IES950988A2 - A test device for rapid diagnosis of disease - Google Patents

Abstract

A device detects polyvalent analytes such as antibody to the AIDS virus, utilizing an inverse sandwich method. The test device comprises a first substance having an epitope, bound to a label and capable of moving within the test device. The test device further comprises a second substance immobilized to the test device and spatially separated from the first substance. The second substance has an epitope substantially similar to the epitope of the first substance. Upon application to the test device, the polyvalent analyte binds to the first substance and moves within the test device to the location of the second substance with both polyvalent analyte and first substance are immobilized at location of the second substance. Polyvalent analyte is detected by the presence of the label at the location of the second substance. Also disclosed is a control substance for use with the device that can be used to determine completion of the test and viability of the device.

Description

A test device For rapid diagnosis of disease Field of the Invention The present invention relates to devices and methods for the detection or quantification of polyvalent substances such as immunoglobulin, and more particularly to devices and methods for performing inverse sandwich assays for the detection or quantification of polyvalent substances such as immunoglobulin. In particular, the invention relates to a test,device for ranid diagnosis of disease.

Backorouno of the Invention There are a wide variety of naturally occurring and synthetically produced polyvalent substances of scientific and commercial interest. Prototypical of such substances are immunoglobulin or antibody, which are proteins usually existing as glycoproteins h vhm. The protein moiety of an immunoglobulin comprises a basic unit of two Gght polypeptidB chains and two heavy polypeptide chains, for a total of four polypeptide chains. An immunoglobulin molecule can have more than one of these basic units or can incorporate polypeptide chains in addition to those found as part of a basic unit.

Ona function of an immunoglobulin molecule is to bind to a substance at a site on the substance called an epitope. The epitope bind'mg site on an immunoglobulin molecule is comprised of a segment of one Gght polypeptide chain and a segment of one heavy polypeptide chain such that the basic unit of four polypeptide chains defines two epitope bind'mg sites.

Naturally occurring immunoglobulins are divided into classes and subclasses on the basis of amino acid sequence differences in the heavy polypeptide chain, in humans, there arp five classes, IgD, IgE. IgG each having two epitope binding sites, IgA having two or four epitope binding sites, and IgM having ten epitope binding sites.

Antibodies are of particular scientific and commercial interest among the types of polyvalent substances because of their role in the mammalian immune response. They are markers of current and past infection, and important in host reactions against foreign substances such as viruses, bacteria, tissue grafts, toxins and drugs. Further, the inappropriate production of antibodies against host tissues contributes to many diseases including myasthenia gravis, hemolytic anemia, scleroderma, Graves' disease and rheumatoid arthritis.

Because of their importance, a variety of methods have been developed for the detection or quantification of antibody, including an antiviral neutralization bioassay and a radioimmunoassay. In most antibody detection or quantification methods, sample containing analyte antibody is applied to the surface of a device having an suitable immobilized antigen on the surface. The analyte antibody is allowed to bind to the immobilized antigen creating immobilized antigen/analyte antibody complexes. A ligand is then applied to the immobilized antigen/analyte antibody complex-coated surface and binds to the analyte antibody of the immobilized antigen/analyte antibody complex at a site other than one of the analyte antibody molecules' epitope binding sites. The ligand contains a chromogen, enzyme or radioactive label. Detection of the chromogen, enzyme or radioactive label remaining on the surface is a measure of the presence or quantity of the analyte antibody.

More recently, there has been described a sandwich-type of enzyme immunoassay for the detection or quantification of antibody, dubbed an inverse sandwich immunoassay. This assay utilizes the presence of the two IE 950988 -2epitope binding sites on an immunoglobulin molecule which allows each immunoglobulin molecule to simultaneously bind to two separate antigen moieties having a substantially-identical epitope.

In the inverse sandwich immunoassays heretofore described, an antigen is immobilized to the surface of plastic beads or a microtitre plate. Disadvantageously, the known techniques for utilizing an inverse sandwich immunoassay for the detection or quantification of immunoglobulin requires specialized equipment not readily available to a consumer or present in a physician's office. A further disadvantage of these prior techniques of performing an inverse sandwich immunoassay using beads or microtitre plates is that tight control is required over several of the parameters involved in performing the assay. Also disadvantageously, using beads or microtitre plates to perform an inverse sandwich immunoassay necessitates one or two washing steps.

Summary of the invention According to one aspect of the present invention, there is provided a device for detecting the presence or amount of analyte antibody in a sample. The device includes a first zone that includes a first matrix and a first substance that includes an epitope that is capable of binding to the analyte antibody. The first substance has a label. The device further includes a second zone spatially separate from the first zone and in fluid communication with the first zone. The second zone includes a second matrix and a second substance that includes an epitope substantially identical to the epitope on the first substance. The second substance is immobilized on the second matrix and the first substance is capable of moving from the first zone to the second zone after application of the sample to the first zone.

According to another aspect of the present invention, the device can be used to detect antibody to Human Immunodeficiency Virus (HIV). In one preferred embodiment, the device can detect the presence or amount of antibody to HIV-1 glycoprotein 41. In another preferred embodiment, the device can detect the presence or amount of antibody to HIV-2 glycoprotein 36.

According to another aspect of the present invention, there is provided a method for detecting the presence or amount of analyte antibody to Human Immunodeficiency Virus in e sample. First, a device according to one of the foregoing aspects of the present invention is provided. Next, sample is applied to the first zone, the antibody in the sample comprising first and second epitope binding sites. After binding the first epitope binding site on the analyte antibody to the first epitope on the first substance, thereby forming an analyte antibody/first substance complex, the complex is allowed to migrate from the first zone to the second zone. Next, the second epitope binding site on the analyte antibody of the first substance/analyte antibody is allowed to bind to the second epitope on the immobilized second substance in the second zone, thereby forming immobilized first substance/analyte antibody/second substance. Finally, the label upon the first substance of the immobilized first substance/analyte antibody/second substance is detected. In a preferred embodiment, the label includes a chromogen and the detecting step includes visually identifying a color change on the second zone. in another aspect of the invention, there is provided a device for detecting the presence or amount of analyte antibody to each c? two antigens, antigen-1 and antigen-2, in a sample. In a preferred embodiment, antigen-1 is Human Immunodeficiency Virus 1 and antigen-2 is Human Immunodeficiency Virus 2. The device includes a first IE 950988 -3zone that includes a first matrix, an antigen-1 first substance and an antigen-2 first substance. The antigen-1 first substance includes an enitooe from antigen-1, and the antigen-2 first substance includes an epitope from antigen-2. Each of the first substances has a label. The device further includes a second zone spatially separate from the first zone and in fluid communication with the first zone. The second zone includes a second matrix, an antigen-1 second substance and an antigen-2 second substance. The antigen-1 second substance includes an epitope from antigen-1 that is substantially identical to the epitope from antigen-1 on the antigen-1 first substance. The antigen-2 second substance includes an epitope from antigen-2 that is substantially identical to the epitope from antigen-2 on the antigen-2 first substance an epitope. Each of the second substances is immobilized on the second matrix. Each of tha first substances is capable of moving from the first zone to the second zone after application of the sample to the first zone. In one particularly preferred embodiment, the first substance and each second substance include at least one substance selected from the group consisting of HIV-1 glycoprotein 41, HIV-2 glycoprotein 36, and a fragment, derivative or combination of any of the foregoing.

