WO1998036278A1

WO1998036278A1 - Multiple-site antibody capture immunoassays and kits

Info

Publication number: WO1998036278A1
Application number: PCT/US1998/002723
Authority: WO
Inventors: Juan G. Alvarez; Gene A. Davis
Original assignee: Beth Israel Deaconess Medical Center, Inc.
Priority date: 1997-02-15
Filing date: 1998-02-13
Publication date: 1998-08-20
Also published as: AU6277498A

Abstract

Devices, assays and kits for quantitating the concentration of an analyte in a sample are disclosed. The devices of the invention include a plurality of detection zones for detecting and quantitating the analyte.

Description

MULTIPLE-SITE ANTIBODY CAPTURE IMMUNOASSAYS AND KITS

Background of the Invention

A variety of immunoassay procedures have been developed to determine the presence and/or concentration of biologically active substances in a sample. For example, in a radioimmunoassay, a sample containing an unknown concentration of antigen is mixed with a fixed amount of antibody and a fixed amount of antigen, which has been radioactively labeled. The resultant antibody/antigen complex is recovered and its radioactivity measured to determine the relative concentrations of the radioactive antigen and the antigen contributed by the sample to determine the concentration of the antigen in the sample. Although highly sensitive, radioimmunoassays suffer from certain disadvantages, including cost, handling, storage and disposal of radioisotopes, and are in general performed by trained personnel.

Enzyme immunoassays (EIA) employ an enzyme labeled antibody conjugate directed against the antigen which is being detected and measured, and a primary antibody directed against the antigen. After appropriate incubation and separation procedures, detection of the enzyme (e.g. based on enzymatic production of color or fluorescence) provides for detection of the antigen. The resulting color intensity or fluorescence may be visually compared to a color guide for a qualitative or semi- qualitative determination. Alternatively, the resulting color intensity or fluorescence may be measured through electronic means, e.g. by density or reflectance photometry for a quantitative determination. To facilitate the separation of bound from unbound reactants, most EIA techniques are enzyme-linked immunosorbent assays (ELISA). Common to ELISA techniques is the binding or attachment of the primary antibody to a solid or impermeable support.

RIAs and EIAs can be performed in a competitive or non-competitive format. In general, competitive immunoassays are performed by adding an appropriate volume of labeled analyte and analyte-specific reagent to a sample containing the analyte, so that the labeled analyte and the analyte in the sample compete for a limited number of analyte-specific reagent binding sites, resulting in the formation of sample analyte/reagent and labeled-analyte/ reagent complexes. By maintaining an appropriate, generally constant concentration of labeled analyte and analyte-specific reagent, the amount of labeled-analyte/reagent complex formed is inversely proportional to the amount of analyte present in the sample. A quantitative determination of the analyte can therefore be made based on the labeled analyte/reagent complex. Competitive assays can be homogeneous (i.e. not requiring separation of antibody bound tracer (e.g. labeled analyte) from free tracer, since the antigen-antibody interaction causes, directly or indirectly, a measurable change in the signal obtained from the label group of the analyte). Alternatively, competitive assays can be heterogeneous (i.e. requiring separation of bound analyte from free analyte prior to determining the amount of analyte in the sample). In contrast to competitive immunoassays, non-competitive assays involve incubating an analyte containing sample with an immobilized analyte-specific (capture) reagent for a period of time sufficient to reach equilibrium with regard to the formation of reagent/analyte conjugates. The analyte-specific reagent can be directly or indirectly labeled. For example, indirect labeling can be carried out after a wash step to remove unbound analyte by contacting the immobilized capture reagent/analyte complexes with a second, labeled reagent that is specific for the capture reagent/analyte complex. Following a second wash step to remove unbound second labeled reagent, the amount of bound second labeled reagent can be detected and measured as an indication of bound analyte. Agglutination immunoassays are also used to detect the presence of an analyte in a sample. An agglutination reaction involves the in vitro aggregation of microscopic carrier particles as a result of the specific reaction between antibodies and antigens, one of which is immobilized on the surface of carrier particles. Agglutination immunoassays can be performed in either a direct or indirect format. In a direct agglutination immunoassay, a sample which may contain an analyte of interest (e.g. antigen or antibody) is contacted with a corresponding analyte-specific reagent (e.g. an antibody or antigen, respectively, attached to the surface of a carrier particle). The presence of the analyte in the sample can be directly detected based on agglutination (e.g. antibody/antigen). In contrast, in the indirect format, the presence of the analyte of interest in the sample inhibits the formation of complexes. The agglutination test is typically performed on a slide and read visually. As a result, it is highly subjective and requires substantial expertise. This is particularly problematic when quantitation of the antigen is important.

Easy-to-use assays for quantitating the concentration of analytes in a sample are needed.

Summary of the Invention

In one aspect, the invention features novel assay devices, which are useful for determining the concentration of an analyte in a sample. In one embodiment, the device includes a substrate including at least two detection zones, each of the detection zones comprising a pre-defined amount of an immobilized analyte-specific capture reagent. In preferred embodiments, a first detection zone includes a first concentration of the immobilized capture reagent, and a second detection zone includes a second concentration of the immobilized capture reagent, the first concentration being greater than the second concentration.

