WO1998013519A1 - Diagnostic test devices with improved fluid movement and resistance to interferences - Google Patents

Diagnostic test devices with improved fluid movement and resistance to interferences

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Publication number
WO1998013519A1
WO1998013519A1 PCT/US1997/017129 US9717129W WO9813519A1 WO 1998013519 A1 WO1998013519 A1 WO 1998013519A1 US 9717129 W US9717129 W US 9717129W WO 9813519 A1 WO9813519 A1 WO 9813519A1
Authority
WO
Grant status
Application
Patent type
Prior art keywords
filter
reagent layer
test device
test
sleeve
Prior art date
Application number
PCT/US1997/017129
Other languages
French (fr)
Inventor
Mary Ann Childs
David Bernstein
Original Assignee
Universal Healthwatch, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date

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Classifications

    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by the preceding groups
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/52Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
    • G01N33/525Multi-layer analytical elements
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by the preceding groups
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366Apparatus specially adapted for solid-phase testing
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by the preceding groups
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366Apparatus specially adapted for solid-phase testing
    • G01N33/54386Analytical elements
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by the preceding groups
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983Viruses
    • G01N33/56988AIDS or HTLV

Abstract

A diagnostic test device contains a filter and exhibits improved transfer of fluid movement between assay components. The filter is an integral part of a strip and can be used for strip-testing of whole blood and other particulate-containing solutions generally. Surfaces of parts within the device are combined in particular ways to improve sample and reagent fluid movement and an optional chemical additive increase test quality. Whole blood HIV tests are exemplified that are easy to carry out and that show improved chemical resistance to false positive results.

Description

DIAGNOSTIC TEST DEVICES WITH IMPROVED FLUID MOVEMENT AND RESISTANCE TO INTERFERENCES

Field of the Invention

The present invention relates to diagnostic test devices that utilize one or more sample/reagent fluids to generate a signal that indicates a positive test result for analyte. Embodiments of the invention relate to improvements in fluid movement within such devices and in improved resistance to chemical interferences.

Background of the Invention

In the development of the medical diagnostics field, there has been explosive growth in the number of substances to be detected in physiological test fluids. Various analytical procedures are commonly used m diagnostic assays to determine the presence and/or amount of these substances of interest or clinical signi icance. Diagnostic assays have become an indispensable means for detecting analytes m test fluids, and for the most part the medical profession has used highly automated clinical laboratories and sophisticated equipment for these determinations. There is, however, an expanding need for having analytical capabilities in doctors' offices, in the home, and in the field where electricity and other semblances of a laboratory are unavailable. Together with the diagnosis of disease or physiological conditions or disorders, there is a growing need to monitor the effects of drug therapy and chronic illness, to detect the use of drugs of abuse and to detect the presence of contaminants . The generation of test results can be followed by appropriate treatment or prophylaxis to prevent further public health problems.

Assay methods, accordingly, have been developed which utilize one or more omding reactions that lead to color development in response to the presence of a test antigen. Exemplary of these methods are antibody-based assays that utilize immobilized antibodies on a reagent material within a test device. During use, a test fluid is added to the device and wickε into a first absorbent material and then is transferred to another absorbent material. Typically one or more test reagents, necessary for one or more binding reactions and a color development reaction become wetted or dissolved within one or more materials in the device. Chemical reactions take place and cause a visual response to the presence or absence of analyte in the test fluid.

Exemplary of these methods and the materials used to carry them out are assays disclosed in IT'S patent No. 3,888,629 issued to Bagshawe on June 10, 1975, US patent No. 5,212,065 issued to Pegg et al . on May 18, 1993, US patent No. 5,518,887 issued to Parsons et al . on May 21, 1996 and US patent No. 4,246,339 issued to Cole et al . on January 20, 1981 all of which are herein expressly incorporated by reference in their entireties.

Unfortunately, these test devices have a number of problems that limit their use with certain types of samples and test analytes. As a result, a truly convenient and simple device for accurate testing of, for example, antibodies that indicate past exposure to infectious disease agents does not exist . A unitized device is needed that contains all mechanical parts and one or more diagnostic reagents, such as sample pre- treatment components (eg. cell filters), binding members (eg. antibodies) and signal development reagents (eg. colloidal gold) and which can render a sufficiently accurate and fast resul .

The problems that have limited the development of this art can be grouped into three categories. One, wicking and seeping of fluids among the solid components housed within a test device as well as uneven dissolution of dried reagent causes non- homogeneous flow of sample and wash fluid. Two, test samples sometimes contain particulate matter which clogs up and creates inconsistent flows within a test device. Three, chemical interferences in test samples may non-specifically bind to physical parts of a test device, or react with chemical reagents such as antibodies or enzymes within the test device to cause false positive and false negative test results. Problems in each of these categories lead to uneven test signal development and concomitant mistakes in test developmen . The first category of problems, relating to wicking and seeping within test devices, frequently arises from the dual motions of test and reagent fluids both downward and in lateral directions. Lateral reagent flow contributes to a higher incidence of inaccurate results due to the tendency of spent reagents to accumulate at the periphery of a reaction zone, for example between an antibody and an antigen. Down directed (transverse) motion perpendicular to a surface and crossing from one surface to another between adjacent parts is affected by contours and contact vagaries between the surfaces . In both cases the reagents tend to interact and produce a test signal that can be mistaken for a true positive or negative result.

Prior art devices often suffer wicking or seeping of fluid through junctions and spaces within the device housing. For example, a reagent layer, such as a membrane may be held in place by compression between two plastic portions. That is, the reagent layer material is sandwiched in a junction between two plastic members. The contact between the reaction portion, termed ("reagent layer") for convenience of discussion, and another absorbent such as a spent reagent reservoir ("absorbent pad") is uneven across the reaction absorbent surface. Unfortunately, the sandwich method of holding these two absorbents together may provide the test fluid, reaction, or wash fluid an additional and undesirable path to move. As a result, a portion of fluid that is applied to the device may go around the reaction portion or penetrate the reaction portion incompletely. An additional problem with these prior art methods is that the compression of the reaction portion will often deform the absorbent layer itself, further causing detection error.

Another aspect of this fluid movement problem in the first category is that application of test fluid (s) to a dry reagent in a device often leads to uneven dissolution of the reagent and inhomogeneity of test signal developmen . The unevenness of dissolution that occurs during the wetting process itself, and differences between samples may play a role in this problem, leading to inaccurate test results.

The second category of problems is uneven fluid flow due to interference by particulate matter and by viscosity of the test sample. Viscosity and/or particulate matter often limit the ability of a test device to give a correct result from blood or blood derived samples that may contain red blood cells, white blood cells, chylomicrons , debris, precipitates or other "cellular" elements. These elements may choke the flow of fluid within the device and affect reaction of test reagents such as an immobilized binding pair member In fact, one of the most important challenges in the diagnostic field is the need to test blood which may comprise one-half by volume such particles. Any improvement to this art would have important consequences m the field of public health by allowing earlier diagnosis from samples such as blood that do not require processing at remote locations.

The third category of problems relates to chemical mterferants that often are encountered in undiluted blood and blood derived products. A chemical interferant m an individual blood sample may give an erroneous positive or negative result for that sample. The biochemical origins of mterferents and of their reactions generally are not known However, attempts have been made to limit the effects of chemical mterferents by adding protein such as gelatin, serum albumin and casein to assay buffer or to absorbents used m test devices

A strategy to overcome the first problem of uneven fluid transfer within a test device is to eliminate the transfer of fluid from one absorbent to another by using a single absorbent. For example, US patent No. 5,212,065, issued on May 18, 1993 to Pegg et al . describes a device that employs a single porous membrane that serves as both a reagent support and spent reagent reservoir. This device directs the flow of test fluid and reagents and alleviates lateral diffusion and reagent backflow. Unfortunately, this device is limited to the use of a single absorbent. Ideally, different absorbents should be used within the same device, to optimize parameters such as porosity, hydrophobicιty, ability to separately impregnate with reagents, etc. as needed for each step of an assay procedure. But housing multiple parts within a test device creates the problem of reproducible fluid transfer between these parts. Thus, the control of fluid flow from one absorbent to another is exacerbated by this strategy.

