WO2007092302A2

WO2007092302A2 - Test device for analyte detection

Info

Publication number: WO2007092302A2
Application number: PCT/US2007/002883
Authority: WO
Inventors: Tanya Schneider; Joan H. Lawton; Murali Bandla; Bruce S. Ferguson
Original assignee: Envirologix Inc.
Priority date: 2006-02-07
Filing date: 2007-02-02
Publication date: 2007-08-16
Also published as: WO2007092302A3

Abstract

As described below, the present invention features test devices and related methods that provide for the detection of an analyte in a sample. Such compositions include test devices that provide for the rapid and accurate detection of a contaminant in a sample from a biological fluid, environmental sample, or agricultural product.

Description

TEST DEVICE FOR ANALYTE DETECTION

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of the following U.S. Provisional Application No.: 60/771,252, which was filed on February 7, 2006. The entire contents of this application is hereby incorporated by reference.

BACKGROUND OF THE INVENTION

Procedures for detecting minute quantities of analytes in solution are widely used in the medical, environmental, and agricultural industries. In the medical field, analytes of clinical interest are detected in a variety of tissues or biological fluids for therapeutic or diagnostic purposes. In the environmental industry, water samples are analyzed for the presence of chemical or biological contaminants. The agricultural industry is also concerned with the detection of contaminants that could be harmful to human or animal health if introduced into the food supply. Contamination of food or water supplies by pathogens, for example, presents serious health and economic consequences. Many currently available methods for detecting contaminants in biological fluids, water, agricultural commodities, or other samples are time-consuming, expensive, or require highly trained laboratory personnel and sophisticated laboratory equipment. Rapid, inexpensive, sensitive, and highly accurate test devices and methods for detecting contaminants are required. Desirably, such tests could be reliably and accurately conducted by personnel without laboratory experience.

SUMMARY OF THE INVENTION

As described below, the present invention features test devices and related methods that provide for the detection of an analyte in a sample. Such compositions include test devices that provide for the rapid and accurate detection of a contaminant in a biological sample, environmental sample, or agricultural product.

In one aspect, the invention generally features a test device for detecting an analyte in a liquid sample (e.g., a biological sample, an environmental sample, or any other test sample). The device contains a liquid permeable material defining the following portions in capillary communication: a first portion that is the site for application of a liquid sample, containing a liquid permeable medium, and an analyte-binding conjugate; a second portion containing a liquid permeable medium; and a third portion that is the site for detecting the binding of the analyte-binding conjugate at the test site, the third portion containing a liquid permeable medium having the analyte fixed to the medium at the test site.

In another aspect, the invention features a test device for detecting an analyte in a liquid sample. The device contains a liquid permeable material defining the following portions in capillary communication: a first portion that is the site for application of a liquid sample, containing a liquid permeable medium, an analyte-binding conjugate and a control conjugate; a second portion containing a liquid permeable medium; and a third portion that is the site for detecting the binding of the analyte-binding conjugate at the test site and the binding of the control conjugate at a control site, the third portion containing a liquid permeable medium having the analyte fixed to the medium at the test site, and having a control conjugate binder present at a control site.

Optionally, the test devices of the previous aspects contain a fourth portion that acts as a wick. The fourth portion contains sorbent material. In embodiments of the previous aspects, the analyte-binding conjugate and the control conjugate coat the surface of the liquid permeable membrane in the first portion. In other embodiments of the previous aspects, the coating is absent from the sample application site. In yet other embodiments of the previous aspects, the analyte is aflatoxin and the analyte binding conjugate is an anti-aflatoxin antibody. In still other embodiments of the previous aspects, the anti-aflatoxin antibody is produced by a P2GA-2D2 cell line. In yet other embodiments of the previous aspects, the second portion contains an agent that alters the composition of the liquid as it contacts the second portion.

In another aspect, the invention features a method for detecting an analyte in a liquid sample. The method involves applying a liquid sample to a device of a previous aspect; and detecting the presence or the absence of an analyte-binding conjugate at a test site, wherein the absence of the analyte-binding conjugate at the test site identifies the presence of the analyte in the sample and the presence of analyte-binding conjugate at the test site identifies the absence of the analyte in the sample.

In another aspect, the invention features a test device for detecting an antigen in a liquid sample. The device contains a liquid permeable material defining the following portions in capillary communication: a first portion that is the site for application of a liquid sample, containing a liquid permeable medium, an anti-antigen antibody conjugate and a control antibody conjugate; a second portion containing a liquid permeable medium; and a third portion that is the site for detecting the binding of the anti-antigen antibody conjugate at a test site and the binding of the control antibody conjugate at a control site, the third portion containing a liquid permeable medium having the antigen fixed to the medium at the test site, and having an antibody that binds the control antibody present at a control site.