In another aspect, the invention features novel assay methods for determining the concentration of an analyte in a sample. In one embodiment, the method includes the step of supplying the sample fluid to an assay device which includes a substrate. The substrate includes at least two analyte detection zones. Each of the analyte detection zones includes a pre-defined amount of an analyte-specific capture reagent. The method includes the further step of allowing the sample fluid to contact the at least two analyte detection zones, such that the concentration of the analyte in the sample fluid is determined.

In another aspect, the invention provides a device for determining a concentration of an analyte in a sample fluid. The device includes a fluid-permeable substrate having a sample application area in fluid communication with a plurality of detection zones. Each of the detection zones includes a capture reagent capable of binding to the analyte, and the concentration of the capture reagent in each of the zones is different, such that the zones bind differentially to different concentrations of an analyte:labeling reagent complex. The device includes a labeling area disposed on the substrate between and in fluid communication with the sample application area and the detection zones. The labeling area includes (e.g., is impregnated with) a labeling reagent which is capable of binding to the analyte to form an analyte:labeling reagent complex. The device is configured to allow flow of the sample fluid from the sample application area through the labeling area, such that the analyte binds to the labeling reagent to form an analyte:labeling reagent complex. The device is configured to permit further flow of the analyte:labeling reagent complex to the plurality of detection zones, where the analyte and/or the complex is bound by the capture reagent, such that presence of bound label in the detection zones is indicative of concentration of the analyte in the sample.

In a further aspect, the invention features novel kits, which are useful for performing the instant claimed assays. Other objects, features and advantages of the invention will be apparent from the following Detailed Description and Claims.

Brief Description of the Figures

Figure 1 is a schematic depiction of a preferred embodiment of the invention. Detailed Description of the Invention

Definitions

As used herein, the following terms and phrases shall have the meanings set forth below: "Analyte" shall refer to a substance (e.g., a molecule or complex) that is to be detected. Thus, an analyte can be any substance capable of detection in an assay method or device, and is preferably a member of a specific binding pair, e.g., one member of a pair such as antigen antibody, hormone/receptor, enzyme/substrate, nucleic acid/complementary nucleic acid, substrate/binding protein, and the like. An analyte can be a compound of interest or can be indicative of a compound of interest, and may be directly or indirectly indicative of a condition, e.g., of a physiological condition such as pregnancy. It will be understood that an analyte can be either member of a specific binding pair, i.e., an analyte can be an antigen (which can be detected by way of an antibody which binds the antigen); or the analyte can be an antibody (which can be detected by use of an antigen to which it binds). In preferred embodiments, an analyte includes one or more sites at which another molecule or complex can bind or associate to form a complex (or a higher-order complex). An analyte can be monovalent (i.e. contain one binding site or epitope) or polyvalent (i.e. contain more than one binding site or epitope). Preferred analytes which can be detected with the devices, kits, and methods of the present invention include proteins (e.g. antibodies (e.g., antibodies against disease organisms, e.g., the causative organisms of AIDS, rubella, hepatitis, Lyme disease, and the like), hormones (such as luteinizing hormone, chorionic gonadotropin, or a steroidal hormone, such as progesterone or glucoronide), surfactant apoproteins (e.g. apoprotein A, B, C or D) or fibrin), nucleic acids (DNA, RNA and analogs thereof), lipids and carbohydrates. Particularly preferred antigens for analysis according to the methods of the invention include: hormones, therapeutic drugs (e.g. theophylline), drugs of abuse (e.g. cocaine, heroin, morphine, amphetamine, PCP, THC, barbiturate and benzodiazepine), infectious agents or other food or water contaminants (e.g. bacteria, fungi, viruses, protists) and sperm antigens. Examples of appropriate sperm antigens include sperm proteins (e.g. sperm flagellar proteins, glycolytic enzymes, antioxidant enzymes (e.g. glutathione peroxidase or superoxide dismutase), nuclear proteins, acrosomal proteins, α-tubulin, lactate dehydrogenase (LDH-X), protamine (sperm histones), acrosomal proteins (e.g. acrosin) or mitochondrial proteins); sperm lipids (e.g. cholesterol, phospholipids, glycolipids, triglycerides, phosphatidylglycerols, seminolipids, and fatty acids, particularly docosahexaenoic acid, which is one of the few fatty acids found in sperm); nucleic acids or a mixture of sperm components (e.g. thiazine blue reacts with sperm proteins, lipids and other sperm components). The term "analyte-specific reagent", as used herein, refers to a molecule or complex that: i) can specifically associate with or bind to an analyte to form a complex; and/or ii) can be detected or quantitated as an indication of the presence and/or quantity (e.g. concentration) of the analyte. Non-limiting examples of analyte-specific reagents for use in the instant invention include: antibodies (e.g. monoclonal or polyclonal) or fragments thereof (e.g. Fab or Fab' fragments), ligands, lectins, receptors, substrates and dyes. Certain preferred analyte-specific reagents are directly or indirectly labeled (i.e. can produce a detectable signal). A labeled analyte-specific reagent that can bind to an analyte and thereby label it is referred to herein as a "labeling reagent". A label is a moiety that can be detected, directly or indirectly, as an indication of the presence or absence of the labeled reagent. Preferred labels include enzymes (e.g., horseradish peroxidase, alkaline phosphatase, urease, β-galactosidase), enzyme co-factors, radioisotopes (e.g. -1H, ^C, ^^ϊ, ^2p_j 131j _an(j 35s)_; fluorescent compounds (e.g. fluorescein, rhodamine, allophycocyanin, phycoerythin, erythrosin, europium, luminol, luciferin and coumarin), dyes, and colored or uncolored beads or particles (e.g. carbon, silica gel, controlled pore glass, magnetic, Sephadex/Sepharose, cellulose, metal (e.g., gold colloid, metal oxides, metal hydroxides, metal salts or polymer nuclei coated with a metal or metal compound, such as gold) or latex. Preferred particles are in the range of about 0.01 to about 5 microns. Many detectable labels are known in the art for use in assay methods, and selection of an appropriate label will be routine to the skilled artisan based on such factors as, e.g., ease of use or detection of the label, the chemical or physical nature of the analyte and/or the analyte-specific reagent, the cost of the label, and the like. For non-limiting examples of labels, see, e.g., U.S. Patent No. 5,252,496 to Kang et al. A labeling reagent will generally be soluble, or will be of a size small enough to permit the labeling reagent, bound to the analyte, to pass through the substrate, urged by fluid flow, to the plurality of detection zones.