The second problem of particulate matter and viscous samples has been addressed by pre- reating the samples by diluting them up to 100 times or more, centrifugmg them, or filtering them before assay. However, dilution causes a corresponding loss in sensitivity and all three methods greatly affect test convenience and increase test cost .

The third problem of chemical interferences and their nonspecific chemical reactions has been addressed by adding a protein or detergent to an assay component. However, each type of assay and assay device may be sensitive to a different set of chemicals. Furthermore, for some tests such as HIV, a false negative or a false positive can cause a major problem and generally is unacceptable. Partly for this reason, until now no sufficiently accurate and simple whole blood HIV test has been available .

Any of the above-discussed problems may cause a test user to misinterpret a test result and lead to an incorrect medical diagnosis or epidemiological public health decision. Thus, advances to the art m the selection of materials and their dimensions or orientation for use in a test device, and in the use of chemical agents to prevent the effects of chemical terferents would provide great benefits overcoming problems in these three areas .

Summary of the Invention The present inventors have discovered that certain advantageous assays can be prepared by orienting components within a test device used to generate a signal in response to a test analyte so that the components contact each other more evenly, and without bridging, leading to more homogeneous fluid flow. In one embodiment, this objective is achieved by mounting two parts that form a mutual surface junction such that primarily the two surfaces, and not a mount or holder, participate in fluid contact with the junction. Another embodiment employs materials that expand upon wetting and which are friction-held, leading to a more even contact between adjacent parts during test performance. Another embodiment employs a filter to remove particulate interferences. Another embodiment provides a choice of components and configurations that limits transverse flow of reagent and sample. Yet another embodiment includes a glycosammoglycan in at least one test component or fluid to prevent effects of chemical interference, particularly for an HIV test . In yet another embodimen , one or more of the above embodiments is combined in a multi-well test device suitable for testing more than one sample simultaneously. Generally, such embodiments serve to: (1) control fluid flow between absorbent materials in test devices,- (2) control the problem of particulates and viscosity of samples,- and/or (3) limit the effects of chemical interferences. More specific details for how embodiments of the present invention improve the reproducibility and accuracy of test results in the three enumerated problem areas are provided below.

Thus, for example, in one embodiment of the invention a test device is provided that comprises a housing,- an absorbent pad held by the housing; a reagent layer that comprises an immobilized test reagent, the layer being m contact with the absorbent pad; a filter in contact with the reagent layer,- an opening in the housing adjacent to the filter for receiving an aqueous sample into the filter; and a sleeve operatively holding the filter in physical contact with the reagent layer, wherein the filter protrudes from the sleeve such that the sleeve does not contact the reagent layer. In other embodiments the test device further comprises a cover for closing the opening. The cover may be a plug adapted to be attached to the opening by friction. In an advantageous embodiment the sleeve is reversibly attached to the housing via a bayonet mount and m another embodiment the sleeve prevents or delays the escape of moisture from the device by covering the reagent layer. In another embodiment the reagent layer of the test device further comprises a binder that immobilizes human antibody as a control to monitor the presence of an adequate sample. In yet another embodiment at least the filter or reagent layer comprises a glycosammoglycan.

In another embodiment the filter of the test device comprises an upper portion for filtering blood and a lower portion for dispersing filtrate to the reagent layer.

In another embodiment the lower portion of the filter m the test device comprises a material that passes fluid at a slower rate compared to the material of the upper portion. In yet another embodiment the upper portion of the filter comprises two separate filters that are held together and which act together to block the flow of particulates (including cells) in a sample. In a further embodiment the filter of the test device has a depth at least one tenth its diameter and in another embodiment the immobilized test reagent comprises a molecule for binding a sample analyte. In other embodiments of the test device the sample analyte is an antigen of an infectious disease agent, which may be a human immunodeficiency virus.

In an embodiment of the test device the sleeve holds the filter by friction and in further embodiments the filter, at least one of its portions, or the sleeve itself is adapted to swell upon wetting. In yet another embodiment of the test device the filter protrudes at least about 0.02 to about 0.25 mm or more from the sleeve. In an acceptable embodiment, the filter protrudes about 0.5 mm (e.g., 0.5 mm) from the sleeve. [* insert I will add a sentence here so I am leaving some space.

In another embodiment a test device is provided comprising a housing having an opening; an absorbent pad within the housing; a reagent layer having an immobilized test reagent, the layer being positioned on top of the absorbent pad; a filter on top of the reagent layer and aligned with the opening so that the filter is adapted to receive a test sample; an opening m the housing above the filter that can admit an aqueous sample; and a sleeve operatively holding the filter in physical contact with the reagent layer, the sleeve being connected to the housing, wherein the filter protrudes from the sleeve so that the sleeve does not contact the reagent layer.

In a further embodiment a test device is provided comprising: a housing having an opening; an absorbent pad held by the housing; a reagent layer having immobilized antigens which may include HIV-1, HIV- 2 and/or HIV-1 subtype o, the layer being positioned in contact with the absorbent pad; at least one filter in contact with the reagent layer and aligned with the opening to receive an aqueous sample into the filter. In other embodiments of this test device at least the filter or reagent layer comprises a glycosammoglycan.

In another embodiment a blood HIV diagnostic assay kit is provided that contains a test device as described in alternate embodiments above. In further embodiments of the assay kit, the test device further comprises a cover for sealing the device and a glycosammoglycan. In yet further embodiments the glycosammoglycan is selected from the group consisting of chondroitm sulfate, dermatan sulfate, heparin sulfate, keratan sulfate, hyaluronic acid and heparin. In another embodiment a process is provided for detecting

HIV antibody from whole blood comprising the steps of: (a) providing a test device that contains a housing having an opening, an absorbent pad held by the housing, a reagent layer having immobilized antigens that may include HIV-1, HIV-2 and/or HIV-1 subtype o, the layer being positioned contact with the absorbent pad, at least one filter in contact with the reagent layer and aligned with the opening to receive an aqueous sample into the filter; (b) applying a blood sample to the opening of the test device; (c) applying at least one wash fluid to the test device; (d) allowing one or more reactions to proceed in the reagent layer of the device; and (e) detecting an optical change m the reagent layer m response to the presence of antl-HIV antibody. In a further embodiment, the wash fluid of the process comprises a glycosammoglycan and in yet a further embodiment the glycosammoglycan is selected from the group consisting of chondroitm sulfate, dermatan sulfate, heparin sulfate, keratan sulfate, hyaluronic acid and heparin. In yet a further embodiment the glycosammoglycan is heparin and is present in the wash fluid m a concentration from about 5 international units per milliliter to about 25 international units per milliliter (e.g., from 5 usp/ml to 25 usp/ml). In yet another embodiment a multiple test device is provided m which each test well comprises a housing; an absorbent pad held by the housing; a reagent layer that comprises an immobilized test reagent, the layer being in fluid communication with the absorbent pad; a filter in fluid communication with the reagent layer,- an opening the housing adjacent to the filter for receiving an aqueous sample into the filter; and a sleeve operatively holding the filter in physical contact with the reagent layer, wherein the filter protrudes from the sleeve such that the filter exerts a greater pressure onto the reagent layer than does the sleeve.

Other embodiments, objects and advantages will become apparent from the following detailed description taken m conjunction with the drawings.

Brief Description of the Drawings FIG. 1 is a perspective view of a test device in accordance with this invention.

FIG. 2 is a side view of the test device shown in FIG. 1. FIG. 3 is a cross sectional view taken along the line of 3-3 shown in FIG. 2.

FIG. 4 is a perspective view of another test device in accordance with this invention.

