately packaged:
(a) the chromatographic assay device of claim 13; and (b) a specific binding partner for each analyte to be tested, each specific binding partner labeled with a detectable label, each specific binding partner packaged separately, and each specific binding partner to be used with the chromatographic assay device.
18. A chromatographic assay device for the detection and/or determination of an analyte comprising:
(a) a first opposable component including:
(i) a sample preparation means; and (ii) a chromatographic medium in operable contact with the sample preparation means;
(b) a second opposable component attachable to the first opposable component including an application means containing a specific binding partner for the analyte labeled with a detectable label in a form that can be resolubilized by the addition of a liquid to the application means; and (c) a conductive barrier attached to the first opposable component;
wherein bringing the first and second opposable components into opposition brings the application means into contact with the conductive barrier such that the labeled specific binding partner for the analyte is resolubilized by liquid passing through the conductive barrier.
19. The chromatographic assay device of claim 18 wherein the first opposable component further comprises a conducting means, and operable contact between the sample preparation means and the chromatographic medium is achieved by having the sample preparation means and the chromatographic medium both in operable contact with the conducting means.
20. The chromatographic assay device of claim 19 wherein the chromatographic medium has first and second ends and the conducting means is in operable contact with the first end of the chromatographic medium, the first opposable component further including an absorbing means in operable contact with the second end of the chromatographic medium.
21. A test kit for the detection and/or determination of an analyte comprising, separately packaged:
(a) the chromatographic assay device of claim 18; and (b) at least one reagent required for extraction of the sample on the sample preparation means, at least one of the reagents to be applied to the sample preparation means.
22. A chromatographic assay device for detection and/or determination of an analyte comprising:
(a) a first opposable component including:
(i) a chromatographic medium having first and second ends;
(ii) a conducting means in operable contact with the first end of the chromatographic medium; and (iii) an absorbing means in operable contact with the second end of the chromatographic medium;
(b) a second opposable component comprising:
(i) a first application means; and (ii) a second application means; and (c) a conductive barrier attached to the first opposable component;
the first and second application means 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;
wherein bringing the first and second opposable components into opposition places the conducting means in operable indirect contact with the first application means through the conductive barrier and places the conducting means in operable contact with the second application means, thereby placing the first and second application means in operable contact with each other.
23. The chromatographic assay device of claim 22 wherein the first application means includes a sample application pad and the second application means comprises a detector application pad, to which detecting reagent can be applied, whereby, when the first and second opposable components are brought into opposition, the contents of the sample application pad and the detector application pad are applied to the conducting means throughthe conductive barrier.
24. A test kit for the detection and/or determination of an analyte comprising, separately packaged:
(a) the chromatographic assay device of claim 23; and (b) a specific binding partner for the analyte labeled with a detectable label, to be applied to the second application means of the chromatographic assay device.
25. A chromatographic assay device for the detection and/or determination of an analyte comprising:
(a) a first opposable component including:
(i) a chromatographic medium having first and second ends;
(ii) a conducting means in operable contact with the first end of the chromatographic medium;

(iii) an absorbing means in operable contact with the second end of the chromatographic medium; and (iv) a detector application pad in direct contact with the conducting means and positioned such that it is in indirect contact with the first end of the chromatographic medium;
(b) a second opposable component including a sample application pad; and (c) a conductive barrier attached to the first opposable component;
whereby bringing the first and second opposable components into opposition causes the sample application pad to apply the sample to be tested to the detector application pad through the conductive barrier and thus to the first end of the chromatographic medium though the conducting means.
26. A test kit for the detection and/or determination of an analyte comprising:
(a) the chromatographic assay device of claim 25; and (b) a specific binding partner for the analyte labeled with a detectable label, to be applied to the second application means.
27. A chromatographic assay device for the detection and/or determination of an analyte comprising:
(a) a first opposable component including:
(i) a chromatographic medium having first and second ends;
(ii) an absorbing means in operable contact with the second end of the chromatographic medium; and (iii) a detector application pad in direct contact with the first end of the chromatographic medium; and (b) a second opposable component including a sample application pad; and (c) a conductive barrier attached to the first opposable component;

