Assay devices and methods of analyte detection

[0002] The present method relates to a solid phase chromatographic assay device, methods, and test kits for use in the detection of one or more analytes in a sample.

[0003] Chromatographic assay systems employed as rapid assay devices are one of several means for detecting the presence of a given analyte in a biological sample. One advantage to these systems is that the execution of these assays does not use additional specialized equipment or trained personnel. Another advantage is the great variety of analytes that can be detected using this type of assay. The use of rapid chromatographic techniques for detection of the presence of an analyte in a biological sample has thus progressed beyond the bounds of the clinical laboratory, as assay devices employing these techniques have been found to be especially valuable in “point of care” situations such as the doctor's office or home settings.

[0004] The typical rapid chromatographic tests utilize either a “sandwich” assay or a “competition” assay to detect the presence of a desired analyte. In the sandwich assay, an analyte is bound, or “sandwiched,” between an unlabeled first binding partner and a labeled second binding partner. For example, an analyte, such as an antibody to HIV, can be captured by a first binding partner, in this case, an HIV antigen immobilized on a membrane. The antibody-antigen complex can then be detected by a second binding partner having a label, such as another HIV antigen tagged with a colored particle.

[0005] In contrast, during the competition assay, the analyte in the sample competes with a labeled analyte, or labeled analogue to the analyte, for a binding partner immobilized on a solid support. A greater concentration of analyte in the sample results in a lower signal in the assay, as the labeled analytes are competed away from the binding partner on the solid support (i.e., the signal produced during a competition assay decreases as the concentration of analyte in the sample increases). Thus, the sandwich assay provides a qualitative assessment with great sensitivity, while the competition assay provides a quantitative measure of analyte concentration.

[0006] Regardless of the analyte-detecting method used, the rapid assay devices currently available are often categorized into one of three basic formats: the “dipstick” format, the “flow through” format, and the “lateral flow” format. The “dipstick” format (exemplified in U.S. Pat. Nos. 5,275,785, 5,504,013, 5,602,040, 5,622,871 and 5,656,503) typically consists of a strip of porous material having a sample receiving end, a reagent zone and a reaction zone. The sample is wicked along the assay device starting at the sample-receiving end and moving into the reagent zone. The analyte to be detected binds to a reagent incorporated into the reagent zone, preferably a labeled binding partner, to form a complex. Typically, these binding pairs are antibody:antigen complexes, or a receptor:ligand complexes having a label such as a colloidal metal incorporated into the reagent portion of the complex. The labeled binding partner-antigen complex then migrates into the reaction zone, where the complex is captured by another specific binding partner firmly immobilized in the reaction zone. Retention of the labeled complex within the reaction zone thus results in a visible readout.

[0007] The “flow through” format (U.S. Pat. No. 402,0046) also utilizes porous solid phase materials. This assay format usually has a porous membrane that contains an immobilized binding partner positioned above an absorptive layer. Once the sample has been added to the membrane surface, the analyte of interest reacts with the immobilized binding partner to form an analyte-binding partner complex. The complex is visualized by addition of a second binding partner having a label, such as an enzyme, dye particles or colloidal metals. The absorptive layer acts as a sink for excess assay reagents, and can be used to regulate the flow rate of the reactants to achieve optimal reaction between the analyte and the binding partner. In this format, the sensitivity of the readout can be improved by “washing” the membrane with additional solution to reduce any nonspecific binding of the label, or remove any other materials which can interfere with the assay readout.

[0008] The “lateral flow” format (see U.S. Pat. Nos. 5,075,078, 5,096,837, 5,354,692 and 5,229,073) utilizes a porous solid phase material and has a linear construction similar to that of the dipstick assay format: a sample application site, a reagent releasing site and a reaction site. However, instead of vertically wicking the samples up the “dipstick,” the lateral flow format allows a sample to flow laterally across the porous solid phase material. The sample is applied directly to the application site and the analyte of interest flows laterally to the reagent-releasing site, and forms a complex with a labeled binding partner. The analyte:binding partner complex then migrates into the reaction site where it is captured by a second, immobilized binding partner and detected.

[0009] The conventional rapid assays are a popular choice for determining the presence of a given analyte in samples provided at the “point of care” sites because they are relatively easy to use, do not use specialized equipment or personnel, and produce results in a short amount of time. For example, simple and rapid immunoassay devices for infectious diseases such as AIDS have been available for almost a decade. However, the existing rapid tests are not without their shortcomings. Most importantly, the sensitivity of such devices has often been questioned, due to various limitations with the currently available formats (Giles et al. (1999) Journal of Medical Virology59:104-109). In addition, there are several practical limitations to the use of these assay devices inherent in the design of the assay format, as exemplified below.

[0010] The dipstick format, which was originally designed for urine analysis, uses a relatively large volume of sample for analysis. This is a considerable limitation to use of such a device for analysis of serum or blood samples. In contrast, assay devices based on the flow-through format reduce the volume requirement of samples significantly. However, the flow-through format cannot be employed in a truly self-contained device. In devices based on the flow-through format, the detecting reagent (i.e. the labeled binding partner) is not directly incorporated into the porous solid matrix of device and thus must be provided separately. This leads to additional limitations regarding reagent stability, if the detecting reagents are provided in liquid form, or issues surrounding the proper preparation and handling of the detecting reagent, if provided in a dried form.

[0011] The lateral flow format overcomes both the sample volume problem of the dipstick format, as well as the detecting reagent issue of the flow-through format. However, the lateral-flow format does not allow for a washing step, as inherent in the flow-through format. Any interfering species, such as particulate or colored material introduced by the sample solution, or unbound label, can potentially interfere with the readout of the assay device. As a result, the lateral flow format often employs filtration (e.g., using specialized filters) during the assay procedure, e.g., using specially coated filters to remove potential interfering species prior to detection of the analyte.

[0012] The present invention provides a novel approach to achieve optimal control of the assay reactions without requiring specially-developed specific antibodies, large volumes of sample, or complicated arrays of reagents or fluid pathways (for example, as compared to that described in U.S. Pat. Nos. 4,960,691 and 5,607,863). The present invention presents assay devices that are particularly suitable for rapid chromatographic assays using a controlled series of reactions. The assay devices use a small volume of sample and achieve a much higher titration-end-point activity than conventional lateral flow assays. In addition, the assay devices of the present invention provide better assay sensitivity, without compromising specificity.

