WO1999047930A1

WO1999047930A1 - Immunoassay device and method

Info

Publication number: WO1999047930A1
Application number: PCT/US1999/005487
Authority: WO
Inventors: Allan D. Pronovost; Hans Boehringer; Michael Imrich
Original assignee: Quidel Corporation
Priority date: 1998-03-16
Filing date: 1999-03-12
Publication date: 1999-09-23
Also published as: AU3185199A; EP1064553A1; JP2002507725A; EP1064553A4

Abstract

An assay device for detecting the presence or absence of an analyte in a liquid sample. The assay device comprising a planar matrix having a sample receiving zone (12) for receiving the liquid sample; a labeling zone (14) in fluid communication with the sample receiving zone; a capture zone (16) in fluid communication with the labeling zone; and an absorbent zone (18) in fluid communication with the capture zone, wherein the absorbent zone includes a dessicant material.

Description

IMMUNOASSAY DEVICE AND METHOD

Field of the Invention The present invention relates to devices and methods for conducting immunoassays. More particularly, the invention relates to lateral flow or flow-through immunoassay devices that incorporate a desiccant material as a functional component of the device assembly.

Background of the Invention Analyte-specific binding assays are important tools for detecting and measuring environmental and biologically relevant compounds, including hormones, metabolites, toxins and pathogen-derived antigens. A convenient version of the binding assay is an immunoassay which can be conducted in a "lateral flow" format. Devices useful for performing lateral flow assays typically include several "zones" that are defined along a length of a matrix. The matrix defines a flow path and provides fluid connection between the various zones, including a sample receiving zone, a labeling zone for specifically labelling the analyte, and a capture zone located downstream from the sample receiving zone and the labeling zone. An absorbent zone typically is located downstream of the capture zone, and provides a means for removing excess sample and unbound label from the matrix. In some applications the matrix of a lateral flow or flow-through assay device is a membrane capable of "non- bibulous lateral flow." In these applications liquid flow occurs such that all of the dissolved or dispersed components in the analyte-containing liquid are carried at substantially equal rates and with relatively unimpaired flow laterally through the membrane. This is distinguished from a situation wherein preferential retention of one or more components occurs, for example, in materials capable of adsorbing or imbibing one or more of the components. A principal advantage of the lateral flow immunoassay is the ease with which the testing procedure is carried out. In this procedure a fluid sample first contacts the matrix following application to the sample receiving zone. Capillary action then draws the liquid sample downstream into a labeling zone that contains a means for indirectly labelling the target analyte. The labelling means generally will be a labelled immunoglobulin, but alternatively may be a non- immunoglobulin labelled compound which specifically binds the target analyte or an analyte analog . After transiting through the labeling zone, the sample continues to flow into the capture zone where it contacts an immobilized compound capable of specifically binding the labelled target analyte. As a specific example, analyte-specific immunoglobulins can be immobilized in the capture zone. Labelled target analytes will bind the immobilized immunoglobulins upon entering the capture zone and will be retained therein. The presence of the labeled analyte in the sample typically will be determined by visual detection of the label within the capture zone. Finally, the procedure is complete when excess sample is taken up by the material of the absorbent zone.

Lateral flow immunoassays typically employ test and procedural control lines in the capture zone. The test line serves to detect an analyte present in a test sample, while the procedural control line conventionally serves to detect a ligand unrelated to the analyte. Rather than being applied in the test sample, the ligand unrelated to the analyte is disposed in the labeling zone of the lateral flow immunoassay device. The test line ordinarily employs specific competitive, sandwich or indirect binding separation principles using a visual label. This requires the use of a labeled detector antibody in the labeling pad of the labeling zone and a capture antibody or ligand immobilized at the capture test line.

The capture zone of lateral flow immunoassay devices may also include a procedure control line useful for indicating that a procedure has been performed. The procedure control line generally is located downstream of the analyte- specific binding compound that is immobilized in the capture zone. Retention of label by the procedural control line indicates that liquid sample has flowed through the capture zone and contacted the immobilized target-specific binding substance. The accumulation of visible label may be assessed either visually or by optical detection devices.

Prior art lateral flow and flow-through immunoassay devices ordinarily are packaged in an air-tight container with a desiccant that is not a functional component of the device construction. These desiccants, which may take the form of a "pill" or a sachet, serve to retard moisture-related decomposition of chemical or immunochemical reagents deposited on the device.

Summary of the Invention

One embodiment of the invention is an assay device for detecting the presence or absence of an analyte in a liquid sample. The assay device includes: a substantially planar matrix having; a sample receiving zone for receiving the liquid sample; a labeling zone in fluid communication with the sample receiving zone, the labeling zone having a mobile agent for binding the analyte; a capture zone in fluid communication with the labeling zone, the capture zone comprising an immobile agent capable of binding the analyte; and an absorbent zone in fluid communication with the capture zone, wherein the absorbent zone includes a desiccant material.

Another embodiment of the invention is, in an assay device having an absorbent zone for absorbing liquid, an improvement including a desiccant material having the absorbent zone.

Brief Description of the Drawings Figure 1 illustrates an immunoassay strip suitable for use with the present invention and the disposition of four separate zones which comprise the strip.

Figure 2 is a graph showing a comparison of immunoassay results obtained using a standard absorbent zone or a desiccant absorbent zone. Lines on the graph represent results from immunoassay strips having either a standard absorbent zone run using samples having PDG concentrations of 0.5 μg/ml (Q) and 1.0 μg/ml (J; or a desiccant absorbent zone run using samples having PDG concentrations of 0.5 μg/ml (") and 1.0 μg/ml (ϊ).

Figure 3 is a graph showing how the visible signal observed at the test line changes with time on immunoassay strips constructed using a thick desiccant pad as the absorbent zone. The PDG concentrations used in the procedure were: 0.0 μg/ml (Q); 0.5 μg/ml (J; 1.0 μg/ml ("); 3.0 μg/ml (1); 5.0 μg/ml (#); 10.0 μg/ml ( ); and 20.0 μg/ml (M).

