WO1995032414A1

WO1995032414A1 - Qualitative surface immunoassay using consecutive reagent application

Info

Publication number: WO1995032414A1
Application number: PCT/US1995/006330
Authority: WO
Inventors: Bingnan Ma; Arepura Gopalaiengar Govindarajan; Victor Y. S. Liu; Tay-Yean Lin
Original assignee: Chemtrak, Inc.
Priority date: 1994-05-20
Filing date: 1995-05-19
Publication date: 1995-11-30

Abstract

Diagnostic devices, and methods for their use in the rapid detection of antibody analytes are provided. The subject devices comprise an assay pathway having a wicking strip (10), a sample pad (12), a detection region (14) comprising at least one detection (16) and control (20) zone, and an absorption strip (22). The detection zone (16) provides an epitope of an antigen of interest for binding to the antibody analyte, while the control zone (20) provides for binding to non-specific antibodies in the sample. A conjugate of a receptor and colloidal particle is employed for detection, whose arrival at the detection zone (16) is subsequent to that of the sample, so as to allow for binding of the antibody in the detector (16) and control (20) zones and removal of non-specifically bound antibody from the detection zone (16). The presence of a signal in the detection zone (16) indicates a positive result.

Description

QUALITATTVE SURFACE IMMUNOASSAY USING CONSECUTIVE REAGENT APPLICATION

INTRODUCTION

Technical Field The field of this invention is diagnostic assays for antibodies.

Background

There is an extensive need for single diagnostic tests in a wide variety of situations. These situations include hospitals, doctors' offices, nursing homes, and the home. In each of these situations, the need will be to rapidly perform a test which is reliable. Frequently, untrained personnel will be called upon to perform the test. This means that the methodology should involve few, if any, measurements of materials, should minimize the number of reagents which have to be handled, and should provide for a simple readout, with a control reading to ensure the performance of the assay.

To achieve this kind of test, it is desirable that all the reagents be packaged together in a way that little, if any, technical capability is required for carrying out the assay. Thus, the assay must be developed in a way that the reagents are present in adequate amounts, their interaction is organized in such a way as to provide for an accurate result, and the reading of the result is simple, desirably visual, so that no additional equipment is required.

In developing such assays, there are many concerns. One must employ reagents which are stable under the conditions in which they are packaged and stored. Any moving parts must be reliable, so that they operate in an efficient and reproducible manner. In addition, the mixing of reagents must occur in such a way as to ensure that the desired reactions occur and that a correct signal will be obtained for a positive result. In determining whether there is an infection in a mammalian host, one may look at either components of the infectious agent or antibodies produced as a result of the immune response to the infectious agent. In many diseases, diagnosis will be at a time when the infectious agent has been present in the host for a sufficient period of time, so as to result in elevated immunoglobulins against the host, particularly elevated immunoglobulin G.

The overabundance of nonspecific antibodies in relation to disease specific antibodies in human test serum presents constraints on developing an immunoassay for specific antibody detection. The background due to nonimmunological adsorption of nonspecific immunoglobulins to solid phases seriously limits the specificity. Short incubation times may be insufficient for immunological reactions to equilibrate and inadequate washing of the solid phase to remove completely the nonimmunological adsorption severely hampers the sensitivity of the specific antibody detection immunoassay. The development of an antibody detection test has to overcome all the above difficulties. Therefore, there is an interest in the development of assay devices which allow for simple, efficient and rapid methodologies, employing devices which do not require technical competence for operation, to detect antibodies to a wide variety of pathogens. Ideally, the subject devices should be prepared from a minimum of components, so that manufacturing costs, such as the cost of individual components and their assembly into the final device, may be held to a minimum Relevant Literature

U.S. Patents of interest include: 4, 987,085; 4,959,324 and 5,260,221, which report a non-instrumented analyte detection device for the detection of serum analytes such as cholesterol. Other references of interest include: Allen et al. , A Noninstrumented

Quantitative Test System and its Application for Determining Cholesterol Concentration in Whole Blood. Clin Chem (1990) 31: 1591-1597; Ishikawa & Kohno, Development and Applications of Sensitive Enzyme Immunoassay for Antibodies: A Review. J. Clin 1Mb Analysis (1990) 3:252-265; Zuk, Enzyme Immunochromatography- A quantitative Immunoassay Requiring No

Instrumentation. Clin Chem 91985) 31: 1144-1150; Craft et a , Antibody Response in Lyme Disease: Evaluation of Diagnostic Tests. J infect Dis (1984) 149:789-795; Talley et al., Serodiagnosis of Helicobacter pylorii: Comparison of EnzymerLinked Immunosorbent Assays. J. Clin Microbiol ( 1991) 29: 1635-1639.

SUMMARY OF THE INVENTION Diagnostic devices and methods of their use in the detection of antibodies to one or more epitopes of an antigen of interest, particularly epitopes diagnostic of a pathogen, are provided. The device employs a source of buffer and an assay path comprising, in the direction of fluid flow, a wicking strip, a sample pad, a detection region comprising detection and control zones, and an absorbent zone. The detection region may be overlapping, or in proximity to, the sample pad. The device further comprises a conjugate of a receptor and a detectable label diffusibly bound to the wicking strip, where the wicking strip may further comprise a means for delaying conjugate flow. In preferred embodiments, the sample flows into the detection region prior to dilution with the wicking buffer.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a exploded diagrammatic view of an assay pathway; Figure 2 is a plan view of the organization of the parts of the assay pathway;

Figure 3 shows diagrammatic side and plan views of an alternative embodiment of the assay pathway; Figure 4 is a diagrammatic plan view of a second alternative embodiment of the assay pathway;

Figure 5 is a diagrammatic plan view of a third alternative embodiment of the assay pathway; and Figure 6 is a diagrammatic plan view of a baseplate and slide of a device incorporating an assay pathway according to any of the above figures.

Figure 7 is a plan view of the organization of the parts of the assay pathway of an alternative, embodiment of the subject device.

Figure 8 is a plan view of the organization of the parts of the assay pathway of a second alternative, embodiment of the subject device.

