WO2005069002A1

WO2005069002A1 - Rapid test for antibodies against hiv in urine

Info

Publication number: WO2005069002A1
Application number: PCT/US2003/041647
Authority: WO
Inventors: Ronald Mink; Toby Gottfried; John Ennis; Paul Smith
Original assignee: Calypte Biomedical Corporation
Priority date: 2003-12-30
Filing date: 2003-12-30
Publication date: 2005-07-28
Also published as: AU2003300109A1

Abstract

The present invention provides devices and methods suitable for the rapid detection of endogenous urine antibodies, particularly antibodies directed against HIV viral coat proteins. Other pathogens giving rise to endogenous urine antibodies, and therefore detectable using the present invention include those organisms known to be causative agents in sexually-transmitted diseases.

Description

RAPID TEST FOR ANTIBODIES AGAINST HIV IN URINE

CROSS-REFERENCES TO RELATED APPLICATIONS [0001] NOT APPLICABLE

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT [0002] NOT APPLICABLE

REFERENCE TO A "SEQUENCE LISTING," A TABLE, OR A COMPUTER PROGRAM LISTING APPENDIX SUBMITTED ON A COMPACT DISK. [0003] NOT APPLICABLE

FIELD OF THE INVENTION

[0004] The present invention relates to immunochemistry and biochemical analysis, providing devices and methods suitable for the rapid detection of endogenous urine antibodies, particularly antibodies directed against HIV viral coat proteins. Other pathogens giving rise to endogenous urine antibodies, and therefore detectable using the present invention include those organisms known to be causative agents in sexually-transmitted diseases.

BACKGROUND OF THE INVENTION

[0005] Many types of lateral flow assays have been used to detect the presence of various substances in body fluids such as urine or blood. These assays typically involve antigen- antibody reactions, synthetic conjugates comprising enzymatic, fluorescent, or visually observable tags, and specially designed reactor chambers. In most of these assays, there is a receptor (e.g. an antibody) which is specific for the selected antigen, and a means for detecting the presence and/or amount of the antigen-antibody reaction product. Most current tests are designed to make a quantitative determination, but in many circumstances all that is required is a positive/negative indication. Examples of such qualitative assays include blood typing, pregnancy testing and many types of urinalysis. For these tests, visually observable indicia such as the presence of agglutination or a color change are preferred.

[0006] The positive/negative assays must be very sensitive because of the often small concentration of the ligand of interest in the test fluid. False positives can be troublesome, particularly with agglutination and other rapid detection methods such as dipstick and color change tests. Because of these problems, sandwich assays and other sensitive detection methods which use metal sols or other types of colored particles have been developed. These techniques have not solved all of the problems encountered in these rapid detection methods however, and a need still exists for detection methods that are both sensitive and selective in detecting target analytes present in body fluids at small concentrations, such as diagnostic assays that detect antibodies endogenous to urine and directed against pathogens.

BRIEF SUMMARY OF THE INVENTION

[0007] The present invention provides devices and methods suitable for the rapid detection of endogenous urine antibodies, particularly antibodies directed against HIV viral coat proteins. The inventors have found that by operating in elevated pH ranges, a much more sensitive and selective assay than is currently available can be carried out. Accordingly, in one embodiment the present invention provides a lateral flow device for the detection of target antibodies in urine, the device comprising an antigen that specifically binds the target antibodies and a sample zone comprising a buffer producing a solution having a pH of at least 8 when contacted with urine. In some aspects of the embodiment the buffer comprises carbonate. Variations of this include devices having buffers comprising carbonate and bicarbonate. A preferred aspect is a device where the buffer comprises potassium bicarbonate and potassium carbonate in a 2:1 molar ratio.

[0008] In another aspect of the above embodiment, the antigen is a human immunodeficiency virus (HIV) protein. In some variations the HIV is HIV-1, in others the HIV is HIV-2. In still other variations the more than one antigen is utilized, with the multiple antigens being from HIV-1 and/or HIV-2. In certain aspects of the embodiment, the protein antigen is recombinantly produced. In preferred aspects the HIV protein is an envelope protein. In another preferred aspect, the protein is a non-native peptide of gpl20 or gp41 from HIV-1 or gp36 from HIV-2, having an amino acid sequence selected from SEQ ID NO: 1 through 4. Still other preferred aspects include multiple HIV proteins including any and all combinations of the pep tides presented in SEQ ID NO: 1 through 4. In some aspects of the embodiment, the antigen is biotinylated, and linked to the matrix of the device through a strepavidin- or avidin-biotin linker.

[0009] In an additional aspect of the embodiment above, the sample zone further comprises avian serum, preferably chicken serum. Other aspects include a conjugation zone comprising labeling agent conjugated to a label. A preferred labeling agents of this aspect are Protein G, more preferably, Protein A. A preferred label is colloidal gold. Additional variations on the above embodiment include the addition of a control zone comprising a capture agent. Preferred capture agents include anti-human IgG antibody, more preferably the anti-human antibody is goat anti-human IgG antibody.

[0010] A second embodiment of the present invention is a lateral flow device for detection of antibodies in urine, the device comprising: (a) a conjugation zone comprising a Protein A/colloidal gold conjugate, and (b) a control line comprising a capture agent, wherein the capture agent has an affinity for a human urine antibody bound to the Protein A/colloidal gold conjugate that is greater than the affinity of Protein A for the human urine antibody bound to the Protein A/colloidal gold conjugate. As with the previous embodiment, this embodiment may have a capture agent that is an anti-human IgG antibody, preferably a goat anti-human IgG antibody, another aspect of the present embodiment is the addition of a sample zone comprising a buffer having pH of at least 8 and an antigen that specifically binds the target antibodies. Preferred buffers of the embodiment include carbonate salts, and or bicarbonate salts, that include a potassium or ammonium counterion. The buffer of these aspects of the embodiment may include both potassium bicarbonate and potassium carbonate in a 2:1 molar ratio.