Figure 4 is a graph comparing the performance of immunoassay strips constructed to incorporate thick and thin desiccant paper absorbent zones. Samples applied to immunoassay strips having thick desiccant pads contained PDG at the following concentrations: 0.0 μg/ml (Q); 1.0 μg/ml {"); 3.0 μg/ml (ft); 5.0 μg/ml (M); 10.0 μg/ml (3). Samples applied to immunoassay strips having thin desiccant pads contained PDG at the following concentrations: 0.0 μg/ml (J; 1.0 μg/ml (1); 3.0 μg/ml ( ); 5.0 μg/ml (ϊ); 10.0 μg/ml (4).

Figure 5 is a graph showing the stability of desiccant-containing immunoassay strips stored at 25EC and run using samples containing PDG at various concentrations. The PDG concentrations used in the procedure were: 0.0 μg/ml (Q); 1.0 μg/ml (J; 3.0 μg/ml ("); 5.0 μg/ml (ϊ); and 10.0 μg/ml (#).

Figure 6 is a graph showing the stability of desiccant-containing immunoassay strips stored at 37EC and run using samples containing PDG at various concentrations. The PDG concentrations used in the procedure were: 0.0 μg/ml (Q); 1.0 μg/ml (J; 3.0 μg/ml ("); 5.0 μg/ml (ϊ); and 10.0 μg/ml (#).

Figure 7 is a graph showing the stability of desiccant-containing immunoassay strips stored at 45EC and run using samples containing PDG at various concentrations. The PDG concentrations used in the procedure were: 0.0 μg/ml (Q); 1.0 μg/ml (J; 3.0 μg/ml ("); 5.0 μg/ml (ϊ); and 10.0 μg/ml (#). Figure 8 is a graph showing the time (in seconds) required to achieve a positive test result for various assay devices that had been stored at 37EC/75% relative humidity and then run using samples containing 25 mlU/ml. The four devices used in the procedure were: "pill" desiccant (Q); Whatman desiccant (!); Multiform desiccant (it); and a QUICK VUE immunoassay device available from Quidel Corp. (J as a control.

Figure 9 is a graph showing the time (in seconds) required to achieve a positive test result for various assay devices that had been stored at 45EC and then run using samples containing 25 mlU/ml. The four devices used in the procedure were: "pill" desiccant (Q); Whatman desiccant (!); Multiform desiccant (it); and a QUICK VUE immunoassay device available from Quidel Corp. (_) as a control.

Detailed Description We have discovered that certain limitations of conventional lateral flow immunoassay and flow-through devices can be overcome by employing an immunoassay strip that incorporates a desiccant as a functional component of the strip assembly. The invention is particularly useful for conducting immunoassays when the analyte being detected is contained in a viscous liquid sample such as serum, or a sample in which the fluid component is reduced by the presence of a cellular

-3- component. Advantageously, the invented construction eliminates the need for packaging a separate desiccant with the immunoassay device during long-term storage of the device.

If the analyte to be detected is contained in a highly viscous sample, then the rate of flow through or across the solid phase matrix can undesirably be reduced in conventional immunoassay strips. If the applied sample is a whole blood sample, the available serum volume may be limited by the volume exclusion effect of the erythrocyte component of blood. This effect is especially noticeable in blood samples with high hematocrits where the volume of available serum is reduced as the proportion of erythrocytes in the sample increases. In such instances, a reduced flow rate resulting from limited fluid transport through or across the immunoassay matrix can reduce the speed of the assay. In addition to lengthening read times and increasing background label in the capture zone, a reduced rate of fluid flow also can adversely promote late appearance positives and false-positives after the recommended read time. Finally, if lateral flow one-step assays are performed in an upright orientation instead of a horizontal orientation, gravity can negatively limit fluid migration from the sample receiving zone to the absorbent zone of the assay device.

The invented assay devices comprise four distinct zones. The device is designed so that the analyte-containing sample is first applied to a sample receiving zone, then flows through a labeling zone and into a capture zone. The capture zone in turn is in contact with an absorbent zone which provides a means for removing excess liquid sample. In conventional immunoassay devices, the absorbent zone consists of an absorbent such as filter paper or glass fiber filter. In the invented devices, the absorbent zone is comprised of a desiccant. Exemplary desiccant materials useful for constructing the invented devices include papers that incorporate a silica gel.

As used herein, the term "pad" refers to the physical material which corresponds to a zone or section of an immunoassay strip. Thus, a sample pad is the material of the sample receiving zone of an immunoassay strip. The labeling pad similarly refers to the material of the labeling zone.

Advantages underlying the use of a desiccant as the absorbent zone in the construction of a lateral flow immunoassay strip include: (1) more rapid fluid transit; (2) more efficient transport of viscous samples or test solutions through the matrix, especially at limiting cell-free fluid volumes associated with samples of whole blood having a high hematocrit; (3) more rapid clearing of the read window of a housing that encloses an immunoassay strip, thereby reducing assay read times; (4) actively driving immunological components of the assay to equilibrium and completion to better separate bound and free labeled antibody; (5) fewer conversions of low positives after the read time; (6) higher sensitivity as low level positives appear sooner and before the read time; (7) longer time-to-read times wherein permanent assay results are achieved earlier and maintain the same result longer; (8) elimination of the need for a separate desiccant compartment; and (9) more efficient and timely bound/free lateral flow separations wherein immunoassays are drawn toward equilibrium.

The invention generally concerns one-step lateral flow or flow-through assays which are conducted on supports which may conduct nonbibulous lateral flow of fluids. As defined herein "nonbibulous" lateral flow refers to liquid flow in which all of the dissolved or dispersed components of the liquid which are not permanently entrapped or "filtered out" are carried at substantially equal rates and with relatively unimpaired flow laterally through the membrane or support. This is distinguished from preferential retention of one or more components as would occur, for example, in materials capable of absorbing or "imbibing" one or more components as occurs in chromatographic separations. "Bibulous" materials include untreated forms of paper, nitrocellulose and the like which effect chromatographic separation of components contained in liquids passed therethrough. Bibulous materials can be converted to materials which exhibit nonbibulous flow characteristics by the application of blocking agents. These agents may be detergents or proteins which can obscure the interactive forces that account of the bibulous nature of the supports. Thus, nonbibulous materials include those which are intrinsically capable of conducting nonbibulous flow, such as porous polyethylene sheets or other inert materials or can be comprised of bibulous materials which have been blocked. Preferred blocking agents include bovine serum albumin, either per se or in methylated or succinylated form, whole animal sera, such as horse serum or fetal calf serum, and other blood proteins. Other protein blocking agents include casein and nonfat dry milk. Detergent-based blocking agents can also be used for rendering a bibulous material capable of nonbibulous flow. The types of detergents appropriate for this purpose are selected from nonionic, cationic, anionic and amphoteric forms, and the selection is based on the nature of the membrane that is being blocked.