The term "capture reagent" refers to an analyte-specific reagent which can be immobilized on the substrate and is capable of specific binding to the analyte, or to an analyte-specific reagent, or to a complex of an analyte with an analyte-specific reagent. In a preferred embodiment, the capture reagent is an immobilized antibody, or fragment of an antibody.

The term "detection site" (also referred to herein as a "detection zone" or "capture zone") refers to a location or zone of the substrate which contains or is impregnated with an immobilized capture reagent, preferably in a predetermined quantity. Such a site can have any shape or dimension consistent with detection of the analyte and readout, in combination with at least one other detection site, of an analyte concentration (quantitative or qualitative). A plurality of capture sites can be configured to form any desired pattern, which can be selected, e.g., to form recognizable visible indicia upon completion of the test.

"Sample" as used herein, shall refer to a sample, such as a fluid, which contains or may contain the analyte to be detected. In a preferred embodiment, the sample is a biological sample, i.e., any material obtained from a living source (e.g. human, animal, plant, bacteria, fungi, protist, virus). The biological sample can be in any form, including solid materials (e.g., tissue, cell pellets and biopsies) and biological fluids (e.g. urine, blood, saliva, amniotic fluid and a mouth wash (e.g., containing buccal cells)). Preferably, a solid material is suspended or dissolved in a liquid prior to analysis according to the invention.

"Substrate" as used herein, refers to an insoluble substance (or a plurality of insoluble components), usually at least partially porous and fluid-permeable, which generally (in preferred embodiments) includes a sample application area (i.e. an area for receiving the applied sample) in fluid communication with an analyte-specific reagent area (i.e., an area which contains or is impregnated with an analyte-specific reagent such as a labeling reagent), which further includes a plurality of detection sites (e.g. 2, 3, 4, 5, 6, 7, 8 or more) each containing an immobilized capture reagent. Preferred substrates for use in the invention include membranes or filters (e.g. polyethylene, polypropylene, polyamide, polyvinylidenedifluoride, nitrocellulose, glass fiber, paper), plates (e.g. microtiter plates), tubes (e.g., glass, plastic or metal capillaries, straws or pipettes) and beads or particles. It will be understood that a substrate can comprise a plurality of parts, which can be composed of the same material or of different materials, if desired, provided that the parts are in fluid communication, and permit the sample fluid to be communicated from a sample application area to a plurality of detection sites. A substrate can include wicking elements, filter elements (e.g., for filtering out solid particles), reservoir elements (e.g., for holding a supplying a sample fluid), and the like. A substrate can be configured for sample application either to a discrete sample application area, or, in certain embodiments, can be configured for dipping into a sample fluid. Dipstick-type assay devices are well-known in the art (see, e.g., EP-A 0 125 118 or EP-A 0 282 192).

It will also be understood that the substrate can be housed in a housing, or otherwise fixed to a supporting member, which can include features such as a viewing window, a handle for easier manipulation by a user, and the like.

The term "concentration" as used herein, refers to a concentration, density, or total amount of an analyte, analyte-specific reagent (such as a labeling reagent) or capture reagent, e.g., a concentration, density, or total amount of a capture reagent which is present in a selected detection site. Assay Devices

A preferred embodiment of the invention is now further described with reference to Figure 1. Figure 1 shows a schematic view of one embodiment of a device which can be used to perform assays in accordance with the methods of the invention. As can be seen in Figure 1 , the device comprises a (preferably substantially flat) substrate 1 of rectangular shape (although any appropriate shape may be employed). Substrate 1 includes a sample application area 2, which is in fluid communication with the detection area 3. The sample application area can include a bibulous reservoir pad, which can absorb and hold a portion of sample liquid. The detection area 3, which is fluid- permeable, includes a plurality of capture sites (detection zones) of a defined width or other dimension (e.g., diameter of a spot) (preferably between about 0.2 mm and 5 mm, more preferably between 0.5 and 2 mm) and each site includes a pre-selected concentration of immobilized capture reagent. In a preferred embodiment, illustrated in Figure 1, the detection sites have a substantially linear shape. Preferably the distance between any two detection sites is between about 0.5 mm and 10 mm, more preferably between about 1 mm and 5 mm. In preferred embodiments, at least two of the detection sites have immobilized capture reagent concentrations which differ from each other. Preferably the concentration of immobilized capture reagent in each detection site in a particular device is an even multiple of the concentration of immobilized capture reagent in another detection site of the device (e.g., 1:1, 1:2. 1 :4, 1 :8, 1:16, 1 :32, etc.). In particularly preferred embodiments, the immobilized capture reagent concentrations of each of a series of detection sites increases in a direction of sample fluid flow. A device in which immobilized capture reagents sites are so arranged (in increasing order of immobilized capture reagent concentration as the sample flows along the substrate) provides accurate results and is easily read.