DESCRIPTION OF SPECIFIC EMBODIMENTS Diagnostic devices and methods of their use in the detection of the presence of antibodies to one or more epitopes of interest, particularly eptitopes of an antigen of a pathogen, are provided. In the subject devices, a buffer source comprising a transport buffer and labeled conjugate are present in conjunction with an assay pathway comprising, in the direction of fluid flow, a wicking strip, a sample pad, a detection region, and an absorbent zone. The detection region may be overlapping, in proximity to, or distal to the sample pad, where the detection region comprises at least one detection zone and one control zone. The substrates or bibulous materials used to make the various components of the assay path may be treated to enhance the rate of transport of the buffer medium and its contents, e.g. the conjugate. In using the devices to detect antibody analyte, sample is introduced into the assay path and, after a period of time, a signal indicative of the presence or absence of analyte is obtained. In further describing the invention, the components of the subject devices will first be described in greater detail, followed by a description of the various assays that may be performed with the subject devices.

The device will comprise a source of wicking buffer or transport solution, where the buffer provides for transport of the various mobile reagents of the assay, described below, down the assay path. The buffer source will be located at the beginning of the assay path and may have any convenient configuration which provides for storage of the buffer prior to use of the device and then release of the buffer at the appropriate time during the assay upon contact with the wicking strip. Configurations which may be employed include wells which may be opened, sealable pouches and the like. Of interest is the buffer source employed in U.S.Pat.No. 5,132,086, the disclosure of which is herein incorporated by reference. As will be described in greater detail below, at the appropriate time following introduction of the sample onto the sample pad, the buffer will be released from the buffer source and contacted with the wicking strip.

The assay path of the subject device is a combination of several components that combine to produce an assay path, down which fluid, e.g. buffer, flows. The first component of the assay path in the direction of fluid flow, the wicking strip, serves to contact the buffer solution or transport solution which serves to move the various components of the assay through the assay path. Various bibulous materials may be employed for the wicking strip, as well as the other strips or components of the assay path described below. Convenient bibulous materials include cellulosic materials, such as paper, glass fiber, silica on a support, alumina on a support, nylon, porous polyethylene and the like, where the particular choice of substrate will be chosen with respect to the desired rate of fluid flow through the material, e.g. for a faster desired rate porous polyethylene may be used and for a slower desired rate, paper may be used. Rates of flow are preferably in the range of about 10 to 40 mm/min, and more preferably in the range of about 15 to 20 mm/min. To enhance the rate of reagent flow down the assay path, e.g. conjugate flow, the bibulous material may be treated. It is found that where nitrocellulose or glass fibers are used as a porous or bibulous substrate for the assay and colloidal particles are used as the detectable label, particularly gold colloidal particles as described below, there is substantial interference with release of the conjugate from the substrate, when the conjugate is stored on the substrate, as well as slow movement of the conjugate through the nitrocellulose substrate during the assay. It is therefore found that, by treating the substrate, one can greatly enhance release of the conjugate from the storage substrate, as well as enhance the rate of transport through the bibulous substrate.

Treatment involves contacting the substrate with an aqueous composition comprising from about 0.2 to 5, more usually from about 0.25 to 2.5% w/v of protein and from about 1 to 7.5, preferably about 1.5 to 5% w/v of trehalose as the primary components of the aqueous medium. In addition, a small amount of a non-ionic detergent is employed, usually ranging from about 0.01 to 0.05% v/v, such as TWEEN^* 20, and an antimicrobial agent, conveniently sodium azide or Proclin-300, generally present in about 0.1 % w/v. The aqueous composition is usually adjusted to a pH of about 8-8.5.

In preparing the nitrocellulose or other substrate, the substrate is conveniently pre-wetted with phosphate buffered saline and the detection and control zones printed with the appropriate reagents. The substrate or strip may then be permitted to dry, conveniently at room temperature, followed by treatment with the solution described above. The treatment may involve incubation in the solution with mild agitation, e.g. rocking, for at least about 0.5 h, and generally not more than about 3 h. The membrane may then be removed from solution and dried under low humidity conditions, preferably below about 10%, preferably below about 7%, until completely dry. The strip is then ready for use. For the glass fiber substrates, the glass fiber may be incubated with mild agitation with the indicated solution, followed by drying at an elevated temperature, generally in the range of about 30-50°C.

The length of the wicking strip is not critical, with the choice of length providing sufficient time for sample to react with the zones in the detection region, where the length and width of the wicking strip can be selected so as to provide an appropriate rate of transport to the sample region upon initiation of the assay. Generally, the wicking strip will be relatively short, ranging from about 5 to 50 mm, more usually ranging from about 10 to 30 mm. In addition to serving as a conduit for buffer flow, the wicking strip will also store conjugate, where the conjugate will be diffusibly bound directly to the wicking strip or associated with a means for delaying conjugate flow, as will be described below.

Immediately downstream from the wicking strip in the direction of fluid flow is the sample pad. The sample pad may be shaped in any convenient shape, round, such as a disk, square, or other shape relevant to the transport of the liquid. Generally, the sample receiving region will be relatively small, usually having a surface area of from about 5-50 mm², usually not more than about 30 mm². Conveniently, the sample pad may be any of the various bibulous materials described above. In some embodiments where it is sufficient for the sample components to move with the buffer front to the detection region, e.g. in embodiments comprising a means for delaying conjugate flow, the sample pad may be fabricated from a bibulous material which does not provide for especially rapid transport of the sample through material, such as paper. In other embodiments where the sample is to move into the detection region prior to the buffer front, e.g. in embodiments where conjugate is diffusibly bound directly to the wicking strip and is not associated with a means for delaying conjugate flow, thereby requiring faster movement of the sample through the sample pad, a bibulous material which provides for rapid fluid flow, such as porous polyethylene, may be employed.

Of particular interest are devices which provide for the sample region to be moved from a first site, where it receives the sample, to a second site, where it is in fluid receiving and transferring relationship in the assay path. These devices will further comprise a means for moving the sample pad from a site out of registry with the assay path into registry with the assay path. See, particularly, U.S. Patent No. 5,132,086, which is specifically incorporated by reference in its entirety.

