WO2000007015A1

WO2000007015A1 - Immunoassay device

Info

Publication number: WO2000007015A1
Application number: PCT/US1998/015916
Authority: WO
Inventors: Jin Po Lee; Guihe Liang
Original assignee: Syntron Bioresearch, Inc.
Priority date: 1998-07-29
Filing date: 1998-07-29
Publication date: 2000-02-10
Also published as: AU8763098A; EP1023600A1; EP1023600A4

Abstract

The invention provides test strip assay devices for detection of an analyte in a fluid analyte sample in which the hook effect is minimized and interference with binding reactions in the assay are substantially avoided, as well as methods for use of the devices to those ends. To these ends, the devices of the invention separate the test zone of the test strip (in which a binder for the analyte is immovably bound) and the tracer zone of the test strip (in which a reconstitutable tracer is movably bound) in horizontal and, in some embodiments, in vertical planes. In all embodiments of the devices and methods of the invention, fluid analyte sample applied to the test strip contacts the test zone before the tracer contacts either the sample or the test zone. The device is especially useful for detection of analyte whose binding with assay binders is especially sensitive to assay conditions, such as antibody analyte.

Description

Immunoassay Device

Field of the Invention

This invention relates generally to the fields of immunology and diagnosis. More particularly, the invention relates to methods for detecting the presence of a given analyte in a sample.

History of the Related Art

Sample migration type of fluid analyte assays (Rosenstein et al, U.S. Pat. No. 4,491,645, Eisinger et al, U.S. Pat. No. 4,943,522, Kang et al, U.S. Pat. No. 5,252,496 and 5,559,041, Charlton et al, U.S. Pat. No. 5,714,389, May et al, U.S. Pat. No. 5,602,040, Chen et al, U.S. Pat. No. 5,384,264) incorporate a visually readable particulate label tracer and a binder immobilized in a porous matrix into a self-contained migration assay device. Such assays offer the convenience of being performable in a single step, consisting of application of an analyte sample to the device. Such devices include a test zone, in which reagents are immobilized that bind to analyte present in the sample and with a tracer to produce signals (usually visually detectable signals) to indicate positive and negative assay results.

In a typical migration assay, a tracer (comprising a binding molecule, which may be combined with a molecule capable of producing a visually detectable signal) is mixed with the analyte sample or sample diluent before contacting the test zone. Since the concentrations of test zone reagent and tracer that can be utilized in the device are limited, application of a high concentration of analyte to the device may produce a false negative result, also known as the "hook effect". Inaccurate test results can also be produced by unintended intereference with binding between the target analyte, tracer and/or binder. Summary of the Invention

The invention provides an analyte assay device having a test strip in which both the hook effect and unintended interference with binding between analyte, tracer and/or binder are substantially avoided. These ends are accomplished in part by separation of test strip zones for sample loading ("sample loading zone"), analyte binding ("test zone"), buffer loading ("buffer loading zone") and tracer reconstitution ("tracer zone") from one another to facilitate flow of fluid analyte sample into the test zone before contact of the test zone by the tracer. In one embodiment, this separation is made in both a vertical and horizontal plane. Vertically, the tracer is bound to a hydrophilic membrane in a tracer zone which lies adjacent to and above the upstream end of a porous reaction membrane. A sample loading zone lies on the reaction membrane.

Horizontally, a test zone in one embodiment of the invention lies between the tracer zone and sample loading zone. On application of a fluid analyte sample to the sample loading zone, the sample diffuses to the test zone and analyte in the sample, if any, contacts a binder specific for analyte in the test zone. On application of a buffer to a buffer loading zone upstream of the tracer zone, the buffer migrates into the tracer zone, reconstitutes the tracer and carries it to the test zone. On reaching the test zone, the buffer displaces unbound analyte from the test zone, minimizing contact between the tracer and unbound analyte.

In another embodiment of the invention, the sample loading zone lies between the test zone and the tracer zone, closer to the former than to the latter.

In another embodiment of the invention, the test zone lies between the sample loading zone and tracer zone, but no vertical separation of the zones is made. In any embodiment of the invention, the sample loading zone and test zone may be located at the same site in the reaction membrane. Also, in preferred embodiments of the invention, a buffer loading zone lies upstream of the tracer zone so buffer migrates downstream to reconstitute the tracer for migration to the test zone.

The devices of the invention may be constructed as assay cassettes or test strips, in uni- or bi-directional form. An especially useful embodiment of the invention is one for use in detecting the presence of antibody in a fluid analyte sample. In this embodiment, the assay device includes an antigen of the antibody and an antibody reactive with the target antibody for use as binders in the test and tracer zones, thereby enhancing the sensitivity of the assay.

The invention further provides assay methods in which a fluid analyte sample and a buffer are applied to a device described herein, then an assay result is read visually on appearance of a visually detectable signal in the test and control zones.

Brief Description of the Drawings

FIG. 1 is an exploded view of a cassette assay device of the invention.

FIG. 2 is an exploded view of a strip assay device of the invention.

FIG. 3 is an exploded view of a bidirectional assay device with two chromatographic strips.