In various embodiments of the above aspects, the invention may be used for the detection of an analyte in a biological sample, environmental sample, or agricultural sample. In other embodiments, the invention is used for the detection of an analyte, such as an antigen having a low molecular weight, where the weight is any integer between 100 g/mol and 1000 g/mol (e.g., 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000). In other embodiments, the antigen is a peptide between 1 kD and 5 kD in size (e.g., 1 kD, 2 kD, 3 kD, 4 kD, and 5 kD). In still other embodiments, the antigen is a protein between IkD and 200 kD in size (e.g., 1, 10, 25, 50, 100, 125, 150, 175, and 200 kD). In particular, a test device of the invention is useful for the detection of aflatoxin Bl, which has a molecular weight of 312.27 g/mol. In other embodiments, the antigen is derived from a pathogen selected from the group consisting of a fungus (e.g., Aspergillus), bacterium, or virus. In various embodiments of the above aspects, the conjugate is a gold particle. In still other embodiments of the above-aspects, the second portion contains a liquid permeable material that acts as a filter to remove particulates or the second portion contains an agent (e.g., buffer, a surfactant, or a salt) that alters a physical characteristic (e.g., pH, salt concentration, or buffering capacity) of a liquid sample flowing through the second portion. In still other embodiments, the anti-antigen antibody is an anti-aflatoxin antibody (e.g., P2GA-2D2, which is publicly available from the U.S. Department of Agriculture). In still other embodiments, the device further includes a fourth portion containing sorbent material that facilitates the flow of liquid through the device. In still other embodiments, the device has increased sensitivity or increased discriminatory power (%Bo) relative to a conventional test device. The discriminatory power of the device is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. Device sensitivity is increased by at least 10%, 25%, 50%, 75%, 100%, 200%, 300% or more.

In another aspect, the method detects an antigen in a liquid sample (e.g., environmental sample, biological sample, or agricultural sample). The method involves applying a liquid sample to a device of any of the above aspects; providing for the flow of the liquid from the site of application to the test site; and detecting the presence or the absence of anti-antigen antibody conjugate binding at the test site, wherein the absence of anti-antigen antibody conjugate binding at the test site identifies the presence of the antigen in the sample and the presence of anti-antigen antibody conjugate binding at the test site identifies the absence of the antigen in the sample. In one embodiment, the method further involves the step of detecting control antibody binding at the control site, wherein detection of the binding indicates that the liquid has flowed past the test site to the control site. In still other embodiments, anti-antigen antibody conjugate binding is detected by visual inspection, for example, where binding results in the development of a color. In one particular embodiment, the anti-antigen antibody conjugate specifically binds aflatoxin. In yet another aspect, the invention provides a kit that includes a test device of any previous aspect. In one embodiment, the kit further includes instructions for the use of the device for the detection of an analyte. In another embodiment, the kit further includes a means for measuring and/or transferring a liquid sample and a test vial.

Other features and advantages of the invention will be apparent from the detailed description, and from the claims.

Definitions

By "aflatoxin" is meant a polyketide-derived secondary metabolite produced by Aspergillus parasiticus, Aspergillus flavus, or Aspergillus nomius.

By "agent" is meant any biological or synthetic chemical compound. Exemplary agents include salts, detergents, buffers, acids, bases, polypeptides, nucleic acid molecules, or fragments thereof.

By "alters the composition of the liquid" is meant produces a change in a physical property of the liquid.

By "antigen" is meant any compound, alone or conjugated to a carrier protein, that induces an immune response when administered to a subject capable of generating an immune response. Exemplary antigens include chemical compounds, haptens, polypeptides, peptides, nucleic acid molecules, and fragments or metabolic derivatives thereof, including low molecular weight secondary metabolites (e.g., mycotoxins, such as aflatoxin).

By "anti-aflatoxin antibody" is meant an antibody that specifically binds an aflatoxin. By "analyte" is meant any compound under investigation using an analytical method.

By "analyte-binding conjugate" is meant a detectable molecule that binds a compound under investigation.

By "capillary communication" is meant facilitating the flow of a liquid between liquid permeable materials. By "competitive assay" is meant any immunoassay that relies on antagonism between a labeled conjugate and an unlabeled conjugate for binding to an analyte of interest. Such binding provides a readout that indicates the presence or absence of the analyte in a sample.

By "a control conjugate" is meant a detectable molecule that does not substantially bind a compound under investigation. By "environmental sample" is meant a sample collected from soil, water, air, a surface wipe, a building material, fabric, or furnishing. Exemplary building materials include wall board, ceiling tiles, wall paper, and floor coverings (e.g., carpet, linoleum, and tiles). Exemplary fabrics include any textile typically found within a building (e.g., draperies, shades, curtains, shower curtains).

By "hapten" is meant a molecule that contains an antigenic determinant. Typically, haptens are low molecular weight molecules that are antigenic only when complexed with a carrier.

By "lateral flow device" is meant a test device that relies on the flow of a liquid via capillary action, wicMng, or wetting a liquid permeable media present in the device.

By "liquid permeable material" is meant a material susceptible to wetting, wicking, or transport of a liquid by capillary action.

By "low molecular weight compound" is meant any weight between 250 g/mol and 5 kD. Aflatoxin Bl is one exemplary low molecular weight compound (312.27 g/mol). By "portion" is meant some fraction of a whole. A portion of a test device, for example, maybe 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 of the length of the interior flow path of the device.

By "sample application site" is meant the portion of the device that contacts a liquid under analysis. By "specifically binds" is meant binds a target analyte without substantially binding to other non-target compounds present in the sample.