To convert a bibulous support such as paper or nitrocellulose to a support capable of effecting nonbibulous lateral flow, the original support is treated with a solution of the blocking agent in an effective concentration to dispose of unwanted reactivities at the surface. In general, this treatment is conducted with a blocking solution, such as a protein solution of 1-20 mg/ml protein at approximately room temperature for between several minutes and several hours. The resulting coated material is then permanently adsorbed to the surface by air-drying, lyophilization, or other drying methods. Both the selection and treatment of carrier porous materials used to construct immunoassay strips of the sort described herein depend on the functional role that each zone performs in the assay device.

The sample-receiving zone serves to begin the flow of analyte-containing sample, and typically will be constructed of a material that exhibits low analyte retention. One means for imparting this property involves impregnating the sample receiving zone with a neutral protein-blocking reagent, followed by treatment to immobilize the blocking agent (e.g., lyophilization). An additional advantage of this treatment is increased wetability and wicking action which speeds transfer of the liquid sample into the labeling zone. The sample-receiving zone may also function as a mechanical filter, entrapping any undesirable particulates present in the sample.

The labeling zone contains visible moieties which can be detected if accumulated in the capture zone. The visible moieties can be dyes or dye polymers which are visible when present in sufficient quantity, or can be, and are preferred to be, particles such as dyed latex beads, liposomes, or metallic, organic, inorganic or dye sols, dyed or colored cells or organisms, red blood cells and the like. The visible moieties used in the assay provide the means for detection of the nature of and quantity of result, and accordingly, their appearance in the capture zone must be a function of the analyte in the sample. In general, this can be accomplished by coupling the visible moieties to a ligand which binds specifically to the analyte, or which competes with analyte for a capture reagent in the capture zone. In the first approach, the visible moieties are coupled to a specific binding partner which binds the analyte specifically. For example, if the analyte is an antigen, an antibody specific for this antigen may be used; immunologically reactive fragments of the antibody, such as F(ab')₂, Fab or Fab' can also be used. These visible moieties, or "test" visible moieties, then bind to analyte in the sample as the sample passes through the labeling zone and are carried into the capture zone by the liquid flow. When the complex reaches the capture zone, it is captured by an analyte-specific capture reagent, such as an antibody. Excess liquid sample finally is taken up by the absorbent zone. In the second approach, the visible moieties are coupled to a ligand which is competitive with analyte for a capture reagent in the capture zone, most typically, other molecules of the analyte itself. Both the analyte from the sample and the competitor bound to the visible moieties are then carried into the capture zone. Both analyte and its competitor then react with the capture reagent, which in this instance is also typically specifically reactive with analyte and its competitor. The unlabeled analyte thus is able to reduce the quantity of competitor- conjugated visible moieties which are retained in the capture zone. This reduction in retention of the visible moieties becomes a measure of the analyte in the sample. The labeling zone of immunoassay devices of the present invention also may include a procedural control which comprises visible moieties that do not contain the specific binding agent or analyte competitor and that are also carried through to a control area of the capture zone by the liquid flow. These visible moieties are coupled to a control reagent which binds to a specific capture partner and can then be captured in a separate procedural control portion of the capture zone to verify that the flow of liquid is as expected. The visible moieties used in the procedural control may be the same or different color than those used for the test moieties. If different colors are used, ease of reading the results is enhanced.

The experimental results of a procedure conducted using an immunoassay strip are read in the capture zone by noting the presence or absence of a visible signal at the location of the capture zone for the test visible moieties. The use of a procedural control region is helpful for indicating the time when test results can be read. Thus, when the expected color appears in the procedural control region, the presence or absence of a color in the test region can be noted. The use of different colors for test and control regions aids in this process.

The use of a matrix which is bibulous inherently, but convertible to a nonbibulous flow characteristic is particularly useful in the creation of the capture zone. Capture reagents can be applied to the matrix before the application of blocking agents and can be immobilized in situ. At this stage, the bibulous nature of the matrix during the coupling of the capture reagents may be advantageous. However, the blocking/washing treatment which converts the bibulous membrane to nonbibulous support provides unimpaired and speedy flow of all components of the system.

The extremely rapid nature of the assay, which typically yields a result in less than one minute, provides in many instances essentially an instantaneous result as the sample flows due to the nonbibulous nature of the zones and of the

-6- short distance the sample must traverse in each zone. Another factor which contributes to the speed of the assay is the absorptive potential of the material used to create the absorbent zone.

Miniaturization of the diagnostic device also contributes to the remarkable speed of the assay. Miniaturization permits instantaneous results which are observable as soon as the sample contacts the capture zone and which occur almost immediately or within 60 seconds of the addition of the sample to the sample receiving zone. The speed of appearance and intensity of the positive visible reaction seen depends on the concentration of analyte in the sample. The speed of appearance of the positive visual reaction can be adjusted to provide the optimal visual result with concentrations of analyte of clinical importance and adjusted to suit the timing needs of the end-user.

Suitable analytes detectable by the invented immunoassay devices are any for which a specific binding partner can be found. In general, most analytes of medical and biological significance can find specific binding partners in antibodies prepared against them or fragments of these antibodies. Suitable analytes include soluble analytes such as hormones, enzymes, lipoproteins, bacterial or viral antigens, immunoglobulins, lymphokines, cytokines, drugs, soluble cancer antigens, and the like. Also included as suitable analytes are hormones such as insulin, glucagon, relaxin, thyrotropin, so atotropin, gonadotropin, follicle-stimulating hormone, gastrin, bradykinin, vasopressin, and various releasing factors. A wide range of antigenic polysaccharides can also be determined such as those from Chlamydia, Neisseria qonorrheae, Pasteurella pestis, Shiαella dvsentereae, and certain fungi such as Mycosporum and Aspergillus. Another major group comprises oligonucleotide sequences which react specifically with other oligonucleotides or protein targets. An extensive list of soluble analytes determinable in the method of the invention is found in U.S. Patent No. 3,996,345, which is incorporated herein by reference. The structure of an exemplary immunoassay strip is shown in Figure 1. The immunoassay strip 10 includes a sample receiving zone 12, a labeling zone 14, a capture zone 16 and an absorbent zone 18. The sample receiving zone 12, label zone 14, and capture zone 16 will be composed of materials capable of receiving liquid samples and other liquid reagents and transporting such samples and reagents in a lateral direction, i.e. from the receiving zone 12 toward the absorbent zone 18. The invented absorbent zone 18 will be composed of a desiccant material that is capable of receiving and absorbing the same liquid samples and reagents. In this way, a liquid sample or other liquid reagent initially applied to the sample zone 12 can flow laterally from the sample receiving zone 12 into and through the labeling zone 14, into and through the capture zone 16, and finally into the absorbent zone 18 which acts as a wick or sink so that the entire sample or reagent volume may flow through the zones 12, 14 and 16 in order to properly complete the assay.