Detailed Description of the Invention A. Definitions

The term "antigen" as used herein refers to any analyte which is capable of binding antibodies. Antigens may comprise, without limitation, chemical compounds, polypeptides, carbohydrates, nucleic acids, lipids, and the like, including viral particles, viral subunits, bacterial and parasite surface antigens, and host proteins that may be diagnostic of the subject's condition. Presently preferred antigens include proteins or protein fragments derived from HIV-1, HIV-2, HCV, dengue, toxoplasma gondii, Chlamydia trachomatis, H. pylori, HSV-1, HSV-2, CMV, rubella. The term "fragment" as used herein refers to a portion of an antigen which exhibits at least one useful epitope. A "useful epitope" is one capable of binding an immunoglobulin generated in response to contact with an antigen. Thus, for example, if a subject develops anti-HSVl antibodies in response to contact with HSV1, a useful epitope is one which binds at least one anti-HSVl antibody derived from said subject. "Fragments" also include fusion proteins, where a "fusion protein" is a polypeptide containing portions of amino acid sequence derived from two or more different proteins.

The term "immunoglobulin" refers generally to any antibody, including without limitation IgA, IgG, IgM, IgD, IgE, and their various subclasses. The term also includes antibody fragments, such as Fab, F(ab)2, and F(ab')2 fragments, single-binding domain antibodies, chimeric antibodies, and the like.

A "binder" refers to a ligand for the analyte as in the format of a sandwich assay, or a ligand for both the analyte and the tracer as in the format of a competitive assay. A binder can be chosen from a group of molecules or compounds capable of binding the analyte, such as an antigen to the antibody analyte, or an antibody to the antigen analyte.

A "test zone" refers to an area in which a binder or the analyte is attached, movably or immovably, to the test strip portion of an assay device. A "tracer" refers to a ligand for the analyte or the binder labeled with a detectable label, preferably a visually readable particulate label, such as colloidal gold, latex and liposomes including dye, carbon black, and the like. The tracer in the device of the invention is dried or lyophilized and movably supported in a "tracer zone". An "analyte sample" refers to a fraction of a fluid of interest, or a dissolved or a liquid extraction of substances of interest. The sample fluid is used in the assay of the invention for detecting an analyte.

A "sample loading zone" refers to an area of a test strip to which a diagnostic volume of analyte sample fluid is applied to the test strip for migration therethrough. The total area through which applied analyte sample fluid will flow is not strictly defined before the assay is carried out, but is limited by the volume of the sample fluid deposited and may slightly vary between different assays. The test zone may be part of the sample loading zone.

A "buffer loading zone" refers to an area of a test strip on which a fluid buffer is applied. The buffer loading zone may be upstream to, or part of, the tracer zone, preferably the former.

A "test strip" of the invention consists of, collectively, all of the zone supporting membranes and any filters of the assay device.

B. Assay Devices and Methods for Their Use

The invention minimizes the hook effect caused by contact between an assay tracer and analyte sample before contact with an assay binder and substantially avoids unintended interference with the binding reactions of the assay. These goals are accomplished by facilitating contact between the fluid analyte sample and the test zone before contact of the latter by the tracer. More particularly, (1) the tracer zone/buffer loading zone and the sample loading zone are preferably separated vertically by placement in different planes of the assay test strip (to avoid unintended mixing by migration of the tracer fluid and analyte sample in proximity within the same plane); (2) the tracer zone/buffer loading zone and sample loading zone are separated horizontally by placement toward opposing ends of the test strip; (3) the tracer dis preferably incorporated within the test strip and reconsistituted by admixture with a buffer solution applied to the tracer zone/buffer loading zone on or after (preferably after) application of the analyte sample to the sample loading zone; and (4) buffer migrating from the buffer loading zone pushes analyte sample fluid out of the test zone to avoid binding between the tracer and unbound analyte.

Further to these ends, the volume of fluid analyte sample applied to the sample loading zone will preferably be limited so as to not exceed the volume of buffer applied to the buffer loading zone. The sample loading zone may also be placed on the test strip in closer proximity to the test zone than the test zone is to the tracer zone/buffer loading zone.

Thus, the assay device comprises a chromatographic strip having, from its upstream end to its down stream end, a buffer loading zone, a tracer zone having a movable tracer, a test zone having an immobilized binder, and an absorbing zone for collection of excess analyte sample fluid.

The chromatographic strip may consist of a single piece of thin porous membrane, but will preferably consist of a porous membrane overlaid at the upstream end by a hydrophilic membrane that includes the tracer zone and buffer loading zone. The sample loading zone, test zone, any control zone and the absorbent zone are included on the porous membrane downstream of the tracer and buffer loading zone(s). A prefilter may also be present in the device to remove cells and other contaminants from the analyte sample before contact with the sample loading zone. Collectively, these zone supporting membranes and any pre-filter are considered to be the test strip of the device. Each zone and membrane is in fluid communication with all other zones and membrane.

The porous membrane preferably has a thickness of less than 0.2 millimeters. The pore size of the membrane is preferably over 1.0 micron so as to allow good migration of fluid through the device. However, when the average pore size of the porous membrane is smaller or similar to the particle size of the tracer, the tracer migrates on the surface of the membrane rather than through its pores. Such surface migration of the tracer allows it to move more quickly toward the test zone, at the risk of contacting the analyte sample before the sample contacts the test zone. Thus, when an insoluble particulate label is used in the assay, the pore size of the membrane should be larger than the size of the tracer particle so as to permit the tracer migrate through the pores of the membrane. In this respect, good results can be expected using nitrocellulose membrane of thickness of 0.05 millimeter and 0.1 millimeter and of pore sizes of 8 microns and 10 microns, with colloidal gold-labeled tracers. Membranes of different composition (e.g., nylons) but comparable pore sizes will also be of use in the invention.