In yet another aspect of the invention, there is provided a device for detecting the presence or amount of a polyvalent analyte in a sample. The device includes a first zone that includes a first matrix and a first substance. The first substance has a label. The device further includes a second zone that includes a second matrix and a second substance. The second substance is immobilized to the second matrix. The second zone is spatially separate from the first zone and in fluid communication with the first zone. The first substance is in relation to the first matrix such that the first substance is capable of moving from the first zone to the second zone after application of the sample to the first zone. A first epitope binding site and a second epitope binding site on the analyte antibody are capable, respectively, of sequentially binding to a first epitope on the first substance and a second epitope on the second substance, such that, after the completion of both bindings, both epitope binding sites are simultaneously occupied. Further, the first epitope on tha first substance and the second epitope on the second substance are substantially identical.

The devices provided according to the several aspects of the present invention may additionally include a control substance.

Brief Description of the Drawings Figure 1 is a schematic representation of a chemical reaction used in manufacturing an embodiment of the present invention.

Figure 2 is a schematic representation of an embodiment of the present invention.

Figure 3 is a schematic representation of an embodiment of the present invention.

Figure 4 is a schematic representation of an embodiment of the present invention.

Detailed Description of the Preferred Embodiments The inverse sandwich enzyme immunoassay method has several advantages over other available antibody detection or quantification methods. First, it detects or quantifies antibody independent of the species-origin of the antibody because equivalent binding sites on an antibody molecule from any species will simultaneously bind two antigen moieties having substantially-identical, appropriate epitopes. This species-independence characteristic of the IE 950988 4inverse-sandwich immunoassay is a great advantage in industries such as the pharmaceutical industry where it is necessary to study the immune reaction of multiple species.

Another advantage of the inverse sandwich immunoassay technique is that human antibody is not required to standardize the assay because the technique is species-independent. This is useful because a suitable preparation of human antibody is often unavailable.

Still another advantage of the inverse sandwich immunoassay is that it requires analyte antibody molecules to specifically bind to two substantially-identical epitopes in order for the analyte antibody to be detected. Thus, non-specific binding of analyte antibody to the solid support w3l not result in a detectible signal which results in reduced background noise.

In one form of inverse sandwich assay, a first antigen is immobilized to the surface of plastic beads or a microtitre plate. Hie immobilized first antigen has en epitope capable of being bound by either of the at least two epitope binding sites on an analyte antibody molecule. Sample potentially containing the analyte antibody is applied to the surface coated with immobilized first antigen and the analyte antibody is allowed to bind to the immobilized first antigen through either of each analyte antibody molecules* at least two epitope binding sites. This creates immobilized first antigen/analyte antibody complexes.

Next, non-analyte sample components and unbound analyte are washed from the surface. A reagent is applied to the surface having the immobilized first antigen/analyte antibody complexes. The reagent contains a second antigen comprising a substantially-identical epitope as the immobilized first antigen and an enzyme label bound to the second antigen. The second antigen-label binds to at least some of the at least two available epitope binding sites on the analyte antibody molecules not bound to the immobilized first antigen, thus creating immobilized first antigen/antibody/second antigen-label complexes. Unbound reagent is washed off and substrate for the enzyme label on the second antigen is applied to the bound immobilized first antigen/antibody/second antigen-label complexes. Tight control is required over the amount of the second antigen-label complex added to the solid support, and over the time at which the reagent containing the second antigen-label is added after applying the sample. Detection or quantification of the antibody is made by detecting a signal produced by the label and substrate.

As discussed above, prior art inverse sandwich assays making use of microtiter plates or solid beads suffer from a number of disadvantages. It is one goal of the present invention to provide a device and method for the detection or quantification of a polyvalent analyte, such as an antibody, which is sensitive, rapid, simple and versatile. In one embodiment, the present invention discloses a device and method which utilize the inverse sandwich principle but avoids some of the disadvantages inherent in using beads or microtitre plates as the solid support through improvements described herein. In one preferred embodiment, the device and method are used to detect the presence of antibody to human immunodeficiency virus. One important improvement made to the inverse sandwich immunoassay by the present invention relates to the spatial separation of the location of the two binding steps.

One aspect of the present invention provides a rapid method for performing an inverse sanwich assay that overcomes the disadvantages of prior art such assays. One inventive device which allows this method to be implemented provides for the detection or quantification of a polyvalent analyte. This device includes a first matrix IE 950988 ·5· having a mobile first substance thereupon and a second matrix having an immobilized second substance thereupon. The first and second matrix are in fluid communication with each other. Sample containing analyte antibody is applied to the first matrix and binds to the first substance. The analyte antibody bound to the first substance moves to the second matrix where it additionally binds to the immobilized second substance. Detection of the presence of the analyte antibody at the location of the second substance is made by a label on the first substance.

As used herein, polyvalent substance and polyvalent analyte* interchangeably refer to a chemical substance having two or more epitope binding sites, each epitope binding site capable of binding to an antigenic determinant or epitope, wherein at least two of the epitope binding sites on a polyvalent substance are capable of binding to substantially-identical epitopes either sequentially or simultaneously so long as after the binding is completed, both epitope binding sites are simultaneously occupied. As used herein, simultaneously bind or a similar phrase refers to the end relationship of the polyvalent substance and epitope, rather than the order m which the binding occurs.

The substantially-identical epitopes can be on either two separate substantially-identical moieties or on two separate substantially non-identical moieties. Thus, a polyvalent substance is capable of at least one of two types of binding. In one type of binding, the polyvalent substance can simultaneously bind an epitope on a first moiety at a first epitope bmding site, while binding a substantially-identical epitope on a substantially-identical second moiety at a second epitope binding site. In a second type of binding, a polyvalent substance can simultaneously bind an epitope on a first moiety at a first epitope binding site, while binding a substantially-identical epitope on a substantially non-identical second moiety at a second epitope binding site.