The next component of the assay path in the direction of fluid flow will be the detection region, which may overlap with the sample pad be proximal, or distal to the sample pad. The detection region will generally be of a size in the range of about 1.0 to 5.0 mm in width, and a length of about 5 to 20, more usually about 5 to 15 mm in length. Various nitrocellulose materials may be employed as available from Schleicher & Schuell, as well as other substrates as described above, such as glass fibers. The bibulous material will have pore sizes varying with the nature of the substrate, generally being in the range from about 2 to 20 μm pore size, with nitrocellulose being in the range of about 5 - 15 μm.

The detection region will comprise at least one first zone serving as the detection zone and at least one second zone serving as the control zone, where each zone will comprise an appropriate reagent or compound for capturing antibody analyte, and in the control zones immunoglobulin, present in the sample. The zones will generally be shaped in the form of narrow bands or stripes traversing the detection region. The narrow bands may be printed onto the detection region with convenient available instrumentation, such as the Bio-Rad SF microfiltration apparatus or the Nouvas liquid reagent dispensing machine. These bands will generally have a width of about .05 mm to 1.0 mm, and conveniently extend across the entire region, although this is not essential. The bands will be separated a convenient distance, so as not to interfere with each other, but allow for a reasonable visual comparison. Usually, the bands will be at least about 0.5 mm apart and not more than about 3 mm apart. After printing, the reagents in the bands will be adhered to the substrate by air drying. The concentration of the reagent will be selected to ensure that the visual reading is clear and sharp at the minimum concentration anticipated for the antibody analyte. Thus, the concentration per unit area and width of the band may vary depending upon the sensitivity required.

Each detection zone in the detection region will comprise a first reagent or capture compound, which will capture antibody specific for the target antigen. Any convenient reagent may be employed in the detection zone. All that is required for detection is that a molecule is available to serve as the capture compound which will specifically bind to the endogenous antibody analyte of the physiological sample with an affinity of at least about 10⁶, preferably with at least about an affinity of 10⁸. The compositions which are employed for detecting the antibodies may be the naturally occurring antigens, fragments thereof, synthetic organic molecules, sugars, nucleic acids, polypeptides, or other mimetics, which provide the desired degree of specificity and affinity. In some instances, where one is interested in the presence of any one or more isoforms, one may employ a common epitope for the different isoforms. By contrast, if one is only interested in a specific isoform, then the epitope should be specific for that isoform and be able to distinguish from the remaining isoforms.

Of particular interest is the detection of antibodies to pathogens, where the pathogens may be viruses, bacteria, protista, fungi, or the like. In each instance, one is only concerned with the ability to provide a compound which will specifically detect antibodies to the particular target. Organisms of interest include Borrelia burgdoiferi, H. pylori, enterobacteriaceae, H. influenza, Pseudomonas ss, M. tuberculosis, Streptococcus, Staphylococci, N. meningitidis, B. pertussis, C. diphtheriae, L. monocytogenes, Salmonella, Shigella, V. cholerae, M. leprae, C. albicans, A. fumigatus, hepatitis A, B, and C, HIV, HTLV, influenza virus, and other common pathogens.

Besides pathogens, there may also be other antibodies of diagnostic interest. In the case of transplants, one may be interested in whether the recipient has antibodies to the major histocompatibility complex (MHC) antigens of the donor. In cancer patients, where there is a general marker which is indicative of the presence of cancer cells in the host, one may perform assays for detecting the presence of antibodies to the particular marker.

The control zone or zones will comprise a second reagent or capture compound which will be a reagent which binds to a conserved region of at least one subclass of the family of antibodies recognized by the anti-antibodies of the conjugate. The conserved region may be the same or different for the reagent of the control region and for the conjugate, preferably different. Thus, one may use antibodies to the constant region of the antibodies of the host, proteins which generally bind to antibodies, such as S. aureus Protein A, or the like. The amount of capture compound can be varied in the control zone, so that the signal which is observed in the control zone can be related to the minimum signal which is a positive result in the detection zone. This can be empirically determined for each antibody analyte, so that a positive result in the detection zone could be at least as intense as the control zone or more intense.

There may be one or more detection bands or zones and control bands or zones, usually a single control band or zone, for simultaneous detection of a plurality of antibody analytes. Where a plurality of detection and control zones are present, the detection and control zones may be alternating or all of the detection zones placed sequentially followed by the control zone or zones, particularly where the control zones detect different immunoglobulin classes or subclasses. For example, if the analytes are different classes or subclasses of antibodies, e.g. IgG, IgM or IgA, one may wish to have the conjugate directed to the particular class or subclass of interest. In effect, each detection zone would have a different epitope directed to a different antibody specificity, and the one or more detection zones, could be related to specific classes or subclasses of antibodies. The control zones may then capture the particular class or subclass of immunoglobulin or be used to capture all classes and subclasses. One would normally ensure that the available antibodies are not significantly depleted by placing the control zones after the detection zones. The plurality of zones would be formed in the same manner as the individual zones and similarly spaced apart. Where the signals are comparable or the intensity of the detection zone is greater than the control zone as visually determined, the result is positive.

The detection and control bands or zones may be positioned at a variety of locations as the detection region. In one embodiment of the subject device, the zones may be positioned about one-half to two thirds the length of the strip serving as the detection region downstream from the end of such detection region adjacent to the sample pad site, i.e. distal from site of the sample pad in the assay. In another embodiment, the zones are positioned at an upstream site of the strip serving as the detection region, i.e. proximal to the site of the assay path occupied by the sample pad, particularly where the configuration of the device ensures that the sample reaches the detection and control zones prior to the buffer front and a rapid result is desired.

In another embodiment of the subject device, the detection region overlaps the sample pad. In this embodiment, the sample pad and the detection region are combined on a single strip or bibulous member component of the device. The sample pad retains the characteristics described previously, where the bibulous material making up the sample pad will generally be nitrocellulose or glass fiber. The final component of the assay path in the direction of buffer flow is an absorbent zone or strip which will serve to store the buffer and aid the flow of the buffer through the assay pathway, thereby ensuring that non-specifically bound antibody is removed from the detection zone. The absorbent strip size will be selected so as to ensure that the entire pathway may be wetted to an end point, which will indicate that the assay is completed. In addition, the absorption capacity of the absorbent strip will ensure that sufficient fluid must flow through the detection and control domains to ensure that the non-specifically or un-bound conjugate is washed from the detection zone, so as to minimize background. To ensure that sufficient fluid may flow through the detection region, the volume storage capacity of the absorbent zone will range from about 0.1 to 5.0 ml, usually from 0.3 to 1.0 ml. Conveniently, the absorbent strip will range from about 10 to 60 mm, more usually about 30 to 50 mm, and may have approximately the same width of the other bibulous members or membranes of the assay path. To enhance the storage capacity of the absorbent strip, the thickness may be varied from about 500 μ to 2,000 μ.