The binder is immobilized in the test zone by means of absorption or covalent binding. The binder may also be immobilized in the test zone indirectly, i.e., through a linker. Two or more binders for different analytes may be immobilized at the test zone. The binders may be mixed or coated separately. For example, HIV-1 antigen and HIV-2 antigen can be immobilized within the same area or in two separate areas of the same device. The first type of device can be used for HIV screening test, while the second type of device is preferably used for differentiation diagnosis. Those of ordinary skill in the art will be familiar with, or can readily ascertain, the identity of particular materials and techniques for attaching particular assay binders to porous membranes; therefore, no further detail regarding such materials and techniques will be discussed here.

A control binder is preferably included on the porous membrane having the test zone. A control binder is a binder of at least one component of the tracer, so that if all the reagents of the device are active, and the assay procedure is correct, the label is always detectable at the control band regardless the presence or absence of the analyte. Binders for use in the device of the invention including, without limitation, antibodies and antigens, may be obtained commercially through sources that will be familiar to those of skill in the art, may be synthesized through immunization of a host and/or may be isolated and purified from a host organism. The membrane which supports the tracer zone/buffer loading zone may be of the same composition as the porous membrane which supports the test zone. However, the membrane which supports the tracer zone/buffer loading zone is desirably a hydrophilic matrix having a pore size larger than the porous membrane which supports the test zone, such as a filter paper. It is also preferably less hydrophilic than the test zone supporting membrane, so that fluid applied to the tracer zone matrix flows somewhat more quickly through the matrix than analyte sample flows to the test zone.

The tracer in the device is a labeled ligand of the analyte, as in the format of a sandwich assay, or a ligand of the binder, as in the format of a competitive assay. The ligand portion of the tracer can be an antibody, antigen, or a binding partner of the analyte or the binder. The label of the tracer portion can be chosen from a category of detectable substances. One group of detectable substances includes enzymes, coenzymes, or enzyme substrates. If an enzyme, coenzyme or an enzyme substrate is used as the label, an enzyme-substrate system is included in the assay. For instance, the ligand may be labeled with an enzyme and an enzyme substrate capable of react with the enzyme and produce a color is impregnated in the porous membrane, or dissolved in the test buffer.

A colored test zone indicates a positive result. An enzyme or enzyme substrate may also be impregnated in the absorbing pad. The methods for using enzyme-substrate systems and other signal producing systems are known to those of ordinary skill in the art (see. e.g., PCT Application. No. WO87/0277 and U.S. Pat. No. 5,030,558).

Conveniently, the label of the tracer portion is chosen from a group of color particulate particles that are visually readable with unaided eye. Such particulate labels include colloidal gold, color latex, dye sol, carbon black, liposome including color dye. The methods for using such particulate labels in migration assays are known in the art. References include (1) Frens, Nature 1973, 241 , 20 and Leuvering et al, J. Immunoassay, 1980, 1, 77 for colloidal gold, (2) May, EP-A 0 280559 and 0 281 327 for colored latex, (3) U.S. Pat. No. 4,373,932 and WO 88/08534 for dye sol, (4) U.S. Pat. No. 5,252,496 for carbon black, and (5) U.S. Pat. No. 4,703,017 for a liposome including a dye. An absorbing pad of a hydrophilic material, such as filter paper, sponge, is preferably included at the absorbing zone of the strip to serve as a fluid sink. The absorbing zone that receives unbound tracer can also be used as a result viewing area. When the label is particulate color particle, the result can be read visually. When an enzyme-substrate system is included in the assay, the substrate of the tracer enzyme can be impregnated within the absorbing pad. The absorbing area can also be used to measure the volume of liquid being migrated through the test zone, for example, to detect the signal at the test zone when certain area of the absorbing zone is saturated with the test solution. For such purpose, a signal producing substance, such as a pH indicator, can be impregnated in the absorbing zone.

A buffer port and a sample port are further included in embodiments of the assay device not designed in the dipstick format. The buffer port provides access to the test strip for application of a buffer to the buffer loading zone. The sample port provides access to the test strip for application of an analyte sample fluid to the sample loading zone. Where the assay device houses the test strip within a cassette, the ports are formed in the housing. Where the assay device consists of the test strip and a hydrophobic tape cover, the ports are formed through the tape. In dipstick format, the buffer port may be eliminated and the upstream end of the device immersed into a buffer during the assay in lieu of applying the buffer to the buffer loading zone through a port. A filter may be included in the sample port. The filter provides a trap for cells or other undissolvable particles and may also contain reagents or buffering agents for admixture with the sample, such as salts or detergents.