The device of the invention can be housed within a fluid-impermeable housing, preferably constructed of a solid, relatively rigid material, such as plastic. The housing will generally include an opening adjacent the sample application area, to permit a liquid sample to be applied to the sample application area (e.g., the reservoir pad). In certain embodiments, a reservoir pad can extend out from the substrate, through an opening in the housing, to permit sample application to the exterior reservoir pad, followed by fluid communication to the portion of the substrate disposed within the housing. The housing can also include a grip or handle portion to permit the user to firmly grasp the device. The grip portion can be textured, e.g., ribbed, to permit easy handling.

The housing also preferably includes at least one window disposed over the detection area to permit inspection of the detection area. A window can be an opening in the housing, or a transparent portion of the housing. More than one window may be present in the housing (e.g., a window over each detection site, or a separate window over a control site).

When a liquid sample is applied to the sample application area, the sample is absorbed, e.g., by a reservoir pad. The sample is then wicked along the substrate (which can be of multi-part construction) to the detection zones, where the analyte concentration is read.

It will be appreciated by one of ordinary skill in the art, in light of the disclosure herein, that the amount (e.g., density or concentration) of the capture reagent present in any detection zone can be selected to provide a detectable amount of bound label when analyte is present in the sample in an amount or at a concentration equal to or greater than a pre-determined concentration. For example, the amount of capture reagent present in each of several detection zones can be selected such that each of the zones has a different (and preferably known) threshold sensitivity for detection of the analyte. In this practice of the invention, if the analyte is present in the sample at or above the threshold sensitivity of a detection zone, the detection zone will provide a detectable signal, e.g., a detectable amount of bound label, e.g., by binding an analyte:labeling reagent complex. The number or pattern of detection zones which provide a detectable signal can then be determined to provide an indication of the concentration of analyte in the sample. The amount of capture reagent in each detection zone is preferably selected to permit the device to measure analyte concentration over a range of values expected for a particular analyte and type of sample fluid. Thus, the amount of capture reagent in a detection zone can be chosen to be sensitive to a pre-selected concentration of analyte in the sample fluid. For example, as described infra, in women the concentration of LH in plasma can vary between about 5 mlU/mL and 40 mlU/mL. Thus, a device for determining the level of LH in plasma is preferably able to determine LH concentrations in the range of 5 to 40 mlU/mL or some subrange thereof. For such a device, each of the plurality of detection zones could have a threshold sensitivity to LH within the selected range or a subrange thereof. For example, a first detection zone can have a concentration of an immobilized anti-LH antibody sufficient to capture a visible amount of label when the LH concentration is at least 5 mIU/ml, while a second detection zone can have a concentration of an immobilized anti-LH antibody sufficient to capture a visible amount of label when the LH concentration is at least 20 mlU/ml. In use, the appearance of a visible indication, such as a colored spot or line, at the position of the first detection zone, would indicate an LH concentration of at least 5 mlU/ml. If a visible spot is present at the first detection zone but not at the second detection zone, the LH concentration of the sample is at least 5 mlU/ml, but less than 20 mlU/ml. Different sensitivities in the plurality of reaction zones can be achieved, e.g., by providing an amount of the capture reagent in each of the detection zones sufficient to permit a detectable amount of a label to be bound to each detection zone at the threshold concentration of LH in a plasma sample. The required amount of capture reagent in each zone will vary depending upon such factors as the affinity of the capture reagent for the analyte (or label) to which it binds. Such concentrations can be readily determined by one of ordinary skill in the art with no more than routine experimentation. Alternatively, different capture reagents (such as two different monoclonal antibodies which bind to a single analyte, or analyte/labeling reagent complex) can be provided, wherein the different capture reagents have differing affinities for the reagent. Similarly, a capture reagent, such as an antibody, can be modified (e.g., chemically) to alter the affinity of the antibody for its antigen. Thus, differing detection zones can be provided with differing sensitivities to the analyte, thereby providing differential detection of the analyte and permitting quantitation of the analyte concentration. For example, in the device of Figure 1, the concentration of immobilized capture reagent is lowest in the capture reagent site (detection zone) closest to the sample application area, and the concentration of immobilized capture reagent increases in successive detection zones in the direction of fluid flow (indicated in Figure 1 by an arrow) from the sample application area. Thus, as shown in Figure 1 , the concentration of capture reagent increases from 1 : 16 at the detection site closest to the sample application area, to 1 :1 at the most distant (i.e., downstream) detection sites. A first analyte detection zone is said to be "upstream" of a second analyte detection zone (which is "downstream" of the first zone) if the first zone is contacted by a fluid sample before the second zone is so contacted. In other words, as fluid flows through the substrate along a defined direction of fluid flow, the fluid will flow through an upstream detection zone before passing through a downstream zone.

