WO2011014673A1

WO2011014673A1 - Automated lateral flow immunoassay cassette with improved flow properties

Info

Publication number: WO2011014673A1
Application number: PCT/US2010/043742
Authority: WO
Inventors: William H. Chapman, Jr.; Richard K.T. Lee
Original assignee: Cholestech Corporation
Priority date: 2009-07-31
Filing date: 2010-07-29
Publication date: 2011-02-03
Also published as: CN102472751A; CN102472751B

Abstract

Methods, devices, and instruments for performing receptor binding assays are described. In particular, the devices described herein provide for an initial wetting process for redissolving assay reagents in a conjugate pad prior to mobilization of those assay reagents into a lateral flow element. This initial wetting preferably occurs along a planar surface of the conjugate pad having the greatest aspect.

Description

AUTOMATED LATERAL FLOW IMMUNOASSAY CASSETTE WITH IMPROVED FLOW PROPERTIES

FIELD OF THE INVENTION

[0001] The present invention relates to systems and methods for conducting assays, including qualitative, semi-quantitative and quantitative determinations of one or more analytes using a lateral flow assay format.

BACKGROUND OF THE INVENTION

[0002] The following discussion of the background of the invention is merely provided to aid the reader in understanding the invention and is not admitted to describe or constitute prior art to the present invention.

[0003] The term "receptor binding assay" refers to methods for generating a detectable signal indicative of the presence or amount of an analyte of interest based upon the ability of the analyte to bind with specificity to a particular binding partner (referred to as a "receptor" for the analyte). A common type of receptor binding assay is the immunoassay, in which antibodies that bind the analyte of interest are used to provide the analyte receptor, and the detectable signal is related to formation of an analyte/antibody complex. In addition to the use of antibodies as receptors for an analyte, the use of other binding partners, including nucleic acids, aptamers, and peptides other than those comprising an immunoglobulin motif. This list is not meant to be limiting.

[0004] Binding of the analyte to its receptor can be detected directly or indirectly, and often employing the use of detectable labels. Exemplary detectable labels, such as colloidal gold, labeled latex micro- or nano-particle conjugates, and labelled paramagnetic particle conjugates, are described hereinafter. Such labels can be conjugated to receptors or to competitive receptor ligands, depending upon the type of assay being performed. In addition, receptors (e.g., antibodies) are often immobilized on solid-phase matrices for use as affinity supports or to simplify sample analysis.

[0005] Numerous competitive, noncompetitive, and sandwich receptor binding assay methods are well known in the art. Additionally, numerous methods, devices, and instruments are also well known in the art for practicing such receptor binding assays. See, e.g., U.S. Patent Nos. 7,220,595; 7,238,5196,143,576; 6,113,855; 6,019,944; 6,007,690, 5,985,579; 5,947,124; 5,939,272; 5,922,615; 5,885,527; 5,851,776; 5,824,799; 5,679,526; 5,525,524; and 5,480,792; and WO03/087401, each of which is hereby incorporated by reference in its entirety, including all tables, figures and claims. One skilled in the art also recognizes that robotic instrumentation, including but not limited to Beckman ACCESS®, Abbott

AXSYM®, Roche ELECSYS®, Dade Behring STRATUS® systems, are among the commercially available analyzers that are capable of performing receptor binding assays. Additionally, certain methods and devices, such as biosensors and optical immunoassays, can be employed to determine the presence or amount of analytes. See, e.g., U.S. Patent Nos. 5,631,171 and 5,955,377, each of which is hereby incorporated by reference in its entirety, including all tables, figures and claims.

[0006] In such assay devices, flow of a sample fluid and other reagents along a desired flow path can be driven passively (e.g., by capillary, hydrostatic, or other forces that do not require further manipulation of the device once sample is applied), actively (e.g., by application of force generated via mechanical pumps, electroosmotic pumps, centrifugal force, increased air pressure, etc.), or by a combination of active and passive driving forces. Additional elements, such as filters to separate plasma or serum from blood, mixing chambers, etc., can be included as required by a particular application.

[0007] Lateral flow-type assay formats (also known as "Lateral Flow

Immunochromatographic Assays") are commonly used for medical diagnostics either for home testing, point of care testing, or laboratory use. Often produced in a dipstick format, lateral flow tests are a form of immunoassay in which the test sample flows along a solid substrate via capillary action. After the sample is applied to the test it encounters a detectably labeled reagent in a "conjugate pad"; this labeled reagent mixes with and transits with the sample through the lateral flow substrate. The "reaction mixture" thus formed encounters lines or zones on a solid phase to which an antibody or antigen has been non-diffusively bound. Depending upon the presence or amount of the analyte(s) of interest present in the sample, the coloured reagent can become bound at the test line or zone.

[0008] A source of variation in the lateral flow-type assay format results from spatial variation in the concentration of detection reagents in the conjugate pad. This variation results from the nature of flow of a liquid sample into the edge of the conjugate pad. The flow of sample partiality carries the dried reagent with the leading edge, leaving a gradient of reagent concentration. The shape of the reagent gradient is determined by the rate at which the reagents dissolve, the strength at which particles of the dry reagent adhere to the stationary phase, as well as many other factors that are difficult to predict or control.

[0009] Consequently, there is a need for methods, devices and assays that allow for the improved detection of analytes. The disclosure provides these and additional benefits.

SUMMARY OF THE INVENTION

[0010] The invention relates to methods and compositions for performing one or more assays for one or more analytes in a fluid sample. The lateral flow devices described herein provide for an improved wetting process for redissolving assay reagents in a conjugate pad prior to mobilization of those assay reagents into a lateral flow element. By transferring fluid to a conjugate pad along its greatest planar aspect, the devices of the present invention avoid differential properties of the various reagents in the conjugate pad, such as the accumulation of detection reagent at the leading edge of reagent flow. In addition, by providing for a time delay to allow for solubilisation of assay reagents in the conjugate pad prior to initiating flow to a lateral flow element, the present devices can improve assay reliability.

[0011] In a first aspect, the present invention relates to assay devices for measuring an analyte in a fluid sample. These assay devices comprise:

a sample application region for receiving said fluid sample; a first fluid transport element in fluid communication with the sample application region; and an analytical element resiliently biased against the first fluid transport element. The analytical element comprises (i) a conjugate pad containing one or more assay reagents for detection of said analyte, and (ii) a second fluid transport element comprising one or more detector region(s) for presentation of an assay signal related to the presence or amount of said analyte.

[0012] Overcoming the resilient bias brings the conjugate pad and the second fluid transport element into fluid communication with the first fluid transport element and causes the conjugate pad to engage and restrict flow of fluid through a region of the first fluid transport element. Preferably, a surface of the conjugate pad having the largest planar aspect engages and receives fluid from the first fluid transport element when the resilient bias is overcome. During conditions of fluid flow through the device, fluid entering the conjugate pad from the first fluid transport element flows from the conjugate pad laterally to enter the second fluid transport element, and fluid entering the second fluid transport element flows laterally to contact said one or more detector regions.