Single or multiple test strips (combined in a housing or attached by, for example, a clip) may be used in single strip, dipstick or cassette housing format. An especially useful format for the assay device of the invention is a bidirectional chromatographic test device as described by Horstman et al, U.S. Pat. No. 4,006,474. Such a device can be constructed by assembling two chromatographic strips of the invention in one device. A bidirectional chromatographic test device is for detecting two or more analytes at the same time. Adapted for use in the invention, one buffer port is disposed in the middle of the device where the upstream ends of the two strips meet. Two sample ports are disposed for each chromatographic strip.

The assay method using a device of the invention includes depositing a diagnostic volume of sample fluid to the sample port and a buffer to the buffer port. The assay process begins simultaneously when the sample and buffer are deposited. First the sample fluid spreads to the sample zone. The buffer migrates to the tracer zone and reconstitutes the tracer. The tracer does not contact the test zone until after the analyte sample fluid has passed through the test zone.

The area of the sample zone at the upstream side of the test zone is related to the assay sensitivity. A relatively large area sample zone at the upstream side of the test zone will provide a relatively large volume of analyte sample fluid to the test zone. Where the analyte sample fluid contains low concentrations of analyte, use of a large volume of anlayte sample fluid enhances the possibility that the analyte will contact the binder in the test zone and produce a detectable assay reaction. However, application of a large volume of analyte sample fluid to the sample loading zone increases the likelihood that the sample fluid will mix with the migrating tracer before the former passes completely through the test zone.

Thus, whenever possible (e.g., where the analyte is believed to be present in relatively high concentration within the fluid sample), the hook effect will be minimized to a greater degree in the assay device of the invention when the volume of sample fluid applied to the device at any one time is limited, either by application of a smaller total volume of sample fluid to the device or restriction of the sample port diameter.

Assay sensitivity may also be enhanced by placement of the sample loading zone downstream of the test zone, so the test zone lies between the tracer zone/buffer loading zone and the sample loading zone. In this embodiment of the invention (see, FIG. 1), a portion of the sample fluid diffuses through the test zone to the upstream side, then is pulled back across the test zone a second time as flow migrates toward the absorbent zone. In this manner, a portion of the sample fluid contacts the test zone twice, and non-specific binding between tracer and excess analyte or sample contaminants is reduced. As such, this embodiment yields a higher assay sensitivity than that when the sample is applied from the upstream side of the test zone (see, FIG. 2, especially for assaying relatively large volumes of analyte fluid.

FIG. 1 is an exploded view of an assay device of the invention, with the chromatographic test strip assembled in a cassette format. The device comprises a two piece housing 100 having a base 101 and cover 103. The base includes a support for test strip 102, shown here as a pair of parallel rails 104 and 105 spaced apart a distance less than the width of test strip 102. Alternative supports may employ a larger number of rails, or a single broad support, a series of perpendicular bars, or the like. The upstream end 106 of the base (including the upstream ends of support rails 104 and 105) is ramped to raise the upstream end of test strip 102 above the downstream end. The entire base can be plastic, metal and the like, and can be fabricated by injection molding, machining, and other processes. A depression 107 is made in the base to accept a bar 120 underlying the cover to assist in sealing the device. Test strip 102 comprises a porous membrane 116. Test zone 112 comprises a binder specific for the analyte, and lies upstream of a control band 115, which comprises a binder for at least one component of the tracer. The arrangement of the test and control binders may vary, for instance, the test band being upstream to the control band, the test band being downstream to the control band, or the control band being in the middle of two test bands. Both binder bands preferably form defined patterns or shapes.

Upstream to test zone 112 is hydrophilic membrane 111, which lies in fluid communication with porous membrane 116. A tracer zone 113 is present on hydrophilic membrane 111 and comprises a tracer (labelled ligand) incorporated in the surface of hydrophilic membrane 111. A buffer loading zone 108 lies upstream of tracer zone 113. The tracer may be a mixture of labeled ligand for the analyte and a labeled ligand for the control binder; for example, where the targeted analyte is human IgG, the tracer may consist of mixed goat anti -human IgG and mouse IgG labeled with colloidal gold. In this embodiment, sample loading zone 114 lies downstream of test zone 112. Alternatively, sample loading zone 114 may lie between test zone 112 and tracer zone 113.

Test strip 102 ends in an absorbent pad 110, which serves to draw the sample in the proper direction and prevent backfiow.

Cover 103 is preferably made to fit firmly over test strip 102, and to seal against base 101 (e.g., by a snap-fit aided by insertion of bar 120 into depression 107). The cover 103 is provided with one or more ports for adding samples and reagents, and for reading the assay result. Sample port 117 provides an opening for applying analyte sample fluid to test strip 102. Sample port 117 is positioned over sample loading zone 114 so that when a defined amount of sample fluid is applied through sample port 117, the sample fluid will diffuse to an area upstream to test zone 112. Buffer port 118 is disposed over buffer loading zone 108 upstream of tracer zone 113.

The ports can be fabricated in any shape, for example rectangular, circular, oval, and the like. The result viewing windows 119A and 119B can be omitted if the cover is transparent or covered with a transparent membrane. Furthermore, sample port

117 can be eliminated in favor of applying sample fluid through viewing window 119A where the sample is applied directly to the test zone 112.