As shown in Figure 1, the device may optionally include a labeling zone 4 (e.g., a zone impregnated with a labeling reagent such as gold-antibody conjugate) between and in fluid communication with the sample application area and the detection area (including the detection zones). The labeling zone comprises a labeling reagent which can bind to the analyte. In preferred embodiments, the labeling reagent is a labeled antibody which can form a complex with the analyte. The complex of labeling reagent and analyte (the analyte labeling reagent complex) can then move to the capture reagent sites (the detection zones) for capture and detection. In preferred embodiments, the labeling reagent is present in excess (i.e., in molar excess, e.g., at least 10%, 20%, 30% or 50%) excess) over the capture reagent (to ensure that there is enough labeling reagent to bind to the analyte and the capture reagent). The label (e.g., as the analyte:labeling reagent complex) is captured by the capture reagent in the detection zones to provide bound label, thereby labeling and visualizing the detection zones. The presence of bound label in a detection zone can then be detected, e.g., by means identified herein, including visual inspection. In preferred embodiments, when the device includes a labeling zone, the device is configured to allow flow of the sample fluid from the sample application area through the labeling zone (which can also be a filter zone) such that the analyte binds to the labeling reagent to form an analyte labeling reagent complex. The sample is preferably filtered to remove large, potentially interfering particles as the sample flows through the (optional)filter zone. The device is also configured to permit flow of the analyte:labeling reagent complex to the detection zones, where the complex is captured by the immobilized capture reagent, so that presence of bound label in the detection zones is indicative of concentration of the analyte in the sample. In preferred embodiments, the labeling reagent comprises a colored particle, preferably colloidal gold, and the concentration of the analyte in the sample is determined visually. In preferred embodiments, the capture reagent comprises an antibody, more preferably a monoclonal antibody.

Assay devices commonly (but optionally) include a control site, typically disposed on the substrate downstream of the capture zones. The control site is not sensitive to the presence or absence of analyte in the sample, but is sensitive to the presence or absence of another component of the sample fluid, or to the labeling reagent, if present. Such a control site provides an indication that the sample fluid has been correctly applied to the assay device by providing a visible indication that the fluid has flowed to the control site (and therefore to the capture sites). In addition, to shorten the assay time, the device may optionally include a "sink"

(i.e. a microporous material of higher capillary suction force and/or greater liquid capacity than the substrate) for absorbing fluid and thereby speeding liquid transport along the substrate. The sink is sited downstream of the sample application area and capture zones. A preferred sink is described in International Patent Application No. WO 92/01226 to Unilever PLC.

In one embodiment, the invention provides an immunochemical assay device for detecting the presence or absence of an analyte of interest in a liquid sample, the device comprising a bibulous reservoir pad for receiving and containing the liquid sample; (optionally) a filter zone adjacent to and in fluid communication with the reservoir pad, the filter zone including a labeled immunochemical reagent (i.e., a labeling reagent such as a labeled antibody) capable of binding to the analyte to form a complex; and a wicking membrane adjacent to and in fluid communication with the reservoir (or the filter zone, if present). The wicking membrane is capable of absorbing a portion of the liquid sample from the reservoir pad (and filter zone if present), and includes a plurality of detection zones, each detection zone including an immobilized capture reagent (such as an immobilized antibody) capable of binding to the complex (i.e., the analyte/immunochemical reagent complex). The reservoir pad, filter zone, and wicking membrane can be constructed of the same or different materials, and can be disposed on solid support or within a housing (preferably a liquid-impermeable housing having at least one window for viewing the plurality of detection zones), as is conventional. The filter zone, if present is preferably capable of filtering out sample particles which could interfere with the assay, while permitting passage of the analyte and the complex from the reservoir pad to the wicking membrane. The antibodies can be polyclonal or monoclonal antibodies.

In preferred embodiments, an assay can be performed as described herein in a short period of time, e.g., in less than 20 minutes, more preferably less than 15, 10, 5, or 3 minutes. When the assay is complete, the results of the assay can be read manually, e.g., by visual inspection, or automatically, e.g., by machine methods such as scanning densitometry (e.g., see Example 1, infra). The analyte concentration can be determined directly, e.g., by reading the number of visualized analyte detection sites, or by comparing signal intensity (e.g., color intensity) to a control scale. Such a control scale can be provided, e.g., as part of the instructions or packaging for an assay kit of the invention.

In certain embodiments, the detection sites can be configured on the substrate so as to permit automated readout, e.g., by a bar-code reader. In this embodiment, the width and separation of the detection sites can be selected to provide a bar-coded assay result which can be read by a conventional bar-code scanner; the resulting bar-code can then be compared, e.g., in a computer memory, to a stored value for determination of analyte concentration.