FIG. 5 is a cross sectional view of another test device in accordance with this invention.

FIG. 6 is a cross sectional view of yet another test device in accordance with this invention.

FIG. 7 is an exploded view of a multiple test device in accordance with this invention. FIG. 8 shows a top view and a side view of an 8 well test device in accordance with this invention. For added clarity, attached hereto is a parts list of some features shown in the Figures m accordance with the invention.

Detailed Description of the Preferred Embodiments

Test devices of the present invention have a housing comprised of a water impermeable material m which other test components such as an absorbent pad, reagent layer, filter and a reagent used to obtain a test result are held. The housing has at least one opening to admit a fluid sample although additional openings may be desired. In some cases, a wick may exist at least partly outside the housing, or the filter may exist at least partly outside the housing. In an advantageous device configuration all mechanical parts are completely held within the housing. Also acceptable in some embodiments is a housing having at least one light transmissive portion to allow detection of a signal either visually, or by instrumentation.

Most acceptable is a housing that comes apart during use so that the user can remove the filter to expose the reagent layer for application of a reagent and/or wash fluid In this embodiment a sleeve that holds the filter is removably attached to the housing such that contact of the filter is favored over contact of the sleeve with the surface of the reagent layer.

Preferably the sleeve is attached to the housing by a bayonet mount. After a sample is applied, and an optional wash solution added, the sleeve s removed and further optional reagent solution and a wash solution are added directly to the reagent layer. In some applications a sample is added to the device and further processing is carried out at a separate location or after storage of the device for a few hours. In these situations, the sleeve remains attached to the housing to prevent or delay the release of moisture from the device until the later processing steps are carried out. The housing also may contain a cover to protect the opening and further guard against the release of moisture.

Multiple housings can be incorporated into a multi-test unit to allow high volume testing. The latter embodiment is acceptable for infectious disease testing of blood samples at blood banks. Especially acceptable in this context is a 96 well multiple-test device that can be used with micropipettor devices. In one embodiment 8 (or 12) test devices that correspond m size to a column (or row) of a microtiter plate are used in applications where intermediate numbers of samples are processed. The invention has particular advantages when used in a multi-test format over other methods such as microtiter plate enzyme linked immunosorbent assays. For example, the invention obviates waste disposal required by these other methods and allows optical perception of several test spots, (including one or more calibrators) in one well. Furthermore, the invention overcomes problems associated with icteric samples, samples that contain debris, or cellular material. In addition, unlike some other methods, the present invention provides detection signals that are stable .

The housing and other parts of the test device may be constructed from well-known materials in accordance with well- known methods of the prior art. Material suitable for the invention should not interfere with the production of a detectable signal and should have a reasonable inherent strength, or strength can be provided by means of a supplemental support. Natural, synthetic, or naturally occurring materials that are synthetically modified, can be used including, but not limited to: cellulose materials such as paper, cellulose, and cellulose derivatives such as cellulose acetate and nitrocellulose; fiberglass; cloth, both naturally occurring (e.g., cotton) and synthetic (e.g., nylon) ; porous gels such as silica gel, agarose, dextran, and gelatin,* porous fibrous matrixes; starch based materials, such as Sephadex (r) brand cross -linked dextran chains; ceramic materials; films of polyvinyl chloride and combinations of polyvinyl chloride-silica; and the like. See, for example, U.S. Patent Nos. 5,075,078, 5,552,276, 4,912,034 and 5,212,065 which are incorporated herein by reference their entireties. Constructions other than those described in these patents readily will be apparent to the skilled artisan. The test device most preferably incorporates the features of (1) positioning parts with a positioning "sleeve" to allow even fluid flow between the parts without interference by the sleeve itself, (2) arranging parts to minimize transverse flow, (3) using friction-held parts and water swellable parts to allow fluid to more evenly flow through junctions between the parts, and (4) using a dispersing layer downstream of the filter to help disperse fluid more evenly to the reagent layer. Each of these four strategies improves test performance and should be considered independently for a particular test device configuration. The physical assembly of components from known materials within the housing generally will be understood to a skilled artisan but for clarity, some details are provided here in the form of definitions of some terms used the claims. A "blood sample" as termed herein means whole blood obtained by, for example, a finger prick or venipuncture either with or without an antl-coagul nt added; a plasma sample; serum sample; or a partly processed or diluted derivative of any of these such as centrifuged blood. A "whole blood" sample is a blood sample that contains red blood cells, white blood cells and other components such as chylo icrons and lipoprotem.

A "filter" removes more than half of particles that are suspended within a test sample and which are larger than the rated sieving size of the filter material The "rated sieving size" of a material is determined by and provided from a manufacturer of the material and typically is stated in microns.

For example, a glass fiber having a porosity of ten microns should remove red blood cells from a blood sample because such cells have a dimension that is greater than 10 microns. In some cases the rated sieving size of the material may be close to or even larger than one or more dimensions of the particle that is to be held back by the cell filter. Depending on the filter thickness, retardation of particle movement and not simply holding back the particles will suffice for a given test device.

How well a given filter material works is best determined empirically, by applying test samples to devices made using the material .

When used for test samples that contain red cells the filter preferably removes at least 90% of the red cells. The filter may comprise one or more materials m physical contact. In practice, the filter should have significant depth to allow entrapment of cells that typically comprise about one half of whole blood volume. When used for testing of saliva samples, the filter preferably retards cells and other debris that may exist in such test specimens. In an advantageous embodiment the filter comprises two glass fiber pads mechanically held together. The filter also may comprise a first portion that traps or slows the movement of cells and a second portion downstream from the first portion. In this case the second portion serves to disperse filtrate to an adjacent material such as a reagent layer.

A "filtrate" of a blood sample or whole blood sample is a portion of the sample that is passed through a filter or component of a filter. For example, a filtrate of a whole blood sample will have had most of the originally present red cells removed by the filter. In some cases the filter may remove blood cells or blood molecules by virtue of binding between the filter surface (s) and the blood cells or blood molecules. Preferably, the filtrate from a filter will lack more than 90% of the originally applied red cells by virtue of the sieving action of one or more portions of the filter.

An "absorbent pad" imbibes water generally and serves as a final repository for much or most of the liquid that enters the housing of a test device. Examples of an absorbent pad include paper, ethyl cellulose, porous plastic, sintered glass , and other polymers that accept water such as polyvinyl pyrrolidone and acrylamide copolymers. An absorbent pad may consist of more than one piece. An acceptable absorbent pad is comprised of ethyl cellulose and is held in place by the device housing, wherein a surface of the pad contacts a reagent layer. An "analyte" is a molecule, cell or cell component that is detected by the test device. Preferably, the analyte is a polypeptide such as, for example, a regular polypeptide, a lipopolypeptide or glycopolypeptide produced by a disease causing organism such as HIV virus, but may be a naturally occurring molecule found in the body, or a molecule produced by the body in response to a disease state. An "analyte" includes any antigenic substance or "antigen", hapten, antibody, and combination thereof. For example, the analyte can be a peptide, an ammo acid, a nucleic acid, a carbohydrate, a hormone, a steroid, a vitamin, a pathogenic microorganism for which polyclonal and/or monoclonal antibodies can be produced, a natural or synthetic chemical substance, a contaminant, a drug including those administered for therapeutic purposes as well as those administered for illicit purposes, and a metabolite of or an antibody to any of the above substances. Examples of the hormones which are suitable as analytes for this invention are the following: thyroid stimulating hormone (TSH) , human choπonic gonadotrop (hCG) , lutemizing hormone (LH) and follicle stimulating hormone (FSH) . The invention s particularly useful for the testing of antibody to one or more HIV viruses from whole blood samples that are involved in AIDS . An "antigen" is a molecule that reacts with an antibody. For tests that detect exposure to an infectious disease organism, the test antigen preferably contains one or more epitopes that are similar or the same as epitopes of the infectious disease agent. When testing for exposure to Human Immunodeficiency Virus (HIV) an advantageous antigen is a polypeptide that contains at least one segment that corresponds in sequence to that of a polypeptide produced by the virus. Exemplary sequences in this context are conserved sequences that have been published by the Los Alamos National Laboratory, Los Alamos, New Mexico 87545 (HUMAN RETROVIRUSES AND AIDS 1996) . Acceptable peptides for use as HIV test antigens comprise one or more of the following sequences: Q-T-H-L-P-I-P-R-G-P-D-R-P-E-G-I -E-E-G, L-Y-K-Y-K-V, P-L-G-V-A-P-T-K-A-K-R-R-V-V, P-L-G-V-A-P-T-R-A-K-R-R-V-V, R-E-K- R-A, S-G-I-V-Q-Q, L-T-V-W-G, D-Q-Q-L-G, W-G-C-S-G-K, Q-Q-E-K-N-E- Q, Q-T-H-L-P-I-P-R-G-P-D-R-P-E-G-I-E-E-G and Q-Q-E-K-N-E-Q .

