WO1992018869A1

WO1992018869A1 - Colorfast reference device for immunoassays

Info

Publication number: WO1992018869A1
Application number: PCT/US1992/003059
Authority: WO
Inventors: Ian D. Wells
Original assignee: Medix Biotech, Inc.
Priority date: 1991-04-17
Filing date: 1992-04-14
Publication date: 1992-10-29
Also published as: AU1879392A

Abstract

The invention comprises the use of a colorfast region contiguous to a test reaction zone in an assay for the detection of an analyte in a test sample. The present invention provides an apparatus for use in the assay for the detection of the analyte, which apparatus comprises: a) a non-porous colorfast region integral to the apparatus and immediately adjacent to and/or surrounding a test reaction zone; and b) the test reaction zone on a porous filter containing a capture agent immobilized on or within the entire test zone, which capture agent is capable of interacting with the analyte, wherein a color indicator is used in the assay to detect the interaction of the analyte and capture agent.

Description

COLORFAST REFERENCE DEVICE FOR IMMUNOASSAYS

BACKGROUND OF THE INVENTION The invention disclosed concerns new materials, apparatuses, devices, kits, and processes for determining the presence of an analyte, particularly through diagnostic immunoassays. Immunological assays or tests, depending on the recognition and measurement of antigens and/or antibodies in body tissues and fluids of patients, are widely used as an aid in clinical analysis. The immunological tests which are in routine use depend on well-established principles of antigen- antibody recognition and the formation of immune complexes. Specific immunoassays have been developed which comprise in part, the utilization of porous solid supports for antigens or immunoglobulins where one of the reactants is immobilized on that support. The immunologic activity or binding reactions are thereby restricted to a particular test zone or area on the support through immobilization.

An inherent difficulty in the preexisting diagnostic tests is that false positive or false negative responses, unrelated to the particular immunologic analysis, may result as a consequence of the inability of the interpreter of the assay to differentiate between a true response and a false response associated with proteins bound up in the reaction site nonspecifically or associated with a failure or error in the assay procedure. In those test formats incorporating two or more distinct reaction zones, i.e. a test reaction zone and a negative and/or positive control zone, it is often difficult to delineate and differentiate the background non-specific binding in the control zone from that of the positive test zone result and to adequately interpret the results in the control and test zones.

For example, when as in a competitive assay, a reduction in signal at the test zone is indicative of analyte in the sample, it is difficult to interpret a partial reduction of the signal in those assays using a color intensity against a white background, particularly when there is variation in the signal or color intensity from one test to another. As a result, in these discrete zonal tests a subjective visual analysis can cause an incorrect false positive or false negative interpretation due to the inherent difficulties in distinguishing the correct color response. These commercially- available assays require the assay interpreter to distinguish between subtle color changes against a white background in order to make a determination of the assay result. Since in many of these tests the assay results are being determined by a laboratory technician or other individual based upon a visual, quantitative reading of color, some individuals cannot accurately assess the assay, especially those who may be colorblind or have difficulty in distinguishing hues.

Furthermore, the materials in a test zone can spread causing the color signal to contaminate other nearby areas or zones, resulting in an incorrect reading. The commercial assays commonly require a number of wash steps during the performance of the assay, which cause the assay procedure to be burdensome and subject to errors.

SUMMARY OF THE INVENTION The present invention is an improvement over the current assay materials, apparatuses, devices, and processes used for interpretation of assay results which will considerably reduce or obviate the subjectivity associated with many of the existing assays. The invention will simplify the visual and instrumental reading and interpretation of those assays, as well as eliminate a number of wash steps.

The invention comprises using as an essential incorporated feature of the assay a colored or colorfast region or reference. This region or reference is situated immediately adjacent to and/or surrounding a test reaction zone, which contains an analyte-capture agent binding reaction, and the directly-associated colored, colorable, or color-producing indicator is within the test zone. The present invention provides an apparatus for use in an assay for the detection of an analyte in a test sample, which apparatus comprises: a. a non-porous colorfast region integral to the apparatus and immediately adjacent to and/or surrounding a test reaction zone; and b. the test reaction zone on a porous filter containing a capture agent immobilized on or within the entire test zone, which capture agent is capable of interacting with the analyte, wherein a color indicator is used in the assay to detect the interaction of the analyte and the capture agent.

An assay method for detecting the presence of analyte in a test sample is also provided wherein the method comprises the steps of: a. applying the test sample, containing an unknown quantity of the analyte of interest, to the apparatus described above, which contains in the test zone a capture agent specific for the analyte or capable of interacting with the analyte, whereby the analyte interacts with the capture agent in the test reaction zone; b. adding a color indicator to the test reaction zone, either before, during, or after the interaction of the capture agent and analyte in the test reaction zone, such that the change in concentration of the indicator at the test reaction zone directly or indirectly correlates with the presence of the analyte interacting with the capture agent; and c. directly or indirectly determining the presence of the analyte interacting with the capture agent by comparing the color associated with the presence of the indicator localized in the test reaction zone with the color in the colorfast region at the end or completion of the assay. The comparison of the colors in the two areas can be performed either quantitatively or qualitatively by instrumentation or by visual means.

The invention also provides for diagnostic kits comprising the apparatus described above and the ancillary reagents used to conduct an assay to determine the presence of an analyte in a test sample. Also provided are diagnostic devices, usually disposable, which comprise the apparatus described above and an absorbent beneath the apparatus, both within a plastic container.