Exemplary desiccants useful for constructing the immunoassay strips of the invention described herein include silica gel-containing desiccant papers that comprise adsorbent particles contained in semi-rigid cellulose fiber matrix. Two different thicknesses of desiccant paper employed in the working Examples disclosed herein are commercially available from Multiform Desiccants, Inc. (Buffalo, New York), and are sold under the DRIKETTE trademark. The DRIKETTE desiccant paper "SG-145" is a paper desiccant having a thickness of 0.054 inches and contains at least 50% activated

-7- silica gel. The DRIKETTE desiccant paper "SG-146" has a thickness of 0.014 inches and contains at least 40% activated silica gel. Another type of desiccant is the Whatman "SG 81" desiccant paper which requires heat activation. The SG 81 paper has a thickness of 0.0095 inches and is made of a semi-rigid cellular fiber matrix containing silica gel.

The invented immunoassay strips can be disposed within a housing that is both protective and functional. In one preferred embodiment the housing is adapted to have at least one port for receiving a liquid sample and guiding fluid flow of the sample to contact the immunoassay strip at the sample receiving zone. The housing also can have windows which allow a user to view portions of the immunoassay strip, including portions of the capture zone and/or the absorbent zone. In another preferred embodiment of the invention the desiccant can serve as a handle portion of the test strip so that the assay can be performed outside of a housing. Our first experiments compared the properties of exemplary immunoassay strips that incorporated either the SG-

145 desiccant pad or the standard Whatman 541 (Whatman Specialty Products, Inc.) pad as the absorbent zone.

Example 1 describes the procedures used to demonstrate that a desiccant could substitute for the standard absorbent in the construction of lateral flow immunoassay strips. The analyte detected with the immunoassay strip in the following Example was a progesterone metabolite called pregnanediol-glucuronide (PDG). All of the immunoassay procedures used to detect PDG were run in a horizontal orientation.

Example 1 Absorbent Zone Prepared from a Desiccant Confers Advantages on Immunoassay Device A capture zone membrane was prepared by spotting a nitrocellulose sheet having a pore size of 8 μm (Schleicher & Schuell) with pregnanediol-glucuronide bovine IgG (PDG-BIgG) (40:1; 2.0 mg/ml) and Goat anti-mouse IgG (0.25 mg/ml). The PDG and BIgG were obtained commercially (Sigma). Pads having the trade name "New Merge" (DuPont) representing the sample receiving zone and the labeling zone were rendered nonbibulous by saturating with 35-40 μl/cm² of a 10mg/ml methylated BSA (mBSA) solution followed by drying/lyophilizing. A strip of the capture zone membrane was affixed centrally on an adhesive transparency strip to begin construction of a panel from which individual immunoassay strips would be cut. The transparency strip was a strip of overhead projection transparency film, made adhesive with double- sided adhesive tape. The labeling zone pad was then affixed next to the capture zone pad with a 1 mm overlap. The sample receiving pad was then placed next to the labeling pad with 1 mm overlap.

The device was then provided with an absorbent pad consisting of a rectangle of material described below affixed to the distal end of the capture zone membrane with a 1 mm overlap. The absorbent zone of the test panel was prepared either from a standard end pad made from cellulose and purchased from Whatman Specialty Products, Inc. or SG- 145 desiccant paper purchased from Multiform Desiccants, Inc. (Buffalo, New York).

Panels were cut into strips 4 mm wide and stored desiccated until use. Red antibody-latex "C1 Red" (Bangs Laboratories, Inc., Carmel IN) at a starting concentration of 0.4 μM was diluted 1/40 with mBSA. PDG containing samples were prepared by mixing 750 μl of male urine with 100 μl soluble capture antibody solution (100 μg/ml), and 100 μl PDG 10x stock solutions (5-200 μg/ml). A 5 μl aliquot of the diluted latex was applied to the interface between the sample pad and the labeling pad. A 35 μl aliquot of the PDG-containing urine sample was then added to the sample pad to initiate the immunoassay procedure. Both flow time and the time for the appearance of the first and the second lines in the capture zone of the immunoassay strip were recorded. The general appearance of the immunoassay strip also was evaluated at 5 and 10 minutes after the addition of the PDG-containing sample.

Results of these procedures indicated that test strips constructed with the desiccant paper dried substantially more quickly when compared to strips having an absorbent zone prepared from paper that did not include a desiccant. Moreover, when strips were evaluated by a technical reader, such as a laboratory technician, we found that signal intensity at the lines in the capture zone remained more stable with time when compared with immunoassay strips that included the standard paper absorbent zone. This latter observation was most significant for the PDG levels near the cut-off concentration of 1.0 μg/ml. Neither the time to achieve a visible positive signal in the assay nor the time required for the liquid front to reach the absorbent pad were significantly different for strips having standard paper or desiccant paper absorbent zones. These findings qualitatively showed that stability of the signal in the capture zone of a lateral flow immunoassay device could be improved by substituting a desiccant material for standard paper in the absorbent zone of the immunoassay strip.

Example 2 describes the quantitative procedures which confirmed that immunoassay strips incorporating a desiccant material as the absorbent zone delivered superior operating results. In this procedure, the properties of immunoassay strips constructed using either the standard Gill pad or the desiccant pad were quantitatively compared using a reflectance monitoring system.