[0011] An other aspect of the second embodiment has an antigen that is a human immunodeficiency virus (HIV) protein. The HIV can be HIV-1 or HIV-2, depending on which alternative aspect is chosen. An additional aspect utilizes multiple antigens from HIV- 1 and/or HIV-2. In some aspects the antigen is an envelope protein. In other aspects the antigen is selected from the group consisting of SEQ LD NO:l, 2, 3 and 4. As in the previous embodiment, antigens of the present embodiment may be isolated from natural sources or recombinantly produced. Several aspects of the second embodiment also have a sample zone further comprising avian serum, preferably chicken serum. [0012] Both first and second embodiments of the invention, as well as all aspects of these embodiments are also provided in kit form, packaged with one or more ancillary articles, as described herein.

[0013] A third embodiment of the invention is a method for detecting antibodies in a urine sample from a patient, the method comprising contacting the sample with a sample zone on a lateral flow device, wherein the sample zone comprises a buffer having a pH of at least 8. Various aspects of this method embodiment utilize the same variety of buffers and antigens as the previous two embodiments.

[0014] A fourth embodiment of the invention is an additional method, this method being for detecting antibodies in a urine sample from a patient, the method comprising contacting the sample with a sample zone on a lateral flow device comprising a conjugation zone comprising a Protein A/colloidal gold conjugate, and a control line comprising a capture agent, wherein the capture agent has an affinity for a human urine antibody bound to the Protein A/colloidal gold conjugate that is greater than the affinity of Protein A for the human urine antibody bound to the Protein A/colloidal gold conjugate. Again, this embodiment shares the same variety of aspects as previous embodiments with regard to antibodies, antigens and buffers.

DETAILED DESCRIPTION OF THE INVENTION

[0015] The present invention provides devices and methods suitable for the rapid detection of endogenous urine antibodies, particularly antibodies directed against HIV viral coat proteins. Other pathogens giving rise to endogenous urine antibodies, and therefore detectable using the present invention include those organisms known to be causative agents in sexually-transmitted and infectious diseases. Exemplary causative agents and disease states detectable using the present invention include Chlamydia, herpes virus, gonorrhea, syphilis, Campylobacter pylori, hepatitis A, C, and H viruses, EBV, CMV, HSV, malaria, influenza, West Nile virus, Rubella, Dengue fever, Lyme disease, Chagas, tuberculosis, toxoplasmosis, Ebola and the like. Utilizing buffers suitable for maintaining the pH of urine solutions at or above 8, these devices can be constructed of simple, cost-effective reagents that are not functional at lower pH values. By maintaining the pH of the urine sample at or above 8, the sensitivity and specificity of the present invention is dramatically increased as the visual signal presented by the label of the invention is substantially enhanced over the signal presented at a lower pH, providing a more accurate determination than current analytical techniques.

[0016] The devices and methods of the present invention are also cost-effective, as they maximize the use of low cost reagents, such as non-specific antibody binding proteins like protein A , protein G or other lectin, in the immunoassays of the invention. A surprising finding from this work is that only one molecule of this type of antibody binding protein can be present in an immunocomplex of the invention.

[0017] Assay devices of the present invention comprise a series of distinct zones defined by the reagents and/or reactions that take place within the respective zones during the operation of the device. The zones may be part of a single continuous matrix, or incorporated into two or more discrete pads that are brought into fluid communication in the claimed device.

[0018] To further ease in understanding the terminology of the present invention, the following definitions are provided:

I. Definitions

[0019] An "analysis zone" is a region of a flow path that includes an immobilized antigen that specifically binds a target antibody endogenous to the urine sample being tested. Specific binding of the target antibody by the antigen retains the target antibody, and any molecule associated with it, in the analysis zone.

[0020] An "antigen" is a substance that, when introduced into a mammal or bird, stimulates the production of an antibody. Subsequently, the antibody recognizes and specifically binds the antigen that stimulated its production. Preferable antigens of the present invention include human immunodeficiency virus (HIV) proteins, particularly the viral envelope proteins gpl20 and gp41 of HIV-1, and gp36 of HIV-2. Target antibodies endogenous to a patient urine sample, as defined below, are also antigens recognized by antibodies of the invention raised against the target antibody type in an species different from that of the patient, for example, a target antibody in a human urine sample is an antigen that is specifically recognized by an anti-human antibody in a non-human species.

[0021] An "antibody" is a polypeptide comprising a framework region from an immunoglobulin gene or fragments thereof that specifically binds and recognizes an antigen. The recognized immunoglobulin genes include the K, λ, α, γ, δ, ε, and μ constant region genes, as well as the myriad immunoglobulin variable region genes. Light chains are classified as either K or λ. Heavy chains are classified as γ, μ, α, δ, or ε, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively. Antibodies of the invention may be polyclonal or monoclonal.

[0022] Antibodies exist, e.g., as intact immunoglobulins or as a number of well- characterized fragments produced by digestion with various peptidases. Thus, for example, pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)'₂, a dimer of Fab which itself is a light chain joined to VH-CHi by a disulfide bond. The F(ab)'₂ may be reduced under mild conditions to break the disulfide linkage in the hinge region, thereby converting the F(ab)' dimer into an Fab' monomer. The Fab' monomer is essentially Fab with part of the hinge region (see Fundamental Immunology (Paul ed., 3rd ed. 1993)). While various antibody fragments, or antigen-binding fragments, are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such fragments may be synthesized de novo either chemically or by using recombinant DNA methodology. Thus, the term antibody, as used herein, also includes antibody fragments either produced by the modification of whole antibodies, or those synthesized de novo using recombinant DNA methodologies (e.g., single chain Fv) or those identified using phage display libraries (see, e.g., McCafferty et al, Nature, 348:552-554 (1990) and Zapata et al., Protein Eng. 8(10): 1057-1062 [1995]. Antibodies of the present invention may be monoclonal or polyclonal. Preparation of both monoclonal or polyclonal antibodies, are known in the art (see, e.g., Kohler and Milstein, Nature, 256:495-497 (1975); Kozbor et al, Immunology Today, 4:72 (1983); Cole et al, pp. 77-96 in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. (1985); (U.S. Patent 4,946,778)).