Primarily the present invention concerns a new means for conducting immunological assays where the interpretation of the colored or colorable end result is made against a preselected color of chosen intensity or gray scale. The result is realized through the color comparison of the two regions in which the colorfast reference is immediately adjacent to the immunological end point color result in the test zone.

BRIEF DESCRIPTION OF THE FIGURES

The figures described below are selected examples of the various aspects of this invention, but should not be construed as being limiting. In Figure 1 there is shown a colorfast reference plate or colorfast region (11) which can be used in the apparatus or device of this invention. The plate is configured as a disk whose surface has one color intensity or hue and which has two ports or apertures (12) cut entirely through the plate to permit the exposure of the test reaction or control zones lying within or beneath the ports. Figure 2 is a cross-section view of a device of this invention wherein the sides of the device (21) are configured to form a funnel, such that the reagents applied to the top opening of the device (20) are directed toward the colorfast regions or reference plate (11) and ports (12) . Immediately beneath the colorfast plate is a porous filter (22) , wherein the portion of the filter that is exposed to the reference plate ports comprises the test reaction zones (23) . These test reaction zones can be used to detect the presence of the analyte in the assay and/or can be used as positive and/or negative control zones.

Figure 3 is a top view of a colorfast plate (11) in the shape of a disk with two ports (12) . Figure 4 is a top view of a colorfast plate (41) , wherein there are two spatially distinct and different colorfast regions (42) and (43), such that each region has a different color, hue, or intensity and between the two regions are two ports (12) , such that each port is immediately adjacent to both regions. In Figures 5 and 6, the colorfast reference plate (11) is configured as a disk with three or four ports (12) , whereby one or more ports expose a test reaction or control zone on a porous filter.

Figure 7 is a top view of a colorfast reference plate (71) in the shape of a disk, wherein there are three spatially distinct and different colorfast regions (72, 73, and 74), wherein each region has a different color, hue, or intensity and situated between each two of the regions is a port (12) , such that the port is immediately adjacent to both regions. The device in Figure 8 is shown in a perspective view, wherein the device is housed in a rectangular container (80) with an opening (81) at one end of the top face of the container for the addition of the sample and reagents. The sides of the container (82) next to the opening are slanted or funneled to channel the test sample and reagents of the assay toward the colorfast reference plate (83) , and the two ports

(12) in the plate are immediately over the test reaction zones on the filter. There is sufficient space within the container, such that the unreacted reagents and test sample can be collected and stored. Figure 9 is a cross-section of the device in Figure

8, wherein the device is housed in a container (80) with an opening (81) for the addition of the sample and reagents. The sides of the container (82) are slanted or funneled to channel the test sample and reagents of the assay toward the colorfast reference plate (83). The two ports (12) within the plate are cut through the entire thickness of the plate. Immediately beneath the ports are two reaction zones (91) in a filter (92) . There is sufficient space (90) within the container, such that the unreacted reagents and test sample can be collected and stored within the container. Immediately beneath the filter is an absorbent (93) , such that the unreacted reagents and test sample, which have permeated the filter by capillary action, can be removed from the test reaction zones. The device is constructed to hold its various components firmly in place throughout the assay procedure.

Figure 10 is a perspective-view of a device wherein the device is housed in a rectangular container (100) and the colorfast reference plate (101) contains six ports (12) arranged in a linear fashion, such that six test reaction zones are within or immediately beneath the ports. This device can be used as a semi-quantitative device, such that the test reaction zones are treated with an ordered succession of increasing concentrations of the immobilized capture agent, or the test zones are utilized to detect multiple analytes.

Figure 11 is a perspective-view of a dipstick device wherein the device can be brought into contact with the test sample in an open container. The device is configured with a handle (111) at one end and an open end (110) at the other end, such that the opening in the container exposes the colorfast reference plate (112) with three ports (12) .

DETAILED DESCRIPTION OF THE INVENTION The invention claims an apparatus comprising a test reaction zone on a porous filter which can contain a capture agent immobilized within the test zone, which capture agent is specific for a particular analyte of interest, and a non-porous colorfast region contiguous or immediately adjacent to and/or surrounding the test zone, which colorfast region is integral to the apparatus.

Colorfast Region

The colorfast reference plate or colorfast region may be made of any essentially impervious or non-porous solid material that retains on its surface a colorant or color that is stable over an indefinite period. There should be no change in color intensity or color degree of matte or gloss before, during, or after, the assay. The colorfast region is essentially waterproof and can be made of heavy paper, light cardboard, glass, plastic, or any other rigid or semi-rigid substance that can be colored or colorable, and preferably is composed of a plastic material. Addition of the test sample or other reactants should not influence or change, to any measurable degree, the color, color intensity or gloss factor at the surface of the colorfast region unless such a change is factored into the selection process. The particular color and intensity of color required for a given assay may be integrated at the surface of the colorfast region by a variety of means, such as spraying, coating, baking, painting, layering, and the like. The color may be an integral part of the region impregnated during manufacture or added afterwards.

The colorfast region also can comprise different colors or color hues surrounding one or more test reaction zones, formed as a color wheel or color sections. Different color and/or shaped regions can be used for different test reaction or control zones depending upon the assay format desired and the analyte or analyte being detected.

The color, color intensity and gloss of the colorfast region may be selected and controlled by the use of commercially-available color standards. These standards are used in the practice of manufacturing paints and printing color of various hues, mattes, and color intensities to exacting specifications; for example, the Pantone* Color Formula Guide (PCFG) .

The non-porous colorfast region or area plays no primary role in the capture agent-analyte reactions but serves to differentiate the results in the test reaction zones. However, the colorfast reference plate may serve to direct the assay reagents into the exposed filter test reaction zones, such that essentially all of the applied reactants of the assay permeate the exposed test zones of the filter.