In addition to any reagents in the detection and control zones, the device will also comprise a conjugate which will provide for the detectable signal. Conjugates which may be used in the subject device will comprise a receptor capable of binding to antibodies of the host, where the receptor may be an antibody which binds to antibodies or another receptor capable of binding to antibodies, such as Protein G, usually an antibody specific to antibodies of the host, normally the conserved region of the antibodies, particularly the constant region. The anti-antibodies of the conjugate will usually bind, desirably specifically, for the various subclasses of IgG, IgA and/or IgM, where the antibodies may be specific for a single epitope or a plurality of epitopes, where the epitopes may be on the same or different subclasses or different constant regions. The anti-antibodies of the conjugate may be monoclonal antibodies or polyclonal antisera, preferably monoclonal antibodies. The antibodies will desirably be labeled with a directly detectable label. (By "directly detectable" is intended a label that does not require any additional reagents for detection, e.g. an enzyme is not directly detectable, since it requires a substrate, while a fluorescer or particle is directly detectable, since no additional reagent is required.) These labels can take many forms, such as colloidal particles, e.g. gold, selenium or graphite, fluorescers, dyes, or the like. Normally, the directly detectable label will be visually detectable without instrumentation, particularly colloidal particles. The gold colloidal particles are described in: Pauly, J. (1989), Advances in Colloidal Gold Technology in: Amer. J. Anatomy, Vol. 185: Issue 2 and 3; and will generally be of a size in the range of about 10 to 100 nm. Conjugation of the colloidal particle to the anti-antibody can be readily achieved by mixing the gold solution with the anti-antibody solution, incubating the mixture and then subjecting the mixture to centrifugation. As for the other labels, they have found general use and extensive description for their conjugation in the literature and do not require description here.

The anti-antibodies of the conjugate can be readily prepared in accordance with conventional ways, using the host antibodies as immunogens. The antibodies may be injected into a species different from the antibody species to be detected and one or more booster shots employed. When a sufficiently high titer has been achieved, the spleen may be removed from the host and the splenocytes immortalized and screened for antibodies specific for the constant regions of interest. See, for example, Antibodies: A Laboratory Manual, eds. Harlow and Lane, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1988.

The conjugate will be positioned in the subject device at a convenient site so that the conjugate arrives at the detection and control zones after the sample comprising the antibody analyte has substantially reacted with the detection and control zones. The amount of reaction which should occur before buffer comprising conjugate is contacted with the detection zone can be readily determined, but usuaually at least about 10% of the sample should have reacted with the detection zone prior to contact with buffer comprising conjugate. It is desirable to have the conjugate arrive at the detection and control zones after antibody analyte so that the presence of the bulky conjugate does not interfere with the binding of the antibody to the reagent in the detection and control zones. In embodiments where the sample front moves through the detection and control zones before the buffer front, the conjugate may be diffusibly bound directly to the wicking strip, either along the entire strip or in a specific region thereof.

In other embodiments, to ensure that the conjugate flows through the detection and control zones after the sample components, the device may further comprise a means for delaying conjugate flow through the assay path. One means of delaying conjugate flow is to include on the wicking strip a storage substrate comprising the conjugate, e.g. a glass fiber substrate. The storage substrate will generally be present on the wicking strip proximal to the site of the assay path occupied by the sample pad. Where the conjugate is stored on a glass fiber substrate, the substrate may be treated as described above, where the amount of protein and trehalose will be in the upper portion of the range as compared to the treatment of the strips of the device, e.g. the detection strip fabricated from nitrocellulose. For example, with a nitrocellulose membrane, the solution may have from about 0.1 to 1% w/v of protein and from about 1 to 3% w/v of trehalose, while with the glass fiber conjugate storage strip, the amount of protein will generally be in the range of about 1 to 3% w/v and the amount of trehalose will generally be in the range of about 3 to 6% w/v. Various proteins may be used, conveniently bovine serum albumin and casein hydrolysate are illustrative. For the glass fiber substrate, desirably a combination of these two proteins is employed, where the ratio will be in the range of about 0.5 -2: 1. When buffer contacts the storage substrate present on the wicking strip, it wicks up into the substrate and thereby releases the stored conjugate into the assay path. Since, during this time, the buffer front has continued wicking down the assay path, by the time the conjugate enters the assay path, the sample components carried on the buffer front will have already contacted the detection region.

Instead of a storage substrate positioned on the wicking strip, the means for delaying conjugate flow may comprise a narrower or restricted passage strip extending from the wicking strip to the site prior to the detection zone, where the restricted passage strip comprises the conjugate. The restricted passage strip may be made of any convenient material, where the transport of fluid through the material is slower than the transport of fluid through the membrane strip. For example, glass fibers, various chromatographic materials, or the like may be employed which will generally impede the flow of fluid and the transport of the conjugate through the restricted passage strip. The flow should not be unduly impeded, so as to unduly extend the time for the assay. Therefore, one may rely solely upon the narrower width of the restricted passage strip for the difference in rate of the transport of the sample to the detection zone in comparison to the rate of transport of the conjugate to the detection zone. Since the buffer flows through the restricted passage strip at a slower rate than through the other strips in the assay path, the conjugate will arrive at the detection zone after the buffer comprising the antibody analyte. Alternatively, the means for delaying conjugate flow may comprise a barrier to fluid flow positioned on the assay path e.g. on at least one wing adjacent to the flow path. When the wicking is initiated, the main flow of the wicking buffer wicks along the central area into the sample region, where components of the sample are then transported into the detection strip. The antibody analyte will then bind in the detection zone and other antibodies in the control zone. At the same time, non-specifically bound antibody will be washed away. During this period, the wicking buffer also wicks into the winged areas and releases the conjugate in an eddy current. The conjugate will then slowly enter the main stream of the buffer. The conjugate will then follow behind the sample components and arrive at the detection and control zones when the two zones are substantially free of non-specifically bound antibody. The conjugate may then bind to any antibody in the detection and control zones, with excess conjugate also washed away, minimizing the background in the two zones.