[0013] In certain embodiments, the first fluid transport element comprises a filter for removing, or retarding flow of, particulate components of a fluid sample, and a porous material in fluid communication with the filter which conducts lateral flow of fluid received from the filter through the porous material. Preferably, the filter is configured to remove or retard particulate components of a blood sample, such as erythrocytes. Examples of suitable filter materials are described hereinafter.

[0014] In these embodiments, engagement of the porous material by the conjugate pad can restrict flow of fluid through the first fluid transport element by compressing a region of said porous material. This compression can restrict fluid flow through the porous material such that a fluid flow path past the compression traverses the conjugate pad. In certain embodiments, fluid flows through the conjugate pad to an uncompressed region of the first fluid transport element downstream of the conjugate pad. The second fluid transport element may be brought into fluid communication with this uncompressed region of the first fluid transport element to receive fluid which has traversed the conjugate pad in a lateral flow manner.

[0015] Fluid received by the second fluid transport element then flows to the detector region(s) by lateral flow through the second fluid transport element. In these embodiments, the second fluid transport element comprises a porous material which conducts lateral flow of fluid received from the conjugate pad and/or the first fluid transport element.

[0016] As described herein, the conjugate pad preferably comprises one or more assay reagents used for detecting the presence or amount of an analyte of interest. Such assay reagents can comprise a detectably labeled receptor which binds to an analyte of interest, and/or a detectably labeled species which competes for binding to a receptor for an analyte of interest. These assay reagents may be dried in the interior of, on the surface of, or both in and on, the conjugate pad.

[0017] Similarly, the detector region of the second fluid transport element preferably comprises one or more assay reagents used for detecting the presence or amount of an analyte of interest. By way of example, the detector regions may comprise receptor which binds to an analyte of interest immobilized therein or thereon, and/or a species which competes for binding to a receptor for an analyte of interest. [0018] Numerous materials may be used to provide the various fluid flow elements of the present device, including porous materials such as papers, membranes, filters, etc., as well as capillary channels which promote fluid movement by capillary forces. Examples of suitable materials are described hereinafter. In the case of materials such as nitrocellulose membranes which become translucent or transparent upon wetting, a reflective layer may be positioned adjacent to a surface thereof to provide for desired optical properties. This may be particularly useful for materials used to form the detector region(s) of the device, where optical detection methods may be used to detect accumulated label.

[0019] The devices of the present invention may further comprise various optional elements, such as a reservoir configured to collect fluid flowing through the second fluid transport element, which are advantageous in a device of the lateral flow type.

[0020] In one preferred embodiment, an assay device of the present invention comprises: a first fluid transport element which comprises a filter for removing, or retarding flow of, particulate components of said fluid sample, and a porous material in fluid communication with the filter, where the porous material conducts lateral flow of fluid received from the filter, and where the porous material comprising glass fibers;

one or more assay reagents which comprise a detectably labeled receptor which binds to an analyte of interest and/or a detectably labeled species which competes for binding to a receptor for an analyte of interest;

a second fluid transport element which comprises a nitrocellulose membrane which comprises one or more detector regions, where the nitrocellulose membrane comprises a reflective layer positioned adjacent to a surface thereof, and where the detector region comprises receptor which binds to an analyte of interest and/or a species which competes for binding to a receptor for an analyte of interest immobilized therein or thereon.

[0021] In another aspect, the present invention relates to assay devices for measuring an analyte in a fluid sample, which assay devices comprise:

a. a first substrate comprising:

(i) a sample application region, and (ii) a first fluid transport element in fluid communication with the sample application region;

b. a second substrate comprising: (i) a conjugate pad containing one or more assay reagents for measuring an analyte of interest, and (ii) a second fluid transport element comprising one or more detector regions for presentation of an assay signal indicative of the presence or amount of said analyte; and c. one or more resilient elements configured to separate the conjugate pad from the first fluid transport element upon removal of a force which overcomes a resilient bias produced by the resilient element(s),

where a surface of the conjugate pad having the largest planar aspect engages the first fluid transport element to receive fluid therefrom when the resilient bias is overcome,

the fluid entering the conjugate pad from the first fluid transport element flows from the conjugate pad laterally to enter the second fluid transport element, and

fluid entering the second fluid transport element flows laterally to contact the one or more detector regions.

[0022] In certain embodiments, such assay devices comprise a soluble adhesive layer lying between the conjugate pad and the first fluid transport element. The soluble adhesive layer acts as a time barrier between the first fluid transport element and the conjugate pad. Upon application of sample fluid to the device, fluid flows through the first fluid transport element to contact the adhesive. Dissolving of the adhesive layer results in wetting of the conjugate pad such that the surface of the conjugate pad having the greatest planar aspect is contacted by fluid at substantially the same time.

[0023] In related aspects, the present invention relates to methods for determining the amount of an analyte in a fluid sample by applying a fluid sample to the sample application region of a device of the present invention, initiating fluid flow through the device, and detecting a signal indicative of the presence or amount of an analyte of interest from a detector region.

[0024] In certain embodiments, these methods comprise:

applying a fluid sample to the sample application region of an assay device as described herein, whereby the fluid sample, or one or more fluid components thereof (for example if the a blood sample is filtered to provide plasma), flow into the first fluid transport element; applying a first force to overcome the resilient bias such that the conjugate pad is brought into fluid communication with the first fluid transport element and receives fluid therefrom, and such that the conjugate pad engages the first fluid transport element; removing the first force to cause separation of the first fluid transport element from the conjugate pad;

after an interval of time applying a second force to overcome the resilient bias such that the conjugate pad and the second fluid transport element are brought into fluid communication with the first fluid transport element. Preferably, the conjugate pad engages and restricts flow of fluid through a region of the first fluid transport element, thereby providing a flow path such that fluid flows through the conjugate pad to an uncompressed region of the first fluid transport element downstream of said conjugate pad, and wherein the second fluid transport element, which is in fluid communication with this uncompressed region of the first fluid transport element receives fluid therefrom;

maintaining the second force for a sufficient interval of time such that the second fluid transport element conducts lateral flow of fluid received from the first fluid transport element, wherein an analyte of interest within the fluid sample, if present, is reacted with one or more of assay reagents in the conjugate pad, and wherein a detectable signal is produced as fluid is drawn past the detector region of the second fluid transport element; and

detecting a signal at the detector region which is indicative of the presence or amount of an analyte of interest present in the fluid sample.

[0025] Other embodiments of the invention will be apparent from the following detailed description, exemplary embodiments, and claims. All references cited herein are

incorporated in their entirety by reference.