Example 2 Quantitative Results Confirming the Advantageous Properties of Immunoassay Strip Construction The procedures described under Example 1 were followed to create and assemble immunoassay test strips and to dilute the antibody-latex solution. Test solutions containing 0.5 μg/ml or 1.0 μg/ml PDG in urine also were prepared as described under Example 1. A 5μl aliquot of the latex solution was applied to the interface between the sample pad and the labeling pad. A 35 μl aliquot of the PDG containing sample was added to the sample pad to initiate the immunoassay procedure. The intensity of the latex signal observed at the test line in three replicate strips was measured using a Minolta optical reading system at 5 minutes, 10 minutes and 20 minutes after addition of the PDG containing sample. Results of these procedures, shown graphically in Figure 2, indicated that the change of signal intensity at the test line was substantially reduced over time for the immunoassay strips that incorporated a desiccant. More specifically, for both PDG sample concentrations tested, the slopes of the lines as shown in the Figure were more shallow for devices that were constructed using a desiccant paper as the absorbent zone. Moreover, there was only minimal change in signal intensity below the cut-off so that negative results did not erroneously become positive over time. For example, inspection of Figure 2 revealed that the line plotted for a 0.5 μg/ml PDG sample applied to a test strip incorporating a desiccant absorbent zone (") showed less change with time when compared with a test strip having a standard absorbent zone (Q). These results quantitatively confirmed that substitution of a desiccant for the standard paper used to construct the absorbent zone of an immunoassay strip could result in improved operating properties of the device. In view of these improvements, we conducted additional experiments to optimize the composition of the desiccant used for construction the immunoassay strip.

Example 3 describes the procedures used to quantitate the rate of signal change over a 30 minute period for tests that were run using urine samples having different PDG concentrations. Example 3

Stability of Immunoassay Signal Intensities Over Extended Periods The procedures described under Example 1 were followed to create and assemble immunoassay test strips and to dilute the antibody-latex solution. Urine sample solutions containing 0.5-20.0 μg/ml PDG in male urine also were prepared essentially as described in Example 1. A 5 μl aliquot of latex solution was applied to the interface between the sample pad and the labeling pad. A 35 μl aliquot of PDG-containing sample was added to the sample pad to initiate the immunoassay procedure. Intensities of the signals observed at the test lines in three replicate strips were measured at 5 minutes, 10 minutes, 20 minutes, and 30 minutes using the Minolta optical reading system.

Results of these procedures, shown graphically in Figure 3, indicated that signal intensities observed for samples containing PDG in the range of from 0.5-20 μg/ml changed only minimally for all time points that were measured. These findings further confirmed the utility of an immunoassay strip constructed to include a desiccant as the absorbent zone.

Example 4 describes the procedures used to demonstrate that properties of the desiccant used to construct an immunoassay strip positively influenced at least some of the operating features of the strip. More specifically, the following Example describes a direct comparison of immunoassay strips constructed to incorporate thick (SG-145; 0.054") and thin (SG-146; 0.014") desiccant paper absorbent zones.

Example 4 Comparison of Immunoassay Strip Properties Using Two Different Desiccant Paper Formats A nitrocellulose sheet having a pore size of 8 μm (Schleicher & Schuell) was spotted with PDG-BIgG (2.5 mg/ml), Goat anti mouse IgG (0.5 mg/ml) and Rabbit anti glucose oxidase (1.0 mg/ml) using an IVEK dispenser, and blocked by immersion in a 1 % BSA solution for 15 minutes. Sample pads were then saturated with 35-40μl/cm² of 0.5% Zwittergent (Calbiochem, La Jolla, CA)/0.1 % Polyvinylpyrrolidone (PVP)-Stabilcoat in mBSA (PVP was obtained commercially from Aldrich, Milwaukee, Wl; Stabilcoat is a trademark of Biometric Systems, Inc., Eden Prairie MN). Labeling pads were

-10- saturated with 35-40μl/cm² of a 1/120 dilution of antibody-latex stock solution (approx. 1.2% EDI-aldehyde red latex, 0.5 μm, Emerald Diagnostics, Eugene, OR), 1/200 glucose oxidase latex (CML blue, 0.5 μm, Seradyn, Indianapolis, IN), and 0.1% PVP-Stabilcoat in mBSA. The pads were then lyophilized. Test panels were assembled using the lyophilized sample and labeling pads, blocked nitrocellulose and either the SG-145 or SG-146 desiccant pad from Multiform Desiccants, Inc. The resulting panels were then cut into individual strips 4 mm wide. Samples (45 μl) containing 0-10.0 μg/ml PDG were assayed in replicates of three. Test line intensity was evaluated with the Minolta system after 5 minutes, 10 minutes and 20 minutes.

Results of these procedures, presented in Table 1 and shown graphically in Figure 4, indicated that assay performance was substantially similar for immunoassay strips that incorporated the different desiccants when tested using samples having low PDG levels. More particularly, these results indicated that signal intensity was substantially stable for at least 10 minutes after initiating the assay. This was particularly true for tests conducted using samples having PDG concentrations of 0.0 and 1.0 μg/ml. However, we found that test strips having the thinner desiccant took substantially longer to dry when compared with the immunoassay strips that incorporated thicker pads. This slower drying characteristic of the test strips constructed with the thinner desiccant was attributed to the lower fluid capacity of the thinner pad when compared with the thicker pad. Although the thin desiccant pad clearly could be used to construct immunoassay strips that gave good results, we chose to conduct additional experiments using only the thick desiccant material.

Table 3

Quantitative Comparison of Thick and Thin Desiccant Pads in Immunoassay Test Strips

(Values expressed as the change (delta) of relative reflective units (dE))

ΨM£mim®t& M

0 μ δlJ % μgM & My WM ugM έ -ik Hϊls t $ tϋe& tϋfϊ ϊhtek $? §!. s tøM w$. $$3$< m& «ss$. ,m& S*|» •r*w*- **tfe : $f$.

5 0.18 0.09 0.76 0.95 1.89 2.17 3.23 5.08 8.01 6.85

10 0.20 0.26 0.90 0.64 2.09 2.65 3.72 5.96 8.62 7.12

20 0.43 0.06 1.06 1.05 3.16 2.49 5.20 588 10.51 8.47

Example 5 describes the procedures used to investigate possible negative effects of the desiccant pad on immunoassay test performance.