[0013] The present invention provides assay devices, test kits, and methods for detecting the presence of one or more analytes in a sample. By controlling the release of the different reagents used in the assay device, the sensitivity of the assay is improved as compared to conventional assays, without compromising the specificity of the assay. Improved sensitivity without loss of specificity is a highly desirable improvement in the field of rapid chromatographic detection. In addition, the assay can be performed by untrained personnel in a minimum amount of time, and without the need for specialized equipment.

[0014] Accordingly, it is a general object of the invention to provide an assay device for use in detecting the presence of an analyte. One embodiment of the assay device of the present invention comprises (a) a chromatographic element comprising a sample receiving end, a reagent releasing end, and a reaction zone; (b) an absorbent pad; and (c) a separator positioned between the chromatographic element and the absorbent pad. Using an assay device of this first embodiment, a sample is applied to the sample-receiving end of the chromatographic element and allowed to migrate laterally by capillary action towards the reagent-releasing end. After the sample covers the reaction zone and the analyte within the sample has interacted with at least one first binding partner immobilized within the reaction zone, an aqueous solution is added to the reagent releasing end of the chromatographic element. The separator is removed from the device, allowing the absorbent pad to come into contact with the chromatographic element. The aqueous solution can be added prior to the removal of the separator, concurrently with the removal, or immediately afterwards. The separator can be removed by pulling the separator entirely from the assay device, or it can be partially removed such that the sample receiving end of the chromatographic element and the absorbent pad come into contact. One or more reagents embedded at the reagent-releasing end, such as a second binding partner labeled with a detectable label such as a naturally colored particle, are released by addition of the aqueous solution and moved toward the reaction zone by the pulling force of the absorber pad. Thus, the device according to this embodiment allows the analyte to form a complex with the first binding partner prior to the reaction between the labeled second binding partner and the bound analyte complex. In addition, the aqueous solution added to the reagent releasing end of the chromatographic element acts not only as a reagent releasing solvent but also as a wash liquid. As a result, a visual readout with a clear background is observed within the reaction zone.

[0015] Another embodiment of the assay device of the present invention comprises (a) a chromatographic element comprising a sample receiving end having a releasable first binding partner, a reaction zone having an immobilized second binding partner, and a reagent releasing end having a releasable third binding partner containing a label; (b) an absorbent pad; and (c) a separator positioned between the chromatographic element and the absorbent pad. This embodiment of the assay device is preferred when a capture assay is desired. Using an assay device of this second embodiment, the analyte (for example, an antibody) reacts with at least one first binding partner (such as an antigen or a recombinant protein) impregnated at the sample receiving end of the chromatographic element. The analyte-binding partner complex then migrates to the reaction zone, where this first complex is captured by an immobilized second binding partner (the “capturing reagent,” such as anti-human IgG or anti-human IgM antibodies) to form a second complex. When the aqueous solution is added and the separator is removed, one or more third binding partners labeled with a detectable label, such as a naturally colored particle, are released from the reagent releasing end of the chromatographic element, and allowed to laterally flow to the reaction zone. Detectable labels include moieties which can be detected by visual inspection (e.g., moieties which include or produce colored elements), or with the aid of artificial detection systems, including. e.g., optical systems, spectroscopic systems, radiographic systems, or the like. For simplicity of operation, visually detectable labels are preferred.

[0016] The third binding partner can interact with the second complex to form a third complex, which can be detected via the label incorporated in the third binding partner. While the first binding partner can be single antigen or a mixture of antigens, a generic reagent can be used as the third labeled binding partner, for example an anti-GST antibody which will react with all GST-constructed recombinant antigens.

[0017] Similarly, a third embodiment of the present invention encompasses the use of two or more reagents interacting at the reagent releasing end of a chromatographic element prior to migration across the reaction zone. In this embodiment of the present invention, the assay device comprises (a) a chromatographic element comprising a sample receiving end, a reaction zone having an immobilized first binding partner, and a reagent releasing end having two releasable binding partners, at least one of which carries a label; (b) an absorbent pad; and (c) a separator positioned between the chromatographic element and the absorbent pad. Using an assay device of this third embodiment, the first complex is formed at the reaction zone between an analyte and a first binding partner bound to the reaction zone. The second reaction occurs at the reagent releasing end between the second and third binding partners once the aqueous solution has been added, to form a second complex bearing a label. The third reaction takes place in the reaction zone, when the analyte-binding partner first complex and the second binding partner- third binding partner second complex interact to form a third, labeled complex which can be detected. As in the embodiment described above, the second (embedded) binding partner can be single or a mixture of antigens, while the third (labeled) binding partner acting as the detector can also be a generic reagent such as an anti-GST antibody.

[0018] The previous embodiments of the present invention address changes in the reagents used in the assay, and in the order in which the reactions take place. Yet another embodiment of the present invention involves the composition of the separator component of the assay device. Rather than using a barrier that must be manually removed during the assay, the separator can be composed of a material that will provide a “time-controlled” barrier, such as a semi-permeable membrane or a material that dissolves over time. When the device is in use as according to this embodiment of the present invention, by the time that the sample added to the sample receiving end has migrated laterally and covered the reaction zone, the separator will be dissolved or permeable, and the absorbent pad is readied for operation. An aqueous solution can then be added and the assay completed.

[0019] In yet other embodiments of the present invention, methods for detecting an analyte in a sample are provided, as are test kits employing the various embodiments of the assay device. Other permutations of the present invention are also possible, such as the simultaneous detection of multiple analytes using a single sample and a single device. Regardless of the embodiment employed, the assay device of the present invention does not need to include any additional filtration techniques using filters with special coatings, as employed in conventional lateral flow devices. The assay device is versatile and can be used to assess a variety of biological fluids including, but not limited to saliva, serum and whole blood samples. This versatility is achieved by controlling the order in which the reactions occur, and by the additional “washing” of the reactants as provided by passage of the aqueous solution though the chromatographic element and into the absorbent pad.