A polyvalent substance can include epitope binding sites in addition to the two epitope binding sites for substantially identical first and second epitopes of a first and second moiety. These additional epitope binding sites can also bind epitopes that are substantially identical to the first and second epitopes, or in the alternate, can bind epitopes that are substantially non-identical to the first and second epitopes. Non-limiting examples of polyvalent substances include antibodies and receptors.

As used herein, immunoglobulin* and antibody are interchangeably intended to refer to a substance or substances having the four amino acid chain basic unit described above, including IgA, IgE, IgG, IgM, which are normally produced by a host organism in response to an antigen, and which have at least two epitope binding sites, each capable of specific binding with an antigen at a binding site on the antigen referred to interchangeably as an antigenic determinant or epitope. Immunoglobulin and antibody also interchangeably refer to cell surface markers and receptors which comprise the four polypeptide chain unit described above, such as IgD, and to structurally substantially similar naturally occurring and synthetically produced compounds, regardless of their function. As used herein synthetic and synthetically encompass both biosynthetic, non-natural production and nonbiosynthetic, non-natural production.

Immunoglobulin” and antibody further interchangeably refer to a fragment or fragments of a substance which, in the non-fragment form has the four amino acid chain unit, and which in the fragment form has the ability to simultaneously bind an epitope on a first antigen at a first epitope binding site on the fragment, while binding a IE 950988 -6substantially-identical epitope on a second antigen, substantially-identical with the first antigen, at a second epitope binding site on the fragment; or wherein the fragment has the ability to simultaneously bind an epitope on a first antigen at a first epitope binding site on the fragment, while binding a substantially-identical epitope on a second antigen, substantially non-identical with the first antigen, at a second epitope binding site on the fragment.

As used herein, epitope binding site refers to a chemical sequence, chemical group or chemical structure capable of specific binding with an epitope or antigenic determinant through non-covalent bonds, regardless of whether the strength of the bond is lesser, equal to or greater than a covalent bond. On an antibody molecule, the epitope binding site refers to one of the at least two chemical structures formed by a segment of one Gght polypeptide chain and a segment of one heavy polypeptide chain which are capable of specific binding to an epitope on an antigen. The epitope binding site of a non-immunoglobulin polyvalent substance can be composed of a chemical structure similar to that of an immunoglobulin molecule or can be of any other chemical structure capable of an equivalent function.

As used herein, epitope and antigenic determinant are interchangeably intended to refer to a chemical sequence, chemical group or chemical structure that is capable of being bound to an epitope binding site on an immunoglobulin molecule or other polyvalent substance.

As used herein, “antigen refers to any substance to which a host immune system mounts an immune response, or more generally, to any substance having an epitope such that a polyvalent analyte is capable of binding the epitope at either of the at least two epitope binding sites present on the polyvalent analyte. Non-limiting examples of antigens comprise one or more of the following, or a fragment or derivative of one or more of the following, whether naturally existing or synthetically produced: proteins, glycoproteins, fatty acids, iipids, polynucleotides, ribonucleic acids, deoxyribonucleic acids, hormones, enzymes, viruses, bacteria, prokaryotic cells and eukaryotic cods.

As used herein, fragment refers to any part or parts of a substance, less than the whole substance.

As used herein, human immunodeficiency virus (HIV) refers to any of the class of retroviruses capable of causing acquired immunodeficiency syndrome in a human or the equivalent disease in an animal, or any fragment, derivative or attenuated form of the virus, such as in an immunization preparation, whether naturally occurring or synthetically or recombinantly produced, or any combination of the foregoing.

In nno aspect, the present invention includes a device for the detection or quantification of a polyvalent analyte. The device includes at least two zones, a first zone and a second zone. The first zone comprises a first matrix and the second zone comprises a second matrix. The first zone is spatially separated from the second zone and in fluid communication with the second zone.

The first zone additionally comprises a first substance. The second zone additionally comprises a second substance. The first substance has a first epitope capable of being bound by either of a first epitope binding site or a second epitope binding site of the at least two epitope binding sites on the polyvalent analyte. The second substance has a second epitope capable of being bound by either of a first epitope binding site or a second epitope binding site of the at least two epitope binding sites on the polyvalent analyte. The polyvalent analyte can bind IE 950988 •7 the first epitope on the first substance at the first epitope binding site and the second epitope on the second substance at the second epitope binding site simultaneously. The polyvalent analyte can also bind the first epitope on the first substance at the second epitope binding site and the second epitope on the second substance at the first epitope binding site simultaneously.

The first substance can be substantially identical or substantially non-identical with the second substance. However, the first epitope end the second epitope are substantially identical The first substance is in relation to the first matrix such that the first substance is capable of moving from the first zone to the second zone. The second substance is immobilized to the second zona.

The first substance additionally comprises a labaL Detection or quantification of the label serves as a means for detecting or quantifying the polyvalent analyte. The label comprises any of a variety of substances known to those with skffl in the art, including but not limited at least one substance selected from the group consisting of an enzyme, a radioactive substance, a florescent compound, a chromogen, colored latex, gold particles such as colloidal gold, a peroxidase, a textile dye, alkaline phosphatase, σ-glucosidase, y9-galactosidase, glucose oxidase, urease, a fragment thereof and a combination thereof. The label can be bound to the first substance by covalent or non-covalent forces, such as by hydrophobic bonding.

One method of use of the device for detecting or quantifying polyvalent analyte according to the present invention is'm the foBowing manner. Sample potentiaBy containing polyvalent analyte is applied to the first zone comprising the first matrix and first substance having a label. If polyvalent-analyte is present in the sample, at least some polyvalent analyte moieties will bind to the first substance at the first epitope by at least one of the epitope binding sites on the polyvalent analyte, thereby, forming polyvalent analyteffirst substance-label complexes.

The first matrix is porous and of such construction that at least some of the polyvalent analyteffirst substance-label complexes will flow through the first zone to the second zone. Flow can be unidirectional, only towards the second zone, or multidirectional Flow in the direction of the first zone to the second zone is considered positive flow. Multidirectional flow, that is positive flow and non-positive flow, can be both towards the second zone and away from the second zone, or towards the second zone and in a direction neither towards nor away from the second zone, or a combination thereof. Flow can be accomplished by capillary action, suction, the addition of nonsample fluid or by other means as well be understood by those with skill in the art.