-11- Example 5

A Desiccant Absorbent Zone Does Not Adversely Influence Flow Time for the Immunoassay Strip Immunoassay strips were prepared by spotting nitrocellulose with PDG-BIgG (2.0 mg/ml) and Goat anti mouse IgG (0.5 mg/ml), and blocking with mBSA according to the procedure described under Example 1. Sample pads were saturated with 35-40μl/cm² of 0.5% Zwittergent in mBSA, while labeling pads were saturated with 35-40μl/cm² of a 1/1 0 dilution of antibody-latex stock solution (approximately 1.2% Bangs C1 red) in mBSA. Pads were then lyophilized. Test panels were assembled using the lyophilized sample and labeling pads, the blocked nitrocellulose and the SG-145 desiccant pad as described above. Panels were cut into strips 4 mm wide and individually pouched. 125 pouches each were then stored at 25EC, 37EC or 45EC. On given test days, the pouches were equilibrated to room temperature before opening, and strips in replicates of three were used to conduct assays with samples (45 μl) containing 0.01 μg/ml, 1.0 μg/ml, 5 μg/ml and 10.0 μg/ml PDG in a phosphate buffered 0.1% gelatin solution. Flow time, measured as the time required for liquid to reach the desiccant pad, was recorded and the intensity of the test line was evaluated visually.

Results presented in Table 2 showed that the flow time was unchanged for a period of 28 days when individually pouched units were stored at 25EC. However, the flow time of the assay increased when the pouched strips were stored either at 37EC or 45EC for 28 days. Visual observation indicated that the signal intensity at the test line was slightly greater as the flow time increased. In other experiments we observed that immunoassay strips constructed to incorporate a standard paper absorbent zone also gave increased flow times when stored at 37EC or 45EC. In aggregate, our results showed that the presence of a desiccant pad in immunoassay strips such as those described herein did not adversely influence flow time in the assay.

-12- Table 2

Stability of Immunoassay Strips that Include a Desiccant Pad When Stored at Various Temperatures

Test Day Flow Time (time for liquid to reach end pad in seconds)

(11=12) stored at 25EC avg. stored at 37EC avg. stored at 45EC avg.

0 45.3

3 44.5 49.4 49.5

7 43.0 49.5 62.9

12 42.4 56.6 71.8

14 43.9 57.4 75.0

17 44.3 54.6 75.6

21 43.3 627 74.8

28 44.9 65.9 88.7

Example 6 describes the procedures used to assess stability of immunoassay strips that incorporated a desiccant as the absorbent zone of the device.

Example 6 Stability of Signal Intensity for Desiccant-Contaiπing Immunoassay Strips Subjected to Different

Storage Temperatures

Capture nitrocellulose was prepared as described in Example 4 except that rabbit anti-glucose oxidase was used at a concentration of 0.5 mg/ml. Sample pads were prepared using 0.3% Zwittergent/0.1 % PVP-Stabilcoat in mBSA.

Labeling pads were saturated with 35-40μl of a 0.0067% antibody-latex, 1/300 glucose oxidase latex, and 0.1 % PVP- Stabilcoat solution in mBSA. Sample and labeling pads were then lyophilized. Test panels were assembled, cut and pouched as described above and then stored at three test temperatures (25EC, 37EC or 45EC) for up to 8 weeks. On given test days, strips were equilibrated to room temperature and used to perform immunoassays using male urine spiked with

PDG at various concentrations. The PDG-containing samples (45 μl) were added to test strips in replicates of three. Test line intensity was measured with the Minolta optical reading system 10 minutes after initiating the immunoassay. The results presented in Figures 5-7 indicated that signal intensity at the lines in the capture zone did not substantially change over the test period for immunoassay strips stored either at 25EC or 37EC. Although there were variations in line intensity for a given PDG concentration, the overall performance as measured by test line intensity after

-13- 56 days was not significantly different from the intensity measured at day 0. Strips stored at 45EC showed a slight decrease in test line intensity for samples having 5 μg/ml PDG after 3-5 weeks of aging, and for 10 μg/ml PDG after 3 weeks. There was no change detectable after 56 days for samples having 3 μg/ml PDG. For both 0.0 μg/ml PDG and 1.0 μg/ml PDG the line intensity increased for immunoassay strips that had been stored for 3-5 weeks. Immunoassay strips stored at 45EC gave stable levels of signal intensity for at least 14 days. These findings showed that stable signal intensities could be obtained using immunoassay strips that incorporated a desiccant absorbent zone and that were stored at 37-45EC for extended periods. Although the signal intensity varied somewhat for immunoassay strips stored at 45EC, the intensity was relatively stable for up to two weeks under these conditions.

Whereas the assay used in all of the procedures described above detected PDG, the second immunoassay used to test the utility of a desiccant paper absorbent zone detected hCG. Moreover, this second immunoassay was run with the strip positioned in a vertical, rather than a horizontal orientation. Since the direction of sample flow along the immunoassay strip was opposite the direction of gravity, the hCG immunoassay allowed us to investigate parameters different from those tested with the PDG immunoassay. Despite these differences, the hCG immunoassay described below is a rapid assay format, having only a one-minute read time. Example 7 describes the procedures used to investigate the performance characteristics of immunoassay strips used in vertical and horizontal orientations.

Example 7 Vertically Oriented Immunoassay Strips Exhibit Improved Properties The sample pad was prepared by saturating a pad of non-woven acrylic fiber adhered to 4 mil polyester with a buffered solution containing carrier and blocking proteins. This saturated pad (30 cm x 30 cm) was freeze dried and cut into 1.1 cm x 30 cm strips. The label pad was prepared by saturating a pad made of non-woven acrylic fiber adhered to 4 mil polyester with the label solution. This solution consisted of dyed anti-hCG antibody (the red test label), Blue Latex coated with Glucose Oxidase (blue control label), and carrier protein in a 50 mM Tris buffer. This material (30 cm x 30 cm sheets) was lyophilized and cut into 1.1 cm x 30 cm strips. The capture membrane was prepared by applying a polyclonal anti-hCG antibody (Test Line) and a polyclonal anti-Glucose Oxidase antibody (Control Line) with a displacement pump onto a 30 cm x 30 cm sheet of 8 micron nitrocellulose (Sartorius Corp, Goettingen Germany).