FIG.2 is an exploded view of an assay device 200 of the invention, a chromatographic strip 202 assembled on backing 201 , and covered with transparent tape 203. The arrangement of reagents (test zone 212, control zone 215 and tracer zone 213) of the chromatographic strip is identical to that of strip 102 in FIG. 1 ; however, in contrast to the device of FIG. 1, sample loading zone 214 and sample port 217 are placed between test zone 212 and tracer zone 213 rather than downstream of the test zone. Buffer port 218 extends through tape 203 over buffer loading zone 208 upstream of tracer zone 213.

FIG. 3 is an exploded view of a bidirctional assay device 300 of the invention, including two chromatographic strips, 302 A and 302B, assembled in the same two piece housing. The housing consists of base 301 and cover 303. A depression 305 in the middle of support rails 304 and 306 of base 301 matches with bar 307 protruding downward from cover 303. Bar 307 defines an opening through cover 303 which comprises buffer port 318. Test strips 302 A and 302B are arranged in the casing with their upstream ends (near buffer loading zones 308A and 308B) meeting in depression 305.

In this embodiment, two sample ports, 317A and 317B, are disposed through cover 303 above sample loading zones 314A and 314B. Sample loading zones 314A and

314B lie, respectively, between test zone 312A and control zone 315 A and between test zone 312B and control zone 315B. The test and control zones in each test strip lie, respectively, downstream of tracer zones 313A and 313B. Result viewing windows,

318A, 319A, 318B, 319B are disposed above the test and control zones of the two strips.

Conveniently, any of the devices of the invention may be supplied in the form of a kit which may include, without limitation, a buffer for use in reconstituting the tracer, a sterile collection cup for analyte sample and a storage container for sterile packaging of the assay device.

The following examples are provided as additional guidance for those of skill in the art, and are not intended to limit the claimed invention in any way. Unless otherwise specified, all procedures were performed at ambient temperature and pressure, and reagents were used following the manufacturer's recommendations.

Example 1 One-Step Assay of Blood for Human anti-HIV-1/2 Antibodies

A. Preparation of the Test Device

1. Preparation of the HIV- 1/2 antigen coated nitrocellulose membrane. A 22 mm wide nitrocellulose membrane strip (Sartorious™ 18501) was horizontally coated with a test line which contains mixed recombinant HIV-1 antigen (Syntron 16030Agl™) and HIV-2 antigen (Syntron 16030Ag2™) solution at a total protein concentration of 1.5 mg/ml in 0.05 mole/liter sodium phosphate buffer, pH7.4 containing 0.1 mole/liter sodium chloride (PBS), and a control line which contains goat anti-mouse IgG antibody at a concentration of 1.0 mg/ml in PBS. The two parallel lines, each of 1 mm in width, located in the middle area of the membrane strip, with a distance of 9 mm from each other. The volume of each solution coated on the membrane was 60 - 70 ul per feet.

The membrane was air dried for four hours in dry room before it was blocked by being soaked in a blocking buffer (PBS containing 1% [w/v] bovine serum albumin,

0.1% tween 20) for five minutes. The membrane was dried in dry room for four hours.

2. Preparation of antibody-colloidal gold conjugate and the antibody- colloidal gold conjugate coated fiberglass strip

Goat anti-human IgG antibody and normal murine IgG were separately conjugated to colloidal gold particles with an average particle size of 20 nm to 30 nm. The antibody and murine IgG - colloidal gold conjugate, suspended in a buffer (PBS containing 0.1 mole /liter sodium chloride, 0.02% (w/v) sodium azid, 0.1%(w/v) PEG 20M, 0.5%) bovine serum albumin, and 0.1% Tween20) was mixed at a ratio of 3:1 (particle number/ particle number) and the mixed colloidal solution was diluted to OD 540 nm (1 cm) of about 2.0. This concentration may vary depending on the specific activity of the antibodies.

The antibody-colloidal gold solution was then horizontally coated on a 31 mm wide and 0.7 mm thick fiberglass (JBC Seals™ 1200-015) by evenly dispensing 0.6 ml of the solution onto every foot of fiberglass strip. The coating line was located at 5 mm away from the downstream edge of the strip. The coated fiberglass was immediately put into the chamber of a lyophilizer and lyophilized for 48 hours. The lyophilized fiberglass containing the tracer was immediately sealed in aluminum pouch in dry room.

B . Assembly of Test Devices

The antigen/ antibody coated nitrocellulose strip was placed on the single glued side of a 62 mm wide vinyl plate, with the upstream edge of the membrane 31 mm above the upstream end of the vinyl plate; the antibody -colloidal gold conjugate coated fiberglass was then placed on the plate with their upstream ends even; a 1 mm thick and 13 mm wide filter paper (Whatman No.1) was placed on the plate with their downstream ends even. It is important for both the fiberglass and the absorbent paper have a section of approximately 2 mm overlapping with the nitrocellulose membrane. The assembled plate was slitted width-wise into 5 mm strips. The test strips were then individually assembled in to a plastic cassette as shown in Figure 1. The cover of the cassette has a buffer port of 5 x 7 mm located at the upstream end of the cassette. The result view window consists of two apertures (4 x 6 mm) separated by a sample port with an aperture of 2.5 mm in diameter.

C. Serum sample test and comparison of two assay methods

1. Steps of assay method of the invention

Apply 5 ul of serum sample or diluted serum sample solution onto the sample port followed by applying three drops (120 ul) of test buffer (PBS containing 0.1% BSA) to the buffer window. Read the result after 10 minutes.