[0020] An additional benefit of the present invention is that the simplicity of the design of the assay device provides a generic platform versatile enough to accommodate the needs and requirements for different product lines. An assay device specific for detection of a particular analyte can be easily adapted to detect a different analyte with minimal modification to the overall design, such as replacing the binding partner immobilized within the reaction zone, but still using a “generic” labeled binding partner for detection purposes. There is not any need for the development of additional specialized reaction reagents for the detection of each desired analyte. This not only reduces the time needed to design and produce new assay devices, but also significantly reduces the costs for product development. Furthermore, since the major components of the assay device are the same, manufacturing parameters can be maintained without major changes. Thus, a production facility for manufacture of a series of products based on the assay device of the present invention utilizes the same equipment and a minimal inventory of raw materials for the manufacture of all of the products, which in turn reduces the cost of operation significantly.

[0021] Additional objects, advantages and novel features of the invention will be set forth in part in the description which follows, and in part will become apparent to those skilled in the art upon examination of the following, or may be learned by practice of the invention.

[0022] FIG. 1A depicts a schematic of a general view of an assay device of the present invention

[0023] FIG. 1B depicts a schematic of an overhead view of a casing containing one embodiment of an assay device of the present invention.

[0024] FIG. 2 depicts a cross-sectional view of a schematic of the casing and assay device of

[0025] FIG. 1B, as viewed along line A-A′.

[0026] FIG. 3A depicts a cross-sectional view of a schematic of an alternative embodiment of the assay device of the present invention.

[0027] FIG. 3B depicts a cross-sectional view showing a schematic of an alternative arrangement of the sample receiving end of the assay device presented in FIG. 3A.

[0028] FIG. 4A depicts a cross-sectional view of a schematic of a third embodiment of the assay device of the present invention.

[0029] FIG. 4B depicts a cross-sectional view showing a schematic of an alternative arrangement of the reagent releasing end of the assay device presented in FIG. 4A.

[0030] FIG. 5A depicts an overhead view of a schematic of a casing containing an assay device of the present invention showing an alternate arrangement of the separator.

[0031] FIG. 5B depicts a schematic cross-sectional view of the casing and device of FIG. 5A, as viewed along line B-B′.

[0032] FIG. 6 is a schematic showing details of assembly of an example chromatographic element.

[0033] DEFINITIONS

[0034] Before describing the present invention in detail, it is to be understood that this invention is not limited to particular compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. As used in this specification and the appended claims, the singular forms “a”, “an” and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “a releasable binding partner” includes a combination of two or more such binding partners, reference to “an analyte” includes mixtures of analytes, and the like.

[0035] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present invention, the preferred materials and methods are described herein.

[0036] In describing and claiming the present invention, the following terminology will be used in accordance with the definitions set out below.

[0037] The term “assay device” is used herein to describe a multi-component chromatographic apparatus used for the detection and/or measurement of one or more analytes of interest.

[0038] The term “chromatographic element” refers to a solid matrix upon which the sample can be applied and allowed to migrate during the assay procedure.

[0039] The term “sample receiving end” refers to the portion of the chromatographic element at which the sample is administered during the assay.

[0040] The term “reagent releasing end” refers to the portion of the chromatographic element distal to the sample receiving end, and at which one or more releasable assay reagents are incorporated.

[0041] The term “reaction zone” refers to the region of the chromatographic element between the sample receiving end and the reagent releasing end, within which one or more binding partners specific to the analyte (or to a complex containing the analyte) have been immobilized.

[0042] The term “absorbent pad” refers to an absorbent or bibulous material usually positioned at the base of the assay device.

[0043] The term “separator” refers to a barrier structure positioned between the chromatographic element and the absorbent pad.

[0044] The term “casing” or “housing” as used herein refers to an optional component of the assay device, which surrounds at least a portion of the chromatographic element, absorbent pad and separator and provides some structural support.

[0045] The term “sample” refers to any desired material for sampling, usually of biological origin.

[0046] The term “analyte” refers to a compound or composition to be detected or measured in a sample.

[0047] The term “binding partner” is used herein to describe a member of a binding pair which interact either chemically or physically to form a complex. An “immobilized” binding partner refers to a binding partner that is adsorbed, embedded or affixed, either permanently or semi-permanently, to a solid substrate or matrix (for example, the reaction zone of the chromatographic element.) A “releasable” binding partner refers to a molecule which is not permanently immobilized or affixed to a solid substrate or matrix, and is capable of migration or movement for example, by diffusion.

[0048] The term “label” as used herein refers to any substance that is capable of producing a detectable signal. Various labels suitable for use in the present invention include, but are not limited to, chromatogens, fluorescent or chemiluminescent compounds, catalysts, enzymes, enzymatic substrates, dyes, colloidal metallic and nonmetallic particles, and organic polymer latex particles. Particularly preferred for use in the present invention are the visually-detectable colored particles, such as colloidal metals and nonmetals, and dye particles.

[0049] The term “bibulous” refers to materials that are absorbent.

[0050] METHODS AND SYSTEMS

[0051] The present invention is directed toward assay devices for detection of one or more analytes in a sample. The assay devices are constructed in a manner to allow for the controlled release and interaction of the assay reagents. Further included in this invention are the methods for detecting the analyte, as well as test kits employing the assay device.