In addition, the said colorfast reference plate communicates directly with and is contiguous to the exposed filter within the open parts of the plate. The exposed test reaction or control zones on a porous filter can be constructed such that they are located within the parts of the colorfast plate or the filter is placed beneath the plate such that the zones are aligned with the apertures or port openings. It is important that the colorfast region or regions are contiguous with, immediately adjacent to and/or surround the zones, so that the colors in the zones are next to the colorfast regions. In using such a device the immunological test results are immediately referenced against the colorfast region which has been specifically selected for color and color intensity (using a color other than white or black) to aid in the interpretation of the assay response, whether it be a positive response, a negative response, or the correct result for a control response. The results are interpreted as lighter, equal to, or darker than the colorfast reference. The colorfast reference technology may be closely equated with the methods of interpretation of shades of white through black by use of the commonly-accepted graded gray-scale measuring process. In a further application of the invention, the colorfast reference plate may also be used to identify a spectral color shift or color change in the assay. The color region may be selected to most effectively visualize a change in the primary color as well as a change in the color intensity. When porous filter or other test reactants imparts a color to the filter, it would be advantageous to select a colorfast reference and label that would best differentiate a color change as well as a change in color intensity.

In some cases, for example, pH indicator dyes, where there is a direct color shift, or for example, where the color in the test zone could be formed from the combination of two distinct colors to form a third distinct color (such as yellow and blue to form green) , the color hue could be compared to the colorfast region.

The colorfast region will be selected to be a hue or color for comparison to or to match the resulting color in the test reaction or control zones, such that the zone can be read to give an assay value. It is important that the color of the colorfast region be selected to correlate or contrast with the color indicator used in the assay.

Filter The porous filter, membrane, matrix, or solid support utilized in the invention may be made of any suitable inert material. The filter should not dissolve the reactants or components of the assay and it should have negligible non¬ specific attraction for these components. However, it must be able to act as a reaction surface for the interaction of the capture agent and analyte, such that the capture agent and analyte can be immobilized. Therefore, although it is porous, it must be able to retain or bind the required components for the assay through physical or chemical means. For example, the capture agent can be coupled to the filter through chemical bonds or reactions; or the capture agent can be trapped, adhered or adsorbed on a particle, for example, a latex particle; which particle is supported, bound, or trapped on or within the filter. The filter can be a matrix or solid support usually composed of a mat of compressed fibers, such as a mat of glass or synthetic fibers or a porous paper mat. It may also be constructed of other porous materials known to those skilled in the art, such as sintered glass, ceramics, synthetic spongy materials, synthetic polymers, gels, mixtures of these substances, and the like, with the preferred material being nylon or nitrocellulose. Nylon 66 cast on a non-woven polyester support with either a co-cast resin containing quartonary ammonium groups or carboxyl groups available from Pall Corporation, Glen Cove, NY is the preferred filter. The filter can be constructed in a variety of shapes, such as films, sheets, plates, cylinders, tubes, dipsticks, and the like, depending upon the particular assay format desired. The filter is from about 100 to about 200 microns (μ) in thickness and the pore size within the filter is from about 0.2 to about 5.0 μ. The preferred filter thickness is about 150 μ with a pore size of 3.0 μ. Where the capture agent is coupled to a particle, such as a latex particle, which is retained, or immobilized, on or within the porous filter, it is required that the pore size of the porous filter support be sufficient to prevent those particles from being passed completely through the filter, but allow the sample and other test reactants to permeate through the filter.

Test Reaction Zone

The test reaction or test zone in the filter is defined as a finite region or site on or in the filter wherein the capture agent and analyte and other reactants in the assay can interact, complex, react, or bind by physical or chemical means. The capture agent can be immobilized in the test zone for interaction with the analyte from the test sample. One skilled in the art will appreciate that the filter can contain one or multiple, or a plurality of, reaction test zones and can range in size and shape. The test zone size is from about 1 millimeter (mm) to about 5 mm, preferably about 3 mm. In addition, the apparatus of this invention is constructed such that the test sample is directed or channeled toward the test reaction zone, increasing the interaction of the analyte contained in the test sample and the capture agent. The test reaction zone is situated such that it is immediately adjacent and/or surrounded by the colorfast region, with no gap or space between the zone and the colorfast region; thereby enabling the accurate detection and determination of a color change in the test reaction zone. One or more zones on the filter can be used as positive and/or negative controls for the assay.

Test Sample

The test sample or specimen for use in the assay methods can be obtained from a variety of sources, preferably from animals in the form of biological samples. Most preferred is a test sample from a human patient or host which is in the form of a liquid, semi-liquid, or fluid, such as whole or fractionated blood, urine, sputum, tears, tissue extracts, and the like. The preferred source of the human sample is plasma, serum, or urine. In addition, the apparatus of this invention can be used to detect analytes in chemical or environmental test samples, such as particular organic chemicals. The test sample can be applied to the apparatus or device of this invention, or the apparatus can be inserted or dipped into the sample. It can also be prefiltered before use in the assay or the label indicator can be added to it.