Flow rate is dependent on temperature. Generally, the higher the temperature, the faster the flow. Therefore, according to one aspect of the present invention, detection or quantification of polyvalent analyte can be accomplished more quickly by raising the temperature surrounding the device.

The second matrix can be the same material and construction as the first matrix, that is, the first and second matrix can form a unitary matrix. In the alternate, the first and second matrix can be of different material or different construction. Either matrix can comprise multiple materials of differing constructions. In addition, glazing can be added to the surface of at least one of the matrices and first substance, second substance or an additional substance applied to the surface of the glazing rather than to the ungfazed matrix, in order to diminish substancematrix interactions.

IE 950988 Glazing can be used, for example, to maintain the mobility of the first substance on the first zone prior to application of the sample. Appropriate glazing for this use can comprise aqueous lactose or sucrose or cellulose solution, applied and then dried, upon which the first substance-label would be then be applied.

Upon entering the second zone, at least some of the polyvalent analyte/first substance-label complexes will encounter the immobilized second substance having a second epitope. At least some of the polyvalent analyte moieties on the polyvalent analyte/first substance-label complexes will bind the second epitopes on the immobilized second substances on at one of the at least two epitope binding sites on the polyvalent analyte moieties that is not binding the first epitope on the first substance. The result is the creation of second substance/polyvalent analyte/first substance-label complexes immobilized to the second matrix through the second substance moieties.

Polyvalent analyte/first substance-label complexes not binding to the second substance, unbound sample components, unbound first substance-label complexes and any additional non-second substance components not part of the second substance/polyvalent analyte/first substance-label complexes will continue to flow away from the immobilized second substance on the second zone.

The label on the immobilized second substance/polyvalent analyte/first substance-label complexes is detected or quantified by any of a variety of means appropriate for the label as are readily understood by those with skill in the art. For example, colored labels or 'direct labels' can be visually detected without special equipment and can be quantified by visual comparison against a reference color chart. Fluorescent labels or radioactive labels can be detected or quantified using appropriate instruments. Enzyme labels can require the addition of an appropriate enzyme substrate. The enzyme substrate can further comprise an additional label allowing detection in the presence of the enzyme.

In one preferred embodiment, the label comprises gold particles. The use of peptides alone as first substance disadvantageous^ promotes aggregation of the gold particles. Therefore, certain modifications of peptides to produce first substances as described below advantageously decreases aggregation of gold particles while retaining activity to be bound by the analyte polyvalent substance.

Conjugation of a peptide-based or other first substance to gold particles to produce first substance-label can be accomplished through adsorption alone or through covalent binding. Adsorption depends upon several effects, including electrostatic interaction between the negatively charged surface of the gold particles and positively charged groups of the peptide-based or other substance, hydrophobic attraction between the surface of the gold particles and peptide-based or other substance, and dative binding between the gold conducting electrons and sulphur atoms of the peptide-based or other substance.

The type of conjugation of the peptide-based or other first substance to the gold particles affects the reactivity of the first substance with analyte polyvalent substance. In the case of analyte antibody, the sterical accessibility or molecular orientation of the epitope in the first substance determines, at least in part, both the sensitivity of the test and the binding rate of the analyte antibody to the first substance. The binding rate determines, at least in part, the time required to complete the test.

IE 950988 -9Peptides containing between about 15 and 30 amino acids vary in their ability to bind to gold particles, depending on the peptides' exact ammo acid composition and isoelectric point. Therefore, various modifications can be made to peptide-based or other first substances to improve the yield of stable, usable first substance.

Referring now to Figure 1, there is illustrated one method of producing the first substance by modifying 5 glycoprotein, whether derived from natural sources or synthetic sources, and linking the modified glycoprotein with the gold particles. Initially, thB carbohydrate moiety of the glycoprotein (a) is oxidized using sodium periodate to produce the aldehyde (b) as shown. Next, a substance containing at least one amino group is linked to the aldehyde forming entity (c). Finally, compound (c) is reduced using sodium cyanborohydride to form stable covalent bonds as indicated in (d).

Alternately, another method of producing the first substance involves a heterobifunctional method employing b reaction between maleimide and sufhydryl groups as is understood by those with skill in the art An example of this method is described in publications by Duncan, RJ.S. et al., Analytical Biochemistry. 132, 68, 1983, Fupwara, K. et al., Journal ofImmunological Method, 45,195,1981, and Kitagawa, T., et aL, Journal Biochemistry, 94, 1160, 1983. In summary, a malemide group is joined to the peptide-based or other substance containing the epitope using Ν-κ-maleidobutyryloxy-succinimide (GMBS). A sulfhydryl group is joined to a carrier substance using N-succinimidyl-S· acetyl-thioacetate (SATA) and hydroxylamine. The two activated molecules are linked together under physiological conditions at room temperature for between about 2 and 10 hours. After preparation of the first substance, it is combined with gold particles by the method described in detail below.

Detection of the label on the immobilized second substance/polyvalent analyte,first substance-label complexes is an indirect indication of the presence of the polyvalent substance bound in the second zone and, hence, present in the sample. Quantification of the label on the immobilized second substance/polyvalent analyte,first substance-label complexes is an indirect indication of the quantity of the polyvalent substance bound in the second zone and, hence, present in the sample or applied to the device. As used herein, amount* can be taken to mean concentration, activity, quantity, titre and a related measure or a combination thereof.

The first zone of the device for the detection or quantification of polyvalent analyte can comprise at least two zones, a sample application region and a separation zone, with or without additional zones. The sample application zone serves as a site for application of the sample. The separation zone serves to separate the sample application zone from the second zone. The first substance can be located in the sample application zone, in the separation zone, in other zones at least partly comprising the first zone or in a combination of zones at least partly comprising the first zone. If the first zone comprises at least two zones, the first matrix can also comprise at least two matrices, each matrix having a unique composition or pore size.

The second zone of the device for the detection or quantification of polyvalent analyte can comprise at least two zones, a detection zone and a collection zone, with or without additional zones. The detection zone serves to contain the immobilized second substance. The collection zone serves to draw away from the detection zone polyvalent analyte,first substance-label complexes not binding to the second substance, unbound sample components, unbound first substance-label complexes and any additional non-second substance components not part of the second IE 950988 -10substance/polyvaient analyte/first substance-label complexes. If the second zone comprises at least two zones, the second matrix can also comprise at least two matrices, each matrix having a unique composition or pore size.

The device for the detection or quantification of polyvalent analyte can be constructed to allow the detection or quantification of multiple polyvalent analytes in a sample or samples. Detection and quantification of multiple polyvalent analytes means that the device can be used to distinguish the individual presence or quantity of each of two or more polyvalent analytes in a sample or samples, or means that the device can be used only to determine if at least one of the polyvalent analytes of two or more polyvalent analytes is present or the amount of a combination of at least two polyvalent analytes.