[0052] As will be understood by the ordinarily skilled artisan upon reading the specification, the analyte can be any specific substance or component that one is desirous of detecting and/or measuring in a chemical, physical, enzymatic, or optical analysis. Analytes of interest include, for example, antigens (such as antigens specific to bacterial, viral or protozoan organisms); antibodies, particularly those induced in response to an infection, allergic reaction, or vaccine; hormones, proteins and other physiological substances (for example, human chorionic gonadotropin, estrogens, progestins, testosterones, corticosteroids, human growth factors, hemoglobin, and cholesterol); nucleic acids; a variety of enzymes; therapeutic compounds and illicit drugs; contaminants and environmental pollutants; or any number of natural or synthetic substances. As is appreciated by one skilled in the art, the number of natural and synthetic substances which can be detected by the assay devices and methods of the present invention is extensive, and include, but is not limited to, the following groups of compounds: ACE inhibitors, adrenergics and anti-adrenergics, alcohol deterrents (for example, disulfiram), anti-allergics, anti-anginals, anti-arthritics, anti-infectives (including but not limited to antibacterials, antibiotics, antifungals, antihelminthics, antimalarials and antiviral agents), analgesics and analgesic combinations, local and systemic anesthetics, appetite suppressants, antioxidants, anxiolytics, anorexics, antiarthritics, anti-asthmatic agents, anticoagulants, anticonvulsants, antidiabetic agents, antidiarrheals, anti-emetics, anti-epileptics, antihistamines, anti-inflammatory agents, antihypertensives, antimigraines, antinauseants, antineoplastics, antioxidants, antiparkinsonism drugs, antipruritics, antipyretics, antirheumatics, antispasmodics, antitussives, adrenergic receptor agonists and antagonists, anorexics, appetite suppressants, cardiovascular preparations (including anti-arrhythmic agents, cardiotonics, cardiac depressants, calcium channel blockers and beta blockers), cholinergics and anticholinergics, contraceptives, diuretics, decongestants, growth stimulants, herbal preparations, hypnotics, immunizing agents, immunomodulators, immunosuppresives, muscle relaxants, neurologically-active agents including anti-anxiety preparations, antidepressants, antipsycotics, psychostimulants, sedatives and tranquilizers, sore throat medicaments, sympathomimetics, vasodilators, vasoconstrictors, vitamins, xanthine derivatives, various combinations of these compounds, and the like.

[0053] The device according to the present invention is particularly useful for detection of analytes in samples of biological origins. Such samples include, but are not limited to blood or serum; saliva, sputum, tears, sweat, or other secreted fluids; urine or fecal matter; as well as biologically derived fluids such as cerebrospinal fluid, interstitial fluid, cellular extracts and the like. A minimal volume of sample is used for the assay device of the present invention, particularly as compared to sample volumes used in a flow-through assay format. Desired sample volumes range from about 1 μL to about 500 μL, preferably from about 1 μL to about 100 μL, more preferably from about 5 μL to about 50 μL, most preferably between about 10 μL and about 30 μL.

[0054] The assay device of the present invention is based on binding assays such as, but not limited to, immunoassays. The binding partners involved in such binding assays include, but are not limited to, the following binding pairs: antibody and antigen or hapten; hormone and receptor; biotin and avidin; carbohydrate and lectin; effector and receptor molecules; enzymes and cofactors, substrates, or inhibitors; and complementary nucleotide sequences. Thus, the descriptions and examples included below are for demonstration purposes and should not be considered limiting to the particular applications addressed.

[0055] The devices of the invention are particularly well adapted to detecting antibody-antigen binding. Thousands of antibody-antigen binding partners are known and can be detected using the devices herein. A number of basic texts describe antibody-antigen interactions, antibody production processes, and other related matters, including, e.g., Borrebaeck (ed.) (1995) Antibody Engineering, 2^ndEdition Freeman and Company, NY; McCafferty et al. (1996) Antibody Engineering, A Practical ApproachIRL at Oxford Press, Oxford, England; Paul (1995) Antibody Engineering ProtocolsHumana Press, Towata, N.J.; Paul (ed.) (1999) Fundamental Immunology, Fourth Edition, Lippincott-Raven, N.Y.; Coligan (1991) Current Protocols in ImmunologyWiley/Greene, NY; Harlow and Lane (1989) Antibodies: A Laboratory Manual Cold Spring Harbor Press, NY; Stites et al. (eds.) Basic and Clinical Immunology(4th ed.) Lange Medical Publications, Los Altos, Calif., and references cited therein; Goding (1986) Monoclonal Antibodies: Principles and Practice(2d ed.) Academic Press, New York, N.Y.; and Kohler and Milstein (1975) Nature256: 495-497.

[0056] FIG. 1A represents a simple illustrative embodiment of the assay device of the present invention. Assay device 2 is composed of chromatographic element 4, absorbent pad 6 and separator 8. Chromatographic element 4 includes three generally contiguous sections: sample receiving end 10, reagent releasing end 12, and reaction zone 14 positioned between sample receiving end 10 and reagent releasing end 12. The device is constructed such that separator 8 is positioned between chromatographic element 4 and absorbent pad 6, and can be removed to allow contact between sample receiving end 10 and absorbent pad 6 during operation of the device. Separator 8 can range in size from extending the entire length of chromatographic element 4, to covering only sample receiving end 10 of chromatographic element 4. In addition, a portion of separator 8 may extend beyond chromatographic element 4, at either sample receiving end 10 (as shown) or alternatively at reagent releasing end 12 of chromatographic element 4.

[0057] Chromatographic element 4 can be a nitrocellulose membrane, a porous matrix, a filter, or other like material. Assay reagents are incorporated into specific portions of chromatographic element 4. Sample receiving end 10 and/or reagent releasing end 12 of chromatographic element 4 can further comprise a layer of absorbent material, such as filter paper or a porous matrix, wherein additional assay reagents are incorporated. Absorbent pad 6 is prepared from any absorbent or bibulous materials (for example, filter paper) that will sufficiently draw and hold aqueous liquid when the assay device is in operation. In one embodiment, separator 8 is formed from an impermeable material, such as a thin piece of plastic, polyester, polycarbonate, or the like. In an alternative embodiment, separator 8 can be prepared from a material which will allow passage of an aqueous solution after a certain period of time, such as a semi-permeable membrane or a material which will dissolve upon exposure to liquid.

[0058] FIG. 1B is a view of the upper face of one embodiment of the assay device according to the present invention. The assay device is enclosed in optional casing 18, formed from plastic, cardboard, treated paper, or other similar materials. Preferably, casing 18 has several windows, or openings, 20, 30 and 40, which are situated over the sample receiving end, the reagent releasing end, and the reaction zone of the chromatographic element, respectively. In FIG. 1B, visible indicator 45 (for example, a colored line) is marked on the reaction zone and can be seen through window 30. Alternatively, visible indicator 45 can be a marking on optional casing 18, at the side of window 40. In this embodiment of the device of the present invention, portion 85 of the separator protrudes from casing 18 to facilitate the removal of the separator during operation of the assay device. Alternatively, a different portion of separator 8 may protrude from the opposite end of casing 18, proximal to reagent releasing end 12, to permit partial removal of separator 8 from assay device 2, thus allowing absorbent pad 6 and sample receiving end 10 to come into contact.