Based on the instant disclosure, one of skill in the art can readily optimize the following variables to design an appropriate device for each particular analyte: number of detection sites, type of substrate, concentration of immobilized capture reagent in each of the capture zones, distance between each of the detection sites, concentration of labeling reagent (if employed), and the width or size of detection sites. For example, if a detection site is configured in a substantially linear form, the width of the detection site will affect the sensitivity and readability of the assay, e.g., a line which is narrow may not be large enough for detection with the unaided eye, while a line which is excessively broad may provide an ambiguous result. Similarly, the concentration of immobilized capture reagent to be employed in each of the detection zones will be determined, at least in part, by considerations such as the concentration of the analyte expected to be present in sample (e.g., as described below, levels of luteinizing hormone in plasma commonly are in the range of from about 5 mlU/mL to about 40 mlU/mL), the nature and sensitivity of the reagent used to detect or visualize the presence of analyte (e.g., colorimetric assay or presence of colored particles), the expense of the capture reagent, and the like.

Kits The invention also provides kits useful for assays, e.g., diagnostic assays as described herein. The kits include a device of the invention, together with instructions for using the device to perform an assay, e.g., to detect the presence or absence of an analyte in a sample. The kits can optionally include a standard chart against which the results of the assay can be compared, e.g., to determine the concentration of the analyte in the sample.

Assay Methods

In general, virtually any assay method can be used in conjunction with the novel device of the invention. In addition to the immunoassay formats, which have been described briefly supra, other immunoassays, which can be used in conjunction with the novel devices of the invention include: fluorescence polarization immunoassay (FPIA), fluorescence immunoassay (FIA) and nephelometric inhibition immunoassay (NIA). General techniques for performing the various immunoassays are known to one of skill in the art. Moreover, a general description of certain procedures is provided in U.S. Patent No. 5,051,361, which is incorporated herein by reference.

In one preferred embodiment, which is particularly suitable for polyvalent analytes, an appropriate amount of a sample containing the analyte is deposited onto the sample application area, so that the sample migrates to the labeling zone, where it binds to a labeling reagent (e.g., an analyte-specific antibody linked to gold colloid or colored particles). The labeled antibody binds to the analyte to form a complex. The complex (e.g., a colored analyte-antibody complex) then migrates through a plurality of detection sites. At each detection site, a portion of the analyte-antibody complex binds to the immobilized capture reagent, producing a pattern of colored bands which is characteristic and/or diagnostic for analyte concentration (at least qualitatively, and more preferably at least semi-quantitatively). The pattern of colored bands can be compared to a reference chart provided with the device, to assist the user in determining the concentration of the analyte in the sample. In another preferred embodiment, which is particularly suitable for monovalent analytes, a sample containing the analyte (e.g., an antigen) is added to a site at which it resuspends an antigen linked to colored particles. This colored particle-antigen complex along with the antigen in the test specimen migrates through several capture sites at which both analyte in the sample and colored particle-antigen complex compete for binding to the capture reagent (e.g., an immobilized antibody). This in turn will produce a pattern of colored bands which is characteristic and diagnostic of antigen concentration.

Exemplary Uses

The devices and kits of the invention are simple and inexpensive to produce, and can be used by non-skilled individuals with a minimum of training. Thus, the devices of the invention can be used by consumers or by professionals such as doctors. The devices and methods of the invention can be used in the following non-limiting assays:

Ovulation diagnostic

Ovulation is physiologically induced by an acute release or surge of LH by the hypophysis. This surge in LH triggers the release of an oocyte from a mature follicle in the ovary. Therefore, determination of the level of LH in plasma could be utilized to predict ovulation, which in turn permits timed intercourse to maximize the chances of achieving a pregnancy. LH levels in plasma generally increase from about 5 miU/mL to about 40 mlU/mL about 24 hours prior to the occurrence of ovulation.

Currently available home diagnostic test kits used for the prediction of ovulation are designed to provide a single band (i.e., a single detection zone) as an indicator of high or low concentrations of LH in urine. However, the difference in color intensity of the bands produced on the test kit when LH is present in urine at low, intermediate or high levels is difficult to discern from one another by the naked eye.

The invention described herein enables the user to assess the concentration of LH present before, during and after the LH surge. This is accomplished by the visualization of an increasing number of colored bands as the concentration of LH increases. As an example of the use of the instant invention for the determination of LH in urine, an assay device is provided according to the invention such that when the concentration of LH in urine is about 5 mlU/mL (baseline levels) no bands will be observed; if the concentration is 10 mlU/mL, one band will be observed; if the concentration is 20 miU/mL, two bands will be observed; and if the concentration is 40 mlU/mL, four bands will be observed. Such a device can be prepared by impregnating bands of substrate (each band having an area in the range of about 1 mm² to about 10mm²) with anti-α-subunit hCG antibody (capture reagent) in amounts from about 0.8 μg to about 52 μg. Exemplary amounts of anti-α-subunit hCG antibody include: band 1, 0.8 to 3.2 μg; band 2, 1.6 to 6.4 μg; band 3, 6.6. to 26 μg; and band 4, 13 to 52 μg. A detectable label is provided by a labeling reagent such as an antibody-gold conjugate (gold-anti-β-subunit hCG antibody) impregnated in the substrate or in a filter pad contacting the substrate (the labeling zone), as described for the device of Example 1, below.

The band pattern provided by the inventive device permits a more quantitative assessment of the concentration of LH in urine than by inspection of a single band in a conventional kit format.