[0023] "Target antibodies" are antibodies endogenous to the urine sample being tested, and detectable by the devices of the present invention. Target antibodies may be antibody fragments, as described above, but more typically are whole immunoglobulins, as they are characterized as having functional variable and constant regions. The functional variable region of a target antibody specifically binds to a corresponding antigen, while the functional constant region is specifically bound by an anti-target antibody antibody (i.e., a secondary antibody) or bound by protein A or G, or other binding moiety that specifically binds the target antibody. [0024] "Bibulous" refers the ability of certain absorbent materials to support differential solute migration rates during fluid flow through the absorbent material. Absorbent materials with bibulous properties are therefore capable of chromatographic separation of solutes based on physical and/or chemical properties.

[0025] A "capture agent" is any molecule that specifically binds a target antibody. Capture reagents of the present invention are preferably immobilized to the matrix in a defined pattern, typically a line perpendicular to the flow path. Preferred capture reagents are anti- target antibody antibodies, protein A and protein G.

[0026] "Colloidal gold" refers to a sol of fine gold particles that are capable of remaining in an aqueous suspension indefinitely.

[0027] "Protein A" refers to a highly stable surface receptor produced by Staphylococcus aureus, which is capable of binding the Fc portion of immunoglobulins, especially IgGs, from a large number of species (Boyle, M. D. P. and K. J. Reis. Bacterial Fc Receptors. Biotechnology 5:697-703 (1987).). One protein A molecule can bind at least 2 molecules of IgG simultaneously (Sjόquist, J., Meloun, B. and Hjelm, H. Protein A is isolated from Staphylococcus aureus after digestion with lysostaphin. Eur J Biochem 29: 572-578 (1972)).

[0028] "Protein G" refers to a cell surface-associated protein from streptococcus that binds to IgG with high affinity. It has three highly homologous IgG-binding domains. (See Lian, et al. 1992. Journal of Mol. Biol. 228:1219-1234 and Derrick and Wigley. 1994. Journal of Mol. Biol. 243:906-918.)

[0029] "Label" refers to a detectable atomic or molecular moiety that specifically associates with an analyte either directly or indirectly through an analyte-specific binding partner. Labels of the present invention may be detected physically or chemically. Preferable labels are visible to the naked eye when associated to indicate a positive assay result.

[0030] "Matrix" refers to an insoluble material capable of supporting fluid flow. Matrix materials may be from natural and/or synthetic sources, bibulous or non-bibulous, fibrous or particulate. Matrices of the invention may be formed as continuous strips of the same material or mixtures of different materials that are distributed consistently along a common strip, or inconsistently such as to form zones having different physical or chemical characteristics in different regions of the strip. Alternatively, a series of discrete pads can be formed from the same or different matrix materials, with reagents for the assay being added to each pad. The pads may then be placed in fluid communication with each other to form a continuous flow path. Materials used to construct matrices of the invention may be inert or may react with one or more reagents of the invention, provided that the materials remain insoluble during the practice of the invention as described herein.

[0031] "Downstream" refers to the directional flow path of a liquid, through a matrix, away from the point of liquid application.

[0032] "Upstream" refers to the directional flow path of a liquid, through a matrix, toward the point of liquid application.

[0033] "Flow path" refers to the route taken by a urine sample as it passes through a matrix. The flow path is preferably a single route, but may include several routes where each route may support liquid flow simultaneously, sequentially or independently relative to other routes.

II. Introduction

[0034] As summarized above, the devices of the present invention are designed to detect the presence of target antibodies endogenous to a patient urine sample. Target antibodies preferably recognized by devices of the invention are those antibodies that specifically bind HIV proteins, preferably gpl20 and gp41 of HIV-1 and gp36 of HIV-2.

[0035] The devices of the invention comprise a series of zones, each distinguished by the chemical reagents, reactions and/or interactions occurring in the respective zone. At a minimum, each device comprises at least three zones; the sample zone; the conjugation zone; and the analysis zone. Devices of the invention may also include a control zone to indicate the device has performed properly in operation and is providing a valid result. Devices also may optionally include a waste region beyond the last zone in the flow path, where excess sample may accumulate. The waste region provides the ability to add additional sample volume to the device when necessary. Typically the waste region includes an absorbent material, which may be the same material as the matrix forming any or all of the zones of the device.

[0036] The following sections provide a detailed description of zones found in devices of the present invention, and how these zones are arranged along a flow path to produce a valid diagnostic result. By way of this description, methodology for operating the devices will also be apparent. III. Device construction

[0037] Generally, the devices of the invention are designed to allow sequential flow of a urine sample along a flow path comprising each zone of the device. Thus a urine sample applied to the sample zone will sequentially encounter the reagents in each subsequent zone thereby allowing a predetermined series of reactions to occur between sample constituents and the reagents present in each zone. Liquid flow through the device is controlled by a matrix material that performs a number of functions, as described below. The matrix material may optionally be placed in a housing, also discussed below. After discussion of the matrix and housing, zones that may be incorporated in each device will be described individually in the order in which they are encountered by a urine sample as it traverses a flow path of the device.

A. Matrix

[0038] Matrices suitable for use in the present invention are insoluble materials capable of supporting fluid flow. Matrix materials may be from natural and/or synthetic sources, are porous, bibulous or non-bibulous, fibrous or particulate. They may be formed as continuous strips of the same material, mixtures of different materials that are distributed consistently along a common strip, or inconsistently distributed mixtures that form regions having different physical or chemical characteristics in different areas of the strip. Alternatively, the matrix may be formed from two or more pads of matrix material. The pads are then orientated in fluid communication with each other to form a flow path of the device. Constructing the flow path from a series of pads is particularly useful when the device requires a plurality of zones, each comprising a different set of reagents, or prepared using mutually exclusive methods, as is the case for several embodiments of the present invention. Materials used to construct matrices of the invention may be inert or may react with one or more reagents of the invention, provided that the materials forming the matrix remain insoluble during the practice of the invention as described herein.