[0059] FIG. 2 illustrates a cross-sectional view of the assay device taken from FIG. 1B along line A-A′. Optional casing 18 consists of top portion 50 and bottom portion 60. Top portion 50 contains openings 20, 30 and 40 seen in FIG. 1B, whereas bottom portion 60 has support 65 which compensates for the difference in thickness between the two ends of the assembly. The design of casing generally ensures that various parts of the assay device are assembled firmly together within casing. Enclosed in casing 18 are three major components of the assay device: chromatographic element 70, which is positioned above separator 80, which in turn is positioned above absorbent pad 90. Chromatographic element 70 consists of sample receiving end 100, reagent releasing end 110, and reaction zone 120. Optionally, chromatographic element 70 can be attached to a backing layer. In this embodiment of the present invention, optional filter 130 is attached to and constitutes part of sample receiving end 100 of chromatographic element 70, while optional reagent-bearing pad 140 impregnated with releasable reagents is attached to and thus constitutes part of reagent releasing end 110. Reaction zone 120 contains colored indicator 150 as well as immobilized binding partner 160. Optionally, reaction zone 120 also contains known antibodies or known antigens (for example, Protein A) for use as control(s) 170.

[0060] A sample is applied to opening 20 that is positioned over sample receiving end 100 of chromatographic element 70. The sample is allowed to migrate laterally via capillary action towards reagent-receiving end 110 of chromatographic element 70. Separator 80 prevents the sample from flowing through chromatographic element 70 and into underlying absorbent pad 90. While the sample passes across reaction zone 120, the analyte (if present in the sample) will be able to bind to its specific binding partner 160 immobilized within reaction zone 120. Once the sample has covered reaction zone 120 (as indicated by the wetting front reaching colored indicator 150), an aqueous solution is added to opening 30 situated over reagent releasing end 110 of chromatographic element 70. Separator 80 is removed by pulling protruding end 85, allowing sample receiving end 100 of chromatographic element 70 and absorbent pad 90 to come into direct contact and reverse the direction of the liquid flow. The aqueous solution releases the assay reagents incorporated within reagent releasing end 110. The aqueous solution can be added prior to the removal of separator 80, concurrently with the removal, or immediately afterwards. A reagent such as a second specific binding partner labeled with a detectable label such as a naturally colored particle can then migrate into reaction zone 120 and react with the analyte-binding partner complex, enabling detection of the analyte. In addition, known antibodies or known antigens can be included in the chromatographic element as control(s) 170.

[0061] FIG. 3A depicts a cross-sectional view of another embodiment of the assay device of the present invention. This embodiment is preferred when a generic capture assay is desired. Similar to the embodiment in FIG. 2, optional casing 200 typically consists of top part 210 and bottom part 215. Top part 210 of casing 200 has openings 220, 230 and 240, whereas bottom part 215 has support 265 to compensate for the difference in thickness between the two ends of the assembly. Enclosed in casing 200 is chromatographic element 270, which is positioned above separator 280, which in turn is positioned above absorbent pad 290. Chromatographic element 270 consists of sample receiving end 300, reagent releasing end 310, and reaction zone 320. Optional filter 330 is attached to and constitutes part of sample receiving end 300 of chromatographic element 270, while optional reagent-bearing pad 340 impregnated with releasable reagents is attached to and constitutes part of reagent releasing end 310. Filter 330 can be subdivided into additional reagent bearing zone 335. Alternatively, as shown in FIG. 3B, additional filter 336 containing releasable reagents can be added for this purpose. Reaction zone 320 contains colored indicator 350 as well as immobilized binding partner 360, such as a capturing reagent specific for the target analyte.

[0062] In this embodiment of the present invention, a sample is applied to opening 220 that is located over sample receiving end 300 of chromatographic element 270. A first reaction takes place at sample receiving end 300 between the analyte (if present in the sample) and a first binding partner, released either directly from sample receiving end 300 or from reagent bearing zone 335 of optional filter 330. The analyte:first binding partner complex is allowed to migrate laterally by capillary action towards reagent-receiving end 310 of chromatographic element 270. Liquid impermeable separator 280 acts as a barrier preventing the sample from flowing through chromatographic element 270 and into underlying absorbent pad 290. When the sample passes across reaction zone 320, a second reaction occurs between the analyte:first binding partner complex and second binding partner 360 immobilized in reaction zone 320. Once the sample has covered reaction zone 320 (as indicated by the wetting front reaching colored indicator 350), separator 280 can be removed by pulling protruding end 285. Removal of separator 280 brings sample receiving end 300 of chromatographic element 270 and absorbent pad 290 into direct contact and reverses the direction of the liquid flow. An aqueous solution is added to opening 230 situated over reagent releasing end 310 of chromatographic element 270, thus releasing the assay reagents incorporated therein. The aqueous solution can be added prior to the removal of separator 280, concurrently with the removal, or immediately afterwards. A labeled reagent such as a third specific binding partner affixed with a detectable label such as a naturally colored particle, usually directed either toward the analyte or the analyte-containing complex, can then migrate into reaction zone 320 and react with the captured analyte-partner complex, thus enabling detection of the analyte. In addition, known antibodies or known antigens can be included in the chromatographic element as controls 370.

[0063] Likewise, yet another embodiment of the assay device of the present invention can be constructed taking a similar approach, as depicted in FIG. 4A. Optional casing 400 typically consists of top part 410 and bottom part 415. Top part 410 of casing 400 has openings 420, 430 and 440, whereas bottom part 415 has support 465 to compensate for the difference in thickness between the two ends of the assembly. Enclosed in casing 400 is chromatographic element 470, which is positioned above separator 480, which in turn is positioned above absorbent pad 490. Chromatographic element 470 consists of sample receiving end 500, reagent releasing end 510, and reaction zone 520. Optional filter 530 is attached to and constitutes part of sample receiving end 500 of chromatographic element 470, while optional reagent-bearing pad 540 impregnated with releasable reagents is attached to and constitutes part of reagent releasing end 510. Reagent-bearing pad 540 can be subdivided into different zones 545, 546 to accommodate different releasable reagents or binding partners. Alternatively, as shown in FIG. 4B, one or more additional optional filters 547 impregnated with additional releasable reagents can be added to reagent releasing end 510 of chromatographic element 470. Reaction zone 520 contains colored indicator 550 as well as an immobilized binding partner 560, for example, a capturing reagent specific for the target analyte.