Alternatively, the means for delaying conjugate flow may comprise a barrier to fluid flow positioned on the assay path, e.g. a groove, where conjugate must flow on eddy currents to move beyond the barrier. Other barriers may be used in place of the groove, such as an aqueous impervious coating, e.g. wax or hydrophobic polymer, compressed membrane, or the like. The only limitation on suitable barriers is that the mainstream of the buffer will move around the barrier and that the conjugate will be transported into the main stream by an eddy current, substantially slowing the rate of introduction of the conjugate into the buffer mainstream. The subject devices find use in methods for detection of any antibodies in a mammalian host, particularly a human host. Thus, by employing a specific binding member which can compete with the antigen which elicited the antibodies in the physiological sample, one can detect whether there are antibodies in the physiological sample to a particular target. The target may take many forms, such as epitopes of pathogens, toxins, major histocompatibility complex antigens, mutant proteins, or the like.

For the most part, the sample will be a complex mixture of antibodies, both as to subclass and idiotype, particularly physiological fluids, such as blood or derivative thereof, e.g. plasma or serum, saliva, and the like. In some embodiments of the device, particularly those comprising a means for delaying conjugate flow, the sample will not be subject to pretreatment, particularly filtration, which may be involved in the removal of red blood cells from blood, and dilution. In preferred embodiments, particularly those embodiments not requiring a means for delaying conjugate flow where the sample reaches the detection and control zones prior to the buffer front, pretreatment of the sample may not be required, i.e. the sample may be introduced directly into the assay path in native form, e.g. whole blood or neat serum. Thus, one can directly assay an untreated sample and thereby reduce the number of procedures in the assay. In carrying out the assay, the method involves applying the sample to the sample pad, where the antibodies in the sample, are transported to a detection zone of the detection region through a bibulous member by capillary action and/or by means of a transport medium, e.g. buffer. As the sample passes through the assay region, antibodies specific for the antigen of interest will be captured in the detection zone, remaining in the detection zone. Antibodies which are not captured, which may include antibodies specific for the antigen of interest, will be transported to the control zone(s), where they may be captured.

Following introduction of the sample to the sample pad, the buffer or transport solution will be released from the buffer source. The period of time from introduction of the sample onto the sample pad to the release of buffer from the buffer source will be one which is sufficient for the sample to saturate the sample pad and for substanial reaction between the sample components and assay reagents, where present on the sample pad, to occur, where "substantial reaction may be readily determined. Usually the time between introduction of the sample and the release of the buffer from the buffer source will range from 15 sec to 2 min, more usually from 30 sec to 2 min., where longer times may be employed when reaction between the sample components and reagents present on the sample pad, such as in the device embodiment where the sample reacts in the detetion region prior to dilution with buffer.

Once buffer is released from the buffer source, it will wick down the assay path through the various components, thereby transporting any reagents and sample components it contacts down the assay path towards the absorbent zone. Depending on the particular embodiment of the device, the sample may have already flowed through the detection region before the buffer front. In other embodiments of the device, such as the ones employing a means for delaying conjugate. flow, the sample may reach the detection region at the same time as the buffer front. In this manner the sample components first reach the detection region, where binding of antibody analyte, if present, to the detection zone occurs. Following this reaction, buffer comprising the conjugate then flows through the detection region, whereby conjugate binds to any antibody present in the detection and the control zones. By providing for a continuing flow of buffer after conjugate has passed through the detection region, non-specifically bound conjugate is removed from the detection region, thereby minimizing background signal.

After conjugate flows through the detection and control zones and non- specifically bound conjugate is removed, one is then able to compare the signals from each of the detection zones with each of the control zones. If desired, one may use an instrument to determine the relative intensity, such as a densitometer for colloidal particles, a fluorimeter for fluorescent labels, a reflectometer for a colored label, etc. Generally, assay times (the time between sample addition and the development of the detectable signal) will range from 2 to 20 min., more usually 2 to 15 min. In some embodiments, where the sample front reaches the detection and control zones prior to the buffer front and a means for delaying conjugate flow is not required, the assay times will generally range from 2 to 5 min. , more usually from 2 to 4 min.

In accordance with the subject invention, antibodies to any particular epitope of interest in a physiological fluid can be determined readily by the subject methodology, regardless of the particular antigen of interest.

For further understanding of the invention, the drawings will now be considered. In Figure 1 is an exploded view of the various parts of one embodiment of the assay pathway, while in Figure 2 is a plan view of the assembled parts. In Figure 1, A is the wicking strip 10, B is the sample pad 12, C is the nitrocellulose membrane detection region 14, and D is the absorbent zone 22. E and F are laminating plastic sealers, 24 & 26 respectively, to enclose a portion of the assay pathway. The detection strip 14 and absorbent strip 22 may be encased in an upper sealing film 24 and a lower sealing film 26, to hold the strips in position and maintain their relationship. The various strips will generally overlie one another by at least about 0.05 mm, and not more than about 5.0 mm, usually not more than about 3.0 mm, to ensure efficient fluid transport from one portion of the assay pathway to another. The assembled components of the assay pathway will usually be placed on a plastic support 31, where adhesives may be employed to ensure the immobility of the various portions. The wicking strip 10 will extend past the plastic support so as to be capable of being dipped into the buffer solution. The entire strip may serve as a stick, where the sample may be placed on the sample pad 12, e.g. a drop of blood, followed by dipping the wicking stick into an appropriate buffer. A nitrocellulose storage member 32 stores the conjugate. Detection zone 16 and control zone 20 are halfway downstream from the sample pad on the detection strip.