2. Steps of conventional assay method

Apply three drops (120 ul) of serum sample or diluted serum sample solution to the buffer window that is the location of the sample port on conventional devices. Read the result after 10 minutes.

3. Results of two assay methods

Two HIV positive and one HIV negative human serum samples (Boston Biomedica Institute, Anti-HIV-1/2 Mixed Titer Performance Panel, PRZ 203) were tested. According to the provider's description, these samples were confirmed with western blotting. Positive samples, No. 9, No. 4, and negative sample, No. 8, were made into series dilutions with the test buffer and each diluent was tested individually with both methods. Using the assay method of the invention, sample No 9 and sample No 4 tested positive over a wide range of dilution factors (1 - 1:512 for No.9, 1 - 1 :216 for No.4). No.8 was tested negative at all concentrations.

Using the conventional method, sample No.4 tested negative at all concentrations. No.9 was tested negative at concentration higher than 1 :64 and lower than 1 : 128. A weak positive result was obtained when No .9 was tested at concentration of between 1:128 and 1:64.

Example 2 One-Step Assay for Hepatitis Virus C (HCV) Antibodies

A. Preparation of the Test Device

Porous membranes including bound reagents for assaying HCV antibodies were prepared and assembled into strips as described in Example 1 above, but substituting

HCV antigen for the HIV-1 and HIV-2 antigens. The strip was covered with a transparent tape having a sample port and buffer port of 2.5 mm in diameter at the locations identified in FIG. 2.

B. Serum sample test and comparison of two assay methods

1. Steps of assay method of the invention

5 ul of serum sample or diluted serum sample solution are applied onto the sample port in the middle of the membrane followed by dipping the upstream end of the strip in a 1 cm x 2.5 cm cup containing 0.5 ml of test buffer. The result is read after 10 minutes.

2. Steps of a conventional assay method

Dip the strip in a 1 cm x 2.5 cm cup containing 0.5 ml of serum sample or serum sample diluent. Read the result after 10 minutes. 3. Results of serum sample test using the two assay methods

Eleven samples of blood were tested. According to the test result of a commercial Enzyme Linked Immunosorbent Assay (ELISA), nine of the eleven samples were HCV positive and two were HCV negative.

Using the assay method of the invention, all nine positive samples tested positive

(100%>) and both negative samples tested negative.

Using the conventional assay method, only four (44%) of the eleven positive samples tested positive. Both negative samples tested negative.

The invention having been fully described, modifications thereof may be envisioned by those of ordinary skill in the art. All such modifications are within the scope of the invention defined by the appended claims.

What is claimed is:

Claims

1. An assay device for detecting analyte in a fluid analyte sample comprising a test strip, the test strip comprising:

(a) at least one porous membrane having an upstream end and a downstream end; (b) a tracer zone in the upstream end of the porous membrane having a tracer movably incorporated therein;

(c) a test zone in the porous membrane downstream of the tracer zone, in which a binder for the analyte is bound;

(d) a sample loading zone in the porous membrane for application of analyte sample to the test strip, which sample loading zone lies upstream of the test zone; and,

(e) an absorbent zone on the porous membrane downstream of the sample loading zone; wherein fluid communication of analyte sample from the sample loading zone to the test zone occurs before fluid communication of the tracer from the tracer zone to the test zone.

2. The test strip of the device of claim 1 , further comprising a buffer loading zone for application of buffer to the test strip, wherein the buffer loading zone is in the porous membrane upstream of the tracer zone.

3. The test strip of the device of claim 1 , further comprising a hydrophilic membrane at the upstream end of the porous membrane, wherein the hydrophilic membrane lies in a plane above, but in fluid communication with, the porous membrane and wherein further the tracer zone is in the hydrophilic membrane.

4. The device of claim 1 , further comprising a housing having a base which supports the test strip and an opposing cover which encloses the test strip on the base, wherein the cover has a sample port which provides access to the test strip for application of sample fluid to the sample loading zone and a buffer port which provides access to the test strip for application of a buffer to the tracer zone.

5. The device of claim 1 , further comprising a base which supports the test strip and an opposing transparent tape which covers the test strip, wherein the tape has a sample port which provides access to the test strip for application of sample fluid to the sample loading zone and a buffer port which provides access to the test strip for application of a buffer to the test strip at or upstream of the tracer zone.

6. The device of claim 1 , further comprising a base which supports the test strip and an opposing transparent tape which covers the test strip, wherein the tape has a sample port which provides access to the test strip for application of sample fluid to the sample loading zone and an area at the upstream end of the test strip for immersion into a buffer.

7. The device of any of claims 3 through 6, wherein the sample port is situated over the test zone.

8. The device of any of claims 3 through 6, wherein the sample port is situated downstream of the test zone.

9. The test strip of the device of claim 1, wherein the porous membrane is 0.2 millimeters or less in thickness and has a pore size greater than 1.0 micron.

10. The test strip of the device of claim 9, wherein the porous membrane is nitrocellulose.

11. The test strip of the device of claim 1 , wherein said binder is selected from the group of binders consisting of antibodies, antigens, antibody fragments and antigen fragments.

12. The test strip of the device of claim 11, wherein the antigen is a synthetic, recombinant or isolated component of a pathogenic microorganism.