[0064] In this embodiment of the present invention, a sample is applied to opening 420 located over sample receiving end 500 of chromatographic element 470. The sample is allowed to migrate laterally by the capillary action towards reagent releasing end 510. The presence of separator 480 provides a liquid impermeable barrier, preventing the sample from flowing through chromatographic element 470 and into underlying absorbent pad 490. When the sample passes across reaction zone 520, a first reaction will take place between the analyte (if present in the sample) and first specific binding partner 560 immobilized in reaction zone 520. Once the sample covers reaction zone 520 (as indicated by the wetting front reaching colored indicator 550), separator 480 can be removed by pulling protruding end 485. Removal of separator 480 brings sample receiving end 500 of chromatographic element 470 and absorbent pad 490 into direct contact and reverses the direction of the liquid flow. An aqueous solution is added to opening 430 positioned over reagent releasing end 510. The aqueous solution can be added prior to the removal of separator 480, concurrently with the removal, or immediately afterwards. This addition of an aqueous solution allows not only release of the assay reactants immobilized within the reagent releasing end, but also enables a second reaction to occur between a second binding partner to the analyte and a third binding partner to the second binding reagent. The complex of second and third binding partners are driven from reagent releasing end 510 and across reaction zone 520, where a third reaction takes place between the two complexes. A label affixed to the third binding partner allows for detection of the complex and determination of the presence of the analyte. In addition, known antibodies or known antigens can be included in the chromatographic element as controls 570.

[0065] Referring to FIGS. 5A and 5B, a fourth embodiment of the present invention can be prepared using a construct similar to that of FIGS. 1B and 2A. In this embodiment, separator 680 is a time-controlled barrier such as a thin piece of semi-permeable material, or a material that will dissolve over time. Alternatively, several compositions which can be used as a dissolving-type separator include, but are not limited to, hydroxypropyl cellulose, polyethylene oxide, polyvinylpyrrolidone, poly(vinyl alcohol), poly(acrylic acid), polyacrylates such as Carbopol 934 (B. F. Goodrich), starch and starch derivatives, polysaccharides, sodium carboxymethyl cellulose, xanthan gum, karaya gum, and gelatin.

[0066] As in the previous embodiments, optional casing 610 consists of top part 650 and bottom part 660. Top part 650 of casing 610 has openings 620, 630 and 640, whereas bottom part has support 665 which compensates for the difference in thickness between the two ends of the assembly. The design of casing 610 ensures that various parts of the assay device are assembled firmly within casing 610. Enclosed in casing 610 is chromatographic element 670, which is positioned above separator 680, which in turn is positioned above absorbent pad 690. Chromatographic element 670 consists of sample receiving end 700, reagent releasing end 710 and reaction zone 720. In this embodiment, separator 680 is a time-controlled barrier such as a thin slide of semi-permeable or time-dissolving material. Separator 680 acts as a barrier to the flow of liquid into underlying absorbent pad 690 for a limited period of time (between 10 seconds and 10 minutes, more preferably between 30 seconds and 5 minutes, most preferably for approximately 1 minute). Optional filter 730 is attached to and constitutes part of sample receiving end 700 of chromatographic element 670, while optional reagent-bearing pad 740 containing one or more releasable reagents is attached to and constitutes a part of reagent releasing end 710. Reaction zone 720 contains colored indicator 750 as well as immobilized binding partner 760.

[0067] When this particular embodiment of the assay device is used, the sample is applied to opening 620 situated over sample receiving end 700 of chromatographic element 670. The sample is allowed to migrate laterally via capillary action towards reagent-receiving end 710. Separator 680 prevents the sample from flowing towards underneath absorbent pad 690 for a predetermined length of time (as determined by the composition and thickness of the separator). A reaction between an analyte (if present in the sample) and its specific binding partner 760 immobilized in reaction zone 720 will take place while the sample passes across this region. By the time that the sample has covered reaction zone 720 (as indicated by the wetting front reaching colored indicator 750), separator 680 will have either become permeable or dissolved completely. In either case, the absence of separator 680 allows sample receiving end 700 of chromatographic element 670 to come into fluid communication with absorbent pad 690, reversing the direction of the liquid flow. An aqueous solution can be added to window 630 positioned over reagent releasing end 710, thus releasing the assay reagents incorporated therein. A reagent such as a second specific binding partner labeled with a detectable label such as a naturally colored particle can then migrate into reaction zone 720 and react with the analyte-binding partner complex, enabling detection of the analyte. In addition, known antibodies or know antigens can be included in the chromatographic element as controls 770.

[0068] FIG. 6 shows details of assembly of example chromatographic element 800. As shown, sample end 810, reagent end 812 and membrane 816 comprising reaction zone 814 are mounted on adhesive backing 818, such that sample end 810 comprises area without backing 820 for subsequent contact with an absorber pad.

[0069] The present invention also addresses assay devices for the detection of multiple disease markers, such antigens to HIV and HTLV, such that the simultaneous detection of different diseases can be performed using a single sample of biological fluid and a single assay device. Multiple disease markers, for example, antigens to different pathogens, can be immobilized within the reaction zone of the chromatographic element. This embodiment of the present invention would enable a single device to be used for detection of multiple analytes from a given sample. Similarly, antibodies such as anti-human IgG or IgM can be immobilized on the reaction zone of the chromatographic element, as shown in FIGS. 3 and 4. Such a construct allows a single device using a generic detector such as a labeled specific binding partner for detection of different types of antibodies to the same pathogen.