To further ensure that there is no reaction, or substantially no reaction, of the conjugate with antibody analyte prior to the reaction of the antibody analyte in the detection zone, a number of alternative structures are provided. In Figure 3 an alternative embodiment is provided, where the wicking strip 10 supports a narrower or restricted passage strip 34, which strips are separated by an inert barrier 42. The conjugate 36 is coated onto the narrower strip 34 at a site 36 proximal to the upstream end of the strip. The ends of the narrower strip 34 will be in fluid transfer contact with the wicking strip 10, so that as the buffer moves through the assay pathway, both the wicking strip 10 and the narrower strip 34 will be wetted and movable components on the two strips transported. The device may be readily assembled by placing the narrower strip 34 on top of the detection region strip 10, where the two strips are separated by inert barrier strip 42, and by using a cover film ensuring the positioning of the two strips in relation to one another.

In Figure 4, an alternative embodiment is depicted to provide for delayed transport of the conjugate. In this embodiment, the wicking strip 10 has dual wings 44 on which the conjugate 46 is coated. An alternative design is depicted in Figure 5, where the wicking strip 10 has a barrier, depicted as an arrowhead groove or cutout 50. The conjugate 52 is coated behind the cutout 50. When the wicking is initiated, the main flow of the wicking buffer bypasses the arrowhead-shaped grooved area 50 and wicks along through the wicking strip 10. During this time, the wicking buffer will also wick behind the arrow-shaped groove and release the conjugate 52, by means of an eddy current. The conjugate will then move around the groove and enter the buffer main stream which bypasses the groove. As indicated previously, the conjugate will then arrive at the detection zone after the sample components have passed through the detection and control zones, so that the two zones will be substantially free of non-specifically bound antibodies.

Of particular interest is the use of the device described in U.S. Patent No. 5,132,086, which is depicted in Figure 6 in substantial part. The base plate 60 supports a slide 62. The base plate 60 consists of a cutout to accept the slide 62 and further comprises a slot 64 with locating pins 66 to position the assay pathway strip 70. The assay pathway strip may be serrated or not-serrated. Serrating appears to improve the linear movement of the buffer front, which is desirable but not necessary for the subject invention. The detection region strip 14 is positioned so as to be separated from the wicking strip 10, where completion of the assay pathway is achieved by moving the sample pad 12 into the gap 72 between the detection region strip 14 and the wicking strip 10. A well 44 is designed to receive the buffer solution, which may be enclosed in a packet positioned in the well 44. Upon opening of the packet, the wicking strip 10 which extends into the well 44 will be wetted and flow of the buffer in the assay pathway will begin.

The slide 62 consists of a vented receptor site 76 into which the sample pad 12 is inserted. The slide also has an arm 80 with shearing action designed to facilitate the release of the buffer solution from the buffer solution pouch or source which is housed in well 44. There is also a snap 82 to lock the slide in place, once pulled.

Above the sample pad may be one or more filtration devices for blood samples. The filtration devices serve to filter out red blood cells, which may dye the detection zones, obscuring detection of the detectable signal. In carrying out the assay, the slide is in a first position, where the sample pad 12 is removed from the gap 72. A drop of blood may be placed over the sample pad and will be absorbed by the sample pad. The volume of sample absorbed by the sample pad is not critical to this invention, since one is primarily interested in the presence or absence of the antibody analyte. However, if semi- quantitation is desired, the pad can serve to control the volume of sample and by providing for a squeegee action as the sample pad is moved from its original position to the gap 72, fairly accurate volumes can be measured in this way.

Once the sample 12 is impregnated with the sample, the slide 62 is then pulled to bring the sample pad into gap 72 and complete the assay pathway, where the sample pad will overlap the wicking strip 10 and the detection region strip 14, so as to complete the fluid transport pathway. At the same time, the shearing action of the arm will open the pouch and release the buffer solution into the well. The wicking strip 10 will become wetted by the buffer solution and the assay will begin with movement of the fluid front through the assay pathway.

Mobile sample components will move with the buffer solution into the detection zone 16 and the control zone 20, where antibody analyte will become bound in the detection zone 16 and non-specific antibody will become bound in control zone 20. With continued flow of the buffer solution, antibody which has not become specifically bound will be washed away from the two zones. Meanwhile, the conjugate will be moving up the assay pathway and will arrive at the detection and control zones, so as to become bound appropriately in these zones in accordance with the presence or absence of the antibody analyte and other antibodies. Continued flow of the buffer solution will remove non-specifically bound conjugate so that by the time the buffer solution front meets a predetermined point in the absorbent zone or strip 22, the assay will be completed, with the detection and control zone substantially free of any non-specifically bound conjugate. One can provide for a color reaction or other signal which indicates completion of the assay. At this point, these zones may be visually read, where the concentration of reagents at both the detection zone and control zone will provide that if the intensity of the detection zone is equal to or greater than the intensity of signal at the control zone, the result will be positive.

In Figure 7 is a plan view of the assay path a preferred, alternative embodiment of the subject device, which provides for rapid flow of buffer and sample through assay path. In this embodiment, wicking strip 10 and sample pad 12 are fabricated from porous polyethylene. The conjugate is diffusibly bound directly to the wicking strip. The detection zone 16 and the control zone 20 of the detection region are positioned near the upstream end of the glass fiber strip (Ahlstrom F-lll or other glas fibers) strip serving as the detection region 14, i.e. proximal to the sample pad. The absorbent zone 22 is 2.60 mm Whatman paper or other suitable material. To test for the presence of antibody analyte in a blood specimen using this assay path embodiment in the cassette design of Figure 6, undiluted sample is placed directly on the sample pad. After one minute the slide is pulled, putting the sample pad in fluid communication with the membrane and wicking strips. The undiluted sample moves freely up the membrane strip through the detection and control zones ahead of the buffer front, and therefore ahead of the conjugate. In this embodiment, total assay times range from 3 to 5 minutes. Figure 8 is a plan view of an alternative preferred embodiment. In this embodiment, the sample pad and the detection region are overlapping so as to provide a combined sample pad/detection strip 83, i.e. a single strip of bibulous material serves as both the sample receiving region and the detection region, where this combined strip is fabricated from glass fiber or nitrocellulose. In using this embodiment of the assay path in the device of cassette configuration of Figure 6 to test for antibody analyte in blood, untreated sample is introduced onto the combined sample pad/detection strip where it contacts the detection and control zones. After thirty seconds the slide is pulled, whereby buffer wicks up the wicking strip 10 carrying conjugate through the detection and control zones, providing a result in 3 to 4 minutes.