Analyte

The analyte to be detected, measured, or determined from the test sample can be a variety of items, such as proteins; peptides; antigens; antibodies; fragments of antibodies, such as the Fc and Fab regions, variable and constant regions, and the heavy and light chains; im unoglobulins; nucleic acid oligomers, such as RNA or DNA; drugs or pharmacological agents; hormones; vitamins; extracts from parasites, allergens, bacteria, viruses, or virus particles; metabolites; organic or chemical compounds; toxins; and the like. The present invention can be used to detect one or multiple analytes in an assay by using one or more test reaction zones on the filter. Preferred analytes for use in this invention include: a. Proteins, such as allergens, protein extracts of pollens, foods, and animal danders; b. Human plasma proteins, such as albumin, microalbumin, and creatinine kinase; c. Immunoglobulins, such as IgG, IgA, IgM, and IgE; d. Protein hormones, such as human chorionic gonadotropin (HCG) , luteinizing hormone (LH) , and parathyroid hormone; e. Haptens; f. Steroids, such as estrogen and digoxin; g. Therapeutic drugs, such as antibiotics and prostaglandins; h. Drugs of abuse, such as benzoylecgonine (BE) , opiates, and cannabinols; and i. Infectious disease agents, such as bacterial agents Streptococcus, Chlamydia, Rubella, Helicobacter, viral agents, Hepatitis viruses, human immunodeficiency virus (HIV) , Herpes viruses, fungal agents, Candida, and Aspergillus. Capture agent

The capture agent is any receptor, complexing agent, binding partner, binding reagent, or reacting agent or is any substance which reacts with, binds to complexes with, or interacts with, the analyte, such that the analyte is bound, fixed, or immobilized by its interaction with the capture agent. For example, when the analyte being detected is an antigen, the capture agent can be a polyclonal or monoclonal antibody which binds to the analyte. A small amount of the solution containing the binding reagent or capture agent is dropped, added, coupled, sprayed, or placed onto the filter at each area designated as a test reaction zone for that capture agent. Enough of the solution is added to completely cover the test reaction zone; the volume of the solution required will vary according to the filter material porosity and the structure and thickness of the test reaction zone. It is important, especially where the filter is a single piece of material in the apparatus with two or more test reaction zones contained therein, that the solution containing the capture agent or binding reagent does not migrate to, or contaminate, another test reaction zone, which may contain a different capture agent for use in the assay. It is also important the capture agent adheres, adsorbs, covalently binds, complexes, or is otherwise immobilized on, or within, the filter test reaction zone for a period of time sufficient to carry out the desired assay.

The concentration of the solution will vary according to the analyte being detected and the assay method and capture agent being utilized. In addition, the solution may also contain a dilution buffer, which is a non-reactive reagent, such as bovine serum albumin.

After the solution is applied to the test reaction zone or zones, the filter can be used immediately or can be dried in order to stabilize the binding reagent on the filter. Typically, solutions containing proteins with volumes of about 1 to about 5 icroliters (μl) will be adequately stabilized within about 10 to about 15 minutes at ambient temperature. Afterwards, each test reaction or control zone is treated with a blocking reagent, which may be bovine serum albumin, milk casein, or other blocking reagent used in conventional techniques to negate any undesirable reactive sites remaining on the filter in the test reaction zone.

The filter can be prepared for use immediately in the assay or it can be stored without loss of activity, provided that the storage conditions are proper for retention of the activity depending upon the materials being stored upon the filter. Typically, one must protect the filter against excessive moisture and excessively high temperatures.

Color Indicator

An indicator (usually an antigen or antibody) capable of specifically participating, in or with the analyte-capture agent binding complex reaction, may be labeled by a variety of means. For the purposes of this invention, the indicator label must result in a color change or color formation in the test or control zone; therefore, the indicator is called the color indicator. Conventional color labels include chromophores, fluorophores, enzymes, dyes, colored or colorable particles or combinations thereof. The color labels to which the indicator may be bound are usually termed direct labels and indirect labels. The direct labels are those which are immediately visible to the naked eye or to an instrument capable of detecting or measuring such labels, such as a spectrophotometer, fluorometer, and the like. These direct labels include, but are not limited to,: a. colored or colorable particles, such as latex particles of various colors, sizes, and chemical characteristics, examples of which are given in US Patent 4,837,168, hereby incorporated by reference; b. gold sol labels, which are available commercially from Sigma Chemical Co., St. Louis, MO as monodispensed colloidal particles, in size ranging from about 5 to about 30 nanometers (nm) , which readily and passively adsorb the assay reagents without additional chemical or physical modification or processes; c. fluorescent labels, such as fluorescein or phycoerythrin; and d. insoluble dye particles, which may be selectively sized by centrifugation and coupled to the reagent by passive absorption at an acid pH, followed by backcoating with bovine serum albumin and which may be contained in vesicles or liposomes, examples of which are given in U.S. Patent 4,703,017, hereby incorporated by reference.

Preferred direct labels for use in this invention are intensely colored latex particles available from Molecular Probes, Eugene, OR or Interfacial Dynamics Corporation,

Portland, OR with diameters from about 0.08 microns (μ) to about 0.2 microns, which particles adsorb the analyte without chemical modification.

The indirect labels include, but are not limited to, enzymes coupled or conjugated to the analyte or other indicator reactants in the assay. The preferred enzymes are alkaline phosphatase and horse radish peroxidase. These enzymes react with a substrate to cause the color change, such as para- nitrophenylphosphate (p-NPP) or bro ocresol indoxyl phosphate/nitroblue tetrazolium (BCIP/NBT) for alkaline phosphatase; or hydrogen peroxide, o-phenylenediamine (OPD) , tetramethylbenzidine (TNBJ, or 2,2'-azino-di(3- ethylbenzthiazolinesulfonic acid (ABTS) for horse radish peroxidase. Regardless of which enzyme and substrate are chosen, pH should be optimized for proper color development.