[0039] Suitable matrix materials are generally hydrophilic, or are capable of being rendered hydrophilic, and include inorganic powders, such as silica and alumina; glass fiber filter paper; natural polymeric materials particularly cellulose-based materials such as filter paper, chromatographic paper, and the like are particularly preferred; synthetic or modified naturally occurring polymers such as nitrocellulose, cellulose acetate, poly(vinyl chloride), polyacrylamide, crosslinked dextran, agarose, etc.; may either be used alone or in conjunction with other materials. The matrix material may also contain functional groups, or be capable of being functionalized to permit covalent bonding of reagents or antigens of the invention.

[0040] The matrix material preferably defines the flow path that will be followed by the urine sample during operation of the device, therefore reagents for use in the assay of the device are typically added directly to the matrix material as a powder or solution, as described below.

1. Buffers

[0041] An aspect of the present invention are buffer systems that maintain urine samples applied to the invention at a pH of at least 8, preferably between 8 and 11, more preferably between 8.2 and 11, advantageously between 8.2 and 10, more advantageously between 8.5 and 9.5 and ideally between 9 and 9.5, during performance of the invention. Buffer systems capable of maintaining a urine sample within the desired pH range are known to those of skill in the art, and are described, for example, as "Biological Buffers" in the 2002 Sigma Catalogue. Preferable buffers include carbonate and bicarbonate salts, and borate salts, particularly potassium and ammonium salts. Other examples of useful buffer systems for the invention include acetate salts, EPPS, Tricine, Gly-Gly, Bicine, HEPBS, TAPS, AMPD, TABS, MOPS, AMPSO, CHES, CAPSO, AMP, CAPS, CABS and several others.

[0042] Buffers used in the invention are preferably applied to the matrix of a device of the invention prior to operation of the device. The buffer may be applied in any manner that allows the buffer to form a solution having the desired pH when the matrix is contacted with a urine sample. For example, the buffer may be applied to the matrix as a dry powder, or preferably applied as a solution, which is subsequently lyophilized in the matrix.

2. Blocking agents

[0043] Although inherently bibulous materials may be used as matrix materials in the present invention, fluid flow through the devices of the present invention is preferably non- bibulous in nature.

[0044] Bibulous materials may be converted to materials which exhibit nonbibulous flow characteristics by the application of blocking agents. These agents may be detergents, sugars or proteins which can obscure the interactive forces giving rise to the bibulous characteristics. Exemplary protein blocking agents include bovine serum albumin, either per se or in methylated or succinylated form, whole animal sera, such as horse serum or fetal calf serum, and other blood proteins. A preferred blocking agent is avian serum such as goose or turkey serum, most preferably chicken serum. Other examples of protein blocking agents include casein and nonfat dry milk. Detergent-based blocking agents are selected from nonionic, cationic, anionic and amphoteric forms, with the selection is based on the nature of the matrix that is being blocked. Tween 20 is a particularly useful detergent for blocking membranes. Exemplary sugars useful as blocking agents include sucrose and fructose.

[0045] Application of the blocking reagent to a bibulous matrix may be carried out by treating the matrix with a solution of the blocking agent in an effective concentration to dispose of unwanted reactivities at the surface. In general, this treatment is conducted with a blocking solution, such as a protein solution of 1-20 mg/ml protein at approximately room temperature for between several minutes and several hours. The resulting coated material is then permanently adsorbed to the surface by air-drying, lyophilization, or other drying methods.

[0046] The use of a matrix that is inherently bibulous, but convertible to a nonbibulous flow characteristic, is particularly useful for immobilizing antigens and capture reagents. For example, a capture reagent may be applied to the matrix before the application of blocking agents and can be immobilized in situ. Once the capture reagent has been immobilized to the matrix, the blocking agent may then be applied.

B. Housing

[0047] Matrices of the invention may be disposed within a housing that is both protective and functional. In one preferred embodiment the housing is adapted to have at least one port for receiving a urine sample and guiding fluid flow of the sample to contact the sample zone. The housing also may have windows allowing access to selected portions of the flow path, preferably the analysis zone and/or the control zone. Embodiments having a housing of this type are termed "cassette devices."

[0048] Alternatively, the matrix may be provided unsupported, or supported by a backing formed from a durable material that is preferably impermeable and maintains the physical integrity of the matrix. Embodiments having this type of construction are termed "dip sticks."

[0049] A third device embodiment includes a protective housing analogous to cassette devices, but with a sample zone that extends outside the housing forming a wick that can be dipped into a urine sample. Other variants on these themes are also contemplated and will be readily identified and appreciated those of skill in the art.

[0050] Housings may be constructed of any suitable material known to those of skill in the art. It will be readily appreciated that housing components in fluid contact with the flow path should not impede fluid flow along the flow path and therefore cannot be too hydrophobic. Conversely, the housing material in contact with the flow path can not be too hydrophilic or the sample may partition to and only traverse the flow path along the walls of the housing.

C. Device zones

[0051] Devices of the present invention have at least three zones that include reagents which may interact with antibodies endogenous to a urine sample applied to the device. The sample zone initially receives the urine sample. Application of urine to the sample zone may be achieved by in stream application, or prior collection of urine followed contact of the sample to the sample zone by dipping, pipetting or pouring the urine sample. The urine sample is preferably applied undiluted, and immediately after collection from the patient. If necessary, urine samples to be analyzed using the invention may be stored for a limited period (e.g., up to a week) at room temperature, or for a more prolonged period refrigerated. Preferably, the sample zone contains a buffer sufficient to raise and/or maintain the pH of the urine sample at pH 8 during operation of the device. The buffer should be readily solubilized by the urine sample in amounts sufficient to provide the desired pH characteristics throughout the operation of the device, as described above.