An "antibody" is IgG, IgE, IgM, IgA and the like and may te obtained from blood, a blood product or saliva. Although the vis of saliva samples is not discussed in great detail it is understood that the invention is used for the detection of analyte from saliva as well as from blood. Advantages of the invention, such as the operation of the filter extend to saliva samples as well as to blood samples.

An "opening in the housing" may be a hole, aperture or simply a region that accepts a fluid sample. In some cases, the housing itself may be partly water permeable and partly water impermeable. In these instances the water permeable portion (s) act as an "opening" in context of the invention. More than one opening may be used in the test device .

The phrase "passes fluid" has its regular meaning that is well known to the skilled artisan. The relative rate of passing fluid can be determined by a number of techniques known to the artisan, including for example, measuring how much time is required for the material to become completely wetted upon contact with water. An advantageous material for trapping cells m the filter is glass fiber and an advantageous material for a dispersant layer in contact with this glass fiber typically is cellulose because cellulose can pass water at a lower rate compared to glass fiber.

The second portion or "dispersing" portion of a filter, when used, may provide a lower flow rate of sample and of wash fluid compared to the cell trapping material the filter. In addition to providing a more reproducible test result, the low flow rate provided by the dispersing portion allows more time for chemical substances to react before these substances contact the reagent layer. Increasing the reaction time, particularly for binding reactions, allows greater test sensitivity. Acceptable materials for use the filter to trap cells include Gelman Cytosep glass fiber filter membrane and Pall corporation blood separation membrane. Acceptable for a "dispersing" portion of a filter, if one is used, are cellulose-based materials such as filter paper. A "reagent layer" is an absorbent or group of absorbents that contains at least one reagent, contacts the filter and receives sample from the filter by virtue of this contact. An advantageous reagent layer is a membrane that comprises an immobilized test reactant such as antibody or antigen. Typically, an absorbent pad that contacts the reagent layer accepts and draws in fluid from the reagent layer. Although the reagent layer preferably comprises an immobilized test reactant, the test reactant may become immobilized during the test itself by for example, precipitation or by binding to the reagent layer during the test procedure. The reagent layer may assume any of a variety of shapes. An advantageous shape is a thin layer between the filter and the absorbent pad, although another shape such as a plug may be used, depending on the features of the other components. Fluid preferably moves traverse to the major surfaces of a reagent layer, although some device configurations may utilize lateral flow along the longest axis of a reagent layer. In an advantageous embodiment for testing of HIV antibody in a blood sample, the reagent layer is a nitrocellulose membrane with HIV antigen (s) bound to it. The reagent layer, however, need not be a "layer" per se, but may consist of a pad, rod or other shaped absorbent . A "sleeve" is a water impermeable solid surface that holds the filter. The sleeve may be a portion of the test device housing or may be a separate part that is separately attached to the housing. In the latter case, the sleeve preferably is reversibly attached to the housing by a bayonet mount . An advantageous construction m this context is to place the filter into the sleeve and to assemble the housing from two portions, an upper housing portion and a lower housing portion. The lower portion holds the absorbent pad and a reagent layer. The two housing portions are snap- fit together and the sleeve is attached by a bayonet mount. During fitting of the sleeve, friction between the sleeve and the filter allows the filter to push onto the reagent layer. "Friction" in this context means that the filter is held in place by mechanical pressure of the filter against the walls of the sleeve In some embodiments, the mechanical pressure is augmented by an adhesive that holds the filter to the sleeve surface. When using friction fit to attach the filter to the sleeve, the filter preferably has a depth that is at least one tenth its diameter. To maintain friction of the filter within the sleeve and also to maintain good contact between the filter and a reagent layer, it is acceptable that the filter (particularly the dispersant portion, if included) , or, (in some cases), the sleeve surface, to swell upon wetting.

The term "swell" in the context of a part or part surface used in the test device means that the part increases its size or that the surface increases its thickness upon wetting. Acceptable in this context is cellulose, although the use of many alternate materials is readily appreciated by a skilled artisan.

An "optical change" in the context of determining the presence or absence of a test analyte means an absorbance, reflectance, fluorescence, phosphorescence, or chemiluminescence change produced within the test device. This change may be determined by eye or by an instrument. One acceptable embodiment employs gold particles to produce a reflectance change that is visually perceived. In an advantageous embodiment the filter is separated from the reagent layer by removal of the sleeve, which exposes a portion of the reagent layer where an additional reagent may be added and a signal is developed from the presence of gold particles.

A "bayonet mount" has the usual meaning in the art and refers to a fastening means whereby a first part firmly attaches to a second part by overlapping indentations along a circular surface that fits into a depression of the second part. After inserting one part into the other, either part is rotated to firmly seal the two together.

A "chemical terferant" in a sample is a chemical that interferes with one or more chemical reactions of the test assay such as a binding reaction or enzymatic reaction. One example of a chemical mterferant is a rheumatoid factor that can enhance certain agglutination reactions. Another example is an auto- antibody that affects some binding interactions. Chemical mterferants may cause false positive assay results and false negative assay results.

A "glycosammoglycan" is a linear heteropolysaccharide possessing a characteristic disacchaπde repeat sequence (as reviewed by Jackson et al . , Physiological Reviews 71: 481-539 (1991) ) which is herein incorporated in its entirety by reference. One monosaccharide of the disaccharide is an amino sugar with D-glucosamme or galactosamine , and the other unit is typically, but not always, a uronic acid residue of either D- glycuronic acid or lduronic acid. Both units are variably N- and O-sulfated, which adds to the heterogeneity of these complex macromolecuies. Examples of glycosam oglycans include chondroitin sulfate, dermatan sulfate, heparin sulfate, keratan sulfate, hyaluronic acid and heparin. Some of these molecules are covalently attached, typically at their reducing ends through an O-glycosidic linkage to a serine residue or N-linked to asparagine in a core protein to form a proteoglycan. In addition, a given glycosaminoglycan can vary by size or degree of sulfation and various subtypes can be prepared by synthesis or by purification of naturally occurring glycosammoglycan. For example, heparin of 11,000 molecular weight and a sulfated pentasaccaride derivative of heparin as described by Jackson et al . ( Id. ) also are include within the ambit of the invention. The inventors have discovered that adding a glycosaminoglycan to assays that contain certain HIV antigen polypeptides will improve resistance to false positive interferences .

For example, glycosaminoglycan prevents false positives when the assay uses a polypeptide antigen that contains four or five basic amino acids within a 10 ammo acid long (decapept de) segment. Further, the improvement by glycosaminoglycan may occur when these basic residues exist in two pairs of basic ammo acids that are separated by between 2 and 6 ammo acids. A "basic amino acid" in this context is arginine or lysine.