The label materials may be used and stored in liquid form or preserved as dry reagents until needed, so long as the proper storage conditions are observed. For example, the targeted antigen or antibody may thereby be directly or indirectly measured or visualized at the test zone by the incorporation of the color indicator in the test zone site. This incorporation of the color indicator can occur prior to, simultaneous with, or after, the addition of the analyte to the test reaction zone.

The intensity of color of the indicator complexed on the exposed test reaction area or zone of the filter or solid porous support, after removal of unbound reagents and when compared with the adjacent colorfast region or reference, is indicative of the presence or absence of the analyte in the sample being assayed.

Device and Kit

In a preferred embodiment of the invention, the assay device has a funnel at the beginning end, which funnel serves the purpose to direct the test sample and other necessary reagents to the porous filter. Since the colorfast region is an integral part of the device, it can be a separate component inserted during device fabrication or device assembly or can be integrated during the device manufacture. The colorfast reference may alternatively be inserted into the device just prior to, during, or immediately upon, completion of the assay. Preferably, the colorfast region or plate is non- porous and has one, two or several ports cut into it. These ports, which pass completely through the colorfast plate, allow the test sample to channel through the openings or ports to come into contact with the exposed porous test reaction zones on the porous membrane or filter situated immediately within or below the color reference plate ports. To the exposed area of the porous filter in the test reaction zones can be bound, trapped or otherwise immobilized, the specific capture agents which are involved in the analyte-capture agent interactions. Also immobilized on or within the filter, can be the latex particles. Optionally, below the filter is situated an absorbent or absorbent material which is configured to facilitate flow of unreacted components or fluids away from the test reaction zones. The absorbent is any moisture or fluid-retaining material and can be composed of a number of fibrous filter materials, such as glass fibers, cellulose acetate fibers, polyester, polyolefin, and the like. The absorbent can comprise one or more layers and can be made up of one or more types of material. A preferred absorbent material is binderless glass fiber sheet available from Hollingsworth and Vose, Westgroton, MA. Additionally, between the filter and the absorbent, a barrier means can be inserted, which allows the absorption of fluids or liquids into the absorbent, but prevents the return or backflow of fluids from the absorbent to the filter and test reaction zone; thereby minimizing contamination of the zone. The components of the device can be housed in a container, usually made of plastic or other rigid or semi¬ rigid substance, which can be disposed of upon completion of the assay. The housing can have a variety of shapes and sizes, such as films, cylinders, tubes, dipsticks, and the like, depending upon the desired assay format.

Another use for the present invention is in a device wherein the test sample is applied to a receiving member and then permeates to a porous carrier and moves through various zones. The colorfast region of the invention could be located above the particular zone being observed. See for example, WO 88/08534 which describes such a device and is hereby incorporated by reference.

The invention also comprises diagnostic kits wherein the apparatus containing the colorfast region and test zones and the ancillary reagents necessary to conduct the assay of interest are part of the kit. Included as ancillary reagents are the color indicator, buffers, diluents, standards, and the like. In most cases, the kits are complete except for the addition on the test sample.

Immunological Binding Assays

There are several well recognized immunological specific binding assays to determine the presence and concentration of particular substances, such as enzyme immunoassays (EIA) . (See for example, U.S. Patents 4,366,241; 4,376,110; 4,517,288; and 4,837,168, which are hereby incorporated by reference.) [See also Basic and Clinical Immunology 7th Edition (D. Stites and A. Terr ed.) 1991, which is hereby incorporated by reference. ] The immunological assays generally fall into two categories. In the competitive binding assay, the sample analyte competes with a labeled analyte, the indicator, for specific binding sites on the binding reagent. The concentration of labeled analyte bound to the capture agent is inversely proportional to the amount of free analyte present in the sample. In the second, the sandwich assay, the sample analyte is bound between two analyte-specific binding reagents, one of which is labeled, the indicator, in order to measure or detect the resultant complex by visual or instrument means.

Commonly, the two specific binding reagents or capture agents are both antibodies with specificities for different epitopes of the antigen analyte and an unlabeled first antibody is immobilized on a solid support or carrier. In an alternative sandwich assay, the first capture agent may be distinctly different from the second labeled binding reactant, the indicator. In this approach the immobilized capture agent may be an antigen, i.e. a bacterial or viral substance, or allergen, which binds an immunoglobulin or antibody in the sample. The labeled reactant, the indicator, will then be directed towards the sample immunoglobulin complexed with the immobilized first reagent. In these sandwich assays, the amount of bound labeled reactant, the indicator, is directly proportional to the concentration of bound sample antibody. In some sandwich assays, the two binding reactants may be two monoclonal antibodies, or one monoclonal and one polyclonal.

These solid phase i munoassay procedures can employ inert porous filters, membranes, or medium supports, where one of the antigen or antibody partners is immobilized within a finite and regulated test reaction zone on the filter, which analyte or antibody is capable of specifically binding and immobilizing the targeted analyte of the reaction. The bound partner is not directly measurable and therefore techniques using color indicators can be employed to facilitate indirect visual or instrumental measurement of the targeted analyte captured at the test reaction zone. Throughout the assays, incubation and/or washing steps may be required after each combination of reagents. Incubation steps can vary from about 5 seconds to several hours, preferably from about 5 minutes to about 2 hours. However, the incubation time will depend upon the assay format, analyte, volume of solution, concentrations, and the like. Usually, the assays will be carried out at ambient temperature, although they can be conducted over a range of temperatures, such as 15 to 40*C. However, this invention allows for fewer or no assay wash steps to be required. It is a preferred embodiment of this invention that the wash steps be eliminated.