The following examples are offered by way of illustration and not by way of limitation.

EXPERIMENTAL Example 1. Diagnosis of infection with B. burgdoiferi or H. pylori

An assay pathway was developed as follows:

A wicking strip of chromatography paper (Whatman, Grade 31ET) was employed of 24 mm in length and 0.35 cm in width. A nitrocellulose test strip (5-10 μm pore size) of dimensions 3.9 cm x 0.35 cm was printed with two protein bands using a Bio-Rad SF microfiltration apparatus, where the strips were of about 0.50 cm width and ran across the width of the nitrocellulose strip. The protein solutions employed were B. burgdoiferi cell lysate and H. pylori cell lysate. After drying of the protein bands, the nitrocellulose membrane was incubated with a solution of 0.5% w/v casein hydrolysate, 2.0% w/v trehalose, 0.02% v/v TWEEN^* 20, 0.01 % w/v Proclin 300 in distilled water adjusted to pH 8.2. The incubation was for about 1 hour on a rocking platform, after which time the membrane was removed from the solution and dried under less than 7% humidity conditions until completely dry.

The assay pathway was then assembled by employing a strip (10 cm x 1.5 cm) of 3M Scotchbrand tape (Core Series 2-3000). The nitrocellulose strip was aligned onto the tape strip so that about 0.20 cm extends beyond the tape strip. A Whatman chromatography paper strip (3.9 cm x 0.35 cm) is aligned above the nitrocellulose strip to overlap the nitrocellulose strip by about 0.20 cm. A strip laminating and assembly instrument such as the one from Kinematic Automation, Inc., Twain Harte, CA 95383, was then used to laminate the whole nitrocellulose membrane and the Whatman chromatography paper, with the extra lamination trimmed off. The sample pad was fitted into the groove as indicated in Figure 6 and the laminated strip was placed between the pins of the base plate as depicted in Figure 6. The exposed nitrocellulose membrane overlapped about one-third of the sample pad, when the sample pad was moved into position into the assay pathway. The wicking strip (2.4 cm x 0.35 cm) was placed on the other side of the sample pad, so that it also overlaps the sample pad by about one-third and touches the bottom of the buffer well. The cassette cover was then placed over the assembly and welded ultrasonically. The cassette was now ready for use.

It was found that with the protein-trehalose solution, a much faster rate of wicking across the nitrocellulose strip was obtained. The detectable signal in the detection zone was observable in 5-7 minutes with the background clearing in about 15 minutes. Without the chemical treatment, the wicking was found to be very slow, non-uniform and required about 30-40 minutes for the signal to appear with background clearing requiring about 60 minutes.

The antibody-gold colloidal particle conjugate was prepared as follows. The antibody composition was dialyzed against 2 mM borax buffer (pH 8.2) overnight at 4°C. The dialysis membrane (SpectraPor, m.w. cutoff 6000-8000) is soaked in deionized water for 5 min. The dialysis buffer is changed twice daily.

Into filtered (0.2 μm) boiling tetrachloroauric acid solution (0.01 %; 100 ml) is added 5 ml of 1 % filtered (0.2 μm) aqueous sodium citrate and the boiling maintained until the color changes to wine red, resulting in about 16 - 20 nm particles. After cooling the solution, the pH is adjusted to 8.2 with 0.2 M potassium carbonate. To 1 mg of dialyzed antibody is added 100 ml of the gold colloidal solution and the mixture stirred for 2 min, followed by the addition of 11.1 ml of filtered 10% aqueous BSA and stirring continued for an additional 2 min. The mixture is centrifuged at 1500 rpm at 4°C for 20 min (Sorval RC-5, HB-4 rotor). The supernatant is pipetted off, centrifuged at 11,500 rpm (10,500 g) for 1 h at 4°C and the supernatant removed. The sediment is resuspended in 1 ml of 20 mM phosphate buffer (1 % BSA and 0.02% sodium azide).

Employing the assay device described above, sera samples were analyzed using either delayed release of the conjugate or simultaneous release of the conjugate. For delayed release, the conjugate was placed on the wicking strip about 5-10 mm upstream of the sample pad. For simultaneous release, the conjugate was placed on the sample pad. Where the conjugate was placed on the sample pad, which sample pad was glass fiber (composed of borosilicate glass fiber of about 39 μm pore size, Lydall), the sample pad was treated with a release solution. The release solution comprised 1.0% w/v bovine serum albumin, 1.0% w/v casein hydrolysate, 5.0% w/v trehalose, 0.02% v/v TWEEN^*20, 0.1% w/v Proclin 300, in distilled water adjusted to pH 8.2. The glass fiber sample pad is incubated on a rocker platform in the above formulation for 1 hour and dried at 45 °C until completely dry. With the above treatment, the glass fiber rapidly releases the colloidal particles substantially completely as contrasted to the incomplete and slow release in the absence of such treatment.

The results with the five Lyme disease sera and ten ulcer patient sera samples using the lysates of the specific pathogens were 100% correlation where the clinical diagnosis of B. burgdoiferi or H. pylori serum samples used the delayed release method By contrast, the simultaneous release method showed very weak signals with differentiation between the positive and negative serum samples only difficultly detected.

Example 2. Detection of antibodies to B. burgdoiferi or H. pylori in alternative device configurations.

A device comprising the assay path shown in Figure 7 was tested for its suitability for use in assays for the presence of antibodies to B. burgdoiferi or H. pylori. Sera both positive and negative for each of these antibody analytes was tested and the following results were obtained. In each case, undiluted sample was introduced into the sample pad and, after 30 seconds, the slide was drawn, thereby bringing the sample pad into the assay path. Results were obtained 2 to 3 minutes later.

Sera # of samples Result Interpretation

H. pylori (+) 3 2 bands positive

H. pylori (-) 2 1 band negative

B. burgdoιferi(+) 3 2 bands positive

B. burgdoiferi (-) 3 1 band negative

The results indicate t t at this device config uration, where the cc >n jugate is placed directly on the wicking strip, is capable of providing rapid and accurate results from untreated samples.