wherein, when the first and second opposable components are brought into opposition, the detector application pad and the sample application pad are in indirect contact through the conductive barrier except for the region of the detector application pad directly adjacent to the first end of the chromatographic medium, and whereby bringing the first and second opposable components into opposition causes the sample application pad to apply the sample to be tested tothe detector application pad and thus to the first end of the chromatographic medium.
28. A test kit for the detection and/or determination of an analyte comprising, separately packaged:
(a) the chromatographic assay device of claim 27; and (b) a specific binding partner for the analyte labeled with a detectable label, to be applied to the second application means of the chromatographic assay device.
29. A chromatographic assay device for detection and/or determination of at least one analyte comprising:
(a) a first opposable component including a plurality of laterally separated reagent pads;
(b) a second opposable component adapted to receive a test card containing a plurality of dried specimens, the second opposable component including:
(i) a chromatographic medium for each sample preparation means on the first opposable component, the chromatographic media being laterally separated, each chromatographic medium having a first and a second end;
(ii) a conducting means in operable contact with the first end of each chromatographic medium and in operable contact with each dried specimen of the test card when the test card is inserted into the second opposable component; and (iii) an absorbing means in operable contact with the second end of each chromatographic medium; and (c) a plurality of conductive barriers each attached to the second opposable component, one for each chromatographic medium;
wherein the first and second opposable components can be brought into opposition so as to cause each reagent pad to be applied to the corresponding dried specimen through the corresponding conductive barrier.
30. The chromatographic assay device of claim 29 wherein each reagent pad includes 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 reagent to the reagent pad.
31. The chromatographic assay device of claim 30 wherein the analyte is human hemoglobin and the specific binding partner is an anti-human hemoglobin antibody.
32. A test kit for the detection and/or determination of an analyte comprising, separately packaged:
(a) the chromatographic assay device of claim 29;
(b) a test card for insertion into the chromatographic assay device that can contain a plurality of dried specimens; and (c) an extraction reagent for the analyte to be assayed, the extraction reagent to be applied to the test card.
33. A chromatographic assay device for detection and/or determination of an analyte in a sample comprising:
(a) a first opposable component including:
(i) a chromatographic medium having first and second ends;