Controls

Sandwich-type assays, which require a control function to distinguish a positive response from a negative response, can be performed using the colorfast region or color scale as the built-in device test control and also to validate assay results. When the result is considered positive where color is intensified and visualized at the test zone, the colorfast region is used to distinguish between a less intense non-specific background color and the more intense specific- binding reaction signal. In this instance a signal at the test zone that is darker than the colorfast reference is scored positive and a signal at the test zone that is lighter than the colorfast reference is scored negative.

Where there is included in the test a positive and/or negative control, which may or may not produce a colored signal according to the assay format, the colorfast region will be used to distinguish a correct positive or negative control signal from an incorrect response.

Where, at the positive or negative control zone, an increase in signal intensity is correct, the result can be confirmed by use of the colorfast reference. The expected control result must be darker than the colorfast reference and if not, then the assay result should be considered invalid.

Alternatively, where there is required to be no (or minimal) increase in signal at the positive or negative control zone, the colorfast reference may be used to confirm the correct response. If the signal at the control zone is lighter than the colorfast reference, then the control result is correct; but if it is darker than the reference, the assay should be scored invalid. The invention as applied to sandwich assays can also be used to perform and monitor the competitive or inhibition assays. In these inhibition tests, the labeled analyte competes with analyte present in the sample for immobilized specific binding sites on the capture agent located at the test reaction zone. The presence of analyte in the sample will result in a reduction in signal intensity which can be confirmed by comparison to the colorfast region. A signal at the test reaction zone of less intensity or lighter than the colorfast reference is indicative of analyte in the sample and is interpreted as a positive result. Where the test reaction zone signal remains darker than or equal to the colorfast reference, the sample is scored as negative for the screened analyte.

The incorporation of positive and/or negative control zones in the test device for competitive assays follow the principles outlined above for the sandwich assays in identification of the correct control zone responses.

Examples The present invention is illustrated by the following examples. These examples are not intended to limit the scope of the invention.

Example 1 This example describes a sandwich type of immunoassay for detecting human chorionic gonadotropin (HCG) in serum, employing two monoclonal anti-HCG antibodies directed against different epitopes of the HCG molecule.

The apparatus was assembled such that the colorfast region's color shade and intensity were approximately PCFG 442U or 537U with a matte finish. Two ports were integral to the plate, each approximately 3 mm in diameter. A filter was situated immediately below the two colorfast plate ports so as to entirely cover the ports. The filter employed was a Biodyne A filter with a 1.2 μ pore size available from Pall Corporation, Glen Cove, NY. Monoclonal anti-HCG as a first antibody was prepared in 0.1 molar (M) borate buffer, pH 8.5, to a concentration of 0.05 milligrams of protein per milliliter (mg/protein/ l) and 2 microliters (μl) of the preparation were applied to the exposed filter through the colorfast plate port designated as the test reaction zone. Normal mouse immunoglobulin (IgG) was diluted to a similar concentration in the borate buffer and 2 μl were applied to the exposed filter area designated to be the control zone.

The proteins were bound to the filter by passive adsorption and air-dried at ambient temperature for 15 minutes and then the residual adsorption sites on the exposed filter areas were blocked with a 0.5% casein solution in the borate buffer. The test apparatuses then were stored at room temperature for further use. The anti-HCG-IgG alkaline phosphatase conjugate

(anti-HCG-ALP) as a second antibody was prepared according to Yonhitaka et al. (1982), J. Biochem. 92:1413, using reagents available through Pierce Chemical Company. This conjugate was diluted in a borate buffer containing protein stabilizers at a pH 9.0.

The HCG samples were prepared using World Health Organization (WHO) referenced HCG in normal human serum at HCG concentrations in the range of zero (0) to one hundred twenty thousand (120,000) illi international units (mlU) per ml. The enzyme substrate, Bromocresol Indoxyl

Phosphate/Nitroblue tetrazolium (BCIP/NBT) , obtained from Kildergard & Perry, was used undiluted.

Five hundred microliters (500 μl) of a test sample was pipetted into the apparatus and allowed to permeate through the filter test reaction and control zones. This was immediately followed by two (2) drops of the second antibody (enzyme conjugate) [approximately one hundred (100) μl] which was allowed to filter through the test reaction zone. Three drops of the substrate were applied to the filter and allowed to react for 2-4 minutes after which time the color intensity in the test reaction zone and the control zone were compared to the colorfast reference. The results were scored and summarized as follows in Table 1:

*******************************

Table 1

DATA FOR VISUAL HCG TEST

Results

+ + +

+ CRC = Colorfast reference comparison

"L" = Lighter than colorfast region "D" = Darker than colorfast region

"-" = no or negative identification of the presence of HCG in the sample

"+" = positive identification of the presence of HCG in the sample

******************************* Example 2 This example describes an alternative sandwich type of immunoassay for the detection of human IgG antibodies to the bacterial organism, Helicobacter pylori (HP) , employing HP antigen and anti-Human IgG as the sandwich pair.

The reference plate color and color intensity were selected to match approximately the PCFG 442U standard with a matte finish.

Two ports (3 mm in diameter) were formed integral to the plate. The filter used was the same as that used in Example 1. Helicobacter pylori antigen, extracted from whole cells, was diluted to approximately 2 milligrams per milliliter (mg/ml) in phosphate buffer at pH 7.4 and 2 μl of the solution were spotted onto the exposed filter designated as the test reaction zone. Affinity-purified human IgG at 0.1 milligrams per milliliter (mg/ml) was spotted on the exposed filter designated as the control zone. The absorbed proteins were dried on the filter for 15 minutes at ambient temperature and backcoated with a one percent bovine serum albumin (1%) (BSA) solution in phosphate buffer.