Example 3. Detection of antibodies to B. burgdoiferi or H. pylori in alternative device configurations.

A device comprising the assay path shown in Figure 8 was tested for its suitability for use in assays for the presence of antibodies to B. burgdoiferi or H. pylori. Sera both positive and negative for each of these antibody analytes was tested and the following results were obtained. In each case, undiluted sample was introduced into the sample pad and, after 30 seconds, the slide was drawn, thereby bringing the sample pad into the assay path. Results were obtained 3 to 4 minutes later.

Sera ft of samples Result Interpretation

H. pylori (+) 3 2 bands positive

H. pylori (-) 2 1 band negative

B. burgdoιferi(+) 3 2 bands positive

B. burgdoiferi (-) 3 1 band negative

The results indicate that this device configuration, where the conjugate is placed directly on the wicking strip, is capable of providing rapid and accurate results from untreated samples. Furthermore, the time in which the sample is contacted with the detection and the control strips may be controlled, thereby providing for the possibility of greater sensitivity.

It is evident from the above results, that a simple, rapid and efficient method is provided for detecting antibody analytes from physiological samples. The device which is used is compact, is able to contain all of the necessary reagents for performing the assay and only the sample need be administered to the device. In addition, no technical training is required and the result may be visually read. In this way, various diseases may be rapidly diagnosed in the doctor's office or at home using non-technically trained personnel, so that a more efficient treatment may be achieved.

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

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

Claims

WHAT IS CLAIMED IS:

1. A device for detecting an antibody to an antigen in an immunoglobulin containing physiological sample employing for detection a conjugate comprising a receptor specific for a conserved region of said antibody and a directly detectable label, wherein said conjugate is present in said device at a position to arrive at said detection region after said sample, said device comprising:

(a) a buffer source; and

(b) an assay path comprising:

(i) a wicking strip for contacting said buffer source; (ii) a sample pad;

(iii) a detection region overlapping said sample pad or downstream from said sample pad comprising a detection zone comprising a first reagent binding specifically to said antibody and a control zone comprising a second reagent for binding to immunoglobulin; and (iv) an absorbent zone for receiving and storing buffer, wherein the storage capacity of said buffer zone is sufficient to substantially remove non- specifically bound immunoglobulin from said detection zone.

2. The device according to Claim 1, wherein said directly detectable label comprises a colloidal particle.

3. The device according to Claim 2, wherein said colloidal particle is gold or selenium.

4. The device according to Claim 1 wherein said device further comprises a means for delaying conjugate flow through said detection zone.

5. The device according to Claim 1, wherein said detection and control zones are narrow bands traversing said detection region.

6. The device according to Claim 1, wherein said device further comprises a means for moving said sample pad from a site out of registry with said assay path into registry with said assay path.

7. A device for detecting an antibody to an antigen in an immunoglobulin containing physiological sample employing for detection a conjugate comprising a receptor for a conserved region of said antibody and a colloidal particle; said device comprising: (a) a buffer source;

(b) an assay path comprising:

(i) a wicking strip for contacting said buffer source, wherein said wicking strip provides for rapid transport of said buffer and comprises diffusibly bound conjugate; (ii) a sample pad;

(iii) a detection region overlapping or in proximity to said sample pad comprising a detection zone comprising a first reagent binding specifically to said antibody and a control zone comprising a second reagent for binding to immunoglobulin, wherein after sample is added to said sample pad, said sample pad is in registry in said assay path and substantial reaction of said antibody with said detection zone occurs prior to contact with buffer comprising said conjugate; and

(iv) an absorbent zone for receiving and storing buffer, wherein the storage capacity of said absorbent zone is sufficient to substantially remove non- specifically bound immunoglobulin from said detection zone; and

(c) means for moving said sample pad from a site out of registry with said assay path and into registry with said assay path.

8. The device according to Claim 7, wherein said detection region is in proximity to said sample pad.

9. The device according to Claim 8, wherein said wicking strip and sample pad are porous polyethylene.

10. The device according to Claim 7, wherein said detection region overlaps said sample pad to provide a combined sample pad.

11. The device according to Claim 10, wherein said wicking strip is porous polyethylene.

12. The device according to Claim 10, wherein said combined sample pad is a material selected from the group consisting of glass fiber and nitrocellulose.

13. A method for detecting an antibody to an antigen in an immunoglobulin containing physiological sample employing for detection a conjugate comprising a receptor for a conserved region of said antibody and a directly detectable label; said device comprising:

(a) a buffer source; and

(b) an assay path comprising:

(i) a wicking strip for contacting said buffer source and comprising diffusibly bound conjugate; (ii) a sample pad;

(iii) a detection region overlapping said sample pad or downstream from said sample pad comprising a detection zone comprising a first reagent binding specifically to said antibody and a control zone comprising a second reagent for binding to immunoglobulin; and (iv) an absorbent zone for receiving and storing buffer, wherein the storage capacity of said buffer zone is sufficient to substantially remove non- specifically bound immunoglobulin from said detection zone;

said method comprising: (a) introducing said immunoglobulin containing physiological sample onto said sample pad, whereby said sample flows downs said assay path through said detection region;

(b) flowing buffer through said detection region to remove substantially all unbound antibody; (c) flowing buffer comprising said conjugate through said detection region, whereby said conjugate binds to said antibody in said detection zone and antibody bound to said second reagent in said control zone; (d) continuing the flow of buffer through said region to remove substantially all unbound conjugate; and

(e) comparing the signals in said detection and control zones, wherein a signal in both of said zones indicates the presence of said analyte in said sample; whereby said antibody is detected.

14. The method according to Claim 13, wherein said antigen comprises an epitope of a pathogen.

15. The method according to Claim 14, wherein said pathogen is B. burgdorferi.

16. The method according to Claim 14, wherein said pathogen is H. pylori.

17. The method according to Claim 14, wherein said pathogen is HIV.

18. The method according to Claim 14, wherein said pathogen is Hepatitis B.

19. The method according to Claim 14, wherein said pathogen is Hepatitis C.

20. The method according to Claim 13, wherein said sample is pretreated.

21. The method according to Claim 13, wherein said sample is untreated.