DESCRIPTION OF THE FIGURES

[0026] Fig. 1 depicts an exemplary lateral flow assay device of the present invention.

[0027] Fig. 2 depicts use of an immunoassay cartridge designed for tangential wetting of a conjugate pad in schematic form.

[0028] Fig. 3 depicts the flow of gold particles in a conjugate pad wetted tangentially (A) and across substantially all of the largest planar surface (B).

[0029] Fig. 4 depicts use of an exemplary lateral flow assay device of the present invention in schematic form. [0030] Fig. 5 depicts results of performing an assay for C-reactive protein (CRP) using an exemplary lateral flow assay device of the present invention, plotted as the relationship between K/S values and reference CRP values.

[0031] Fig. 6 depicts a direct comparison between CRP concentrations determined using an exemplary lateral flow assay device of the present invention and a commercially available CRP assay.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0032] Disclosed herein are methods, devices, and instruments for performing receptor binding assays. In particular, the devices described herein provide for an initial wetting process for redissolving assay reagents in a conjugate pad prior to mobilization of those assay reagents into a lateral flow element. This helps to avoid differential properties of the various reagents in the conjugate pad, such as the accumulation of detection reagent at the leading edge of reagent flow, thereby improving assay reliability.

[0033] Device Components [0034] 1. Device Substrate

[0035] The present devices preferably comprise one or more substrates or scaffolds which hold the various components which are active in fluid transport and/or assay-specific reactions. An exemplary configuration is shown in Fig. 1.

[0036] Generally in this configuration, a first substrate (Fig. IB) provides a sample application region in the form of an open well which is fluidly connected to a first fluid transport element. This first fluid transport element provides a filter for removing particulate material from the sample. The filter is fluidly connected to a porous material which provides for lateral flow of fluid received from the filter. A second substrate (lateral view in Fig. IA, top view in Fig. 1C) is held in resilient bias adjacent to the first substrate. This second substrate provides a porous conjugate pad and a second porous material which provides for lateral flow of fluid and which comprises a detection region. These various components, as well as additional optional elements such as a reagent reservoir lying downstream of the second porous material, will be described in more detail hereinafter. Other configurations will be readily apparent to those of skill in the art. [0037] The term "resiliently biased" as used herein refers to two elements of an assay device, each of which comprise one or more regions which, in the absence of a force which overcomes the resilient bias, are maintained physically separated from one or more regions of the other element. Application of the necessary force results in contact of physically separated regions(s) on the two elements, and the release of the applied force results in the region(s) again becoming separated. The resilient bias can be provided, for example, by positioning a "spring" between the two elements such that the application of force overcomes the spring effect so that one or more regions on the two elements contact one another, but release of the force permits the spring to separate the two elements. The term "spring" used in this context refers to any element which provides the desired resilient arrangement between the two elements of the assay device. In exemplary embodiments described hereinafter, the spring is provided by blocks of resilient foam positioned between two elements, depicted as element 101 of Fig. 1. In this figure, the two elements A and B are depicted as being separated from one another; in use, part A is preferably affixed to the resilient foam blocks using an adhesive. Other arrangements for providing the resilient bias between two elements will be apparent to those of skill in the art. The term "overcoming resilient bias" used in this context refers to application of the necessary force so as to cause contact of physically separated regions(s) on the two elements.

[0038] The term "fluid transport element" as used herein refers to a portion of an assay device which communicates fluid therethrough during use. The term includes porous materials such as papers, membranes, filters, etc., as well as capillary channels which promote fluid movement by capillary forces. Fluid transport elements typically comprise hydrophilic surfaces, but may comprise hydrophobic surfaces which are rendered hydrophilic by materials in the sample volume or reaction mixture.

[0039] Preferably, the devices of the present invention utilize one or more lateral flow elements for fluid transport. The term "lateral flow" as used herein refers to flow of reagents in a longitudinal direction through a substantially flat porous material. Such porous material is "substantially flat" if the thickness of the material is no more than 10% of the length and width dimensions.

[0040] The term "downstream region" as used herein relative to a first region of a device refers to which receives fluid flow after that fluid has already reached the first region. [0041] The substrate components of the device (i.e. a physical structure of the device whether or not a discrete piece from other parts of the device) can be prepared from copolymers, blends, laminates, metallized foils, metallized films or metals, polyolefins, polyesters, styrene containing polymers, polycarbonate, acrylic polymers, chlorine containing polymers, acetal homopolymers and copolymers, cellulosics and their esters, cellulose nitrate, fluorine containing polymers, polyamides, polyimides, polymethylmethacrylates, sulfur containing polymers, polyurethanes, silicon containing polymers, glass, and ceramic materials, elastomers, latex, silicon chip, polyethylene, polypropylene, silicon elastomers, TEFLON®, polycarbonate, etc.

[0042] The structure of the various substrate components can be defined by a variety of ways, for example, machining the surfaces to the appropriate shapes or by injection molding or other molding techniques. One skilled in the art will recognize that various techniques can be used to render an otherwise hydrophobic material hydrophilic if necessary, such as plasma treatment of hydrophobic surfaces.

[0043] 2. Sample Addition Region

[0044] The term "sample application region" as used herein refers to a portion of an assay device into which a fluid sample is introduced. A sample application region can be provided, for example, in the form of an open chamber in a housing, in the form of an absorbent pad, etc. Exemplary embodiments of the sample addition region are depicted as element 102 of Fig. 1. The sample addition region can be a port of various configurations, that is, round, oblong, square and the like or the region can be a trough in the device.

[0045] The term "fluid sample" as used herein refers to a sample in the liquid phase. Preferred are body fluid samples are selected from the group consisting of urine, blood, serum, plasma, tears, saliva, and cerebrospinal fluid.

[0046] The volume of the sample addition region can be at least the volume of the second device region or greater. The volume or capacity of the sample addition region can be 1 to 5 times the volume of the downstream regions into which fluid flows. In certain embodiments, a volume or capacity of this sample addition region may be selected such that excess sample provides a wash to thoroughly remove any unbound reagents from the detection region where the analyte-dependent signal is generated. [0047] The sample addition region can also contain certain dried reagents which are used in the assay process. For example, a surfactant can be dried in this sample addition region which dissolves when sample is added. The surfactant in the sample would aid in the movement of the sample and reaction mixture through the device by lowering the surface tension of the liquid.

[0048] 3. Particulate Filter

[0049] As noted, a filter element can be placed in, on, or adjacent to the sample addition region to filter particulates from the sample, such as to remove or retard blood cells from blood so that plasma can further travel through the device. Exemplary embodiments of such a filter element are depicted as element 103 of FIG. 1. Filtrate can then move into a porous member fluidly connected to the filter, depicted as element 104 of Fig. 1.