An affinity-purified goat IgG anti-Human IgG was conjugated to alkaline phosphatase by a procedure similar to that used in Example 1.

Human sera, previously screened for the presence of anti-HP antibodies by another assay or test method (ELISA- enzyme-linked immunoassay) , were evaluated by the method of the present invention.

Two hundred and fifty (250) μl of each human serum sample, diluted 1:100 (volume:volume) in 20% normal goat serum in phosphate buffered saline, were added to the device and allowed to transfuse through the exposed filter zones. Then two (2) drops of the conjugate were added and allowed to filter through the exposed test and control zones. This was immediately followed by the addition of three (3) drops of the substrate reagents (BCIP/NBT) which reacted for three (3) minutes. The results were scored by comparing the color intensity on the test and control zones with the color of the colorfast region. The results are provided below in Table 2 using the same symbols or abbreviations as Example 1.

*******************************

Table 2

DATA FOR HP IgG TEST

******************************* Example 3

This example describes a competitive type of immunoassay for the screening of benzylecgonine (BE) in urine in accordance with the present invention. The apparatus was assembled using a colorfast reference plate with a color of medium intensity [PCFG 534U] in order to determine a reduction in signal rather than an increase in signal.

Monoclonal anti-BE was diluted to 200 μg protein/ml in borate buffer, pH 8.6, and 2 μl of solution were applied to the exposed test reaction zone.

The control zone was treated with 2 μl of monoclonal anti-alkaline phosphatase at approximately 50 μg protein/ml. The filter was dried and backcoated with one-half percent (0.5%) casein solution.

BE was conjugated to alkaline phosphatase by practiced methods and diluted to 1:100 (volume:volume) in 0.05 M borate buffer at pH 9.2. The BE samples were prepared using crystalline BE dissolved in normal human urine at BE concentrations in the range of zero (0) to ten thousand nanograms (10,000) (ng) per ml.

Approximately 250 μl of a human urine sample was applied to the device and allowed to permeate through the exposed filter zones. Two (2) drops of the conjugate were added and allowed to filter through the reaction sites. This was followed by four (4) drops of BCIP/NBT substrate and the color reaction was allowed to proceed until the control spot was darker then the color reference at which time the result in the test zone was interpreted. Where the test zone is also darker then the color reference, the test is recorded negative for the presence of BE. When the test zone remains lighter then the color reference, it is indicative of the presence of BE in the sample. The results are shown in Table 3: *******************************

Table 2

DATA FOR BE TEST

Results

+ +

+

*******************************

Example 4

This example is similar to Example 1 except that a colored latex-second antibody conjugate is substituted for the IgG anti-HCG-alkaline phosphatase conjugate.

Intensely colored blue-sulfate latex particles of 0.1 μ in diameter (available from Molecular Probes) were washed in 0.01 M borate buffer and diluted to 0.25% solids. Monoclonal anti-HCG antibody diluted to 200 μg/ml in the same buffer was added in equal volume to the latex particles and agitated at room temperature for 6 hours. A one-half (0.5) mg of BSA solution at one-tenth the volume was then added and the reactants further incubated for 30 minutes. The latex particles were then separated from the supernatant by centrifugation at 13,000 revolutions per minute (RPM) and washed three times in a 0.05 M borate buffer before being suspended at 0.05% solids in 1 mg/ml BSA in 0.1 M phosphate buffered saline pH 7.4 and stored at 5^βC until used.

Approximately 250 μl of a human urine sample was added to the device and allowed to permeate through the exposed filter zones. Four (4) drops of latex suspension were added and allowed to filter through the reaction sites. The results were then immediately interpreted and scored as follows: *******************************

Table 4 DATA FOR VISUAL HCG TEST

Results

+ +

*******************************

Claims

WHAT IS CLAIMED IS:

1. An apparatus for use in an assay for the detection of an analyte in a test sample, which apparatus comprises: a. a non-porous colorfast region integral to the apparatus and immediately adjacent to and/or surrounding a test reaction zone; and b. the test reaction zone on a porous filter containing a capture agent immobilized on or within the entire test reaction zone, which capture agent is capable of interacting with the analyte.

2. The apparatus of Claim 1 wherein the porous filter is selected from the group consisting of nylon and nitrocellulose.

3. The apparatus of Claim 1 wherein the capture agent is selected from the group consisting of an antigen and an antibody.

4. The apparatus of Claim 1 comprising multiple test reaction zones and multiple colorfast regions, in which each test reaction zone has one or more corresponding colorfast regions immediately adjacent to and/or surrounding it.

5. The apparatus of Claim 4 wherein each test reaction zone contains a capture agent different from the capture agents in the other test reaction zones.

6. The apparatus of Claim 4 wherein each colorfast region is a different color or shade of color.

7. The apparatus of Claim 4 wherein at least one of the test reaction zones is utilized as a positive or negative control zone.

8. The apparatus of Claim 7 wherein at least one of the test reaction zones is utilized as a positive control zone and at least one other test reaction zone is utilized as a negative control zone.

9. The apparatus of Claim 4 which additionally comprises an absorbent beneath the filter.

10. The apparatus of Claim 9 wherein the absorbent is selected from the group consisting of glass fiber, cellulose acetate fiber and polyester, and the indicator is selected from the group consisting of latex particles; horse radish peroxidase and o-phenylene diamine; and alkaline phosphatase and bromocresol indoxyl phosphate/nitroblue tetrazolium.