One way for the device to be used to distinguish the individual presence or quantity of multiple oolyvalent (0 analytes in a sample or samples containing at least two polyvalent anahrtes. is to provide at least one second substance immobilized in the second zone for each oolwalent analyte to ba individually detected or quantified, The second substances can occupy the same portion of the second zone or different portions of the second zone. If they occupy different portions of the second zone, they can be linearly arrayed, horizontally arrayed, vertically arrayed or a combination or variation thereof. If they occupy the same portion of the second zone then the labels on the corresponding first substances must be distinguishable from one another when the labels occupy the same space.

For example, in order to determine the presence or quantity of polyvalent analyte #1 and polyvalent analyte #2 from a single or multiple samples, the device can be constructed with two first substances, first substance #1 and first substance #2, each having a label, label #1 and label #2, respectively, in the first zone. First substance #1 and first substance #2 can occupy the same space in the first zone, different spaces in the first zone or overlapping spaces in the first zone. Application of the sample or samples containing polyvalent analyte #1 and polyvalent analyte #2 to the first zone produces polyvalent analyte #1/first substance #1-label #1 complexes and polyvalent analyte #2/first substance #2-label #2 complexes. At least some of these two complexes will flow from the first zone to the second zone where they will encounter second substance #1 and second substance #2 immobilized in the matrix of the second zone. At least some of polyvalent analyte #1 and polyvalent analyte #2 will bind to immobilized second substance #1 and immobilized second substance #2 respectively through one of their at least two epitope binding sites to produce immobilized second substance #1/polyvalent analyte #1/first substance #1label #1 complexes and immobilized second substance #2/polyvalent analyte #2/first substance #2-label »2 complexes. Polyvalent analyte/first substance-label complexes not binding to the second substance, unbound sample components, unbound first substance-label complexes and any additional non-second substance components not part of the second substance/polyvalent analyte/first substance-label complexes will continue to flow away from the immobilized second substances.

Polyvalent analyte #1 and polyvalent analyte #2 are detected or quantified indirectly by detecting or quantifying label #1 and label #2 of the immobilized second substance #1/polyvalent analyte #1 /first substance #1label #1 complexes and immobilized second substance #2/polyvalent analyte #2/first substance #2-label #2 complexes, by analogous methods described above for detecting or quantifying a single polyvalent analyte, as will be understood by those with skill in the art.

IE 950988 •11A second way for the device to be used to distinguish the individual presence or quantity of multiple polyvalent analytes in a sample or samples containing at least two polyvalent analytes, is to provide at least one label for each polyvalent analyte to be individually detected or quantified, even though there is only a single second substance immobilized in the second zone. In this aspect of the present invention, detection or quantification of the label corresponding to each polyvalent analyte among the immobilized second substance,polyvalent analyte,first substance-label complexes will indirectly detect or quantify the amount of each polyvalent analyte present in the sample or samples. As will be readily appreciated by those with skill m the art, the labels can ba of differing types, such as a fluorescent label and a chromogen, or can ba of the same type, such as two chromogens, which produce additive effects that are distinguishable for the effect for either label individually.

Three or more polyvalent analytes in a sample or samples can similarly ba detected using a device having three or more second substances, three or more labels or a combination thereof, in analogous methods to that used to detect two polyvalent analyte substances. Further, the modifications of the device and method of using the device described above and below for detecting or quantifying a single polyvalent analyte can be adapted for use in detecting or quantifying multiple polyvalent analytes.

Nonlimiting examples of multiple polyvalent analytes suitable for detection or quantification by the device and method according to a preferred embodiment of the present invention include antibodies of significance in pregnancy such as antibodies to toxoplasma, rubella, cytomegalovirus and herpes, antibodies to various common allergens such as antibodies to various pollens, animals, food or components of cosmetics, antibodies to common sexually transmitted diseases such as herpes, AIDS, syphilis, gonorrhea and chlamydia, antibodies involved in autoimmune diseases, and polyvalent analytes produced by damaged cardiac or neurologic tissues.

In a particularly preferred embodiment, the device and method of the present invention is used to detect multiple types of antibodies having specifities for various gene products or modified gene products of HIV-1, HIV-2 or a combination, thereof. Nonlimiting examples of suitable polyvalent analytes include antibodies to HIV-1 core protein 24, transmembrane glycoprotein 41, reverse transcriptase 51 and envelope glycoprotein 120. Using a device according to one aspect of the present invention to detect or quantify these antibodies simultaneously allows an antibody profile to HIV-1 to be determined, similar to a Western blot, as is understood by those with skill in the art. An equivalent antibody profile can be determined for HIV-2 as is understood by those with skill in the art.

A sample can originate from multiple sources, can be taken from the same source at different times, and can be applied together, separately or intermittently. Samples can be derived from any of a variety of fluids, or can be derived from non-fluids and constituted or reconstituted into fluids before application to the device. Non-limiting examples of samples include blood, serum, plasma, urine, sputum, cell culture supernatant, cell culture suspension, tissue culture supernatant, tissue culture suspension, cerebrospinal fluid, bone marrow, aqueous fluid, lacrimal fluid, saliva, pus, seminal fluid, amniotic fluid, sweat, mammary gland secretion, lymph, intraarticular fluid, pericardial fluid, peritoneal fluid and combinations thereof.

IE 950988 -12The appropriate pore size or range of sizes present in the first and second matrices, or additional matrices, is determined by a number of factors. In addition to varying pore size, pore configuration can also be varied to promote unidirectional flow, bidirectional flow, or to limit flow in another manner.

Generally, the larger the pore size of the first matrix, the more rapidly the polyvalent analyte/first substancelabel complexes flow towards the second zona, which is generally advantageous. However, the larger the pore size, the less surface area in the second matrix is available to immobilize the second substance and contain the first substance, which is generally disadvantageous.

Also a factor in determining an appropriate pore size »the viscosity and particle size present in the sample fluid. If the sample contains particles significantly larger than the polyvalent analyte/first substance-label complexes, it can be advantageous to have a first matrix pore size large enough to pass the polyvalent analyte/first substancelabel complexes but small enough to filter out unwanted particles present in the sample such that they do not reach the second zone.

For example, in a sample of blood where the analyte is immunoglobulin and the first substance a suitable protein, first matrix pore size can be less than about 5μ in order to retain rad cells and white cells in the first zona. Non-limiting examples of sample fluids that can use the matrix pore size to filter out unwanted particles include sputum, bone marrow, pus, seminal fluid, mammary gland secretion, lymph and combinations thereof.