[0050] Blood is predominantly composed of two parts: (a) plasma, and (b) blood cells and platelets suspended in the plasma. The plasma accounts for about 55% of the total volume of blood, and is about 92% water, 7% protein, and less than 1% other substances. Removal of certain proteins from the plasma by a clotting reaction results in a fluid referred to as serum. Suitable filters for removing or retarding cellular material present in blood are well known in the art. See, e.g., U.S. Patents 4,477,575; 5,166,051; 6,391,265; and 7,125,493, each of which is hereby incorporated by reference in its entirety. Many suitable materials are known to skilled artisans, and can include glass fibers, synthetic resin fibers, membranes of various types including asymmetric membrane filters in which the pore size varies from about 65 to about 15 μm, and combinations of such materials. In addition, a filter element can comprise one or more chemical substances to facilitate separation of red blood cells from blood plasma. Examples of such chemical substances are thrombin, lectins, cationic polymers, antibodies against one or more red blood cell surface antigens and the like. Such chemical substance(s) which facilitate separation of red blood cells from plasma may be provided in the filter element by covalent means, nonspecific absorption, etc.

[0051 ] 4. Assay Reagents

[0052] The term "assay reagents" as used herein refers to reagents which are employed in an assay for detection of an analyte. These assay reagents can include, for example, one or more detectably labelled receptors for performing a sandwich or competitive receptor binding assay, buffers, enzymatic substrates, wetting agents, blocking agents for reducing nonspecific or unintended binding reactions, etc.

[0053] The term "reaction mixture" as used herein refers to the mixture of fluid sample suspected of containing target analytes, and one or more reagents for determining the presence or amount of analytes in the sample. For example, the reaction mixture might comprise one or more ligand analogue conjugates or receptor conjugates corresponding to one or more analytes of interest, and/or the magnetically responsive particles comprising receptors corresponding to one or more analytes of interest. As used herein, the reaction mixture can comprise additional components, including for example buffering agents, HAMA inhibitors, detergents, salts (e.g., chloride and/or sulfate salts of calcium, magnesium, potassium, etc.), proteinaceous components (e.g., serum albumin, gelatin, milk proteins, etc.). This list is not meant to be limiting.

[0054] The term "receptor" refers to a particular binding partner (referred to as a "receptor" for the analyte). A common type of receptor binding assay is the immunoassay, in which antibodies that bind the analyte of interest are used to provide the analyte receptor, and the detectable signal is related to formation of an analyte/antibody complex. Numerous competitive, noncompetitive, and sandwich receptor binding assay methods are well known in the art. In addition to the use of antibodies as receptors for an analyte, the use of other binding partners, including nucleic acids, aptamers, and peptides other than those comprising an immunoglobulin motif. This list is not meant to be limiting.

[0055] With regard to receptors and/or labeled conjugates, the phrase "corresponding to an analyte of interest" refers to a receptor and/or labeled conjugate used in the method to generate a signal indicative of the presence or amount of the analyte in the reaction mixture. Depending on the receptor binding assay format being performed, the labeled conjugate can comprise a detectable label conjugated to a receptor that binds the analyte of interest (e.g., an anti-analyte antibody), can be a detectable label conjugated to a molecule that competes with the analyte of interest for binding to a receptor (e.g., an analyte analogue), or can be a detectable label conjugated to a binding partner that binds to a receptor for the analyte of interest (e.g., a secondary antibody, such as a goat anti-mouse IgG that binds to a mouse anti- analyte antibody). This list is not meant to be limiting.

[0056] The term "detectable label" as used herein refers to a chemical moiety from which a signal can be developed in an assay, but which does not itself bind to the analyte of interest. Detectable labels can be conjugated to receptors or to competitive receptor ligands, depending upon the type of assay being performed biological assays utilize various methods for detection, and one of the most common methods for quantitation of results is to conjugate an enzyme, fluorophore or other detectable label to the molecules under study (e.g., one or more analyte analogues), which can be immobilized for detection by receptors that have affinity for the molecules. Alternatively, the receptors to the one or more analytes of interest (e.g., an antibody or binding fragment thereof made or selected using the analyte of interest) can be conjugated to an enzyme, fluorophore or other detectable label. Enzyme conjugates are among the most common conjugates used. Detectable labels can include molecules that are themselves detectable (e.g., fluorescent moieties, electrochemical labels, metal chelates, etc.) as well as molecules that can be indirectly detected by production of a detectable reaction product (e.g., enzymes such as horseradish peroxidase, alkaline phosphatase, etc.) or by a specific binding molecule which itself may be detectable (e.g., biotin, digoxigenin, maltose, oligohistidine, 2,4-dintrobenzene, phenylarsenate, ssDNA, dsDNA, etc.). The term "direct label" as used herein refers to a signal development element from which a signal can be generated without the addition of a further binding molecule that specifically binds one or more components of the analyte/receptor complex being detected. Examples of such direct labels include enzyme labels, fluorescent labels, electrochemical labels, metal chelates, colloidal metal labels, and biosensors relying on optical detection such as surface plasmon resonance and ellipsometry. Conversely, the term "indirect label" refers to a signal development element that binds, not to the analyte, but to a molecule that is itself bound to the analyte. A labeled secondary antibody, for example a detectably labeled goat anti-mouse IgG that binds to a mouse antibody directed to the analyte of interest, is an example of an indirect label.

[0057] 5. Conjugate Pad

[0058] The term "conjugate pad" as used herein refers to a porous member into which one or more assay reagents are emplaced for incorporation with a fluid sample to create an assay reaction mixture. Typically, a conjugate pad will receive assay reagents during manufacture of an assay device and then dried. The reagents are dispersed within the conjugate pad, and are solubilized when the pad is wetted by fluid during use of the assay device. Preferably, the conjugate pad comprises one or more detectably labelled reagents, such as a receptor conjugate, corresponding to an analyte of interest. [0059] In accordance with the present invention, the conjugate pad, depicted as element 105 of Fig. 1, is initially wetted with a small volume of fluid, most preferably along the substantial entirety of its largest planar aspect, in order to solubilize the assay reagents contained therein prior to those reagents flowing into a downstream lateral flow fluid transport element. This wetting is affected by transiently overcoming the resilient bias which separates the conjugate pad from a first porous member which has received fluid flow from the sample application region. Under these conditions, the conjugate pad engages the first porous member and a fluid volume is transferred to the conjugate pad. The term "engage" as used herein with regard to two elements within an assay device of the invention refers to elements which are initially separated but that are brought into physical contact with one another. The resilient bias is then released to disengage (physically separate) the conjugate pad from the first porous member, and the conjugate pad is incubated for a period of time with the fluid volume. The conjugate pad is not fluidly connected to a downstream lateral flow porous member, depicted as element 106 of Fig. 1, during this incubation time.