By "test device" is meant a device used in the detection of an analyte in a sample. By "wick" is meant sorb a liquid.

BRIEF DESCRIPTION OF THE DRAWINGS

Figures IA and IB are schematic diagrams that depict a test device of the invention in cross-section. Figure IA shows that a positive sample (sample containing an analyte of interest) is applied to the first portion of the lateral flow device (LFD); it flows from the application site to the test site. The absence of a signal at the test site indicates the presence of an analyte in the test sample. Figure IB shows that a negative sample is applied to the first portion of the LFD; it flows from the application site to the test site, where the labeled antibody binds to the immobilized antigen resulting in a signal. The presence of a signal at the test site indicates the absence of an analyte in the test sample. Figures 2 A and 2B are schematic diagrams showing the dimensions and design of a test device of the invention and of a conventional test device, respectively. Figure 2A shows a test device of the invention having an Antibody Conjugate Pad positioned in the first portion of the device (dark shading) and having a Sample Pad (light shading) in the second portion of the device. Figure 2B shows a conventional test device having the Sample Pad in the first portion of the device (light shading) and an Antibody Conjugate Pad positioned in the second portion of the device.

Figure 3 is a graph showing that the test device of the invention (having the Antibody Gold Conjugate Pad in the first portion of the device) has increased discriminatory power relative to a conventional test device (having the Sample Pad in the first portion of the device).

DETAILED DESCRIPTION OF THE INVENTION

The present invention features compositions and methods that are useful for the detection of an analyte in a test sample. In particular, the invention provides a test device, such as a lateral flow device, that comprises a liquid permeable media that provides for the flow of a liquid sample through the device. Test devices of the invention can be used for the detection of virtually any analyte of interest by. a detectably labeled reactant capable of specifically interacting with the analyte. The test device described herein is particularly suitable for the detection of an antigen of interest using an antibody that specifically binds the antigen. While particular examples describe the use of a lateral flow device for the detection of aflatoxin, a fungal metabolite that contaminates a variety of agricultural commodities, one skilled in the art appreciates that test devices and methods of the invention are not so limited. The test devices and methods of the invention are generally useful for the detection of virtually any antigen or hapten capable of detection using conventional immunoassay procedures.

Detection of Food Contaminants in Agricultural Commodities

Agricultural commodities are susceptible to contamination with a variety of undesirable microbes that are hazardous to human and animal health. Contamination of agricultural food products with fungi, for example, results in the production of mycotoxins. Mycotoxins are highly stable compounds that can contaminate agricultural commodities before harvest or during storage. Contamination by toxigenic fungi occurs not only in hot and humid climates, but in temperate conditions as well. The fungi Aspergillus flavus and A. parasiticus, which produce a mycotoxin known as aflatoxin (e.g., aflatoxin type Bl, B2, Gl, G2, Ml and M2), are common contaminants of tree nuts, peanuts, and other oilseeds, including corn and cottonseed. Milk, eggs, and meat products are sometimes contaminated when animals consume aflatoxin-contaminated feed. The toxicity of mycotoxins varies with the toxin, and with the animal species exposed to it. While the various forms of aflatoxin can occur in a single sample, aflatoxin B 1 is usually predominant and is the most toxic.

Humans are typically exposed to aflatoxins by consuming foods contaminated with products of fungal growth. Animal exposure to aflatoxin typically results from consuming contaminated feed. Human or animal exposure to toxic amounts of aflatoxin may result in acute aflatoxicosis. In humans this condition is characterized by vomiting, abdominal pain, pulmonary edema, convulsions, and coma. Because aflatoxins are carcinogenic, human exposure to low levels of aflatoxin may result in an increased risk of neoplasm. In one embodiment, the present invention provides test devices and methods for the sensitive, accurate and reliable detection of aflatoxin in a variety of food and agricultural products.

Design of a Test Device

The test device can take any form desired that provides for the flow of a liquid test sample from the point of contact with the test sample past the test and/or control sites. In general, the test device of the present invention includes an interior flow pathway that includes one or more liquid permeable materials. A schematic diagram showing an exemplary test device is provided at Figures IA and IB. In a first portion, the device includes a site for the application of a liquid sample. This first portion of the device also includes an analyte-binding conjugate, such as an antibody that specifically binds an antigen of interest. The analyte binding conjugate typically binds the analyte to form a complex. Complex formation (e.g., formation of an antigen/antibody conjugate complex) may occur at any point in the interior flow pathway after the analyte contacts the analyte-binding conjugate. For example, complex formation may occur or continue as the sample flows from the first portion to the second portion of the device.