As an alternate to adapting the pore size to act as a filter, the sample can be filtered prior to application to the device or can pass through an adjunct filter covering the sample application site on the first zone. The filter can be made of the same or different material as a matrix, only having a pore size appropriate for the filtering function. The first matrix, second matrix, any additional matrix and the filter, if one is present, can comprise at least one of the following, nitrocellulose, polystyrene, polyvinyl, .polycarbonate, nylon, cellulose acetate, glass fiber, filter paper or a combination thereof, as would be appreciated by those with skill in the art In a preferred embodiment, the matrices are comprised of nitrocellulose, as nitrocellulose is available commercially in a range of pore sizes.

The overall configuration of the device can be any of a number of forms including substantially rectangular, square, oval, round, trapezoidal or any other shape suitable for purposes as would be understood by those in the art. For example, the first and second zone can be in horizontal fluid communication with the first zone occupying one end of a rectangular overall configuration and the second zone occupying the other end of a rectangular overall configuration. Alternately, the first and second zone can be in horizontal fluid communication with the first zone occupying the center of a disc-like overall configuration and the second zone occupying a circumferentially area around the first zone.

In another aspect of the present invention, the device can have first and second zones in vertical fluid communication with each other or in a combination of horizontal and vertical communication with each other.

In another aspect of the present invention, the device for detecting or quantifying the amount of a polyvalent analyte has Y-shaped or other multi-pronqed shaped configuration. In this aspect of the present invention, the first zone occupies the lower part of the Y, or central portion of the multi-pronged shape, and the second zone occupying the two upper parts of the Y or multiple prongs of the multi-pronged shape. These configurations can be IE 950988 •13especially suitable for the simultaneous detection of at least two polyvalent analytes, where each of the corresponding at least two second substances are immobffized in the matrix on one of the prongs. In this form, the label on the multiple first substances can be identical and individual detection or quantification of the multiple polyvalent analytes accomplished by presence or quantity of the label corresponding to each polyvalent analyte in a specific portion of the various prongs. Other variations for the position of the second substances, and hence immobilization site for the label, are described above. in another aspect of the present invention, the device for detecting or quantifying the amount of a polyvalent analyte also has Y-shaped or other multi-pronged shaped configuration. In this aspect of the present invention, the second zone occupies the lower part of the Y, or central portion of the multi-pronged shape, and multiple first zones occupy the two upper parts of the Y or multiple prongs of the multi-pronged shape. These configurations can bo especially suitable for the simultaneous detection of at least two polyvalent analytes, where each of the corresponding at least two first substances and labels are immobilized in the matrix on one of the prongs. In this form, the second substances corresponding to each polyvalent analyte can bo identical and individual detection or quantification of the multiple polyvalent analytes accomplished by presence or quantity of the differing label corresponding to each polyvalent analyte in the second zone. Other variations for the position of the second substances, end hence immobilization site for the label, are described above.

Application of the sample to the device can be applied by any of a number of methods as are understood by those with skill m the art. Non-limiting examples include at least one of dipping the device in the sample, dropping the sample on the device, such as for example by a pipette, and inserting the device into a tube containing sample. Further, one end of the device can contain a wick. Dipping the wick in the sample would allow an amount of sample, determined by the properties of the wick, to flow into the first matrix.

Alternately, a non-fluid sample or sample containing insufficient fluid to cause the polyvalent analyte,first substance-label complexes to flow can be applied to the first zone and non-sample fluid applied to the device in at least one manner as would fluid sample be applied, in order to cause the polyvalent analyte,first substance-label complexes to flow to the second zone.

In another aspect of the present invention, the device can contain one or more control zones. Each control zone comprises a control matrix having a control substance immobilized thereupon. The control zone can be part of second zone. In one particularly preferred embodiment, the collection zone comprises the control zone.

In one preferred embodiment, the control substance has the property of binding a corresponding first substance-label, at either the epitope binding site or another site on the first substance or label, and hence immobilizing the first substance-label to the control matrix. The control substance can comprise at least one of a monoclonal antibody, a polyclonal antibody, the polyvalent analyte and a fragment thereof.

The control substance can be the same as the polyvalent analyte, but does not need to be the same. Further, the control substance can be an antibody having an equivalent binding specificity as the polyvalent substance but coming from a different species than the polyvalent analyte. Using a species difference advantageously allows IE 950988 .μ. the selection of a noninfectious control substance in cases where the polyvalent analyte itself is infective or dangerous to persons manufacturing the device.

In another preferred embodiment, the control substance comprises an anhydrous substance that produces a color when contacted with fluid from the sample. An example of a suitable control substance according to this aspect of the present invention is anhydrous copper sulphate.

In one preferred embodiment of the present invention, the control zone is downstream from the second zone in a direction of sample flow from the first zone to the second zone. Thus, to reach the control zone, the first substance-label must pass through at least a portion of the second zone.

Incorporation of a control substance into the device downstream from the second zone advantageously allows an objective means of determining that the test for detection or quantification of a polyvalent analyte has been completed. Further, a control substance can be used as an indication that the first substanca-labei complexes retained activity to be recognized by the polyvalent analyte.

The control substance can also be other than downstream from the second zone in order to demonstrate the retained activity of the first substrate label without mdicating whan the test is completed. Additional control substances can be incorporated to indicate, for example, exposure to the ah or humidity where appropriate by incorporating a control substance that changes color when exposes to air or moisture.

Where a control zone is present, a collection zone as described above, can be placed downstream from the control zone, up stream from the control zone or split into both upstream, and downstream segments.

In a preferred embodiment of the present invention, the matrices are backed by, sandwiched between or incased in a housing. The housing preferably comprises a moisture-proof material such as any of a variety of plastics known to those with skill in the art and supply handling strength and a means of handling without directly touching the matrices.

The housing can be opaque and can contain words or symbols, thereupon, such as to indicate the first zone or sample application area, the second zone or detection area and control zone or to give instructions to the user of the device. If the housing is opaque and the label is detected by visual means or means requiring the transmission of visible spectrum radiation, it is contemplated by the present invention that non-opaque areas of the housing would be incorporated over one or more areas of the second zone, such as the detection zone, or control zone. The housing can further contain apertures for application of the sample or application of additional substances needed for detection of the label.

In a preferred embodiment, the device is packaged in a moisture proof, opaque package, such as sealed aluminum foil, and stored in a temperature ranging from about 2°C to about 30°C. A desiccant can also be provided as is known to those with skill in the art.