(ii) a detector application pad in operable contact with the first end of the chromatographic medium, the detector application pad containing at least one reagent for detection of the analyte;
(iii) a conductor for allowing the passage of fluid in operable contact with the detector application pad and in indirect contact with the first end of the chromatographic medium; and (iv) an absorber for absorbing fluid in operable contact with the second end of the chromatographic medium;
(b) a second opposable component including a sample preparation zone for receiving a sample to be tested, the second opposable component being attachable to the first opposable component so that the first and second opposable components are brought into opposition and fluid is transferredfrom the second opposable component to the first opposable component; and (c) a conductive barrier attached to the first opposable component;
wherein the first and second opposable components are configured so that a sample can be applied to the sample preparation zone on the second opposable component when the first and second opposable components are not in opposition and so that bringing the first and second opposable components into opposition results in the sample preparation zone being in indirect contact with the conductor to apply the sample to be tested through the conductive barrier to theconductor for flow through the conductor and then to the first end of the chromatographic medium through the detector application pad to add the reagent for detection of the analyte to the sample, the flow from the conductor through the detector application pad to the first end of the chromatographic medium being aided by absorption of fluid by the absorber.
34. A test kit for the detection and/or the determination of an analyte in a sample comprising, in separate containers:
(a) the chromatographic assay device of claim 33; and (b) a specific binding partner for the analyte labeled with a detectable label to be applied to the detector application pad.
35. A chromatographic assay device for detection and/or determination of an analyte in a sample comprising:
(a) a first opposable component including:
(i) a chromatographic medium having first and second ends;
(ii) a first detector application pad in operable contact with the first end of the chromatographic medium, the first detector applicationpad containing a first specific binding partner to the analyte in a form that can be resolubilized by the addition of a liquid to the first detector application pad, the first specific binding partner being labeled with a detectable label;
(iii) a conductor for allowing the passage of fluid in operable contact with the first detector application pad so that the first detector application pad bridges the conductor and the first end of the chromatographic medium to allow fluid flow from the conductor through the first detector application pad and to the first end of the chromatographic medium; and (iv) an absorber for absorbing fluid in operable contact with the second end of the chromatographic medium;
(b) a second opposable component attachable to the first opposable component so that the first and second opposable components are brought into opposition and fluid is transferred from the second opposable component to the first opposable component, the second opposable component including:
(i) a sample preparation zone for receiving a sample to be assayed; and (ii) a second detector application pad in operable contact with the sample preparation zone, the second detector application pad containing 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, the second specific binding partner being labeled with a detectable label, the second detector application pad being located adjacent to the sample preparation zone on the second opposable component such that application of the sample to the sample preparation zone resolubilizes the second specific binding partner so that the sample preparation zone contains a mixture of the sample and the second specific binding partner; and (c) a conductive barrier attached to the first opposable component;
wherein the first and second opposable components are configured so that a sample can be applied to the sample preparation zone on the second opposable component when the first and second opposable components are not in opposition and so that bringing the first and second opposable components into opposition results in the sample preparation zone on the second opposable component being in operable indirect contact with the conductor on the first opposable componentthrough the conductive barrier to apply the sample to be tested and the second specific binding partner to the conductor for flow through the conductor and then to the first end of the chromatographic medium through the first detector application pad to add the first specific binding partner to the sample and the second specific binding partner, the flow from the conductor through the first detector application pad to the first end of the chromatographic medium being aided by absorption of fluid by the absorber.
36. The chromatographic assay device of claim 35 wherein the chromatographic medium further comprises a detection zone substantially smaller in area than the chromatographic medium, the detection zone containing a third specific binding partner to the analyte immobilized thereto, such that a ternarycomplex comprising: (1) one of the first and second specific binding partners; (2) the analyte; and (3) the immobilized third specific binding partner forms at thedetection zone if analyte is present in the sample.
37. A test kit for the detection and/or the determination of an analyte in a sample comprising, in separate containers:
(a) the chromatographic assay device of claim 35; and (b) a liquid for resolubilizing at least one of the first specific binding partner to the analyte and the second specific binding partner to the analyte, the liquid to be applied to at least one of the first detector application pad on the first opposable component and the second detector application pad on the second opposable component.
38. A chromatographic assay device for the detection and/or determination of an analyte in a sample comprising:
(a) a first opposable component including:
(i) a chromatographic medium having first and second ends;
(ii) a conductor for allowing the passage of fluid in operable contact with the first end of the chromatographic medium and (iii) an absorber for absorbing fluid in operable contact with the second end of the chromatographic medium;
(b) a second opposable component attachable to the first opposable component so that the first and second opposable components are brought into opposition and fluid is transferred from the second opposable component to the first opposable component, the second opposable component including an applicator for applying fluid to the conductor on the first opposable component when the first and second opposable components are brought into opposition, the applicator divided into two sectors:
(i) a first sector containing a first specific binding partner for the analyte in a form that can be resolubilized by the addition of aliquid to the applicator when the first and second opposable components are not in opposition, the first specific binding partner being labeled with a detectable label; and (ii) a second sector lacking a first specific binding partner for the analyte; and (c) a conductive barrier attached to the first opposable component;
wherein 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 on the second opposable component into indirect contact with the conductor on the first opposable component through the conductive barrier, the second sector of the applicator being in indirect contact with the conductor through the first sector, to apply the contents of the first sector of the applicator to the chromatographic medium, and subsequent to the application of the contents of the first sector of the applicator to the chromatographic medium, to apply the contents of the second sector of theapplicator to the chromatographic medium, the absorber withdrawing fluid from the chromatographic medium to aid fluid flow from the applicator through the conductor and the chromatographic medium.
39. The chromatographic assay device of any of claims 23, 25, 27, 33, or 38 wherein the chromatographic medium further comprises a detection zone substantially smaller in area than the chromatographic medium, the detection zone containing a second specific binding partner to the analyte immobilized thereto, such that a ternary complex comprising of first specific binding partner, the analyte, and the second specific binding partner forms at the detection zone if analyte is present in the sample.
CA002221125A 1995-06-02 1996-05-23 Opposable-element assay device employing conductive barrier Abandoned CA2221125A1 (en)

Applications Claiming Priority (2)

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US08/458,132 US5869345A (en) 1991-05-29 1995-06-02 Opposable-element assay device employing conductive barrier

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JP (1) JP3585933B2 (en)
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