11. The apparatus of Claim 10 wherein the absorbent is slanted away from the filter thereby restricting the fluid portion of the test sample which is not retained in the filter from returning to the test reaction zone.

12. An assay method for detecting the presence of an analyte in a test sample which comprises the steps of: a. applying the test sample, containing an unknown quantity of analyte, to the apparatus of Claim 1, which contains in the test reaction zone a capture agent specific for the analyte, whereby the analyte interacts with the capture agent in the test reaction zone; b. adding a color indicator to the test reaction zone, either before, during, or after the interaction of the capture agent and analyte in the test reaction zone, such that the change in concentration of the indicator at the test reaction zone directly or indirectly correlates with the presence of the analyte interacting with the capture agent; and c. directly or indirectly determining the presence of the analyte interacting with the capture agent by comparing the color associated with the presence of the indicator localized in the test reaction zone with the color in the colorfast region at the end or completion of the assay.

13. The method of Claim 12 wherein the assay method is selected from the group consisting of competitive and sandwich immunoassay methods.

14. The method of Claim 13 wherein the analyte is selected from the group consisting of: a. Proteins, such as allergens, protein extracts of pollens, foods, and animal danders; b. Human plasma proteins, such as albumin, microalbumin, and creatinine kinase; c. Immunoglobulins, such as IgG, IgA, IgM, and IgE; d. Protein hormones, such as human chorionic gonadotropin (HCG) , luteinizing hormone (LH) , and parathyroid hormone; e. Haptens; f. Steroids, such as estrogen and digoxin; g. Therapeutic drugs, such as antibiotics and prostaglandins; h. Drugs of abuse, such as benzoylecgonine (BE) , opiates, and cannabinols; and i. Infectious disease agents, such as bacterial agents Streptococcus, Chlamydia, Rubella, Helicobacter, viral agents. Hepatitis viruses, human immunodeficiency virus (HIV) , Herpes viruses, fungal agents, Candida, and Aspergillus.

15. The method of Claim 14 wherein the color indicator is added after the interaction of the analyte and capture agent and consists of an enzyme and a substrate, whereby the interaction of the enzyme and the substrate results in a change in the color of the indicator.

16. The method of Claim 15 wherein the comparison between the color in the test reaction zone and the colorfast region is determined visually.

17. The method of Claim 15 wherein the comparison between the color in the test reaction zone and the colorfast region is determined by instrumentation.

18. The apparatus of Claim 1 wherein a color indicator is reversibly immobilized on the capture agent.

19. An assay method for detecting the presence of an analyte in a test sample which comprises the steps of: a. applying a test sample, containing an unknown quantity of analyte, to the apparatus of Claim 18, which contains, in the test reaction zone a capture agent specific for the analyte, whereby the analyte interacts with the capture agent in the test reaction zone; such that the change in concentration of the indicator at the test reaction zone directly or indirectly correlates with the presence of the analyte interacting with the capture agent; and b. directly or indirectly determining the presence of the analyte interacting with the capture agent by comparing the color associated with the presence of the indicator localized in the test reaction zone with the color in the colorfast region at the end of the assay.

20. The method of Claim 19 wherein the color indicator is a latex particle.

21. An assay method for detecting the presence of an analyte in a test sample which comprises the steps of: a. adding a color indicator to the test sample such that the indicator binds to the analyte; b. applying the test sample to the apparatus of Claim l, which contains a capture agent specific for the analyte in the test reaction zone; such that the change in the concentration of the indicator at the test reaction zone directly or indirectly correlates with the presence of the analyte interacting with the capture agent; and c. directly or indirectly determining the presence of the analyte interacting with the capture agent by comparing the color associated with the presence of the indicator in the test reaction zone with the color in the colorfast region at the end of the assay.

22. An assay method for detecting the presence of an analyte in a test sample which comprises the steps of: a. adding a color indicator to the test sample such that the indicator competes with the analyte; b. applying the test sample to the apparatus of Claim 1, which contains a capture agent specific for the analyte in the test reaction zone; such that the change in the concentration of the indicator at the test reaction zone directly or indirectly correlates with the presence of the analyte interacting with the capture agent; and c. directly or indirectly determining the presence of the analyte interacting with the capture agent by comparing the color associated with the presence of the indicator in the test reaction zone with the color in the colorfast region at the end of the assay.

23. A diagnostic device which comprises the apparatus of Claim 1 and an absorbent beneath the apparatus, both within a disposable plastic container.

24. The device of claim 23 which has the configuration as shown in Figures 8 and 9.

25. A diagnostic kit comprising the apparatus of Claim 1 and ancillary reagents used in conducting an assay to determine the presence of an analyte in a test sample.

26. An assay method for detecting the presence of an analyte in a test sample which comprises the steps of: a. adding a color indicator and a capture agent bound to a particle to the sample, such that the capture agent is specific for interaction with the analyte, and the indicator also interacts with or competes with the analyte; b. then applying the test sample to an apparatus comprising a non-porous colorfast region integral to the apparatus and immediately adjacent to and/or surrounding a test reaction zone; and wherein the test reaction zone is on a porous filter; and c. directly or indirectly determining the presence of the analyte interacting with the capture agent on the filter within the test reaction zone by comparing the color localized in the test reaction zone with the color in the colorfast region at the end of the assay.

27. An apparatus for use in an assay for the detection of an analyte in a test sample, which apparatus comprises a non- porous colorfast region integral to the apparatus and immediately adjacent to and/or surrounding a test reaction zone on a porous filter.