The second portion of the device has a variety of features that enhance functionality. In one embodiment, the second portion is composed of a material capable of filtering the sample to prevent the flow of particulate matter through the device. In another embodiment, the second portion facilitates complex formation by increasing the time required for the liquid to flow from the site of application to the test site. Accordingly, the dimensions of the second portion may be altered (e.g., increased or decreased) to empirically determine for each application those dimensions that enhance sensitivity while reducing false positives, i.e., optimizing the signal-to-noise ratio. In yet another embodiment, the second portion of the device can be used to deliver a desired agent to the liquid as it flows through the device. For example, the second portion may be impregnated with a buffer (e.g., TRIS, sodium carbonate), surfactant (e.g., Tween, Triton), preservative (e.g., Na azide, thimerosol), salt, or other agent, such that contact of the sample with the second portion of the device alters the sample. Exemplary alterations include an increase or decrease in the pH of the sample, in the salt concentration, in the buffering capacity, or in the binding between the conjugate and the analyte. The third portion of the device includes a test site, which acts as a readout zone that provides for detection of an analyte in the sample. Various means for detecting the presence of an analyte at a test site are known in the art. In a competitive assay, a labeled probe competes with an analyte of interest for binding to a detector at the test site. The more analyte that is present in the sample, the more effectively it will be able to compete with, and/or displace, the binding of a detector. The hallmark of most competitive assays is that an increase in the amount of analyte in the sample results in a decrease of signal in the readout zone. In contrast, a "sandwich" format typically involves mixing the test sample with a detection probe conjugated with a specific binding member (e.g., antibody). The conjugate and the analyte bind to form a complex. These complexes are then allowed to contact a receptive material (e.g., antibody) that is immobilized at the test site. The analyte/conjugate complex binds to the immobilized receptive material to form a "sandwich complex" (e.g., antibody coηjugate/antigen/antibody). In this approach, detection of the "sandwich complex" indicates the presence of analyte in the sample.

It may be desirable to include a positive control to indicate that the liquid sample has traversed the interior flow path from the site of application past the test site. In a competitive assay format, the first portion of the device further includes a control conjugate and the third portion of the device includes a control site with a receptive material that binds the control conjugate. The control site is situated in the third portion of the device downstream from the test site. Detection of control conjugate binding at the control site indicates that the liquid sample flowed from the application site past the test site to the control site. In a sandwich assay format, a control antibody that binds the anti-antigen antibody is fixed at the control site. In the presence or absence of an antigen, excess anti-antigen antibody is detected at the control site. The device may also include in a fourth portion a wicking pad that contains sorbent material capable of absorbing or adsorbing excess liquid present in the liquid sample.

In one embodiment, the test device contains a liquid permeable material defining the following portions in capillary communication: a) a first portion that is the site for application of a liquid sample, comprising a liquid permeable medium, an anti-antigen antibody conjugate and a control antibody conjugate, where the first portion is between 5 mm and 20 mm in length; for example, the length of the first portion is equal to any integer between 5 and 20 (5, 10, 15, 20 mm in length); b) a second portion comprising a liquid permeable medium, where the second portion overlaps the first portion by at least 1, 2, 3, 4, or 5 mm; and the length of the second portion is between 10 mm and 40 mm; for example, the length of the second portion is any integer between 10 and 40 (e.g., 10, 15, 20, 25, 30, 35, 40); and c) a third portion that is the site for detecting the binding of the anti-antigen antibody conjugate at a test site and the binding of the control antibody conjugate at a control site, the third portion comprising a liquid permeable medium having the antigen fixed to the medium at the test site, and having an antibody that binds the control antibody present at a control site, wherein the third portion is between 15 and 40 mm in length; for example, is any integer between 15 and 40 (e.g., 15, 20, 25, 30, 35, 40); and the second portion overlaps the third portion by at least 1, 2, 3, 4, or 5 mm. In a fourth portion the device contains sorbent material. The sorbent material has a length between 25 and 75 mm. For example, the length is an integer between 25 and 75 (e.g., 25, 35, 50, 60, 70, 75). In one embodiment, the fourth portion overlaps the third portion by at least 1, 2, 3, 4, or 5 mm.

In general the interior flow path is between 1 mm and 10 mm in width; for example, the width of a test device (e.g., test strip) is any integer between 1 and 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10). In one embodiment the width of the strip is 3.8 mm. The design and dimensions of an exemplary test strip of the invention are shown in Figure 2 A. The design and dimensions of a conventional test strip are shown in Figure 2B. Desirably, a test device of the invention has increased sensitivity relative to a conventional test device. Sensitivity of a test device of the invention is increased by at least 5%, 10%, 25%, 50%, 75%, 100%, 150%, or 200% relative to a conventional test device. Antibodies

As described herein, the test device includes a conjugate that binds an analyte. In one approach, the conjugate is an antibody capable of binding an antigen capable of generating an immune response, either alone or when conjugated to another compound, as in the case of a hapten. Any antibody, antibody conjugate, or fragment thereof that binds an antigen of interest may be used in the present invention. Such antibodies or antibody conjugates are present within the interior flow path of the test device. Suitable antibodies include, but are not limited to, polyclonal antibodies, monoclonal antibodies, or fragments thereof. Such antibodies and their fragments are well known in the art. As used herein, the term "antibody" means not only intact immunoglobulin molecules but also the well-known active fragments F(ab')_2» and Fab. Preferably, the selected antibody binds an antigen of interest with high affinity. Many such antibodies are in the public domain or are commercially available. For example, antibodies against aflatoxin and methods of using them are described, for example, in U.S. Patent No. 5,212,065, 5,166,078, 4859,611, 4,835,100, and 4,818,687. Antibodies having a desired binding characteristic may be identified using methods known to the skilled artisan. One method of obtaining antibodies is to immunize a suitable host animal with an immunogen and to follow standard procedures for polyclonal or monoclonal antibody production. The desired antibodies are then purified from the host. Antibody purification methods may include salt precipitation (for example, with ammonium sulfate), ion exchange chromatography (for example, on a cationic or anionic exchange column preferably run at neutral pH and eluted with step gradients of increasing ionic strength), gel filtration chromatography (including gel filtration HPLC), and chromatography on affinity resins such as protein A, protein G, hydroxyapatite, and antiimmunoglobulin.