[0070] The assay devices of the present invention are able to provide improved sensitivity for analyte detection over current available rapid chromatographic assays without compromising in the specificity. This advancement is demonstrated in the example section of the present invention. However, the advantages of the present invention are not limited to the functional aspects of the assay device, but address the practical aspects as well. Regardless of the particular embodiment employed, an assay device based on the present invention does not need to include additional filtration devices, such as filters with special coatings, to handle a wide variety of biological fluids. This versatility is achieved by the design of the assay device, which allows staged reactions and sufficient washing without involving additional steps.

[0071] An additional advantage of the assay devices of the present invention is in the ease of manufacture of the assay device. The devices of the present invention employ a generic construct, which can be modified with minimal alteration from one application to another. This generic platform is versatile enough to accommodate the needs and requirements for multiple product lines. A product specific for detection of a particular analyte can be easily adapted to another product for a different analyte with minimal modification of the overall design of the assay device, such as replacement of the binding partner to one particular analyte. Accordingly, it is not necessary to develop additional specific detecting reagents for each specific product. Rather, the specificity of the reaction is determined by the first binding partner while the labeled binding partner can be a multipurpose generic construct (for example, anti-human antibodies or anti-GST antibodies labeled with a detectable label such as a naturally colored particle). This is a huge advantage as compared to the development of traditional rapid assays, in which a specific detector for a specific product must be developed for each assay, in order to maintain an acceptable sensitivity. The present invention therefore reduces both the time and the cost used for product development. Furthermore, since the major components of the assay device can be used across a variety of assays, production parameters can be maintained without changes. A production facility manufacturing a series of products based on the present invention would use a single set of manufacturing equipment and a minimal array of inventories of raw materials, which in turn significantly reduces the costs of operation.

[0072] The following examples are offered for illustration. One of skill in the art will recognize a variety of noncritical parameters that can be changed.

[0073] Assay devices for the detection of human antibodies to HIV types 1 and 2 were prepared as follows. Recombinant HIV 1 antigens p24 and gp41, and recombinant HIV 2 antigen gp36, were immobilized, or “slotted,” at a concentration range of about 0.08 to about 0.3 mg/ml onto a nitrocellulose membrane of 8 μm average pore size (Whatman, Ann Arbor, Mich.) using an IVEX striping machine. Protein A was immobilized in the same manner for use as an assay control line. The membrane was dried for approximately 10 minutes before addition of a blocking buffer (Milli-Q purified water with 0.3% casein and 0.25% sucrose). The membrane was exposed to the blocking buffer for approximately 1 minute, after which the membrane was dried at 37° C. for another 15 minutes. The membrane was finally affixed to a membrane backing (Adhesives Research Inc., Glen Rock, pa.).

[0074] A reagent-bearing pad was prepared using a porous matrix (Hollingsworth & Vose, Inc., East Walpole, Mass.). The pad was sprayed with goat anti-human IgG antibodies that were labeled with colloidal gold particles of approximately 40 μm, and dried at 37° C. for 30 minutes. A chromatographic element was prepared by affixing an untreated porous matrix to one end of the nitrocellulose strip and the reagent-bearing pad to the other end of the nitrocellulose strip. The assembly was then cut into strips of about 4 mm by about 56 mm in size to form a chromatographic element having the untreated porous matrix at the sample receiving end, the reagent-bearing pad attached at the reagent releasing end, and the antigens immobilized in the reaction zone region. An assay device was assembled by placing an absorbent pad in the bottom half of a casing, then laying a separator above the absorbent pad, such that one edge of the separator extended from the casing. The chromatographic element was situated on top of the separator (such that the sample receiving end was positioned above the separator) and the top half of the casing was attached.

[0075] A serum sample (approximately 15 μl) was added to the sample receiving end of the chromatographic element via a first opening, or window, on the casing. The sample was allowed to migrate laterally and cover the reaction zone region of the membrane, as determined by viewing the progression of the wetting front through an opening in the casing directly above the reaction zone. Any human antibodies to the three HIV antigens present in the sample were bound to these antigens as the sample fluid crossed the region at which the antigens are bound to the nitrocellulose membrane (the reaction zone). When the sample reached the indicator in the reaction zone after approximately 1 minute, three drops (approximately 120 μL) of aqueous solution (reagent releasing buffer, comprised of 0.01M phosphate buffered saline pH 7.4 plus 0.4% SDS) were added to a second opening on the casing located above the reagent releasing end of the chromatographic element. Addition of the aqueous solution solubilized the releasable binding partner (in this example, the colloidal gold-labeled goat anti-human IgG antibodies). Immediately after addition of the aqueous solution, the separator was removed from the assay device by pulling on the protruding end, thus allowing the chromatographic element and the absorbent pad to come into contact. The labeled goat anti-human IgG antibodies were then allowed to migrate across the reaction zone of the chromatographic element and bind to any human IgG antibodies immobilized in this region. The results were readable in approximately 5 minutes through the opening in the casing. Typically, a negative result is indicated by the appearance of a single control line in the reaction region. Bands representing either one or both of the disease markers will also appear if the analyte(s), in this case anti-HIV antibodies, are present.

[0076] To demonstrate the sensitivity of this assay, a titration-end point activity test was performed. Samples positive for HIV 1 (SBA033) or HIV 2 (SBB043) were serially diluted to generate a series of sample concentrations. These diluted samples were tested in parallel with both the device described in Example 1 and two commercially-available test kits (Instant-check “flow-through” device, Genelabs Diagnostics, Singapore; HIV 1/2 Stat-Pak “lateral flow” device, Chembio Diagnostics Systems Inc., New York, USA). As a negative control, two HIV-negative samples (NAA196 and NAA 237) were employed. The intensity of the resulting band or bands were scored visually, and recorded as 3+ or 2+ if the intensity was greater than that of the control band, and 1+, +/− and +/−− if the intensity was less than that of the control band.

[0077] The results of this experiment, as tabulated in Table 1, clearly demonstrate the improved sensitivity of the present invention as compared to commercially available test kits employing conventional lateral flow or flow-through technologies.