[0052] Urine sample applied to the sample zone migrates first to the conjugation zone where antibodies endogenous to the urine sample interact with a labeling reagent that is coupled to a label, as described below. This interaction forms an labeled antibody conjugate.

[0053] The labeled antibody conjugate then migrates into the analysis zone, where an antigen that specifically binds the target antibody is immobilized to the matrix. If an antibody in the labeled antibody conjugate is a target antibody, the immobilized antigen specifically binds the target antibody, immobilizing the labeled antibody conjugate to the matrix. In this manner label accumulates in the analysis zone, where it can be detected, indicating the presence of the target antibody in the urine sample. If the labeled antibody conjugate does not include the target antibody, it continues along the flow path and label does not accumulate in the analysis zone.

[0054] Devices of the invention may optionally include a control zone. Within the control zone is a capture reagent immobilized to the matrix. The capture reagent is deposited to form a control line within the control zone, and binds the labeled antibody conjugate regardless of the nature of the antibody associated with it. This allows the labeled antibody conjugate to accumulate along the control line, accumulating label in the control zone, which indicates that the device is working properly. When present, the control zone is downstream from the conjugation zone, preferably downstream from the analysis zone.

[0055] Devices may also optionally include a waste region downstream from all of the zones noted above. The waste region may simply be an extension of the matrix material discussed above, but is preferably constructed from an absorbent material that helps maximize the amount of urine sample that can be applied to the device.

[0056] To better describe the invention, each of the zones mentioned in this section is discussed more fully below.

1. Sample zone

[0057] The sample zone receives the urine sample from the operator of the invention. The sample zone is typically constructed of a material that exhibits low target antibody retention. Accordingly, blocking agents of the invention applied to the sample zone in amounts sufficient to prevent target antibody interaction with the matrix material during operation of the invention. A particularly advantageous blocking agent for use in the sample zone is avian sera, more preferably chicken sera. In a preferable embodiment, the sample zone is prepared from a glass fiber pad that is impregnated with a solution containing polyvinylpyrollidone, bovine serum albumin, avian sera, borate and/or carbonate buffers (~0.5M), and triton X-100 or tween-20 detergent. The pad is squeezed to remove excess buffer and the pad is dried overnight at 30°C. An advantage of this approach is increased wetability and wicking action of the sample zone. In some embodiments the sample zone may also function as a mechanical filter, entrapping any undesirable particulates.

2. Conjugation zone

[0058] The conjugation zone is downstream from the sample zone and contains a label moiety comprising a labeling agent coupled, directly or indirectly, with a label. Methods for coupling labeling agents and labels, as described herein, are well known to those of skill in the art.

[0059] The label moiety is deposited in the matrix of the conjugation zone in a manner that allows it to be readily mobilizable in the fluid flow upon contact with a liquid sample, such as a urine sample. To accomplish this, the matrix of the conjugation zone is formed from a spun-bonded polyester and blocked by dipping it in a buffer containing polyvinlypyrollidone, chicken serum, bovine serum albumin, carbonate and/or borate buffers. The conjugation zone is then dried at 50°C and forced air for 50 minutes. The label moiety is striped onto the pad using, for example, either a contact tip or a aerosol tip. Prior to striping, the conjugate is preferably stabilized. For example, the label moiety may be placed in a simple or complex sugar solution, e.g., sucrose at 20% w/v and trehalose at 5% dextrin at 10% w/v.

[0060] As the urine sample flows through the conjugation zone, the label moiety is solublized and joins the fluid flow through the device. Both suitable labeling agents and labels are discussed further below. a) labeling agents

[0061] It is preferable for labeling agents of the invention to specifically bind antibody endogenous to the urine sample. Suitable labeling agents capable of binding any antibody endogenous to the urine sample include bacterial proteins, such as protein G and protein A, and antibodies that recognize particular antibody types. For example, goat anti-human IgG may be used to bind any IgG antibody endogenous to a urine sample from a human patient.

[0062] Regardless of the labeling reagent used, a labeled antibody conjugate will always arise from the conjugation zone, even in the absence of target antibody in the urine sample. If the labeling agent is in the absence of target antibody, then the labeled antibody conjugate will include antibodies generally endogenous to the urine sample, as urine samples are known to include enough endogenous antibody to form a detectable labeled antibody conjugate. b) labels

[0063] Suitable labels for use in the present invention may or may not be visible, but can be detected if accumulated in the analysis zone. Labels suitable for use in the present invention include, but are not limited to, particulate moieties and enzymes. Visible labels may be dyes or dyed polymers that are visible when present in sufficient quantity. Preferable labels are particles such as dyed latex beads, liposomes, or metallic, organic, inorganic or dye solutions, fluorescent particles, dyed or colored cells or organisms, red blood cells and the like. The metal sol particles, dyed or fluorescent labeled microparticles should be visible to the naked eye or able to be read with an appropriate instrument (spectrophotometer, fluorescent reader, etc.). Alternatively, radioactive isotopes may also be used.

[0064] A preferred label of the present invention are colloidal gold particles that are preferably larger than 20 urn, more preferably in the range of about 20 to 100 nm, and most preferably in the range of 20 to 40 nm. The gold sol particles used in accordance with the present invention may be prepared by methodologies that are well known, e.g., G. Frens, Nature, 241, 20-22 (1973). In addition to gold metal sol, particles may be made of platinum, gold, silver, selenium, or copper or any number of metal compounds which exhibit characteristic colors. Coupling metal, metal compounds and polymer nuclei coated with metals or metal compounds is known in the art and described in U.S. Pat No. 4,313,734. Other methods well known in the art may be used to attach the analyte to gold particles. The methods include but are not limited to covalent coupling and hydrophobic bonding.