13. The test strip of the device of claim 12, wherein the pathogenic microorganism is selected from the group of pathogenic microorganisms consisting of HIV-1, HIV-2, HCV, HBV, flavivirus, toxoplasma gondii, Chlamydia trachomatis, H. pylori, HSV-1, HSV-2, CMV and rubella.

14. The test strip of the device of claim 1, wherein the tracer consists of a labelled ligand and the ligand is selected from the group of ligands consisting of antibodies, antigens, antibody fragments, antigen fragments and analyte analouges.

15. The test strip of the device of claim 1 , wherein the label of the tracer is a selected from the group of labels consisting of colloidal gold, latex, carbon black and liposomes containing colored dye.

16. The test strip of the device of claim 1, wherein the tracer is a ligand labeled with an enzyme or a substrate of an enzyme, and wherein the device further comprises an enzyme-substrate system bound in the test and control zones.

17. The device of any of claims 3 through 6, wherein the sample port contains a filter.

18. The device of claim 1 consisting of two test strips arranged end to end on the base.

19. A test strip assay device for detecting analyte in a fluid analyte sample comprising at least one test strip, the test strip comprising:

(a) a porous membrane having an upstream end and a downstream end;

(b) a hydrophilic membrane lying above and in fluid communication with the upstream end of the porous membrane;

(c) a tracer zone in the hydrophilic membrane, wherein a tracer is movably incorporated into the tracer zone;

(d) a test zone in the porous membrane downstream of the tracer zone, in which a binder for the analyte is bound; (e) a sample loading zone in the porous membrane for application of analyte sample to the test strip, which sample loading zone lies upstream of the test zone; and, (f) an absorbent zone on the porous membrane downstream of the sample loading zone; wherein fluid communication of analyte sample from the sample loading zone to the test zone occurs before fluid communication of the tracer from the tracer zone to the test zone.

20. The test strip of the device of claim 19, further comprising a buffer loading zone upstream of the tracer zone.

21. The device of claim 19, further comprising a housing having a base which supports the test strip and an opposing cover which encloses the test strip on the base, wherein the cover has a sample port which provides access to the test strip for application of sample fluid to the sample loading zone and a buffer port which provides access to the test strip for application of a buffer to the test strip upstream of or at the tracer zone.

22. The device of claim 19, further comprising a base which supports the test strip and an opposing transparent tape which covers the test strip, wherein the tape has a sample port which provides access to the test strip for application of sample fluid to the sample loading zone and a buffer port which provides access to the test strip for application of a buffer to the tracer zone.

23. The device of claim 19, further comprising a base which supports the test strip and an opposing transparent tape which covers the test strip, wherein the tape has a

5 sample port which provides access to the test strip for application of sample fluid to the sample loading zone and an area at the upstream end of the test strip for immersion into a buffer.

24. The device of any of claims 21 through 23, wherein the sample port is situated over the test zone.

10 25. The device of any of claims 21 through 23, wherein the sample port is situated downstream of the test zone.

26. The test strip of the device of claim 19, wherein the porous membrane is 0.2 millimeters or less in thickness and has a pore size greater than 1.0 micron.

27. The test strip of the device of claim 26, wherein the porous membrane is 15 nitrocellulose.

28. The test strip of the device of claim 19, wherein said binder is selected from the group of binders consisting of antibodies, antigens, antibody fragments and antigen fragments.

29. The test strip of the device of claim 28, wherein the antigen is a synthetic, 0 recombinant or isolated component of a pathogenic microorganism.

30. The test strip of the device of claim 29 , wherein the pathogenic microorganism is selected from the group of pathogenic microorganisms consisting of HIV-1, HIV-2, HCV, HBV, flavivirus, toxoplasma gondii, Chlamydia trachomatis, H. pylori, HSV-1, HSV-2, CMV and rubella.

31. The test strip of the device of claim 19, wherein the tracer consists of a labelled ligand and the ligand is selected from the group of ligands consisting of antibodies, antigens, antibody fragments, antigen fragments and analyte analouges.

32. The test strip of the device of claim 19, wherein the label of the tracer is selected from the group of labels consisting of colloidal gold, latex, carbon black and liposomes containing colored dye.

33. The test strip of the device of claim 19, wherein said tracer is a ligand labelled with an enzyme or a substrate of an enzyme, and wherein the device further comprises an enzyme-substrate system bound in the test and control zones.

34. The device of any of claims 21 through 23, wherein the sample port contains a filter.

35. An assay device for detecting analyte in a fluid analyte sample comprising a test strip, the test strip comprising:

(a) at least one porous membrane having an upstream end and a downstream end;

(b) a tracer zone in the upstream end of the porous membrane having a tracer movably incorporated therein;

(d) a sample loading zone encompassing the test zone in the porous membrane for application of analyte sample to the test strip; and, (e) an absorbent zone on the porous membrane downstream of the sample loading zone; wherein fluid communication of analyte sample from the sample loading zone to the test zone occurs before fluid communication of the tracer from the tracer zone to the test zone.

36. The test strip of the device of claim 35, further comprising a buffer loading zone for application of buffer to the test strip, wherein the buffer loading zone is in the porous membrane upstream of the tracer zone.