In one approach, antibodies are produced from hybridoma cells engineered to express a desired antibody. Methods of making hybridomas are well known in the art. The hybridoma cells can be cultured in a suitable medium, and spent medium can be used as an antibody source. Polynucleotides encoding the antibody of interest can in turn be obtained from the hybridoma that produces the antibody, and then the antibody may be produced synthetically or recombinantly from these DNA sequences. For the production of large amounts of antibody, it is generally more convenient to obtain an ascites fluid. The method of raising ascites generally comprises injecting hybridoma cells into an immunologically naive histocompatible or immunotolerant mammal, especially a mouse. The mammal may be primed for ascites production by prior administration of a suitable composition (e.g., Pristane). In order to detect the antibody/antigen complex within the test device, a detector reagent, or conjugate, must be coupled to the antibody or antigen. Exemplary conjugates include colored reagents, fluorescent compounds, enzymes, and radioactive isotopes. Colored or fluorescent compounds include gold particles, colored or fluorescent latex particles, polystyrene beads, and dyes, such as fluorescein isothiocyanate, BODIPY FL, Oregon Green, Alexa Fluor 488, phycoerythrin and phycocyanin. Colloidal metals, metal sols and other types of colored particles useful as marker substances in immunoassay procedures are known in the art. See, for example, U.S. Pat. No. 4,313,734. Antibody conjugates are widely available, for example, from a variety of well-known commercial sources (e.g., Molecular Probes (e.g., Zenon® labeling technology), Nanoprobes (e.g., Nanogold® Gold-Antibody Conjugates).

Enzymes that may be coupled to an antibody include peroxidases (such as horseradish peroxidase), phosphatases (such as acid or alkaline phosphatase), β-galactosidase, urease, glucose oxidase, carbonic anhydrase, acetylcholinesterase, glucoamylase, lysozyme, malate dehydrogenase, glucose-6-phosphate dehydrogenase, β-glucosidase, proteases, pyruvate decarboxylase, esterases, luciferase, or any other enzyme known to the skilled artisan. Enzymes are not in themselves detectable but must be combined with a substrate to catalyse a reaction the end product of which is detectable.

Antibodies, antibody conjugates, protein-antigen conjugates, and protein-hapten conjugates are fixed within the interior flow path using standard methods known to the skilled artisan. Protein immobilization protocols are known to the skilled artisan. See, for example, Laboratory Techniques in Biochemistry and Molecular Biology, Tijssen, Vol. 15, Practice and Theory of Enzyme Immunoassays, Chapter 13, The Immobilization of Immunoreactants on Solid Phases, pp. 297-328, and the references cited therein. In one approach, an antibody is immobilized directly on a solid support by physical adsorption or is bound covalently or through bridging molecules such as protein A, polylysine or to a solid support.

Interior Flow Path The test device comprises an interior flow path that facilitates the flow of a liquid sample through the device. This interior flow path contains one or more liquid permeable materials or membranes composed of any relatively inert material or a combination of materials suitable for transporting a liquid (e.g., glass fibers, polyester, nitrocellulose, fibers of cellulose or derivatives thereof, non-cellulose hydrocarbon materials, ceramics) from the contact site past the test and/or control sites and, optionally, into a reservoir. Suitable materials for use in the interior flow path are wettable and exhibit low non-specific binding. Materials having increased sorptivity promote the flow of liquid. Different materials having different absorption characteristics or sorptivities may be used in various portions of the flow path. If desired, the materials to be used are screened for optimal pore size and density in order to facilitate the controlled distribution of an antibody within a membrane, to optimize reaction kinetics, or to optimize the sensitivity, discriminatory ability, or signal-to-noise ratio of the device.

Solid Supports

For most applications, the test device includes an interior flow pathway fixed to a solid support. The physical shape of the solid support is not critical, although some shapes may be more convenient than others for the present purpose. Accordingly, the solid support may be in the shape of a paper strip, dipstick, membrane (e.g. a nylon membrane or a cellulose filter), a plate (e.g. a microtiter plate) or solid particles (e.g. latex beads). The solid support may be made of any suitable material, including but not limited to a plastic (e.g., polyethylene, polypropylene, polystyrene, latex, polyvinylchloride, polyurethane, polyacrylamide, polyvinylalcohol, nylon, polyvinyl acetate, or any suitable copolymers thereof), cellulose (e.g. various types of paper, such as nitrocellulose paper and the like), a silicon polymer (e.g. siloxane), a polysaccharide (e.g. agarose or dextran), or an ion exchange resin (e.g. conventional anion or cation exchange resins).