3. Analysis zone

[0065] The analysis zone lies downstream in the flow path from conjugation zone. The analysis zone contains an immobilized antigen that specifically binds the target antibody and in so doing immobilizes the labeled antibody conjugate to the matrix. The immobilized antigen may be any antigen that specifically binds the target antibody, but is preferably an HIV protein, preferably an HIV envelope protein, more preferably one or more peptides from gpl20 or gp41 of HIV-1, or gp36 of HIV-2, most preferably at least one antigen selected from SEQ ID NO:l through 4. [0066] Antigen suitable for use in the invention may be obtained from any source including native, chemical synthesis or recombinant production, using methods well known to those of skill in the art. For example, the peptide portion of the preferred SEQ LO NO:l through 4 may be chemically synthesized using solid-phase peptide synthesis techniques, or recombinantly produced by operably linking a nucleic acid encoding the desired peptide into an expression vector, and expressing the nucleic acid in a suitable host. Once isolated, the peptide may be biotinylated using known techniques.

[0067] Suitable antigens may be immobilized to the matrix using any method known to those of skill in the art that does not destroy specific binding of the antigen to the target antibody. Preferably, the antigen is immobilized to the matrix using a biotin/strepavidin linker, most preferably, the antigen is coupled to biotin and complexed with strepavidin prior to coupling strepavidin to the matrix. Coupling strepavidin of the complex to the matrix is typically done prior to blocking, for bibulous matrices, using techniques well known to those of skill in the art. Preferably coupling is achieved in a solution containing at least a 2:1 ratio of strepavidin binding site equivalents to each biotin moiety, although other ratios such as 0.5:1, 1 :1, 3:1, 4:1 and 5:1, among others and all intermediate (fractional) ratios, are also contemplated as being part of the present invention. For bibulous matrices, the final complex is may simply be applied to the matrix material and dried followed by blocking with a suitable blocking agent. The amino acid sequences of exemplary antigenic peptides suitable for use in the present invention are provided as SEQ ED NO:l through 4, below. The line connecting cysteine residues in SEQ ID NO: 1,3 and 4 indicates that the side chains of these residues are prone to undergoing an oxidation reaction to form cystine

[0068] Immobilization of the antigen to the matrix is preferably performed in a manner that serves to concentrate labeled antibody conjugate that specifically binds to the immobilized antigen. By concentrating labeled antibody conjugate, the signal produced by the label is strengthened, improving sensitivity and minimizing the potential of obtaining an erroneous result.

[0069] Typically label signals may be observed between 15 and 60 minutes, more preferably between 15 and 45 minutes, most preferably between 15 and 30 minutes after the urine sample is applied to the sample zone. Signals produced by colored labels, as described above, can generally be detected directly from the device without further processing. Fluorescent labels may require a fluorimeter to detect. Signals produced by metal sol labels may be enhanced using silver salt solutions in methods well known to those of skill in the art. Similarly, when enzymes are used, the label must be contacted with a substrate of the enzyme label that produces a detectable product. Thus these enhanced methods deviate from the routine, single-step assay performed with colored particulate labels and sols, as the matrix must be contacted with a developing solution (a silver salt or substrate solution) before the label is detected.

4. Control zone

[0070] Devices of the present invention optionally include a control zone. When present, the control zone is down stream in the flow path from the conjugation zone, preferably downstream from the analysis zone.

[0071] The control zone contains a capture reagent that specifically binds antibodies endogenous to the urine sample, and is preferably immobilized within the control zone to form a control line that concentrates any labeled antibody conjugate bound by the capture reagent. The capture reagent may be a protein having affinity for a class of antibodies, such as protein A or G, but these antibody binding molecules can only be used as the capture reagent of the invention when they are not being used as the inventions labeling agent. Preferred capture reagents have an affinity for endogenous urine antibody that is greater than that of protein A under the operating conditions of the invention. Preferred capture reagents include anti-IgG antibodies from a species other than the one contributing the urine sample, as described above.

[0072] Capture reagents suitable for use in the present invention are immobilized to the matrix using known techniques, including those described above for the immobilized antigen. The capture reagent is preferably immobilized to the matrix using a biotin/strepavidin linker, most preferably, the capture reagent is coupled to biotin and complexed with strepavidin prior to coupling strepavidin to the matrix, as described above. For bibulous matrices, the matrix material must again be blocked using, for example, a solution containing 0.01M potassium phosphate solution, with 0.25% BSA and 0.025% tween-20. The membrane is then dried overnight at 50°C.

[0073] When operating correctly, capture reagent will continue to bind all labeled antibody conjugate until the unbound labeled antibody conjugate is depleted, or the capture reagent is saturated. As even urine samples from healthy mammals contain endogenous IgG, and the molar amount of labeling agent coupled to label preferably exceeds the molar amount of immobilized antigen, labeled antibody conjugate should always be available to bind to capture reagent, producing a signal at the control line. Therefore, failure to detect a signal at the control line is indicative of a faulty device or poor operation of the device.

IV. Kits

[0074] The present invention also provides kits that include the one or more devices described above. Each kit may optionally include a package insert providing instruction on the use of the enclosed device(s), vials containing positive and negative control solutions for quality testing the device(s), a timer that may be used to determine when the assay of the invention is complete, a urine collection container (e.g., a small test tube), one or more transfer pipettes and/or a biohazard disposal container.

[0075] All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.

[0076] Although the foregoing invention has been described in some detail by way of illustration and example for clarity and understanding, it will be readily apparent to one of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit and scope of the appended claims.

[0077] As can be appreciated from the disclosure provided above, the present invention has a wide variety of applications. Accordingly, the following examples are offered for illustration purposes and are not intended to be construed as a limitation on the invention in any way. Those of skill in the art will readily recognize a variety of noncritical parameters that could be changed or modified to yield essentially similar results. EXAMPLES

Example 1: Comparison of selectivity and sensitivity of the immunoassay device in evaluating refrigerated and freshly collected specimens

[0078] This example demonstrates that the immunoassay devices according to the present invention have extremely high sensitivity and specificity towards AIDS over diverse sample sources.