The use of glycosaminoglycan to improve diagnostic assay performance is advantageous in combination with use of a recombinant polypeptide that has been designed with four or more positive charged amino acids within a short region of twenty amino acids. The glycosam oglycan advantageously is used with an antigen having four or more ammo acids within a short region of ten amino acids. The glycosaminoglycan is advantageously used with a recombinant polypeptide that has been designed by deleting a portion of a pre-existing sequence, yielding a "new" segment which forms a tertiary structure that has not been determined by natural selection. Such a new structure often is unstable and tends to bind other substances. This binding problem is accentuated when a large proportion of the new polypeptide segment acquires a net positive charge. It is known that proteins which contact blood are primarily negatively charged and that surfaces which contact blood also are negatively charged. Forming a cluster of positive charges on a polypeptide segment can facilitate the binding of other substances to the segment and to a surface in an unpredictable manner. Although not wishing to be bound by a particular theory of the invention, the inventors have discovered that glycosam oglycans stabilize basic ammo acid clusters of such proteins and prevent undesirable false positive assay results with such assays. This discovery solves a general problem of using engineering polypeptides as antigens in diagnostic assays.

An advantageous device configuration comprises an absorbent pad, reagent layer and a two-part filter within a single housing. Acceptable materials for the housing include water impermeable plastics such as polystyrene, polypropylene, polyvinyl chloride and the like. Acceptable materials for the filtering portion of the filter, the dispersing portion of the filter, the reagent layer and the absorbent pad are glass fiber, ethyl cellulose, nitrocellulose and ethyl cellulose respectively. When nitrocellulose is used for the reagent layer, however, the material of the filter should be chosen for its ability to premix the test sample and any test reagent that may be present in the filter. Two or more materials can be physically combined to make up a filter, reagent layer or absorbent pad. Two materials are acceptable for the filter, an upper filtering material and a lower dispersal material . Most acceptable is a dispersal layer that passes fluid at a slower flow rate compared to passage of fluid through an upper filtering material. The applicants have found that the use of a dispersal layer is surprisingly superior over the use of a single part filter. Although applicants wish not to be bound to one particular theory of how their dispersal layer works in the invention, the layer may remove irregularities in the flow that arise from irregular collection of particles in the upper regions of the filter. A second advantage of using a two-part filter is that the dispersal layer, by virtue of its slow fluid flow rate, can prolong one or more reaction times for substances that react before entering the reaction layer, thereby increasing sensitivity.

A skilled artisan will readily appreciate that one or more portions of the device summarized above can be replaced by a single piece of a suitable material. In this context, many absorbent, porous or capillary possessing materials through which a solution containing the analyte can be transported by a wicking action are suitable for construction of the test device and are known to the skilled artisan.

Referring now to the Figures, some of the embodiments will be explored in greater detail. FIG. 1 depicts device housing 10 that consists of sleeve assembly 20 and container 30. Container 30 consists of top half 40 and bottom half 50. Sleeve assembly 20 consists of top surface 60 and opening 70 made by sleeve 80. Sleeve assembly 20 is reversibly mounted onto container 30 and can be removed after application of a sample for further processing of the sample by adding wash and/or reagent solution to the middle of the top surface of the container. FIG. 2 is a side perspective drawing of device housing 10

FIG. 3 shows a cross section of the device shown FIG. 1 and FIG. 2. Top half 40 and bottom half 50 of container 30 snap together to form a solid structure. Nestled within bottom half 50 is absorbent pad 90. On top of absorbent pad 90 is reagent layer 100. On top of reagent layer 100 is circular washer 110. Circular washer 110 is comprised of a water impermeable plastic and forms a tight seal with container top half 130, leaving space 200 at the bottom of top half 130.

Sleeve assembly 20 consists of lip region 120 that forms funnel 130. Assembly 20 is a single body that forms sleeve 140 underneath funnel 130. Within sleeve 140 are first filter portion 150, second filter portion 160 and dispersant layer 170. First filter portion 150, second filter portion 160 and dispersant layer 170 form a "filter" having an upper surface 180 and a lower surface 190. Lower surface 190 protrudes out of sleeve 140 to contact the upper surface of reagent layer 100, leaving space 200 between the side edge of dispersant layer 170 and circular washer 110.

FIG. 4 depicts an embodiment of the device shown in FIG. 3 that further contains cover 210 attached at location 220 of sleeve assembly 20. FIG. 5 is a cross section view of an alternate device that differs from the device of FIG. 1 - FIG. 4 by having reagent layer 100 inside sleeve 140. This figure depicts an embodiment wherein reagent layer 100 is not compressed by sleeve 140 because it is held mside sleeve 140 along with filter portions 150 and 160. In this embodiment, the reagent layer is in fluid contact with both the filter and absorbent pad 90 ( i . e . it may contact both directly or via an intervening layer (s) ) . Preferably, the reagent layer protrudes beyond the sleeve to the absorbent pad as shown in this figure. When using a reagent layer in th s manner, it s acceptable that the filter, one of the filter portions, or the sleeve be removable to allow direct optical detection of a signal that forms below the filter. Most acceptable in this context is a reagent layer comprised of a clear polycarbonate porous membrane in which antigen immobilized on polystyrene microparticles have been spotted. According to this embodiment, an optical signal develops at the bottom of the polycarbonate filter and can be seen by inspecting the bottom of the polycarbonate filter after removal of the filter. Also acceptable is to place a colloidal gold detection reagent in the filter a dried form so that the detection reagent may become resuspended during application of a test sample.

In some embodiments, an optional wick may be used to bring a fluid sample into the container. In these cases, a portion of the wick that is exposed to the outside of the container is contacted with the sample, which then wicks into the container. The wick may itself have filtering properties and can replace the filter. FIG. 6 shows an alternate etribodiment that uses lateral flow within a reagent pad and two openings. Housing 200 is made up of top half 202 and bottom half 204 which snap together. The housing has openings 210 and 220. Opening 210 is formed by cylinder 230 which holds filter 240. Filter 240 contacts reagent layer 250. End 260 of reagent layer 250 contains immobilized HIV antigen. Above end 260 is opening 220. Opening 220 holds dispersant layer 270 which contacts end 260 of the reagent layer. End 260 is situated above and contact with absorbent pad 280. Dispersant layer 270 is held in contact with reagent layer 260 by virtue of friction between the dispersant layer and sleeve 290. Spaces 300 are optional and may be used to help direct the flow of fluid into absorbent 280.

In this embodiment a sample is applied to opening 210, enters filter 240 and then enters reagent layer 250. Fluid moves across reagent layer 250 to end 260 above absorbent pad 280. An optional reagent and wash fluid are applied to opening 220 and an optical signal is developed at the reagent layer surface below this opening. The reagent may be present within the filter or the reagent layer and dissolves or becomes suspended upon application of fluid to the device.

FIG. 7 shows the use of the invention in a 96 well multiple test format. A 96 well cassette 310 consists of upper housing 320 and lower housing 330. Each well position of upper housing 320 contains a filter and dispersant layer. Each well position of lower housing 330 contains reagent layer and absorbent pad. In an advantageous embodiment the lower housing contains a continuous sheet of absorbent layer and a continuous absorbent pad. Optional multiple test device assembly 340 contains 8 individual tests and can be assembled into 96 well cassette 310 for test sequences in which up to 8 tests are performed at once. Alternatively, cassette 310 can be assembled with 96 tests as a single unit . FIG. 8 shows a top view and a side view of optional multiple test device assembly 340. The top view shows tab 360 for grasping a section of 8 tests that can be moved together. The side view shows individual sleeves 370 attached directly and indirectly to tab 360. Inside each sleeve and exposed to that sleeve's opening is filter 380. Filter 380 contacts, at its lower surface, dispersant layer 390. Dispersant layer 390 protrudes from sleeve 370 to form protrusion 400. During use, assembly 340 is inserted into a row of a 96 well plate that contains reagent layer and absorbent pad. Up to 8 samples can be processed at the same time using an 8 position pipetter. During use, sample and wash fluid are added to the 8 well sections of multiple device assembly 340, followed by removal of this assembly. Removal exposes the reagent layer for direct addition of fluid and also direct optical detection of a test signal from the reagent layer. In another embodiment (not shown) individual absorbent pads are replaced with a large surface continuous absorbent sheet inside lower housing 420.