The membrane was blocked with a solution of carrier protein and laminated to 4 mil polyester with an acrylic based adhesive. The laminated membrane was cut into 2 cm strips (parallel to the spotted lines) with the test and control lines centered in each strip. These materials were separately laminated with the following adsorbents to form test panels: Ahlstrom ED 939 (Ahlstrom Filtration, Mt. Holly Springs, PA), SG-145 or SG-146 (Multiform Desiccants, Inc.). The process for lamination and cutting of test panels to form individual immunoassay strips was as described in the #2795 protocol Label Pad strip was placed into the machine with the mylar backing facing down. Absorbent strip (2 cm x 30 cm) material was places into the machine. A gate was then moved over the label and absorbent strips. This permitted the placement of

-14- the sample pad strip such that the mylar backing faced up and overlapped the label pad by 1 mm. Then the spotted capture strip was placed into the gauge so that the mylar backing faced up and overlapping of 1 mm on the label pad and absorbent occurred.

A plastic (PVC) backing material was secured to a swinging platen via vacuum. This backing had an area coated with adhesive. The gate aligning the various components was removed (carefully to ensure maintenance of overlap) and the platen with the PVC backing was "swung" onto the overlapped materials. The adhesive on the backing secured all materials into place. A cover tape was applied over the absorbent for cosmetic appearance. Another tape was placed over the label pad, overlapping slightly onto the capture membrane and sample pad. This tape contained a set of arrows indicating the depth to which the strip should be placed in urine to perform the assay. An hCG standard was prepared by diluting a stock solution of hCG to 100 mlU/ml in phosphate buffered saline/bovine serum albumin (PBS.BSA).

Assays were separately performed using both vertical and horizontal formats. According to the vertical format, dipsticks made from the immunoassay strips described above were added to a test tube that contained the hCG standard. Care was exercised to ensure that the level of the liquid sample did not extend beyond the "dip line" marked on the immunoassay strip. As is known, the dip line indicates the depth at which the test strip should be immersed in urine. It is set at a level on the sample pad to permit absorption and flow of urine without interfering with the function of the other test strip components. According to the horizontal format, dipsticks were "dipped" for ten seconds into the hCG standard and then placed flat on a horizontal surface. For both assay formats we: (a) measured the time required for the appearance of a visually detectable signal at the test line; and (b) quaπtitated the signal intensity of the test and control lines using a Gretag Densitometer five minutes after initiating the assay. Values for the signal intensity are reported herein as Gretag Density Units (GDU).

The results presented in Table 3 indicated that superior assay performance was obtained in both the vertical and horizontal formats of the immunoassay when a desiccant was used as the absorbent. The immunoassay strip constructed using the thin desiccant paper produced better test and control line intensities in both the vertical and horizontal assay formats when compared with strips that incorporated the standard paper absorbent zone. In the horizontal assay format the thin desiccant paper also gave superior results when compared with the standard paper absorbent zone. Dipsticks constructed with the thick SG-145 desiccant paper were not evaluated in this horizontal system because no difference was observed in the vertical assay when compared to the control. These results confirmed that lateral flow or flow- through immunoassay devices that incorporated a desiccant advantageously provided superior operating performance when compared with standard devices that did not incorporate a desiccant.

-15- Table 3

Comparison of Vertical Immunoassay Results

Vertical Assay

Absorbent Type lOO mlU/ml FIT Positive Horizontal Control (seconds) Assay 100 GDU

MlU/ml Test

GDU

Ft Positive Test GDU Control GDU (seconds)

Standard (Paper) 30 0.1 0.24 42 0.06 0.27

Desiccant (Thin) 33 0.13 0.29 40 0.14 0.29

Desiccant (Thick) 29 0.09 0.25

Example 8 describes the procedures used to investigate whether the paper desiccant which functioned as the absorbent zone of an immunoassay strip could substitute for a separate desiccant that conventionally maintained the functional integrity of the strip.

Example 8

The Desiccant Paper Absorbent Zone Circumvents the

Need for a Separate Desiccant We next conducted a study to determine whether an immunoassay strip constructed to include a desiccant paper absorbent zone such as that described in the preceding Example would remain stable when packaged in an air-tight container in the absence of a separate desiccant. As part of this investigation we tested two different paper desiccants purchased from different vendors. More specifically, immunoassay strips were constructed essentially as described under

Example 7 except that the absorbent zone was prepared either from SG-146 (Multiform Desiccants, Inc.) or from Whatman paper desiccants. The resulting immunoassay strips were sealed into plastic bottles (100 strips per bottle). Control immunoassay strips were prepared using absorbent material without desiccant to form the absorbent zone, and packaged together with two sachet desiccants containing silica gel. Other test and control strips were sealed in foil pouches.

Control strips were packaged together with a molecular sieve desiccant "pill." Test strips were sealed in the pouch without desiccant. The bottles and pouches containing the test or control strips were stored either at room temperature, at 37EC with 75% relative humidity or at 45EC with ambient humidity. At specified intervals immunoassay strips were removed from the specified storage condition and used to perform assays using samples having 25 mlU/ml and 100 mlU/ml.

The results presented in Tables 4-6 and in Figures 8 and 9 indicated that, at the 12 week time point the paper desiccants from both of the commercial sources forming the absorbent zone on the test strip were functionally equivalent to a desiccant sachet or "pill" separately packaged with the strip. In the Tables, results are presented for the standard paper desiccant, Whatman and Multiform desiccants, and the QUICK VUE product commercially available from Quidel

-16- Corp., as a control. Also presented in the Tables, "GDU" represents Gretag Density Units, as indicated above. As indicated by our results, the time required to produce a positive result in the assay, and the intensity at the test and reference lines for the two paper desiccant constructions were consistent with results obtained using the pill or sachet desiccant. Our accelerated stability - shelf life protection model predicted a product shelf life of two years for individually pouched strips. These results indicated that immunoassay strips constructed to incorporate a desiccant paper absorbent zone could be packaged without a separate desiccant without compromising functional integrity of the strip. Moreover, these results were obtainable with different desiccant papers.