Sorbent Reservoir

The test device optionally includes a fourth portion that forms a reservoir of adsorbent or absorbent material (See Figures IA and IB). This reservoir sorbs excess liquid as it flows through the test device. For some applications, such as where the concentration of antigen in a test sample is particularly low, it may be desirable to apply large volumes of a liquid test sample to the test device. In such cases, the presence of the adsorbent material may enhance the sensitivity of antigen detection. Optionally, the region of the flow path in the test cell defining the test and control sites is restricted in cross-sectional area relative to other regions of the flow path. This feature produces a "bottle-neck" effect wherein the antigen in the entire volume of adsorbed sample must pass through an area of restricted flow immediately above the test site. This "bottle-neck" may facilitate sandwich formation. Suitable sorbent materials include virtually any commercial material (e.g., synthetic or natural materials, such 83

as cotton) capable of absorbing many times its weight in water. Such materials are widely available in commerce.

Methods of Using the Test Device The invention provides methods of using a test device of the invention for the detection of an analyte (e.g., an antigen) in a test sample. In one example, the assay is conducted by placing the leading edge (first portion) of a lateral flow device in contact with a liquid test sample. In another example, the sample is brought into contact with the device by applying a liquid test sample to the first portion of the lateral flow device in a drop-wise fashion.

Test Samples

Methods and compositions of the invention are useful for the identification of an analyte (e.g., chemical or biological in origin) in a test sample. In one embodiment, the methods of the invention are suitable for detecting analytes of biological origin. Test samples include, but are not limited to, any liquid containing a dissolved or dispersed analyte of biological origin. Exemplary test samples include body fluids (e.g. blood, serum, plasma, amniotic fluid, sputum, urine, cerebrospinal fluid, lymph, tear fluid, feces, or gastric fluid), tissue extracts, culture media (e.g., a liquid in which a cell, such as a pathogen cell, has been grown), environmental samples, agricultural products or other foodstuffs, and their extracts. In one embodiment, a test device of the invention detects the presence of a pathogen in a sample. Exemplary pathogens include fungal, bacterial, or viral proteins or metabolites, including secondary metabolites, such as toxins, in a test sample. Exemplary toxins include, but are not limited to aflatoxin, cholera toxin, diphtheria toxin, Salmonella toxin, Shiga toxin, Clostridium botulinum toxin, endotoxin, and mycotoxin. In another embodiment, a test device of the invention detects a peptide or protein, such as a hormone or an allergen. Peptide and protein hormones include, but are not limited to, angiotensin I and II, bradykinin, chorionic gonadotropin, corticotropin, insulin, erythropoietin, follicle-stimulating hormone, gastrin, glucagon, human growth hormone, human placental lactogen, gonadotropin, luteinizing hormone, luteotropin, melanotropin, relaxin, somatotropin, thyrotropin, prolactin, oxytocin, parathyroid hormone, secretin, thyrocalcitonin, and vasopressin. Allergens include but are not limited to; arachnid allergens, such as house dust mite allergens (e.g., Der p I, Der f I, etc.); storage mite allergens; Japanese cedar pollen/hay fever; mold spore allergens; animal allergens (e.g., dog, guinea pig, hamster, gerbil, rat, mouse, etc., allergens); insect allergens; venoms: (Hymenoptera, yellow jacket, honeybee, wasp, hornet, fire ant); environmental insect allergens (e.g., cockroaches, fleas, and mosquitoes); bacterial allergens (e.g., streptococcal antigens); parasite allergens (e.g., Ascaris antigen); viral antigens; fungal spores; drug allergens (e.g., antibiotics; penicillins and related compounds; enzymes (streptokinase); drugs and their metabolites capable of acting as incomplete antigens or haptens; industrial chemicals and metabolites capable of acting as haptens and functioning as allergens (e.g., the acid anhydrides, such as trimellitic anhydride, and the isocyanates, such as toluene diisocyanate); occupational allergens, such as flour (e.g., allergens causing Baker's asthma), castor bean, coffee bean, and industrial chemicals, and allergens derived from Dermataphagoides farinae, Alternaria alternata, Aspergillus fumigatus, Cladosporium herbarum, Fusarium vasinfectum, Helminthosporium sativum, Mucor recemosus, Penicillium notatum, Pullularia pullulans, Rhizopus nigricans and/or Tricophyton spp.

If the test sample is not in itself sufficiently fluid for the present purpose, it may be admixed with a suitable fluid to the desired fluidity, for instance by homogenization. In one example, an agricultural sample is homogenized in any suitable liquid to test for the presence of contaminants, such as pathogens or their toxins. For environmental applications, test samples may include water, liquid extracts of air filters, soil samples, building materials (e.g., drywall, ceiling tiles, wall board, fabrics, wall paper, and floor coverings), environmental swabs, or any other sample suitable for use in a liquid assay.

Kits

The invention provides kits that include a test device for the detection of an analyte in a sample. In one embodiment, the kit includes a lateral flow device described herein. In some embodiments, the kit comprises a container, which contains the lateral flow device; such containers can be boxes, ampoules, bottles, vials, tubes, bags, pouches, blister packs, or other suitable container forms known in the art. In one embodiment, such containers may be sterile. Such containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding medicaments.