As shown in FIG. 3, one of the advantageous features of a test device in accordance with this invention is that the surface of a filter that protrudes beyond a sleeve within the test device indirectly mates m a flush manner with a surface of an absorbent pad. More particularly, a diffuser (lower portion of filter that disperses filtrate) mates with the reagent layer such that the total surface of the diffuser contacts a surface of the reagent layer. Most acceptably, the diffuser protrudes beyond the sleeve so that a space is maintained when the surface of the diffuser contacts the surface of the reagent layer.

During use, a single test device is obtained and sample and wash fluids are added directly to either the filter or the reagent layer of the device. An additional wick also can be used to bring a fluid sample into the device, if desired. Alternately, if desired, a sample can be introduced into the device by leaving a portion of the filter exposed to the outside, where it can be used to directly absorb sample fluid and then bring this fluid, or a filtrate into the housing.

After adding the fluid sample, an aqueous wash solution is applied. The wash solution preferably contains a non- ionic detergent such as Tween 20 (r) and may also contain a protein such as bovine serum albumin. Most acceptably, after adding a wash solution, the filter is removed and then a reagent solution is added directly to the reagent layer. An advantageous reagent solution in this context contains a detection agent such as colloidal gold coated with protein A. The detection agent is optionally washed with a wash solution.

For the acceptable embodiment whereby the filter is removed, it is most acceptable that the sleeve be attached to the device reversibly by, for example a bayonet mount According to this embodiment, contact between the filter and a reagent layer is established upon attaching the sleeve that holds the filter to the device housing. This contact is broken when the sleeve is removed after washing a sample into the reagent layer. In a further embodiment, the sleeve remains place and the opening at the top of the sleeve is covered after addition of sample (and optional wash) . The cover (and optionally the sleeve) can be removed later for further processing if needed.

Reagents needed for operation of the test device may be placed in the filter, reagent layer, dispersant (if used) , and/or an aqueous fluid that is applied to the device. For convenience manufacturing, a reagent such as gold sol can be prepared and used as a liquid phase reagent and added during operation of the device. For optimum convenience to the test user, however, it is acceptable to place all reagent s except for the wash reagents within the device a dried form.

As seen in FIG. 3, the lower portion of the filter, i.e. the "dispersant layer, " extends beyond the sleeve that holds the filter such that when the filter is attached to the container, the dispersant layer portion exerts a greater pressure onto the reagent layer than does the sleeve. "Greater pressure" in this context means that the mechanical force per unit area exerted by the filter onto the reagent layer exceeds the mechanical force per unit area exerted by the sleeve onto the reagent layer Preferably, the filter exerts at least twice as much mechanical pressure onto the reagent layer compared to the sleeve More preferably, the sleeve does not contact the reagent layer. By relying principally on the filter itself to contact the reagent layer, the sleeve does not deform the reagent layer. This allows a more even and reproducible transfer of fluid from the filter to the reagent layer.

Although not willing to be bound by one particular theory of the invention, the applicants believe that an improvement in assay performance, which is seen as a more perpendicular flow of fluid through the reagent layer and into the absorbent pad, results from maintaining a smooth and undeformed reagent layer. Applicants have found and/or realized that preventing a mechanical support for the holder from contacting the reagent layer, or limiting the contact, allows less radial movement of fluid in the reagent layer, and particularly in the periphery of the reagent layer. This discovery, initially made while testing real HIV test specimens, was important to improve test sensitivity and selectivity.

In describing the physical manifestation of these advantageous effects, applicants have chosen the phrase "substantially in a direction perpendicular to the surface of the reagent layer" because initial wetting as well as subsequent color development indicates that fluid initially flows more perpendicular to the surface of the reagent layer according to an advantageous embodiment. The term "substantially," as used in this context means that fluid from the edges of the filter

(away from the center) initially penetrate the reagent layer to the reagent pad more vertically than horizontally within the reagent layer. A skilled artisan in this field who makes and uses such diagnostic devices visually can perceive these effects.

In one embodiment, a detection agent such as colloidal gold is placed in an optional lower portion ("dispersant") of the filter during manufacture of the device. In this case, a user would apply a blood sample to the device, followed by a wash, removal of the filter, and then another optional wash.

Adding sample and wash solution starts sequential and automatic reactions in the test device. These reactions include one or more binding reaction (s) , an optional separation step and an optional enzymatic reaction step according to the test format employed. Many types of chemistry formats are useful for the test device and are contemplated for the invention. One acceptable format is a binding assay in which an immobilized antigen in the reagent layer binds to an analyte molecule. In this format the analyte has at least one epitope or binding site for which there exists a naturally occurring, complementary specific binding member or for which a specific binding member can be prepared. In such a binding assay two molecules that bind specifically (i.e. with an association constant of at least 100,000 under typical assay conditions) form the basis of collecting a signal producing substance to form an optical signal . An advantageous signal producing substance is colloidal gold. Molecules that act as "binding partners" with an analyte m a test sample typically are antibodies specific to the analyte, but, as the skilled artisan will readily appreciate, other molecules that bind specifically to the analyte molecule also can be used.

After adding one or more fluids to the test device and then allowing one or more reactions to proceed, an optical change in the reagent layer is determined. When used in a binding assay format to form this optical change, the test device may contain a chemical label to generate the signal or the user may add this label. The label generally is any substance which s attached to a specific binding member and which is capable of producing a signal that is detectable visually or by an instrument. An acceptable label is further described in US patent application No. 08/577,108 (CHILDS et al . , entitled PARTICLE ASSISTED IMMUNOASSAY) which is herein incorporated m its entirety by reference . The above -summarized procedure can be modified by the use of an aqueous "pre- reatment " solution that is applied to the device before test fluid application. The pre-treatment solution comprises a protein and a detergent The protein may be, for example, bovine serum albumin, human serum albumin, casein, non- fat milk product or gelatin. The protein is preferably at a concentration of from 0.05% to 10% and more preferably between 0.2% and 2% (wgt/vol) . Most acceptable is a 1% solution of bovine serum albumin. The detergent is an amphoteric molecule or composition such as triton X-100, Tween 20, Tween 80, NP-40, zwitterionic detergents and the like. The detergent is preferably at a concentration above its critical micelle concentration and typically should be at a concentration of between 0.05% and 3% (wgt/vol) . The use of Tween-20 detergent is most acceptable at a concentration of between 0.2% and 2%. The pre- treatment solution should have a pH in the range of 5.0 to 10.0 and preferably contains a buffering component such as sodium phosphate or Tris-Cl at a concentration of between ImM and 1M and more preferably between lOmM and 200mM. Most acceptable is a 50mM concentration of Tris-Cl at pH 8.0.

For a test such as that for antibody against HIV 1 antigen, the applicants surprisingly discovered that better results are obtained by adding a glycosaminoglycan such as heparin or chondroitm sulfate to the pretreatment solution, the filter, the reagent layer, or the wash fluid in a concentration of more than about 0.1 ug/ml (i.e. 0.1 ug/ml) . The problem solved was that an occasional whole blood test fluid can give a positive response. Surprisingly, even when glycosammoglycan has already been added to the device and improves performance, adding glycosammoglycan to the wash solution further improves test performance and is most acceptable.