[0079] Immunoassay devices of the invention was constructed from the following components:

[0080] A glass fiber sample zone pad, blocked and loaded with buffer by impregnating the pad with a solution containing polyvinylpyrollidone, bovine serum albumin, avian sera, and carbonate buffers (0.5M) and Triton X-100. The pad was squeezed to remove excess liquid and allowed to dry overnight at 30°C.

[0081] Analysis zone pads include HIV antigens coupled to a spun polyester membrane using a strepavidin/biotin linkage. Briefly, avidin was prepared at a lOOmg/ml solution. The HIV-1 and HIV-2 peptides were prepared each at lOmg/ml. Avidin and the HIV peptides were mixed together at a ratio of 2.1 avidin binding site equivalents to 1 biotin moiety. The reaction was carried out at room temperature (25°C) for five minutes. The solutions were brought to their final solumes using a DI water/5 % isopropyl alcohol solution. These solutions were then striped to the membrane using a linear striper. The membrane was dried for four hours at 50°C and blocked overnight in blocking solution (0.01M potassium phosphate solution, with 0.25% BSA and 0.025% tween-20) overnight at 50°C.

[0082] The conjugation zone pads were prepared from spun-bonded polyester membranes by striping label moiety (components?) onto the pad using an aerosol tip. Prior to striping, the label moiety was stabilized using sucrose at 20% w/v and trehalose at 5% w/v. The pad was then dipped in a buffer containing polyvinlypyrollidone, chicken serum, bovine serum albumin, and carbonate buffer and dried at 50°C using forced air for 50 minutes.

[0083] Control lines in the control zones of the devices were prepared by diluting a f(AB) fragment of a goat antibody specific to the fC fragment of Human antibodies in water. The resulting solution was then sprayed onto spun-bonded polyester membranes using an u aerosol tip. The membrane was dried for four hours at 50°C and blocked overnight in blocking solution (0.01M potassium phosphate solution, with 0.25% BSA and 0.025% tween- 20) overnight at 50°C.

[0084] The resulting membranes were then aligned in fluid communication relative to each other, with the Sample zone being upstream from the conjugation zone; the conjugation zone upstream from the analysis zone; and the analysis zone upstream from the control zone.

[0085] The device is operated by adding four drops (50-100μl) of urine to the sample zone. After 20 minutes at room temperature the result can be read from the device. If a positive signal (e.g., colored line) appears at the control line, then the test is functioning properly. If there is no positive signal at the control line, then the device is faulty, should be discarded and the immunoassay redone with a new device.

[0086] Assuming the device is functioning properly, a positive signal in the analysis zone corresponding to the antigen is indicative of the presence of antibodies in the urine sample directed against the antigen. In the present analysis, this result indicates that the urine sample donor is infected with HIV. If a positive signal fails to appear in the analysis zone, this result is indicative of an absence of antibodies in the urine sample directed against the antigen, i.e., the donor of the urine sample is not infected with HIV.

[0087] Samples taken from ten different sources, as indicated in table 1, were then analyzed according to the methodology described above, and the results displayed in Table 1 :

Table 1.

Example 2: One-step RIC test format results

[0088] This example illustrates that the cassette test format is suitable for the detection of HIV antibodies in urine. Urine has been successfully used as a specimen for the detection of HIV-1 antibodies (Ab) by EIA and Western Blot despite the fact that urine antibodies are at lower levels than found in blood or serum. Rapid immunochromatography (RIC) tests have been developed for blood/serum HIV Ab testing and are considered a vital element in programs to reduce the rates of HIV transmission. The study described below was conducted using an HIV-1 RIC assay to determine the potential of using this format for HIV-1 Ab detection in urine.

Methods: The rapid test prototype was evaluated using a total of 11 archived, refrigerated urine specimens, of which 6 were HIV-1 Ab positive and 4 were HIV-1 Ab negative as determined by the Calypte HIV-1 Urine EIA, and confirmed by Calypte's Cambridge Biotech HIV-1 Urine Western Blot performed per the manufacturer's test algorithm. Urine samples were briefly mixed prior to addition to the RIC test device at room temperature. 150ml of sample was added to the device for analysis and the intensity of the signal present at the sample and control lines of the device determined. Sample to control ratios were calculated as an indicator of relative intensity. Ratios above 1.0 were determined to be HIV positive. The appearance of a control line on the rapid test also confirms test performance, and identifies a reactive sample. Results were read at 10, 20 and 30 minutes using a cassette-type format of the assay device described above. Results: The RIC test gave H1N-1 antibody reactive results in 6 of the 6 positive specimens, and non-reactive results in 4 of the 4 negative specimens, yielding a sensitivity and a specificity of 100% when compared to EIA and Western Blot. Overall accuracy for the test was 100%.

Table 2

Conclusions: The one-step RIC test format is suitable for the detection of HIV-1 antibodies in urine, with all refrigerated samples testing as predicted in a blind study. The advantages of this test format are the ease of use, requiring minimal specialized training, equipment or collection devices, and excellent stability under unrefrigerated storage making it ideal for distribution to remote areas where AIDS is prevalent. The absence of infectious HIV in a urine specimen presents a significantly lower risk of HIV exposure. In addition, urine specimens provide sufficient volume for confirmatory HIV or tests for other diseases or conditions. SEQUENCE LISTING

SEQ ID NO: 1 Single-Lysine linker H1N-1 (gp41 peptide) i 1

Biotin-KILAVERYLKDQQLLGIWGCSGKLICTTAVPWNASGKLI-COOH

SEQ ID NO:2 biotinylated HIV-1 gpl20 peptide

Biotin-KTEPLGVAPTKAKRRVVQREKR-COOH

SEQ ID NO: 3 Single-Lysine linker H1N-2 (gp36 peptide) i 1

Biotin-KVTAIEKYLQDQARLNSWGCAFRQVCHTVPWVNDS-CONH₂

SEQ ID NO:4 Double-Lysine linker HIV-1 (gp41 peptide) I I

Biotin-KKILAVERYLKDQQLLGIWGCSGKLICTTAVPWNASGKLI-COOH

SEQ ID NO: 5 Double-Lysine linker HIV-2 (gp36 peptide) I I

Biotin-KKVTAIEKYLQDQARLNSWGCAFRQVCHTTVPWVNDS-CONH₂

Claims

WHAT IS CLAIMED IS: 1. A lateral flow device for the detection of target antibodies in urine, the device comprising an antigen that specifically binds the target antibodies and a sample zone comprising a buffer producing a solution having a pH of at least 8 when contacted with urine.