Preferably, engagement of the first porous member by the conjugate pad restricts fluid flow through the first porous member. The term "restrict flow" refers to an action which reduces, and optionally stops, flow through at least a region of a fluid transport element. In exemplary embodiments described hereinafter for example, flow through a porous material is restricted by compression or the pores when a conjugate pad engages the porous material. Preferably, this restriction remains even when the engagement is released. In the examples, this restriction is used to alter the fluid flow path.

[0060] Fluid is caused to flow through the downstream lateral flow porous member by overcoming the resilient bias which separates the conjugate pad from the first porous member for a second time. In this exemplary embodiment, fluid flow is directed through the porous conjugate pad, as the compression of the region of the first fluid transport element engaged by the conjugate pad impedes flow therethrough. The downstream lateral flow porous member is brought into fluid communication with said first porous member, thereby providing a flow path such that fluid flows through the conjugate pad to an uncompressed region of the first porous member downstream of the conjugate pad, and then into the downstream lateral flow porous member. [0061] 6. Analyte Detection Region

[0062] Referring to Fig. 1, the analyte detection region(s) 107 of the lateral flow porous member 106 captures for detection labeled conjugates corresponding to one or more analytes of interest. These regions are depicted in Fig. 1C as "windows" in substrate A which allow observational access to the lateral flow porous member 106. In a sandwich assay, a sandwich complex is formed comprising a first analyte receptor immobilized on the analyte detection region, the analyte, and a second analyte receptor bound to a detectable label (the labeled conjugate). In a competitive assay, detectably labeled analyte (the labeled conjugate) and analyte in the sample compete to form a complex with an analyte receptor immobilized on the analyte detection region; or analyte immobilized on the analyte detection region and analyte in the sample compete to form a complex with detectably labeled analyte receptor (the labeled conjugate). In any case, the labeled conjugate is delivered to the analyte detection region for generation of a signal from the detectable label. This description is not meant to be limiting, and other suitable assay formats are well known to those of skill in the art. In certain embodiments, a plurality of spatially distinct analyte detection region(s) can provided on a single lateral flow porous member 106. If necessary, the lateral flow porous member 106 may be backed by a reflective layer 109, which may also act to hold a reservoir 108 in fluid communication with lateral flow porous member 106.

[0063] The term "immobilized" as used herein refers to a reagent which does not flow through the assay device. In performing receptor binding assays, receptors (e.g., antibodies) are often immobilized on solid-phase matrices for use as affinity supports or to simplify sample analysis. The term "solid phase" as used herein refers to a wide variety of materials including solids, semi-solids, gels, films, membranes, meshes, felts, composites, particles, papers and the like typically used by those of skill in the art to sequester molecules. The solid phase can be non-porous or porous. Suitable solid phases include those developed and/or used as solid phases in solid phase binding assays. See, e.g., chapter 9 of Immunoassay, E. P. Dianiandis and T. K. Christopoulos eds., Academic Press, New York, 1996; Leon et al, Bioorg. Med. Chem. Lett. 8, 2997 (1998); Kessler et al., Agnew. Chem. Int. Ed. 40, 165 (2001); Smith et al., J. Comb. Med. 1, 326 (1999); Orain et al., Tetrahedron Lett. 42, 515 (2001); Papanikos et al., J. Am. Chem. Soc. 123, 2176 (2001); Gottschling et al., Bioorg. Med. Chem. Lett. 11, 2997 (2001), each of which is hereby incorporated by reference in its entirety. Such solid phase matrices can be modified to provide linkage sites, for example by bromoacetylation, silation, addition of amino groups using nitric acid, and attachment of intermediary proteins, dendrimers and/or star polymers. This list is not meant to be limiting, and any method known to those of skill in the art can be employed.

[0064] Once such a complex is delivered within the analyte detection region, the signal representative of the presence or amount of the target analyte in the test sample can be measured. One skilled in the art can appreciate that various means can be used for the detection of signal in the analyte detection region. Exemplary types of optical detection means include, but are not limited to visual and instrumental means, such as reflectance, spectrophotometric, fluorescence, luminescence, etc. Such methods can rely on the use a photodiode, a CCD camera, a fluorometer, a spectrophotometer, etc. for detection of an optical signal. Other means of detection known to those skilled in the art can be employed. In detecting an optical label, the analyte detection region can be interrogated with a light source that illuminates the region with an appropriate wavelength for the label being employed, and an optical detector can be positioned to receive transmitted, reflected, or emitted light, depending on the detection method.

[0065] The foregoing is described in terms of optically detectable labels, but the skilled artisan will understand that numerous other detection modalities may be employed, depending on the nature of the label. Suitable other detection modalities include

amperometric, conductimetric, potentiometric, impedimetric, acoustic, electrochemical luminescence (ECL), interferometric, and surface plasmon resonance (SPR) methods. This list is not meant to be limiting.

[0066] 7. Conjugation of Receptors

[0067] A variety of linkage chemistries have been described for the attachment

("conjugation") of a detectable label to a particular molecule of interest (a detectable label, a solid phase, etc.), often for purposes developing binding assay (e.g., immunoassay) reagents. Thus, molecules may be coupled via a selected linkage chemistry for solid-phase

immobilization, preparation of antibody-detectable label conjugates and other labeled protein and nucleic acid reagents, etc. Such linkage chemistries often provide the molecule of interest with one or more functional groups that couple to amino acid side chains of peptides. Among other characteristics, these "linkage reagents" may be classified on the basis of the following: 1. Functional group(s) and chemical specificity;

2. length and composition of the cross-bridge;

3. whether the functional group(s) react chemically or photochemically; and

4. whether the resultant linkage is cleavable.

[0068] Reactive groups that can be targeted using linkage chemistries include primary amines, sulfhydryls, carbonyls, carbohydrates and carboxylic acids. In addition, many reactive groups can be coupled nonselectively using a cross-linker such as photoreactive phenyl azides.

[0069] Linkage chemistries may be provided with a variety of spacer arm (or "bridge") lengths for spacing the molecule of interest from its conjugate partner. The most apparent attribute of the bridge is its ability to deal with steric considerations of the moieties to be linked. Because steric effects dictate the distance between potential reaction sites, different lengths of bridges may be considered for the interaction. Suitable linkers are well known in the art, and are commercially available from companies such as Pierce Biotechnology, Inc. (Rockford, IL).

[0070] Preferred detectable label conjugates are less than about 100 nm in size, more preferably less than about 70 nm in size, still more preferably less than about 40 nm in size, and most preferably less than about 20 nm in size. The term "about" as used in this context refers to +/- 10% of a given value. Certain preferred detectable labels comprise latex particles such as those described in U.S. Patent Nos. 5,763,189; 6,238,931; and 6,251,687; and International Publication WO95/08772, each of which is hereby incorporated by reference in its entirety, which particles are themselves detectably labeled by incorporating a detectable label therein or thereon.