The glycosam oglycan typically may be chondroitm sulfate, dermatan sulfate, heparin sulfate, keratan sulfate, hyaluronic acid or a heparin, although other glycosaminoglycan compounds not recited here can be used. The glycosaminoglycan compound typically may be at a concentration of from about lug/ml to about lOmg/ml (e.g., lug/ml to lOmg/ml) and more acceptably at a concentration of from about 0. lmg/ml to about lmg/ml (e.g., 0. lmg/ml to lmg/ml) upon dissolution m the assay or in the pretreatment solution. The compound may be a heparin salt such as sodium heparin, ammonium heparin or lithium heparin. If used, the heparin advantageously is at a concentration of from about 1 usp/ml to about 50 usp/ml (e.g., 1 usp/ml to 50 usp/ml) and more advantageously from about 5 usp/ml to about 25 usp/ml (e.g., 5 usp/ml to 25 usp/ml) . The glycosaminoglycan compound, particularly heparin or a heparin derivative, does not work when present at a high concentration. Heparin, for example, should not be used at a final dissolved concentration that exceeds 25 usp/ml although chondroitm sulfate was found to remain active at 10 mg/ml final concentration. Optimal levels of the glycosam oglycan compound are determined by adding the compound to an assay at various concentrations and determining test results from different test samples.

The acceptable embodiment whereby all solutions are added to a single horizontal location at the top of the device (either before or after separating the top and bottom halves) is particularly suited for automated instrumentation. The inventors realized that the filter, reagent pad and absorbent can be sized to allow their manufacture into a 96 well microtiter plate. Furthermore, 8 (or 12) devices can be manufactured into a vertical (or horizontal) strip of multiple test devices that can be assembled to become part of a microtiter plate. Such microtiter plate embodiments can be processed by existing microtiter plate instruments. In these embodiments the user can add sample, wash and/or reagent solutions by hand or by automated instrumentation.

A skilled artisan will readily appreciate that a device of the present invention can be packaged with other components such as an instruction pamphlet, a wash reagent that may be either a dried material that is redissolved water or buffer , or already present as a fluid, and optionally one or more reagents to add to the device such as gold particles treated with a binding partner for a binding reaction. Accordingly, the invention allows the production and use of kits for infectious disease testing from blood. One advantage of these kits, according to an advantageous embodiment is that they can contain a glycosam oglycan to eliminate false measurements.

The present invention will now be illustrated by the following examples, which are not intended to be limiting n any way . EXAMPLE ONE

In this example, a test fluid preparation filter holder was constructed with a filter composed of the following components m sequential order from top to bottom: a blood separation filter; a backup blood separation filter; and a dispersant layer. The dispersant layer was situated in direct contact with a reagent layer in which a member of a binding pair, namely HIV antigen, was immobilized. The blood separation filters and the dispersant layer were chemically treated by exposure to a solution of buffer, detergent, protein and anticoagulant.

The preparation of the reagents used is generally known to the skilled artisan and is described in co-pending US patent application nos. 08/577,108 (Childs et al . , entitled PARTICLE ASSISTED IMMUNOASSAY) and 08/577,630 (Childs et al . , entitled STRIP TEST FOR HIV) which are herein incorporated m their entireties by reference.

In the test procedure, two drops of pre-treatment solution are added to the filter of the device. Then one to two drops of test fluid (whole blood, EDTA treated blood, hepaπnized blood, acid citrate dextran treated blood, plasma or serum) are added to the filter. Three drops of pre-treatment solution are then added to wash the test fluid from the top of the filter (the primary filter) into the middle (backup filter) region, the lower

(dispersant layer) region and the reagent layer. The test fluid preparation filter holder is then removed from the detection device which contains the reagent layer and an absorbent pad. After removing the filter holder, the reagent layer of the test device is exposed. Exposing the reagent layer allows for the addition of wash fluid. Two drops of wash buffer are added to the reagent layer on which HIV antigens have been immobilized. Then two drops of labelled colloidal gold labelled protein A are added, followed by two drops of wash buffer. The presence of anti-HIV antibody in the sample is detected as color formation from the deposition of colloidal gold. More than 100 whole blood samples that were obtained from patients suspected of carrying HIV were tested for antibodies to HIV. The test results correlated 100% with results obtained by a western blot method.

EXAMPLE TWO In this example a device is constructed as described in example one. The device is used in accordance with the procedure of Example One except that after sample application and wash, the test device is maintained at room temperature for 4 hours. The remaining steps of the assay are then carried ou . Blood samples tested in accordance with this device and method and which contain anti-HIV antibody accurately formed color from the deposition of colloidal gold. Blood samples that lacked anti-HIV antibody did not cause the deposition of colloidal gold.

Further details for preparing the materials and reagents employed herein are provided in the above-cited patent documents or are known to those skilled in this art . Each of the above-cited patents and pending patent applications, in its entirety, is specifically incorporated herein by reference. Although the present invention has been described with reference to certain acceptable embodiments, modifications or changes may be made therein by those skilled in this art without departing from the scope or spirit of the present invention, as defined by the appended claims. Further, although the inventors have described certain embodiments, designs, materials, etc. as preferred, it is understood that such preferred embodiments merely represent the preferred embodiments that have resulted from the inventors' work to date and that many other acceptable embodiments can be discovered and employed through practice of the invention and routine trial and error, and that some of such other embodiments ultimately may be more preferred than those specifically described.

In the appended claims, the articles "a" and "an'1 shall mean "at least one" unless otherwise indicated.