Table 4 Stability Testing: Room Temperature

Week Week l Week Week Week Week Week Week Week

0 2 3 4 5 6 8 12

CONDITION

Standard

25mlU/ml 55.5 55 63 63 50 51 55.5 54 63

100mlU/ml 36 35.5 43 31 29.5 34.5 34 33.5 34.5

GDU Control 0.275 0.265 0.275 0.265 0.27 0.255 0.255 0.305 0.29

GDU Test 0.22 0.175 0.15 0.195 0.175 0.17 0.165 0.21 0.195

Whatman

25mlU/ml 59 60.5 60.5 56.5 47.5 54 46.5 53.5 57

100mlU/ml 32 36 35 31 28 40 35 34.5 39.5

GDU Control 0.295 0.265 0.23 0.275 0.28 0.27 0.29 0.375 0.31

GDU Test 0.185 0.21 0.205 0.21 0.23 0.18 0.225 0.26 .21

Multiform

25mlU/ml 58 58 54.5 56.5 50.5 58 60.5 50.5 60

100mlU/ml 38.5 34.5 35 32.5 38.5 34.5 37 35.5 37.5

GDU Control 0.23 0.275 0.305 0.33 0.33 0.265 0.31 0.38 0.265

GDU Test 0.195 0.195 0.19 0.185 0.235 0.18 0.235 0.25 0.19

QUICK VUE

25mlU/ml 60 74 91 74 67 79 81 56.5 101.5

. OOmlll.ml 39.5 38.5 44.5 36 46.5 48.5 54 41.5 48

GDU Control 0.14 0.15 0J5 0.15 0.17 0.14 0.14 0.12 0.135

GDU Test 0.075 0.07 0.07 0.07 0.06 0.055 0.065 0.062 0.065

-17- Table 5

Stability Testing: 37EC/ 75% Relative Humidity

Week l Week 2 Week 3 Week 4 Week 5 Week 6 Week Week

8 12

CONDITION

Standard 25mlU/ml 55 63.5 68 62.5 63 64 56.5 79 .OOmlU/ml 35.5 40 42.5 53 43.5 39 42.5 53.5 GDU Control 0.28 0.315 0.28 0.285 0.22 0.16 0.25 0.25 GDU Test 0.195 0.185 0.195 0.2 0.165 0.14 0.19 0.135

Whatman 25mlU/ml 54 59 68.5 55 58 66.5 57 79.5 lOOmlU/ml 33.5 37.5 34 43 39.5 48.5 38.5 54.5 GDU Control 0.275 0.25 0.255 0.315 0.215 0.29 0.28 0.21 GDU Test 0.19 0.205 0.19 0.23 0.125 0.175 0.18 0.115

Multiform 25mlU/ml 55 71 58 55.5 66.5 66.5 56.5 73 100mlU/ml 36.5 41.5 39.5 39.5 42 40.5 36.5 60 GDU Control 0.285 0.255 0.31 0.27 0.255 0.305 0.285 0.23 GDU Test 0.16 0.165 0.165 0.16 0.175 0.23 0.175 0.12

QUICK VUE 25mlU/ml 82 99.5 82 67.5 84 81 74.5 102.5 lOOmlU/ml 40.5 42.5 46.5 38.5 44.5 51 49 56 GDU Control 0.145 0.16 0.165 0.15 0.17 0.15 0.15 0.135 GDU Test

0.045 0.065 0.05 0.08 0.05 0.05 0.06 0.06

Example 9 describes the procedures used to measure uptake of moisture for immunoassay strips constructed using either standard or desiccant materials as the absorbent zone.

Example 9 Uptake Studies Moisture uptake studies were performed on five lots of Multiform and two lots of Whatman paper. The original lots of Multiform and Whatman served as controls. In one study, strips manufactured with the respective paper types (the standard absorbent, and Multiform or Whatman desiccant papers) were placed in a moisture chamber overnight and the weight gain was evaluated. In the second study, equal sized strips were weighed, placed in a humidity chamber for 24 hours, and then reweighed to determine the amount of moisture absorbed by each. Strips manufactured with the standard absorbent paper gained an average of 3.75g/10 strips (0.375g/strip). The average weight gains for strips manufactured with the Whatman absorbent paper was 3.46 g and 3.34 g (2 lots). These

-18- strips absorbed less moisture than did the standard strips. Units made with Multiform absorbent paper gained an average of 4.15 g and 4.5 g (2 lots), indicating that the capacity to absorb moisture exceeds both Whatman and absorbent paper.

The average (5 lots) water uptake from a 13/16"x2' swatch of Multiform absorbent paper was 13.84% by weight. The average (2 lots) gain for Whatman paper was 3.09%. Clearly, the capacity for the Multiform desiccant paper to absorb water is greater than that of Whatman paper.

The aforementioned performance, stability and uptake studies suggest that the production of devices containing one-step immunoassay strips can include a one strip-pouched format. The Multiform desiccant paper, which replaced the standard paper absorbent, provided adequate protection from moisture. This type of product will have a projected shelf life of two years. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.

-19-

Claims

WHAT IS CLAIMED IS:

1. An assay device for detecting the presence or absence of an analyte in a liquid sample, comprising: a substantially planar matrix having; a sample receiving zone for receiving the liquid sample; a labeling zone in fluid communication with the sample receiving zone, said labeling zone comprising a mobile agent for binding the analyte; a capture zone in fluid communication with the labeling zone, said capture zone comprising an immobile agent capable of binding the analyte; and an absorbent zone in fluid communication with the capture zone, wherein said absorbent zone comprises a desiccant material.

2. The assay device of Claim 1, wherein the desiccant material comprises an activated silica gel.

3. The assay device of Claim 2, wherein the desiccant material further comprises a semi-rigid cellulose fiber matrix.

4. The assay device of Claim 3, wherein the activated silica gel is present in an amount of between 1% and 100% by weight of the desiccant material.

5. The assay device of Claim 3, wherein the activated silica gel is present in an amount of between 15% to 75% by weight of the desiccant material.

6. The assay device of Claim 1, further comprising a housing that contains said substantially planar matrix.

7. The assay device of Claim 1, wherein the absorbent zone also functions as a handle.

8. The assay device of Claim 1, sealed within an air-tight packaging material.

9. In an assay device having an absorbent zone for absorbing liquid, wherein the improvement comprises: a desiccant material comprising the absorbent zone.

10. The assay device of Claim 9, wherein the desiccant material is an active silica gel.

11. The assay device of Claim 10, wherein the desiccant material further comprises a semi-rigid cellulose fiber matrix.

12. The assay device of Claim 11, contained in an air-tight container that does not also contain a separately packaged desiccant.

-20-