[0071] 8. Selection of Receptors

[0072] A ligand-receptor pair refers to a ligand and receptor that are chemical moieties capable of recognizing and binding to each other. The ligand and receptor can be any moieties that are capable of recognizing and binding to each other to form a complex.

Additionally, the ligand and receptor can interact via the binding of a third intermediary substance. Typically, the ligand and receptor constituting the ligand-receptor pair are binding molecules that undergo a specific noncovalent binding interaction with each other. The ligand and receptor can be naturally occurring or artificially produced, and optionally can be aggregated with other species.

[0073] Examples of ligands and/or receptors include, but are not limited to, agonists and antagonists for cell membrane receptors, toxins and venoms, viral epitopes, hormones such as steroids, hormone receptors, peptides, enzymes and other catalytic polypeptides, enzyme substrates, cofactors, drugs including small organic molecule drugs, opiates, opiate receptors, lectins, sugars, saccharides including polysaccharides, proteins, and antibodies including monoclonal antibodies and synthetic antibody fragments, cells, cell membranes and moieties therein including cell membrane receptors, and organelles. Examples of ligand-receptor pairs include lectin-carbohydrate; peptide-cell membrane receptor; protein A-antibody; hapten- antihapten; digoxigenin-anti-digoxigenin; enzyme-cofactor; enzyme-substrate; and antibody- antigen. As used herein, analytes can be ligands or can be associated with a ligand. Thus, where the analyte is an antigen, an antibody that binds to the antigen is a receptor.

[0074] The generation and selection of antibodies can be accomplished several ways. For example, one way is to purify polypeptides of interest or to synthesize the polypeptides of interest using, e.g., solid phase peptide synthesis methods well known in the art. See, e.g., Guide to Protein Purification, Murray P. Deutcher, ed., Meth. Enzymol. VoI 182 (1990); Solid Phase Peptide Synthesis, Greg B. Fields ed., Meth. Enzymol. Vol. 289 (1997); Kiso et al, Chem. Pharm. Bull. (Tokyo) 38, 1192 (1990); Mostafavi et al, Biomed. Pept. Proteins Nucleic Acids 1, 255 (1995); Fujiwara et al., Chem. Pharm. Bull. (Tokyo) 44, 1326 (1996). The selected polypeptides can then be injected, for example, into mice or rabbits, to generate polyclonal or monoclonal antibodies. One skilled in the art will recognize that many procedures are available for the production of antibodies, for example, as described in Antibodies, A Laboratory Manual, Ed Harlow and David Lane, Cold Spring Harbor

Laboratory, Cold Spring Harbor, N.Y., 1988. One skilled in the art will also appreciate that binding fragments or Fab fragments which mimic antibodies can also be prepared from genetic information by various procedures (Antibody Engineering: A Practical Approach, Borrebaeck, C, ed., Oxford University Press, Oxford, 1995; J. Immunol. 149, 3914 (1992)).

[0075] In addition, numerous publications have reported the use of phage display technology to produce and screen libraries of polypeptides for binding to a selected target. See, e.g., Cwirla et al., Proc. Natl. Acad. ScL USA 87, 6378 (1990); Devlin et al., Science 249, 404 (1990); Scott & Smith, Science 249, 386 (1990); and Ladner et al, U.S. Pat. No. 5,571,698. A basic concept of phage display methods is the establishment of a physical association between DNA encoding a polypeptide to be screened and the polypeptide. This physical association is provided by the phage particle, which displays a polypeptide as part of a capsid enclosing the phage genome which encodes the polypeptide. The establishment of a physical association between polypeptides and their genetic material allows simultaneous mass screening of very large numbers of phage bearing different polypeptides. Phage displaying a polypeptide with affinity to a target bind to the target and these phage are enriched by affinity screening to the target. The identity of polypeptides displayed from these phage can be determined from their respective genomes. Using these methods a polypeptide identified as having a binding affinity for a desired target can then be synthesized in bulk by conventional means. See, e.g., U.S. Patent No. 6,057,098, which is hereby incorporated in its entirety, including all tables, figures, and claims.

[0076] The antibodies that are generated by these methods can then be selected by first screening for affinity and specificity with the purified polypeptide of interest and, if required, comparing the results to the affinity and specificity of the antibodies with polypeptides that are desired to be excluded from binding. The screening procedure can involve immobilization of the purified polypeptides in separate wells of microtiter plates. The solution containing a potential antibody or groups of antibodies is then placed into the respective microtiter wells and incubated for about 30 minutes to 2 hours. The microtiter wells are then washed and a labeled secondary antibody (for example, an anti-mouse antibody conjugated to alkaline phosphatase if the raised antibodies are mouse antibodies) is added to the wells and incubated for about 30 minutes and then washed. Substrate is added to the wells and a color reaction will appear where antibody to the immobilized polypeptide(s) is present.

[0077] The antibodies so identified can then be further analyzed for affinity and specificity in the assay design selected. In the development of immunoassays for a target protein, the purified target protein acts as a standard with which to judge the sensitivity and specificity of the immunoassay using the antibodies that have been selected. Because the binding affinity of various antibodies can differ; certain antibody pairs (e.g., in sandwich assays) may interfere with one another sterically, etc., assay performance of an antibody may be a more important measure than absolute affinity and specificity of an antibody. [0078] Those skilled in the art will recognize that many approaches can be taken in producing antibodies or binding fragments and screening and selecting for affinity and specificity for the various polypeptides, but these approaches do not change the scope of the invention.

[0079] 9. Used Reagent Reservoir

[0080] Referring to Fig. 1, an optional used reagent reservoir 108 can receive the reaction mixture, other reagents and any excess sample from the upstream regions of the device. The term "reservoir" as used herein refers to a terminal device element which collects fluid downstream from a detector region. Fluid collected into such a reservoir no longer participates in any analyte detection reaction. The volume of the used reagent reservoir can be at least the volume of the sample and extra reagents which are added to or are in the device. The used reagent reservoir can take many forms using an absorbent, such as a bibulous material of nitrocellulose, porous polyethylene or polypropylene and the like or the used reagent reservoir can be comprised of a series of capillary grooves. In the case of grooves in the used reagent reservoir, the capillary grooves can be designed to have different capillary pressures to pull the reagents through the device or to allow the reagents to be received without a capillary pull and prevent the reagents from flowing backwards through the device.

[0081] Examples

[0082] The following examples serve to illustrate certain embodiments of the present invention. These examples are in no way intended to limit the scope of the invention.