If desired the device is provided together with instructions for using it to identify the presence or absence of an analyte in a sample. The instructions will generally include information about the use of the device for the identification of a particular analyte, such as an antigen in a liquid sample (e.g., a environmental sample, biological sample, or liquid sample extracted from an agricultural commodity). The instructions may be printed directly on the container (when present), or as a label applied to the container, or as a separate sheet, pamphlet, card, or folder supplied in or with the container. If desired, the kit may also include a standard measure pipet, a test vial, and/or a liquid (e.g., ethanol, methanol, organic solvent, suitable buffer, such as phosphate buffered saline, or water) to be used in the extraction of a sample.

EXAMPLES

Components of Lateral Flow Device: 1. Antibody Conjugate Pad

Gold conjugate preparation and application to pad The lateral flow device includes an Antibody Conjugate Pad that is positioned at the lower edge of the strip. The Antibody Conjugate Pad was prepared as follows.

Anti-aflatoxin monoclonal antibody was purified from cell line P2GA-2D2 using a protein A column. The purified antibody was diluted to 0.25 mg/mL in 50 mM Tris buffer, pH 8.6. This antibody was titrated with gold particles to determine the point at which the antibody completely coated the particles. While many coating levels were tested, an optimized coating level of antibody on gold conjugate was empirically determined.

Using BioDot Reel-to-Reel equipment, gold conjugate spray solution, including 10% control gold-goat IgG conjugate, was applied to polyester (Ahlstrom Filtration - MHS PN: Grade# 6613, 10 mm wide, on a 3" paper core). The volume of gold sprayed onto the polyester was varied to achieve an optimal range of sensitivity for the test. This was determined empirically for each lot. Under optimal conditions, at 20 ppb aflatoxin no signal at the test site is observed. For each lot of strips prepared, test gold sprays were performed and matched to the sprayed nitrocellulose.

2. Sample Pad

Treated glass fiber

The lateral flow device includes a Sample Pad composed of fiberglass (Ahlstrom Filtration - MHS, Grade# 8975, 25 mm wide on a 3" core) treated with a buffer. 3. Nitrocellulose Membrane with Test and Control Lines

Treatment of nitrocellulose membrane

The capture line solution contained 0.125 mg/mL BSA*Aflatoxin Bj conjugate (Sigma A-6655) in a buffer. This was positioned 13 mm from the leading edge of the lateral flow device, as shown in Figure IA.

The control line solution contained rabbit anti-goat IgG (Jackson ImtmmoResearch Labs, PN 305-001-003) at 2 mg/mL in phosphate buffered saline with Na azide. Using BioDot Reel-to-Reel equipment, both solutions were sprayed onto nitrocellulose membrane (Schleicher and Schuell AE99, 8 μm pore size, 25 mm x 100 M, PN 10548081); the control line was sprayed at 18 mm from the lower edge.

4. Wicking Pad

A cotton linter absorbent pad (#470, 39 mm, Schleicher and Schuell) was used.

Assembly of new anatoxin lateral flow strip

The components described above were mounted onto polystyrene backing in accordance with the design shown in Figures IA and B and using the dimensions shown in Figure 2A. The nitrocellulose membrane with test and control lines was laminated onto the polystyrene backing first. The antibody conjugate pad was positioned in the first portion of the device. The nitrocellulose membrane with test and control lines (in the third portion of the device) and the antibody conjugate pad were overlaid with a bridging treated glass fiber sample pad (in the second portion of the device), such that the sample pad overlapped the nitrocellulose by 4 mm. A cotton linter absorbent pad (#470, 39 mm, Schleicher and Schuell) was placed at the edge furthest from the antibody conjugate pad.

Sample testing

The strip was evaluated using commercially available ground corn samples that were naturally contaminated with known amounts of aflatoxin (Trilogy, certified reference material). Ten grams of corn were combined with 20 mL of 70% methanol, shaken for 1 minute at high speed on an orbital shaker. The contents were then allowed to settle and the supernatant was removed for analysis.

Each sample was tested in a 1.5 mL reaction vial. Sixty microliters of tap water was placed in the vial followed by 60 μL of extract; and the mixture was stirred. .The aflatoxin strip was then placed into the sample in the reaction vial. The results of the analysis were T7US2007/002883

evaluated after 5 minutes. The intensity of the lines was quantitated using an EnviroLogix QuikStix Reader.

The results of this analysis are shown in Table 1 and in Figure 3.

Table 1

%B₀ = [intensity of test line/intensity of negative sample test line] X 100. The test device, where the gold conjugate pad is located in the first portion of the device (as shown in Figures IA and IB), has increased discriminatory power (greater change in %Bo) relative to a conventional device as noted in Table 1. For example, the difference in %B<> for the test device between 0 ppb (parts per billion) and 20 ppb is 98.25%; the difference in %Bo for a conventional device is merely 72.9%.

Other Embodiments

From the foregoing description, it will be apparent that variations and modifications may be made to the invention described herein to adapt it to various usages and conditions. Such embodiments are also within the scope of the following claims.

The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

All patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each independent patent and publication was specifically and individually indicated to be incorporated by reference.