37. The test strip of the device of claim 35, further comprising a hydrophilic membrane at the upstream end of the porous membrane, wherein the hydrophilic membrane lies in a plane above, but in fluid communication with, the porous membrane and wherein further the tracer zone is in the hydrophilic membrane.

38. The device of claim 35, further comprising a housing having a base which supports the test strip and an opposing cover which encloses the test strip on the base, wherein the cover has a sample port which provides access to the test strip for application of sample fluid to the sample loading zone and a buffer port which provides access to the test strip for application of a buffer to the tracer zone.

39. The device of claim 35, further comprising a base which supports the test strip and an opposing transparent tape which covers the test strip, wherein the tape has a sample port which provides access to the test strip for application of sample fluid to the sample loading zone and a buffer port which provides access to the test strip for application of a buffer to the test strip at or upstream of the tracer zone.

40. The device of claim 35, further comprising a base which supports the test strip and an opposing transparent tape which covers the test strip, wherein the tape has a sample port which provides access to the test strip for application of sample fluid to the sample loading zone and an area at the upstream end of the test strip for immersion into a buffer.

41. The device of any of claims 38 through 40, wherein the sample port is situated over the test zone.

42. The device of any of claims 38 through 40, wherein the sample port is situated downstream of the test zone.

43. The test strip of the device of claim 35, wherein the porous membrane is 0.2 millimeters or less in thickness and has a pore size greater than 1.0 micron.

44. The test strip of the device of claim 43, wherein the porous membrane is nitrocellulose.

45. The test strip of the device of claim 35 , wherein said binder is selected from the group of binders consisting of antibodies, antigens, antibody fragments and antigen fragments.

46. The test strip of the device of claim 45, wherein the antigen is a synthetic, recombinant or isolated component of a pathogenic microorganism.

47. The test strip of the device of claim 46, wherein the pathogenic microorganism is selected from the group of pathogenic microorganisms consisting of HIV-1, HIV-2, HCV, HBV, flavivirus, toxoplasma gondii, Chlamydia trachomatis, H. pylori, HSV-1, HSV-2, CMV and rubella.

48. The test strip of the device of claim 35, wherein the tracer consists of a labelled ligand and the ligand is selected from the group of ligands consisting of antibodies, antigens, antibody fragments, antigen fragments and analyte analouges.

49. The test strip of the device of claim 35, wherein the label of the tracer is selected from the group of labels consisting of colloidal gold, latex, carbon black and liposomes containing colored dye.

50. The test strip of the device of claim 35 wherein said tracer is a ligand labeled with an enzyme or a substrate of an enzyme, and wherein the device further comprises an enzyme-substrate system bound in the test and control zones.

51. The device of any of claims 38 through 40, wherein the sample port contains a filter.

52. The device of claim 35 consisting of two test strips arranged end to end on the base.

53. The test strip of the device of claim 11 , wherein the ligand portion of the tracer is a human anti-immunoglobulin antibody.

54. The test strip of the device of claim 28, wherein the ligand portion of the tracer is a human anti-immunoglobulin antibody.

55. The test strip of the device of claim 45, wherein the ligand portion of the tracer is a human anti-immunoglobulin antibody.

56. An assay method for detecting the presence of at least one analyte in a fluid analyte sample comprising applying a fluid analyte sample to the sample loading zone, and a buffer to the tracer zone, of the device of claim 1, claim 19 or claim 35; then, observing whether a band appears in the test zone, which band in the test zone is indicative of the presence of analyte in the fluid analyte sample; wherein buffer migrating from the tracer zone pushes unbound analyte out of the test zone, thereby minimizing contact between the tracer and unbound analyte in the fluid analyte sample.

57. The assay method according to Claim 56 , wherein the buffer is applied to the tracer zone after the fluid analyte sample is applied to the sample loading zone.

58. The assay method according to Claim 56, wherein the volume of fluid analyte sample applied to the sample loading zone is no larger than the volume of buffer applied to the tracer zone.

59. An assay method for detecting the presence of at least one analyte in a fluid analyte sample comprising applying a fluid analyte sample to the sample loading zone, and a buffer to the buffer loading zone, of the device of claim 2, claim 20 or claim 36; then, observing whether a band appears in the test zone, which band in the test zone is indicative of the presence of analyte in the fluid analyte sample; wherein buffer migrating from the buffer loading zone pushes unbound analyte out of the test zone, thereby minimizing contact between the tracer and unbound analyte in the fluid analyte sample.

60. The assay method according to Claim 59, wherein the buffer is applied to the buffer loading zone after the fluid analyte sample is applied to the sample loading zone.

61. The assay method according to Claim 59, wherein the volume of fluid analyte sample applied to the sample loading zone is no larger than the volume of buffer applied to the buffer loading zone.

62. An assay kit comprising the device of claim 1 and a buffer for application to the tracer zone of the device of claim 1 to reconstitute the tracer incorporated in the test strip at the tracer zone.

63. An assay kit comprising the device of claim 19 and a buffer for application to the buffer loading zone of the device of claim 19 to reconstitute the tracer incorporated in the test strip at the tracer zone.

64. An assay kit comprising the device of claim 35 and a buffer for application to the tracer zone of the device of claim 35 to reconstitute the tracer incorporated in the test strip at the tracer zone.