Referring now to Figure 2, a device 100 for the detection or quantification of polyvalent substance according to one aspect of the present invention is shown having a sample application end 200, a left side 210 a right side 220 and a sample collection end 230. The device is divided into a first zone 110 and a second zone 120, IE 950988 -15wherein the first zone is spatially separate from the second zone and in fluid communication with the second zone. The relative proportions illustrated in Figure 2 were chosen for clarity rather than necessarily drawn to scale.

The first zone 110 comprises a sample application zone 112 and a separation zone 114. The separation zone 114 comprises First substance-label 116 thereupon or therewithin, as indicated by the 45° crosshatching extending from left side 210 to right side 220.

The second zone is divided into two zones, a detection zone 122 and a collection zone 124. The detection zone 112 comprises second substance 126 immobilized thereupon or therewithin, as indicated by the speckled pattern extending from loft side 210 to right side 220.

The collection zone 124 comprises control substance 128 thereupon or therewithin as indicated by the 135° cross-hatching extending from loft side 210 to right side 220. In one preferred embodiment, not illustrated, the control substance Is part of a control zone spatially separated from the second zone and downstream from the second substance.

Referring now to Figure 3, a device 300 for the detection or quantification of multiple polyvalent substances according to one aspect of the present invention is shown having a sample application end 310, a left side 330, a right side 340 and a sample collection end 320. The device is divided into a first zone 350 and a second zone 360, wherein the first zone is spatially separata from the second zone and in fluid communication with the second zone. The relative proportions illustrated in Figure 3 were chosen for clarity rather than necessarily drawn to scale.

The first zone 350 comprises a sample appFication zone 352 am) a separation zone 354. The separation zone 354 comprises 8t least one first substance-label 356 thereupon or therewithin, as indicated by the 45° crosshatching extending from left side 330 to right side 340. When used to detect multiple polyvalent analytes, the at least one first substance can comprise multiple first substance-labels, such as one first substance-label for each polyvalent analyte to be detected or can comprise a single first substance-label depending on the multiple polyvalent analytes, as will ba understood by those with skill in the art with reference to the disclosure herein.

The second zone is divided into two zones, a detection zone 362 and a collection zone 364. The detection zone 362 comprises at least one second substance 328 immobilized thereupon or therewithin, as indicated by the speckled pattern extending from left side 330 to right side 340. As shown, the detection zone 362 comprises multiple second substances 327, 328 and 329 immobilized thereupon. As is understood by those with skill in the art with reference to the disclosure herein, the device can comprise one second substance for each polyvalent analyte to be detected or less second substances for each polyvalent analyte to be detected.

According to a preferred embodiment of the present invention, when multiple second substances are utilized to detect multiple polyvalent analytes, the multiple second substances are separated by at least about 3 mm of second matrix and more preferably by about 5 mm of second matrix. In another preferred embodiment of the present invention, the at least one second substances is present over an at least a 1mm wide (measuring in the direction of positive flow) horizontal strip across the device and more preferably is present over an at least a 2 mm wide strip across the device. -16The collection zone 364 comprises at least one control substance 332 thereupon or therewithin as indicated by the 135° cross-hatching extending from left side 330 to right side 340. In one preferred embodiment, not illustrated, the control substance is part of a control zone spatially separated from the second zone and downstream from all of the at least one second substance.

According to a preferred embodiment of the present invention, when multiple second substances are utilized to detect multiple polyvalent analytes, the multiple second substances are separated by at least about 3 mm of second matrix and more preferably fay at least about 5 mm of second matrix. This separation is particularly useful when the label or labels are to be detected visually as this spacing is conducive to determining tha spatial separation of second substances.

In another preferred embodiment of the present invention, the at least one second substance is present in at least a 1mm wide fin the direction of positive flow) horizontal strip across the device and more preferably is present in at least a 2 mm strip across the device. This strip size is particularly useful when the label or labels are to be detected visually as this size is conducive to distinguishing the presence of a colored label against a background.

The overall size of a device made according to the disclosure herein will vary with the type of use, such as whether one or multiple polyvalent analytes are to be detected. In a preferred embodiment, the overall size would be large enough to handle and use manually, while small enough to manufacture effectively. For example, the device in strip configuration as shown in Figures 2 and 3, without any housing cap be at least about 4 mm wide and more preferably at least about 6 mm wide. Similarly, in a preferred embodiment, the device in strip configuration as shown in Figures 2 and 3, without any housing can be at least about 20 mm long and more preferably at least about 50 mm long. In a particularly preferred embodiment, the device is at least about 60 mm to 80 mm long.

In one preferred embodiment of the present invention, the method of detecting or quantifying polyvalent analyte present in a sample does not require the use of instrumentation after applying the sample to the device and does not require the addition of reagents to the test device after application of the sample. It is therefore, a onestep method, as the term is understood by those with skill in the art. The use of a standardized color chart does not qualify as the use of instrumentation, as is understood by those with skill in the art.

In another preferred embodiment, the method of detecting or quantifying polyvalent analyte present in a sample does require the use of instrumentation after applying the sample to the device or does require the addition of fluids to the test device after application of the sample. It is therefore, a two-step or multi-step method, as the term is understood by those with skill in the art. The additional steps can include the application of non-sample fluid for a sample containing insufficient fluid and the addition of enzyme substrates or developing solutions to the location of the second substance as required to detect or quantify the label.

IE 950988 -17Example 1: Manufacture of Device for the Detection of Antibody to HIV-1 glycoprotein 41 In accordance with one aspect of the present invention, a device for the detection and quantification of immunoglobulin to HIV-1 envelope glycoprotein 41 was manufactured bs follows. Initially, first substance-label was prepared using modified synthetic peptides and gold particles, and was impregnated onto a suitable first matrix. Next, second substance was prepared using modified synthetic peptides and immobilized onto a suitable second matrix. Finally, the test device was assembled from the first matrix impregnated with first substance-label the second matrix containing immobilized second substance and other components. Each of these steps are discussed in greater detail below. All steps in the construction of the device were performed using clean hands techniques so as not to contaminate the device with proteins present on the skin.

(A) PREPARATION AND IMPREGNATION OF FIRST SUBSTANCE-LABEL Colloidal gold used in one aspect of the present invention, were gold particles coated with modified synthetic peptides. The gold particles had a characteristic reddish color and a diameter of between about 5 (five) and 50 (fifty) nanometers. The reddish color of the gold particles were utilized to visually detect and quantify analyte immunoglobulin. Therefore, according to one aspect of the present invention, analyte antibody were detected and quantified by a visual signal without either using additional instrumentation after applying the sample or without adding substances to the test device after applying the sample.