[0083] Example 1: Flow in the absence of pre-wetting of the conjugate pad

[0084] The immunoassay cassette described in U.S. Patents 7,220,595 and 7,238,519 has been developed to measure C-reactive protein ("CRP") in samples of whole blood, serum, or other fluid sample. The cassette is designed to be compatible with the Cholestech LDX device, a point-of-care analyzer that was developed for cholesterol testing. The volume requirements of the cassette have been chosen to be 50 μL or less, so that samples for testing can be obtained using a simple finger stick. Test time is less than 10 minutes. [0085] The cassette was also designed to be a lateral flow device in which each of the three assay steps: 1) red blood cell separation, 2) incubation of the separated plasma with a gold particle labeled antibody and 3) flow of the sample and labeled antibody through a capture zone in a porous media, were mechanically isolated. The Cholestech LDX contains a mechanism which applies mechanical pressure to an inserted cassette, allowing physically separate assay components to be brought into physical contact. In this design (see Fig. T), a sample is introduced into sample well 206, which flows into a blood separating layer 201 (sample is depicted in the figure as an amorphous gray material). The LDX (Fig. 2, step 2) applies a light pressure that tangentially connects a blood separating layer 201 (a lectin impregnated fiber glass) to a conjugate pad (202, made of rigid Porex) which contains gold nanoparticles conjugated to a monoclonal antibody to hs-CRP dried therein. After a predefined period of time, the instrument applies additional pressure to the cassette (Fig. 2, step 3) which brings a nitrocellulose lateral flow member 203 into fluid communication with the conjugate pad. A downstream absorbent pad 204 serves as a terminal reservoir. The nitrocellulose is laminated to a strip of opaque vinyl tape 207 which acted as a light reflecting layer and to hold the absorbent sponge in fluid communication with the nitrocellulose. During step two, the anti-CRP antibodies bound to the gold particles capture the CRP in the plasma volume that wets the conjugate pad. In step 3, these gold particles flow through a capture zone on the nitrocellulose lateral flow member that contains anti-CRP antibodies. Gold particles that have CRP bound to them become immunochemically bound 205 at the capture zone, and an optical signal is then generated from the gold particles and measured in reflectance mode using a photodiode detector.

[0086] Detailed study of the flow of the gold conjugate through the cassette showed that during the tangential wetting of the conjugate pad, the flow of serum caused the formation of a gradient of concentration of gold label in the conjugate pad (Fig. 3A). The result of this reagent gradient in the hs-CRP cassette is irregular flow patterns of the gold particles (depicted as dark gray material in the figure; also depicted graphically, with the arrow showing the direction of fluid flow) through the nitrocellulose. A more even flow pattern is obtained by wetting of the conjugate pad in a more uniform manner than can be provided by tangential wetting (Fig. 3B). [0087] Example 2: Flow following non-tangential wetting of the conjugate pad

[0088] A new type of cassette, hereinafter referred to as V 2.0, was designed to avoid the problems with the formation of a gradient in the concentration of reagents in the conjugate pad.

[0089] Cassettes V 2.0 were built using material similar to those used in the production of cassettes described in Example 1. The cassette consist of two parts; (i) a "reaction bar" 401 that includes a conjugate pad 402, a nitrocellulose strip lateral flow member 403, and absorbent sponge 404; and (ii) a "main body" 405 that includes a lectin impregnated glass fiber member 406 (the blood separation layer) and a glass fiber porous member 407 (see Figure 4). These two substrates 401 and 405 are attached to one another in the configuration shown in Figure 1 using adhesives and two blocks of foam rubber 410 (not shown in Fig. 4; 101 in Fig. 1).

[0090] For formation of the nitrocellulose strip lateral flow member 403, strips of nitrocellulose (about 12" in length) were striped (Biodot XYZ) with anti-CRP antibody (Unipath anti-CRP 6929) and dried in a desiccated foil pouch for 48h at 48⁰C. The nitrocellulose was laminated to a strip of opaque vinyl tape 408 which acted as a light reflecting layer and to hold the absorbent sponge 404 in fluid communication with the nitrocellulose. The laminate was cut perpendicular to the direction of the antibody strip into pieces that were about 1/8" wide. The cut pieces were attached to reaction bar 401. The antibody stripe (capture zone) was placed in a window of the reaction bar so that its color could be measured with the LDX as flow occurs during the assay. The conjugate pad 402, made of POREX® porous plastic (Porex Corporation), was coated with a formula that contains a gold anti-CRP conjugate (manufactured by BBI), TRITON® X-100 octophenol ethylene oxide condensate (Union Carbide Corp.), bovine serum albumin, and sucrose. The conjugate pad 402 was attached to the reaction bar with a piece of water insoluble adhesive about 0.02" from the edge of the nitrocellulose strip lateral flow member 403.

[0091] The conjugate pad is wet from the surface having the greatest planar aspect (e.g., the "top" or "bottom" plane of a substantially flattened shape, rather than the narrower edge plane), thus providing a more uniform concentration of re-suspended reagent. A sample (e.g., blood) is first introduced into sample well 409, from which it flows into the blood separation layer 406 (Fig. 4, Step 1). Plasma flows from the blood separation layer into glass fiber porous member 407 lying below conjugate pad 402. Pressure is applied to the cassette in the LDX for several seconds (Fig. 4, Step 2) and the conjugate pad 402 is wet from the bottom surface with a volume of serum that is controlled by the nature and dimensions of the porous member 407 chosen. The pressure is released (Fig. 4, Step 3) and the wet conjugate pad 402 is separated from the porous member 407 and allowed to incubate (from about 15 seconds to about 2 minutes). Pressure is again applied to the cassette (Fig. 4, Step 4) and conjugate pad 402 and nitrocellulose membrane 403 are brought into fluid communication with porous member 407. Fluid is allowed to flow though the conjugate pad 402, into a downstream portion of porous member 407 and then into and through the nitrocellulose membrane 403 to absorbent sponge 404 acting as a terminal reservoir. Preferably, the applied pressure is sufficient to compress the porous member 407 below the conjugate pad 402 such that fluid flow cannot occur through the compressed region of porous member 407, but rather occurs through the conjugate pad 402 exclusively.

[0092] The performance of the cassette V 2.0 was determined by measuring the reflectance generated by five human serum samples with the following CRP levels (mg/L): 0.45, 0.99, 3.15, 5.58 and 7.81. The samples were prepared from pooled serum by mixing a high and low CRP samples. The reference values were determined using a Dade-Behring BN- 100 analyzer. K/S values were calculated from the measured reflectance values using the Kubelka-Munk equation. Coefficients for calculation of CRP values from the K/S values were determined by regression analysis assuming a linear relationship between the K/S values and sample CRP values. (Fig. 5). Final CRP values calculated from LDX

measurements correlate favorable with reference values (Fig. 6) determined from the Dade- Behring BN- 100 analyzer.