Claims

CLAIMSWhat is claimed is:

1. A test device for detecting an analyte in a liquid sample, the device comprising a liquid permeable material defining the following portions in capillary communication: a) a first portion that is the site for application of a liquid sample, comprising a liquid permeable medium, and an analyte-binding conjugate; b) a second portion comprising a liquid permeable medium; and c) a third portion that is the site for detecting the binding of the analyte-binding conjugate at the test site, the third portion comprising a liquid permeable medium having the analyte fixed to the medium at the test site.

2. A test device for detecting an analyte in a liquid sample, the device comprising a liquid permeable material defining the following portions in capillary communication: a) a first portion that is the site for application of a liquid sample, comprising a liquid permeable medium, an analyte-binding conjugate and a control conjugate; b) a second portion comprising a liquid permeable medium; and c) a third portion that is the site for detecting the binding of the analyte-binding conjugate at the test site and the binding of the control conjugate at a control site, the third portion comprising a liquid permeable medium having the analyte fixed to the medium at the test site, and having a control conjugate binder present at a control site.

3. The test device of claim 1 or 2, wherein the analyte-binding conjugate and the control conjugate coat the surface of the liquid permeable membrane in the first portion.

4. The test device of claim 3, wherein the coating is absent from the sample application site.

5. The test device of claim 1 or 2, further comprising a fourth portion that acts as a wick, the fourth portion comprising sorbent material.

6. The test device of claim 1 or 2, wherein the analyte is aflatoxin.

7. The test device of claim 1 or 2, wherein the analyte binding conjugate is an anti- aflatoxin antibody.

8. The test device of claim 1 or 2, wherein the anti-aflatoxin antibody is P2GA-2D2.

9. The test device of claim 1 or 2, wherein the second portion comprises an agent that alters the composition of the liquid as it contacts the second portion.

10. A method for detecting an analyte in a liquid sample, the method comprising: a) applying a liquid sample to the device of claim 1 or 2; and b) detecting presence or absence of an analyte-binding conjugate at a test site, wherein the absence of the analyte-binding conjugate at the test site identifies the presence of an analyte in the sample and the presence of analyte-binding conjugate at the test site identifies the absence of the analyte in the sample.

11. A test device for detecting an antigen in a liquid sample, the device comprising a liquid permeable material defining the following portions in capillary communication: a) a first portion that is the site for application of a liquid sample, comprising a liquid permeable medium, an anti-antigen antibody conjugate and a control antibody conjugate; b) a second portion comprising a liquid permeable medium; and c) a third portion that is the site for detecting the binding of the anti-antigen antibody conjugate at a test site and the binding of the control antibody conjugate at a control site, the third portion comprising a liquid permeable medium having the antigen fixed to the medium at the test site, and having an antibody that binds the control antibody present at a control site.

12. The test device of claim 11 , wherein the antigen is a low molecular weight compound between 100 g/mol and 1000 g/mol.

13. The test device of claim 12, wherein the antigen has a molecular weight between 200 g/mol and 400 g/mol.

14. The test device of claim 11 , wherein the antigen has a molecular weight between 1 kD and 200 kD.

15. The test device of claim 11 , wherein the antigen is derived from a pathogen selected from the group consisting of a fungus, bacterium, or virus.

16. The test device of claim 15, wherein the fungus is Aspergillus.

17. The test device of claim 16, wherein the antigen is aflatoxin.

18. The test device of claim 11, wherein the conjugate is a gold particle.

19. The test device of claim 11 , wherein the second portion comprises a liquid permeable material that acts as a filter to remove particulates.

20. The test device of claim 11 , wherein the second portion comprises an agent that alters a physical characteristic of a liquid sample flowing through the second portion.

21. The test device of claim 20, wherein the agent is a buffer, a surfactant, or a salt.

22. The test device of claim 21, wherein the altered physical characteristic is pH, salt concentration, or buffering capacity of the liquid sample.

23. The test device of claim 11, wherein the anti-antigen antibody is an anti-aflatoxin antibody.

24. The test device of claim 11, wherein the device further comprises a fourth portion comprising absorbent material that facilitates the flow of liquid through the device.

25. The test device of claim 11, wherein the device has increased sensitivity relative to a conventional test device.

26. The test device of claim 24, wherein sensitivity is increased by at least 10%.

27. A method for detecting an antigen in a liquid sample, the method comprising: a) applying a liquid sample to the device of claim 11; b) providing for the flow of the liquid from the site of application to the test site; and c) detecting the presence or the absence of anti-antigen antibody conjugate binding at the test site, wherein the absence of anti-antigen antibody conjugate binding at the test site identifies the presence of the antigen in the sample and the presence of anti-antigen antibody conjugate binding at the test site identifies the absence of the antigen in the sample.

28. The method of claim 27, further comprising the step of detecting control antibody binding at the control site, wherein detection of the binding indicates that the liquid has flowed past the test site to the control site.

29. The method of claim 27, wherein anti-antigen antibody conjugate binding is detected by visual inspection.

30. The method of claim 29, wherein anti-antigen antibody conjugate binding results in the development of a color.

31. The method of claim 29, wherein the anti-antigen antibody conjugate specifically binds aflatoxin.

32. A kit comprising a test device of claim 1 or claim 11.

33. The kit of claim 32, further comprising instructions for the use of the device for the detection of an analyte.

34. The kit of claim 32, further comprising a means for measuring a liquid sample and a test vial.