Pregnancy Diagnostics

Determination of the concentration of human chorionic gonadotropic hormone (hCG) in plasma or urine is commonly used to assess the occurrence of a normal pregnancy or diagnose an ectopic pregnancy. Monitoring the concentration of hCG is particularly useful in the diagnosis of an ectopic pregnancy when the concentration of hCG is < 2,000 mlU/mL, since at this stage in embryo development, the pregnancy sac is too small to be seen by ultrasound. Currently available home diagnostic test kits used for the determination of hCG in urine are designed to provide a single-band endpoint as an indicator of high or low concentrations of hCG in urine. However, the difference in color intensity of the bands produced at different hCG concentrations is often difficult to discern by the naked eye.

The invention described herein permits the user to better assess the concentration of hCG in urine. This is accomplished by the visualization of an increasing number of colored bands as the concentration of hCG in urine increases. A typical example of the band pattern obtained using the multiband kit at different concentrations of hCG, is shown in Table 1 : when the concentration of hCG in urine is about < 50 mlU/mL, no bands are observed; if the concentration is about 250 mlU/mL, one strong and one very weak band are observed; if the concentration is about 500 mlU/mL, two strong and one weak band are observed; if the concentration is 1,000 mlU/mL, three strong and one moderate intensity band are observed; if the concentration is 2,500 mlU/mL, four strong bands are observed; and if the concentration is 5,000 mlU/mL, five strong bands are observed. Therefore, a more quantitative assessment of the concentration of hCG in urine can be obtained by viewing the pattern of the number and strength of colored bands. Respiratory Distress Syndrome Diagnostics

Surfactant apoproteins are obligate components of mature fetal lung surfactant. Four different apoproteins have been identified in mature fetal lung surfactant isolated from amniotic fluid: A, B, C and D. These apoproteins have been shown to play a key role in the in vivo adsoφtion of monolayers of dipalmitoyl phosphatidylcholine (DPPC) onto the air-liquid interface of the alveoli. Efficient apoprotein-induced coating of the alveoli wall with DPPC decreases surface tension in the alveoli which, in turn, prevents the development of respiratory distress syndrome in preterm infants. Therefore, testing for surfactant apoproteins in amniotic fluid can be useful for predicting the occurrence of respiratory distress syndrome in pregnancies at risk for preterm delivery. For example, concentration of 1.7 μg/mL of apoprotein A in amniotic fluid is diagnostic of respiratory distress syndrome.

The use of the multisite capture technology described above should permit more quantitative estimation of apoprotein and thus a more accurate prediction of respiratory distress syndrome.

Male Fertility/Infertility Diagnostics

Sperm antigens can be analyzed, for example, to (i) obtain the total count of sperm in a sperm-containing sample (e.g., semen); or (ii) evaluate sperm properties related to the corresponding antigen (e.g., acrosin is a sperm acrosome-specific enzyme that plays a key role in the induction of the acrosome reaction in mammalian sperm; low acrosin levels in sperm may be associated with male infertility). For example, International Patent Application Serial No. WO 96/13225, entitled Assays and Kits for Determining Male Fertility, by Juan G. Alvarez, describes and claims means for identifying fertile sperm samples (e.g. for use in an assisted reproductive technology or as an indication of an ineffective male contraception (vasectomy)) and sub-fertile samples (e.g. as an indication of a potentially infertile male donor or an effective male contraception), based on quantitation of a sperm antigen.

The present invention is further illustrated by the following Example, which is intended merely to further illustrate the invention and should not be construed as limiting. The entire contents of all of the references (including literature references, issued patents, published patent applications, and co-pending patent applications) cited throughout this application are hereby expressly incoφorated by reference. Example 1 Assay For Determining Human Chorionic Gonatotropin (hCG) Concentration in a Sample

Aliquots of 4 μL of different concentrations (1:16, 0.42 μg/μL; 1:8, 0.84 μg/μL; 1:2, 3.35 μg/μL; 1:1, 6.70 μg/μL) of goat anti-α-hCG antibody (capture reagent) (O.E.M. Concepts Inc., Toms River, NJ) were applied to 0.5 x 5 cm strips of nitrocellulose (Schleicher & Schuell, Keene, NH, 5μm in pore size) with a capillary pipette. The capture reagent was applied as five bands, each 1-3 mm wide, at 10, 14, 18, 22 and 26 mm from the origin. Each band contained about 4 microliters of solution. The strips were then allowed to dry at room temperature for about 15 minutes, dipped in a 0.5% solution of powdered milk in phosphate-buffered saline for about 5 minutes and then dried at room temperature for about 15 minutes. The proximal end of the nitrocellulose strip (the sample application area or origin) was then placed under a 0.5 x 0.5 cm filter pad embedded with colloidal gold/anti-β-hCG monoclonal antibody complex (labeling reagent) (O.E.M. Concepts Inc., Toms River, NJ). The pad was impregnated with colloidal gold/anti-β-hCG monoclonal antibody complex at a ^■concentration of 8.62 μg/μL. A macroporous filter was placed on top of the filter pad, and then a test solution of 150 μL of hCG (Sigma Chemical Co, St. Louis, MO) at a selected concentration was placed on the filter. The flow of colloidal gold/anti-β-hCG antibody complex through the nitrocellulose strip was complete after about 3 minutes. The nitrocellulose strip was then rinsed with distilled water, dried at room temperature for about 15 minutes, and the resulting bands scanned at 500 ran in the reflectance mode using a Shimadzu CS-9000 spectrodensitometer

The results obtained are set forth in Table 1.