Claims

We claim:

1. An assay device for measuring an analyte in a fluid sample, comprising:

a sample application region for receiving said fluid sample;

a first fluid transport element in fluid communication with the sample application region;

an analytical element resiliently biased against the first fluid transport element, said analytical element comprising (i) a conjugate pad containing one or more assay reagents for detection of said analyte, and (ii) a second fluid transport element comprising a detector region for presentation of an assay signal related to the presence or amount of said analyte; wherein overcoming said resilient bias brings said conjugate pad and said second fluid transport element into fluid communication with said first fluid transport element and causes said conjugate pad to engage and restrict flow of fluid through a region of said first fluid transport element.

2. An assay device according to claim 1, wherein said first fluid transport element comprises a filter for removing, or retarding flow of, particulate components of said fluid sample, and a porous material in fluid communication with said filter which conducts lateral flow of fluid received from said filter through said porous material.

3. An assay device according to claim 2, wherein said conjugate pad restricts flow of fluid through said first fluid transport element by engaging and compressing a region of said porous material.

4. An assay device according to claim 3, wherein overcoming said resilient bias provides a flow path such that fluid flows through said conjugate pad to an uncompressed region of said first fluid transport element downstream of said conjugate pad, and wherein said second fluid transport element is in fluid communication with said second uncompressed region of said first fluid transport element.

5. An assay device according to claim 4, wherein said second fluid transport element comprises a porous material which conducts lateral flow of fluid received from said first fluid transport element.

6. An assay device according to claim 1, wherein said one or more assay reagents comprise a detectably labeled receptor which binds to said analyte or a detectably labeled species which competes for binding to a receptor for said analyte.

7. An assay device according to claim 6, wherein said one or more assay reagents are dried in, on, or both in and on, said conjugate pad.

8. An assay device according to claim 1, wherein said detector region comprises receptor which binds to said analyte immobilized therein or thereon.

9. An assay device according to claim 2, wherein said porous material comprises glass fibers.

10. An assay device according to claim 1, wherein said second fluid transport element comprises a nitrocellulose membrane which comprises said detector region, wherein said nitrocellulose membrane comprises a reflective layer positioned adjacent to a surface thereof.

11. An assay device according to claim 1, further comprising a reservoir configured to collect fluid flowing through said second fluid transport element.

12. An assay device according to claim 1, wherein:

said first fluid transport element comprises a filter for removing, or retarding flow of, particulate components of said fluid sample, and a porous material in fluid communication with said filter which conducts lateral flow of fluid received from said filter through said porous material, said porous material comprising glass fibers;

said one or more assay reagents comprise a detectably labeled receptor which binds to said analyte or a detectably labeled species which competes for binding to a receptor for said analyte;

said second fluid transport element comprises a nitrocellulose membrane which comprises said detector region, wherein said nitrocellulose membrane comprises a reflective layer positioned adjacent to a surface thereof;

and said detector region comprises receptor which binds to said analyte immobilized therein or thereon.

13. A method for determining the amount of an analyte in a fluid sample, comprising: applying a fluid sample to the sample application region of an assay device according to claim 1, wherein said fluid sample, or one or more fluid components thereof, flow into said first fluid transport element; applying a first force to overcome said resilient bias such that said conjugate pad is brought into fluid communication with said first fluid transport element and receives fluid therefrom, such that said conjugate pad engages said first fluid transport element;

removing the first force to cause separation of the first fluid transport element from the conjugate pad;

after an interval of time applying a second force to overcome said resilient bias such that said conjugate pad and said second fluid transport element are brought into fluid communication with said first fluid transport element such that said conjugate pad engages and restricts flow of fluid through a region of said first fluid transport element, thereby providing a flow path such that fluid flows through said conjugate pad to an uncompressed region of said first fluid transport element downstream of said conjugate pad, and wherein said second fluid transport element is in fluid communication with said second uncompressed region of said first fluid transport element;

maintaining the second force for a sufficient interval of time such that said second fluid transport element conducts lateral flow of fluid received from said first fluid transport element, wherein said analyte within the fluid sample is reacted with one or more of said reagents in the conjugate pad, and wherein a detectable signal is produced as fluid is drawn past the detector region; and

detecting a signal at the detector region, wherein the signal is indicative of the presence or amount of said analyte present in the fluid sample.

14. A method according to claim 13, wherein said one or more of said reagents in the conjugate pad comprise a detectably labeled receptor which binds to said analyte, said detector region comprises receptor which binds to said analyte immobilized therein or thereon, and said detectable signal is produced by sandwich complexes formed between said detectably labeled receptor, said analyte, and said immobilized receptor.

15. A method according to claim 13, wherein said one or more of said reagents in the conjugate pad comprise a detectably labeled species which competes for binding to a receptor for said analyte, said detector region comprises receptor which binds to said analyte immobilized therein or thereon, and said detectable signal is produced by competition between said analyte and said detectably labeled species for binding to said immobilized receptor.

16. A method according to claim 13, wherein said fluid sample is blood, and said first fluid transport element receives plasma separated therefrom.

17. A method for determining the presence or amount of an analyte in a fluid sample, comprising:

applying a fluid sample to an assay device, wherein said fluid sample, or one or more fluid components thereof, flow into a first porous member;

transferring fluid from said first porous member to a conjugate pad along substantially all of the largest planar surface of the conjugate pad, wherein dried reagent in the conjugate pad reconstitutes into the transferred fluid to yield a substantially uniform concentration of the reagent throughout the volume of the conjugate pad;

initiating fluid outflow from a lateral surface of the conjugate pad into a lateral flow element, said lateral flow element comprising one or more detector regions for presentation of an assay signal indicative of the presence or amount of said analyte; and

determining the presence or amount of said analyte based on a signal generated from one or more of said detector regions.

18. An assay device for measuring an analyte in a fluid sample, comprising:

a. a first substrate comprising:

(i) a sample application region, and

(ii) a first fluid transport element in fluid communication with the sample application region;

b. a second substrate comprising

(i) a conjugate pad containing one or more assay reagents for measuring said analyte, and

(ii) a second fluid transport element comprising one or more detector regions for presentation of an assay signal indicative of the presence or amount of said analyte; and c. one or more resilient elements configured to separate said conjugate pad from said first fluid transport element upon removal of a force which overcomes a resilient bias produced by said resilient element(s), wherein a surface of said conjugate pad having the largest planar aspect engages said first fluid transport element to receive fluid therefrom when said resilient bias is overcome, wherein fluid entering said conjugate pad from said first fluid transport element flows from said conjugate pad laterally to enter said second fluid transport element, and

wherein fluid entering said second fluid transport element flows laterally to contact said one or more detector regions.

19. An assay device according to claim 18, wherein said conjugate pad is adhered to the first fluid transport element by a soluble adhesive layer, wherein the soluble adhesive layer acts as a time barrier between the first fluid transport element and the conjugate pad, such that the dissolving of the adhesive layer results in wetting of the conjugate pad along the longitudinal axis of the conjugate pad such that the surface of the conjugate pad having the greatest planar aspect is contacted by fluid at substantially the same time.