[0078] Assay devices for the detection of human antibodies to Helicobacter pyloriwere prepared in a manner similar to that described in Example 1. Briefly, a nitrocellulose membrane of 8 μm average pore size (Whatman, Ann Arbor, Mich.) was slotted with a proprietary recombinant antigen toward H. pylonat a concentration of approximately 0.6 mg/ml using the IVEX striping machine. The membrane was dried for 10 minutes before blocking with a blocking buffer (Milli-Q purified water with 0.3% casein and 0.25% sucrose) for 1 minute. The blocked membrane was then dried at 37° C. for another 15 minutes before being affixed to a membrane backing (Adhesives Research Inc., Glen Rock, pa.). A reagent-bearing pad was prepared using a porous matrix (Hollingsworth & Vose Inc., East Walpole, Mass.) and sprayed with goat anti-human IgG antibodies that were labeled with colloidal gold particles of approximately 40 μm. The reagent-bearing pad was also dried at 37° C. for 30 minutes prior to use. The chromatographic element was prepared by affixing a porous matrix to the sample receiving end of the membrane-backed nitrocellulose strip and the reagent-bearing pad to the reagent releasing end of the strip, such that the immobilized H. pyloriantigen was situated in the reaction zone between the two ends. The assembly was then cut into a strip approximately 4 mm by 56 mm in size. An assay device was assembled by placing an absorbent pad in the bottom half of a casing, followed by a separator, and lastly the chromatographic element before closing the top half of the casing.

[0079] Approximately 30 μl of serum sample was added to the sample receiving end of the chromatographic element via a first window on the casing. The sample was allowed to migrate laterally and cover part of the nitrocellulose membrane. When the sample reached the indicator in the reaction zone (after approximately 1 minute), three drops (approximately 120 μL) of reagent releasing buffer (of 0.01M phosphate buffered saline pH 7.4 plus 0.4% SDS) were added to a second window on the casing, releasing the colloidal gold labeled goat anti-human IgG antibodies (the releasable binding partner) incorporated therein. The separator was then removed by pulling the end protruding from the device casing, to allow the chromatographic element and the absorbent pad to come into contact. The labeled goat anti-human IgG antibodies were then allowed to migrate across the reaction zone of the chromatographic element and bind to any human IgG antibodies immobilized in this region. The results generated by the assay device can be read in approximately 5 minutes through the third window that is directly situated on the reaction zone. Typically, a negative result will be indicated by the appearance of a control line only in the window. Another band representing the [H. pylori antigen:human antibody: labeled goat anti-human IgG] complex will also appear if the analyte, in this case the anti-H. pyloriantibody, is present in the sample.

[0080] In a titration-end point activity test, a sample from an individual infected with H. pylori(sample W003) was serially diluted, and the diluted samples were tested with both the device of the present invention and a device constructed according to conventional lateral flow assay design. As a negative control, a sample negative for H. pylon (sample H5) was employed, The results of the experiment are presented in Table 2. The data clearly demonstrate the improved sensitivity of the assay device of the present invention as compared to a device of the conventional lateral flow design.

[0081] In another comparison study, a panel of samples either positive or negative for H. pyloriwas assayed using both devices of the present invention and a commercially available Western Blot assay (Helico Blot 2.1, Genelabs Diagnostics, Singapore). The negative samples were from healthy donor, whereas the positive samples were from patients infected with H. pylorias confirmed by at least two of the following methods: histology, culture, and rapid urease test. The results of the assays are presented in Table 4. Devices of the present invention were shown to provide a slight improvement of detection specificity without compromising the sensitivity of the assay.

[0082] An experiment was also performed with the H. pyloriassay device as prepared in Example 2 using saliva samples instead of serum samples. Saliva samples from a healthy individual and an H. pylon infected individual, as confirmed by a Western blot test, were collected and diluted 1:5 in phosphate buffer saline (0.01M, pH 7.4). The samples were centrifuged for 5 minutes at 12,000 rpm and then stored in a freezer at −20° C. before use. Approximately 30 μl of each sample was applied to separate assay devices and tested according to the assay procedure described in Example 2. As a negative control, an assay using only phosphate buffer saline (0.01M, pH7.4) was included in the experiment. The saliva sample from the infected individual produced a defined control band as well as an infection-indicating band with intensity in the range of 1+ to 2+. Neither the saliva sample from the healthy individual the PBS experiment control gave rise to an infection-indicating band. The saliva sample at the same 1:5 dilution was not detected by a conventional lateral flow format using the same reagents. The results of this experiment, therefore, demonstrated not only the improved sensitivity of device of the present invention, but also that the device can be used for detection of anti H. poyloriantibodies in saliva without the need for any structural modification to the device.

[0083] In another experiment performed with the assay device as prepared in Example 2

[0084] whole blood samples were used in place of serum samples. Whole blood samples were from healthy individuals and an H. pyloninfected individual, as confirmed by a Weste, test. Approximately 50 μl of each of the samples were applied to separate assay device tested as according to the assay procedure described in Example 2. The whole blood from the infected individual produced a defined control band as well as an infection-indicating band having an intensity of 3+. In contrast, the whole blood samples obtained from healthy individuals (n=13) produced only the control band and not the infection-indicating band. For comparison, the whole blood samples were also tested using a conventional lateral flow assay prepared using the same reagents. In the conventional lateral flow assay, higher backgrounds were produced, rendering interpretation of the assay results difficult. This comparison demonstrated that the assay device of the present invention can be used for detection of anti H. pyloriantibodies in whole blood samples without the need of a structural modification to the device. In addition the results showed that the assay device based on the present invention was not affected by the high background problems that arose in the conventional lateral flow format.

[0085] The above described devices can be packaged and sold as kits for detection of analytes. Indeed, the above devices, being self-contained and convenient for use, are themselves kits. Other kit elements can include containers for packaging one or more device elements, instruction sets for directing a user in the use of the device, i.e., according to the methods set forth herein, packaging materials, aqueous solutions for use with the device, and the like.

[0086] While the foregoing invention has been described in some detail for purposes of clarity and understanding, it will be clear to one skilled in the art from a reading of this disclosure that various changes in form and detail can be made without departing from the true scope of the invention. For example, all the techniques, methods, compositions, apparatus and systems described above may be used in various combinations. All publications and patent documents cited in this application are incorporated by reference in their entirety for all purpose to the same extent as if each individual publication or patent document were individually indicated to be incorporated by reference for all purposes.