Table 1 : Band Patterns at Different Concentrations of hCGi

ND: not detected

1 : Values correspond to the integration areas obtained following densitometric scanning at 500 ran of the bands generated on nitrocellulose strips.

2; The concentrations of capture antibody that correspond to the different dilutions are:

1 :16, 0.42 μg/μL; 1 :8, 0.84 μg/μL; 1:2, 3.35 μg/μL; 1 :1, 6.70 μg/μL. The bands thus contained the following total amounts of antibody: 1:16, 1.68 μg; 1:8, 3.36. μg; 1 :2, 13.4 μg; and 1 : 1, 27 μg. The 1 :16 band is closest to the sample application zone (origin) and the remaining bands are listed in order along the direction of fluid flow.

As expected, in Table 1 the intensity of bands for a given analyte concentration generally increases as the concentration of immobilized capture reagent increases (without wishing to be bound by any theory, the drop-off in intensity at the second, downstream, high-concentration (1:1) detection band is believed to be due to exhaustion of the analyte, which has been captured by the other, "upstream" bands). Also as expected, the bands having low concentration of immobilized capture reagent are not sensitive to low concentrations of analyte. Thus, at low analyte concentration, the analyte detection sites having low capture reagent concentration provide little or no signal, while bands having higher concentrations of capture reagent show a detectable signal. The concentration of the analyte can be determined, at least approximately, by noting which of the bands provides a detectable signal, and (optionally) comparing the pattern seen with a standard or control pattern. A second test strip was made as described above, except that each of the five analyte detection zones (bands) had the same concentration of capture reagent (the 1 : 1 concentration of capture antibody described above) applied. Two analyte concentrations were tested; the samples contained hCG at 1000 and 5000 mlU/mL, respectively. When a sample was applied to the test strip, and the developed strip analyzed as described above, the resulting band pattern showed substantially constant band intensity at a given analyte concentration, as would be expected. The bands were of greater intensity at the higher of the two analyte concentrations. These results show that a test strip in which all analyte detection zones (capture reagent sites) have a constant capture reagent concentration is useful for the detection of analyte, but less useful for quantitation of analyte than is a device in which the capture reagent sites contain differing concentrations of analyte capture reagent.

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of this invention and are covered by the following claims.

Other embodiments are within the following claims.

What is claimed is:

Claims

1. An assay device for determining a concentration of an analyte in a sample fluid, the device comprising a substrate including at least two detection zones, each of the detection zones comprising a pre-defined amount of an immobilized analyte-specific capture reagent.

2. The assay device of claim 1, wherein a first detection zone comprises a first concentration of the immobilized capture reagent, and a second detection zone comprises a second concentration of the immobilized capture reagent, the first concentration being greater than the second concentration.

3. The assay device of claim 2, wherein the substrate is configured to provide a direction of fluid flow of the sample fluid, and the first detection zone is configured to be upstream of the second detection zone along the direction of fluid flow.

4. The assay device of claim 1, wherein the device comprises at least three detection zones.

5. The assay device of claim 1, wherein the substrate further comprises a labeling zone which comprises a labeling reagent.

6. The assay device of claim 5, wherein the labeling reagent is a labeled antibody or a labeled antigen.

7. The assay device of claim 6, wherein the labeling reagent comprises an antibody reactive with hCG.

8. The assay device of claim 1, the immobilized capture reagent being operable to detect a drug of abuse.

9. The assay device of claim 1, wherein the substrate further comprises a filter element.

10. The assay device of claim 1, wherein the substrate further comprises a reservoir element.

11. A method for determining a concentration of an analyte in a sample fluid, the method comprising the steps of: supplying the sample fluid to an assay device comprising a substrate including at least two analyte detection zones, each of the analyte detection zones comprising a pre- defined amount of an analyte-specific capture reagent; and allowing the sample fluid to contact the at least two analyte detection zones; such that the concentration of the analyte in the sample fluid is determined.

12. The method of claim 11 , wherein the supplying step comprises applying the sample fluid to a sample application area of the assay device.

13. The method of claim 12, wherein the device further comprises a labeling zone which comprises a labeling reagent.

14. A device for determining a concentration of an analyte in a sample fluid, comprising: a fluid-permeable substrate having a sample application area in fluid communication with a plurality of detection zones, wherein each of the detection zones comprises a capture reagent capable of binding to an analyte, and wherein the concentration of the capture reagent in each of the zones is different, such that the zones bind differentially to different concentrations of an analyte labeling reagent complex; a labeling area disposed on the substrate between and in fluid communication with the sample application area and the detection zones, the labeling area comprising a labeling reagent which is capable of binding to the analyte to form an analyte:labeling reagent complex; the device being configured to allow flow of the sample fluid from the sample application area through the labeling area such that the analyte binds to the labeling reagent to form an analyte:labeling reagent complex, and flow of the analyte:labeling reagent complex to the plurality of detection zones, such that presence of bound label in the detection zones is indicative of concentration of the analyte in the sample.

15. The device of claim 14, wherein the labeling reagent comprises a colored particle, and the concentration of the analyte in the sample is determined visually.

16. The device of claim 15, wherein the colored particle is colloidal gold.

17. The device of claim 14, wherein the capture reagent comprises an antibody.

18. The device of claim 14, wherein the analyte is hCG.

19. The device of claim 14, wherein the analyte is LH.