2. The lateral flow device of claim 1, wherein the buffer comprises carbonate.

3. The lateral flow device of claim 2, wherein the buffer further comprises bicarbonate.

4. The lateral flow device of claim 3, wherein the buffer comprises potassium bicarbonate and potassium carbonate in a 2: 1 molar ratio.

5. The lateral device of claim 1, wherein the antigen is a human immunodeficiency virus (HIV) protein.

6. The lateral flow device of claim 5, wherein the HIV is HIV-1.

7. The lateral flow device of claim 5, wherein the HIV is HIV-2.

8. The lateral flow device of claim 5, wherein the protein is recombinantly produced.

9. The lateral flow device of claim 5, wherein the HIV protein is an envelope protein.

10. The lateral flow device of claim 9, wherein the protein is selected from the group consisting of SEQ ID NO:l, 2, 3 and 4.

11. The lateral flow device of claim 1, wherein the sample zone further comprises avian serum.

12. The lateral flow device of claim 11, wherein the avian serum is chicken serum.

13. The lateral flow device of claim 1, further comprising a conjugation zone comprising labeling agent conjugated to a label.

14. The lateral flow device of claim 13, wherein the labeling agent is Protein A.

15. The lateral flow device of claim 13, wherein the label is colloidal gold.

16. The lateral flow device of claim 1, further comprising a control zone comprising a capture agent.

17. The lateral flow device of claim 16, wherein the capture agent is an anti-human IgG antibody.

18. The lateral flow device of claim 17, wherein the anti -human antibody is goat anti-human IgG antibody.

19. A lateral flow device for detection of antibodies in urine, the device comprising: a. a conjugation zone comprising a Protein A/colloidal gold conjugate, and b. a control line comprising a capture agent, wherein the capture agent has an affinity for a human urine antibody bound to the Protein A/colloidal gold conjugate that is greater than the affinity of Protein A for the human urine antibody bound to the Protein A/colloidal gold conjugate.

20. The lateral flow device of claim 19, wherein the capture agent is an anti-human IgG antibody.

21. The lateral flow device of claim 20, wherein the anti-human IgG antibody is a goat anti-human IgG antibody.

22. The lateral flow device of claim 19, further comprising a sample zone comprising a buffer having pH of at least 8 and an antigen that specifically binds the target antibodies.

23. The lateral flow device of claim 22, wherein the buffer comprises carbonate.

24. The lateral flow device of claim 23, wherein the buffer further comprises bicarbonate.

25. The lateral flow device of claim 24, wherein the buffer comprises potassium bicarbonate and potassium carbonate in a 2: 1 molar ratio.

26. The lateral device of claim 22, wherein the antigen is a human immunodeficiency virus (HIV) protein.

27. The lateral flow device of claim 26, wherein the HIV is HIV-1.

28. The lateral flow device of claim 26, wherein the HIV is HIV-2.

29. The lateral flow device of claim 26, wherein the protein is recombinantly produced.

30. The lateral flow device of claim 26, wherein the HIV protein is an envelope protein.

31. The lateral flow device of claim 30, wherein the protein is selected from the group consisting of SEQ ID NO: 1, 2, 3 and 4.

32. The lateral flow device of claim 22, wherein the sample zone further comprises avian serum.

33. The lateral flow device of claim 32, wherein the avian serum is chicken serum.

34. A kit comprising a lateral flow device of claim 1, or claim 19.

35. A method for detecting antibodies in a urine sample from a patient, the method comprising contacting the sample with a sample zone on a lateral flow device, wherein the sample zone comprises a buffer having a pH of at least 8.

36. The method of claim 35, wherein the buffer comprises carbonate.

37. The method of claim 36, wherein the buffer further comprises bicarbonate.

38. The method of claim 37, wherein the buffer comprises potassium bicarbonate and potassium carbonate in a 2: 1 molar ratio.

39. A method for detecting antibodies in a urine sample from a patient, the method comprising contacting the sample with a sample zone on a lateral flow device comprising a conjugation zone comprising a Protein A/colloidal gold conjugate, and a control line comprising a capture agent, wherein the capture agent has an affinity for a human urine antibody bound to the Protein A/colloidal gold conjugate that is greater than the affinity of Protein A for the human urine antibody bound to the Protein A/colloidal gold conjugate.

40. The method of claim 39, wherein the capture agent is an anti-human IgG antibody.

41. The method of claim 40, wherein the anti -human IgG antibody is a goat anti-human IgG antibody.

42. The method of claim 39, wherein the sample zone comprises a buffer having pH of at least 8.

43. The method of claim 42, wherein the buffer comprises carbonate.

44. The method of claim 43, wherein the buffer further comprises bicarbonate.

45. The method of claim 44, wherein the buffer comprises potassium bicarbonate and potassium carbonate in a 2: 1 molar ratio.

46. The method of claim 39 or claim 35, wherein the patient is a human suspected of being infected with human immunodeficiency virus (HIV) and the lateral flow device comprises an HIV antigen.

47. The method of claim 46, wherein the HIV is HIV- 1.

48. The method of claim 46, wherein the HIV is HIV-2.

49. The method of claim 46, wherein the protein is recombinantly produced.

50. The method of claim 46, wherein the HIV protein is an envelope protein.

51. The method of claim 50, wherein the protein is selected from the group consisting of SEQ LO NO: 1, 2, 3 and 4.