Claims

What is claimed is :
1 . A test device comprising : a housing; an absorbent pad held by the housing; a reagent layer that comprises an immobilized test reagent, the layer being in fluid communication with the absorbent pad; a filter in fluid communication with the reagent layer; an opening the housing adjacent to the filter for receiving an aqueous sample into the filter; and a sleeve operatively holding the filter in physical contact with the reagent layer, wherein the filter protrudes from the sleeve such that the filter exerts a greater pressure onto the reagent layer than does the sleeve.
2. The test device of claim 1, wherein the filter contacts the reagent layer, and wherein the sleeve contacts but substantially does not deform the reagent layer.
3. The test device of claim 1, wherein the sleeve does not contact the reagent layer.
4. The test device of claim 1, wherein the filter comprises an upper portion suitable for filtering blood and a lower portion for dispersing filtrate to the reagent layer.
5. The test device of claim 4, wherein the lower portion of the filter comprises a material that passes fluid at a slower rate compared to the material of the upper portion.
6. The test device of claim 4, wherein the upper portion of the filter comprises two separate filters that are held together.
7. The test device of claim 4, wherein at least one of the filter portions is adapted to swell upon wetting.
8. The test device of claim 1, wherein the filter has a depth equal to at least one tenth its diameter.
9. The test device of claim 1, wherein the immobilized test reagent comprises a molecule for binding a sample analyte.
10. The test device of claim 9, wherein the molecule is an antigen of an infectious disease agent .
11. The test device of claim 10, wherein the infectious disease agent is a human immunodeficiency virus.
12. The test device of claim 1, wherein the sleeve holds the filter by friction.
13. The test device of claim 1, wherein the filter is adapted to swell upon wetting.
14. The test device of claim 1, wherein the absorbent layer is adapted to swell upon wetting.
15. The test device of claim 1, wherein the filter protrudes at least 0.05 mm from the sleeve.
16. The test device of claim 1, wherein the sleeve is separable from the housing.
17. The test device of claim 16, wherein the sleeve is separable
from the housing by means of a bayonet mount .
18. The test device of claim 1, wherein at least the filter or reagent layer comprises a glycosam oglycan.
19. A test device comprising a housing having an opening; an absorbent pad within the housing; a reagent layer having an immobilized test reagent, the layer being positioned on top of the absorbent pad; a filter on top of the reagent layer and aligned with the opening so that the filter is adapted to receive a test sample ; an opening the housing above the filter that can admit an aqueous sample; and a sleeve operatively holding the filter in physical contact with the reagent layer, the sleeve being connected to the housing, wherein the filter protrudes from the sleeve such that the filter exerts a greater pressure onto the reagent layer than does the sleeve.
20. The test device of claim 19, wherein the filter contacts the reagent layer, and wherein the sleeve contacts but substantially does not deform the reagent layer.
21. The test device of claim 19, wherein the sleeve does not contact the reagent layer.
22. A test device comprising: a housing having an opening; an absorbent pad held by the housing; a reagent layer comprising an immobilized antigen selected from the group consisting of HIV-1,
HIV-2 and HIV-1 subtype o, the layer being positioned in fluid communication with the absorbent pad; a filter in fluid communication with the reagent layer and aligned with the opening to receive an aqueous sample into the filter.
23. The test device of claim 22, wherein the reagent layer physically contacts the absorbent pad and the filter physically contacts the reagent layer.
24. The test device of claim 22, further comprising a sleeve that holds the filter, and wherein the sleeve covers the reagent layer.
25. The test device of claim 24, wherein the cover is a plug adapted to be held in the opening by friction.
26. The test device of claim 22, wherein the reagent layer further comprises immobilized goat anti-human antibody.
27. The test device of claim 22, further comprising a glycosaminoglycan .
28. The test device of claim 27, wherein said antigen further comprises a cluster of four basic amino acids within a decapeptide portion of the antigen.
29. An HIV diagnostic assay kit for testing blood, said kit comprising a test device comprising: a housing having an opening; an absorbent pad held by the housing; a reagent layer comprising an immobilized antigen selected from the group consisting of HIV-1, HIV- 2 and HIV-1 subtype o, the layer being positioned in fluid communication with the absorbent pad; and a filter in fluid communication with the reagent layer and aligned with the opening to receive an aqueous sample into the filter.
30. The assay kit of claim 29, wherein the test device further comprises a cover for sealing the device.
31. The assay kit of claim 29, that further comprises a wash fluid that contains a glycosaminoglycan.
32. The assay kit of claim 31, wherein the glycosammoglycan is selected from the group consisting of chondroitm sulfate, dermatan sulfate, heparin sulfate, keratan sulfate, hyaluronic acid and heparin.
33. A process for detecting HIV antibody in blood comprising the steps of:
(a) providing a test device that contains a housing having an opening, an absorbent pad held by the housing, a reagent layer comprising an immobilized antigen
selected from the group consisting of HIV-1, HIV- 2 and HIV-1 subtype o, the layer being positioned in fluid communication with the absorbent pad, and a filter in fluid communication with the reagent layer and aligned with the opening to receive an aqueous sample into the filter;
(b) applying a blood sample to the opening of the test device ;
(c) applying at least one wash fluid to the test device;
(d) allowing one or more reactions to proceed in the reagent layer of the device; and (e) detecting the one or more reactions in the reagent layer in response to the presence of anti-HIV antibody.
34. The process of claim 33, wherein an optical change is used to detect the one or more reactions in the reagent layer.
35. The process of claim 33, wherein the reagent layer physically contacts the absorbent pad and the filter physically contacts the reagent layer.
36. The process of claim 33, wherein the wash fluid comprises a glycosaminoglycan.
37. The process of claim 36, wherein the glycosaminoglycan is selected from the group consisting of chondroitm sulfate, dermatan sulfate, heparin sulfate, keratan sulfate, hyaluronic acid and heparin.
38. The process of claim 36, wherein the glycosaminoglycan is heparin and is present in the wash fluid m the concentration of between 5 and 25 international units per milliliter.
39. The process of claim 33, wherein the HIV-1 immobilized antigen comprises a sequence selected from the group consisting of Q-T-H-L-P-I-P-R-G-P-D-R-P-E-G-I -E-E-G, L-Y-K- Y-K-V, P-L-G-V-A-P-T-K-A-K-R-R-V-V, P-L-G-V-A-P-T-R-A-K-R- R-V-V, R-E-K-R-A, S-G-I-V-Q-Q, L-T-V-W-G, D-Q-Q-L-G, W-G-C- S-G-K, Q-Q-E-K-N-E-Q, Q-T-H-L-P- I -P-R-G-P-D-R-P-E-G- I -E-E-G and Q-Q-E-K-N-E-Q.
40. A test kit that contains a wash reagent and that is used for detection of HIV-1 from a body fluid, wherein the wash reagent comprises a glycosaminoglycan.
41. The test kit of claim 40 wherein the body fluid is whole blood.
42. The improved test kit of claim 40, wherein the kit contains immobilized HIV-1 antigen that comprises four or more basic ammo acids within a ten ammo acid long sequence.
43. A process for detecting HIV-1 antibody from blood comprising the steps of:
(a) providing a test device that contains immobilized HIV-1 antigen;
(b) applying a blood sample to the test device;
(c) applying at least one fluid that comprises a glycosam oglycan to the test device;
(d) allowing one or more reactions to proceed in the reagent layer of the device; and
(e) detecting an optical change the reagent layer in response to the presence of antl -HIV-1 antibody.
44. A test device comprising: a housing; an absorbent pad held by the housing; a reagent layer that comprises an immobilized test reagent the layer being in fluid communication with the absorbent pad; a filter in fluid communication with the reagent layer
that comprises a filtering portion and a dispersant portion, the flow rate of fluid in the dispersant portion being slower than the flow rate in the filtering portion; and an opening to admit a fluid sample to the filter.
45. The test device of claim 44, wherein the reagent layer contacts the absorbent pad and the filter contacts the reagent layer.
46. A test device comprising a housing having an opening,- an absorbent pad within the housing; a reagent layer having an immobilized test reagent, the layer being positioned on top of the absorbent pad; a filter on top of the reagent layer and aligned with the opening so that the filter is adapted to receive a test sample; and an opening in the housing above the filter that can admit an aqueous sample, wherein when a test sample is added to the filter, filtrate from the filter passes through the reagent layer into the absorbent pad substantially in a direction perpendicular to the surface of the reagent layer.
47. A multiple test device comprising a multiwell plate comprising multiple absorbent pads, a reagent layer being positioned on top of each absorbent pad and an opening positioned on top of each reagent layer; and a multiple sleeve comprising a plurality of sleeves held together in a row, each sleeve comprising a filter wherein the multiple sleeve reversibly fits into the multiwell plate.
48. A multiple test device comprising a multiwell plate comprising a single absorbent pad having (1) multiple areas of reagent layer disposed in a regular pattern on its surface and (ii) an opening above each reagent layer; and a multiple sleeve comprising a plurality of sleeves held together a row, each sleeve comprising a filter wherein the multiple sleeve reversibly fits into the multiwell plate.
49. A multiple test device comprising a plurality of test units, each test unit comprising a sleeve having an opening; a reagent layer having an immobilized test reagent; a filter on top of the reagent layer and aligned with the opening so that the filter is adapted to receive a test sample; an opening in the sleeve above the filter that can admit an aqueous sample; an opening in the sleeve under the reagent layer; and an absorbent pad that can receive fluid from multiple test units wherein when a test sample is added to a test unit filter, filtrate from the filter passes through the reagent layer into the absorbent pad substantially a direction perpendicular to the surface of the reagent layer.
50. The multiple test device of claim 49 wherein the filter protrudes from the sleeve such that the filter exerts a greater pressure onto the reagent layer than does the sleeve .
51. A kit for detecting HIV-1 antibody from a blood sample comprising a device, an optional fluid, an immobilized HIV- 1 antigen reagent having four or more basic ammo acids within a ten amino acid long sequence and a glycosam oglycan.
52. A diagnostic assay kit comprising a device, an optional fluid, a recombinant antigen having four or more basic amino acids within a twenty amino acid long segment, and a glycosaminoglycan .
PCT/US1997/017129 1996-09-25 1997-09-25 Diagnostic test devices with improved fluid movement and resistance to interferences WO1998013519A1 (en)

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JP51586698A JP2001502052A (en) 1996-09-25 1997-09-25 Testing device for diagnosis with improved resistance to liquid transfer and interference
EP19970942680 EP0972073A1 (en) 1996-09-25 1997-09-25 Diagnostic test devices with improved fluid movement and resistance to interferences

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CA2266747A1 (en) 1998-04-02 application

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