The preparation of first substance-label involved three procedures. First, gold particles of the proper size were prepared. Next, synthetically produced HIV-1 transmembrane or envelope glycoprotein 41 (gp41) was covalently linked to Bovine Serum Albumin (BSA) or Bovine Serum Albumin-Lactosyl (BSA-L) forming gp41BSA or gp4,BSA-L, respectively. Finally, the gp4,BSA or gp41 BSA-L were conjugated with the gold particles, thereby, forming first substance. The details of these procedures are described below. (i) Preparation of the Gold Particles Gold particles of the proper size were produced by chemical reduction of tetrachloroauic acid with sodium citrate in a manner known to those with skill in the art. In summary, 100 ml glass distilled, 0.2 μ filtered water was heated to approximately 85 to 90*C while constantly stirring. 0.1 ml of 10% HAuCI4 was added and the resultant solution boiled for about 2 minutes. Next, 0.15 ml of freshly prepared 12% sodium citrate (trisodium citrate dihydrate) was added and the solution kept boiling for about 5 minutes. The solution, which was now intensely reddish colored, was allowed to cool and stored at between 4 and 8°C until needed.

In the alternate, gold particles were also purchased commercially from a variety of known sources, including British Biocell International of Cardiff, United Kingdom, Sigma of St. Louis, MO or Amersham of Arlington Heights, Illinois. ,Ε950988 -18(iia) Covalent Linkage of HIV-1 Envelope Glycoprotein 41 to Bovine Serum Albumin to Produce GP„BSA Using Glutaraldehyde Next, HIV-1 envelope glycoprotein 41 (“gp41) was linked to Bovine Serum Albumin (BSA) in preparation for conjugation with the gold particles as follows. Synthetically produced HIV-1 peptide derived from the highly conserved N-terminal region of the transmembrane glycoprotein 41 containing twenty (20) amino acid residues 594613 (used interchangeably with gp41) was obtained from Ferring Diagnostics AB, Mahno, Sweden. BSA was obtained from Sigma of St. Louis, MO but can be obtained from other sources known to those with skill in the art, including United States Biochemical Corporation, Cleveland, Ohio, Miles Laboratories, Elkhart, IN and Irvine Scientific, Santa Ana, CA. Electron-microscopy grade Glutaraldehyde (GA) 25% was also obtained from Sigma of St. Louis, MO but can be obtained from other sources known to those with skill in the art, such as E. Merck, Darmstadt, Germany and ICN Biomedicals, Inc, Costa Mesa, CA. 6.0 mg of gp41, 13.0 mg of BSA in 2.0 ml 0.01 M PBS (pH 7.2±0.2) and 50//11% GA were added to a 12 x 75 mm glass tube. The air was displaced with nitrogen gas and the tube was sealed with Parafilm1. The resultant solution was continuously stirred on a magnetic stir plate at room temperature for between 4 and 8 hours until moderate turbidity developed.

Fresh reducing solution of sodiun cyanoborohydride (NaCNBHJ was prepared containing 20 mg NaCNBHj/ml distilled water. 50 pi of this reducing solution was added to the stirred mixture and stirring continued for 15-30 minutes at room temperature while the mixture was shielded from light. The resultant mixture containing conjugated gp41BSA and unconjugated peptide was concentrated and partially purified by. centrifugal ultrafiltration method at 1500 x g for 15 minutes at 8°C, using Centriprep-30 concentrator from Amicon, Inc., Beverly, MA.

Finally, the concentrated solution of about 1.5 ml of conjugated gp41BSA was purified by applying it to a 1 x 25 cm Sephadex G-25 column equilibrated with 10 mM sodium phosphate, pH 7.2, containing 145 mM sodium chloride and 0.05% sodium azide (PBS). 1.0 ml fractions were collected at room temperature and the first peak containing gp41BSA conjugate was evaluated to determine immunoreactivity with the specific human anti-gp41 antibody. Fractions comprising the center 75 to 80% of the immunoreactive peak, were then pooled and stored at 4 to 8°C. (fib) Covalent Linkage of HIV-1 Envelope Glycoprotein 41 to Bovine Serum Albumin-Lactosvl to Produce GP„BSAL In an alternate method to that disclosed in section |A)(iia), HIV-1 envelope glycoprotein 41 (gp41) was 30 covalently linked to Bovine Serum Albumin-Lactosyl (BSA-L) in preparation for conjugation with the gold particles as follows. Gp41 was obtained from the same source identified above. BSA-L was obtained from Sigma, St. Louis, MO, but can be obtained from other sources known to those with skill in the art. mg of BSA-L was activated in a 12 x 75 mm glass tube by first dissolving it in 750 pi of distilled water. 50 pi of 3 molar sodium acetate was used to adjust the pH to 4.5. 100 pi of 0.1 molar periodate solution was added. The resultant solution was well mixed and allowed to incubate at room temperature for about 5 minutes. •19IE 95093g Next, 150 //1 of 0.1 molar periodate was added. The glass tube was sealed and allowed to incubate at room temperature for about 35 minutes, completing the activation step.

The activated BSA-L was purified by applying the mixture to a 1 x 25 cm Sephadex G-25 gel permeation column equilibrated with 0.5 mM sodium acetate and having a pH of 4.5. Fractions were collected in 1.0 ml increments. Protein containing fractions were identified by Coomassie* Brilliant Blue G-250 dye reagent supplied by BioRad in form of a single solution as BioRad Protein Assay Dye Reagent, BioRad, Richmond, CA. Center fractions comprising 70 to 80% of the protein peak were pooled to yield purified, activated BSA-L Conjugation of activated BSA-L with gp41 was accomplished by dissolving gp4, in distilled water and combining this solution with activated BSA-L at a ratio of 1.0 mg of gp« for each mg of activated BSA-L 2.5 mg activated BSA-L in 0.6 ml 0.5 mM sodium acetate ware mixed with 2.5 mg gp», dissolved in 125 pf distilled water and adjusted with 50/d 0.5 M sodium carbonate to pH 9.5. The pH of the resultant solution was adjusted to 9.5 using 0.5 M sodium carbonate buffer. The solution was then incubated at room temperature for 2 hours, and followed by incubation at 2-8°C for between 12 and 18 hours. The resultant Schiff base was stabilized by adding 0.32 M sodium cyanoborohydride solution to obtain a final concentration of 8-10 mM of sodium cyanoborohydride and incubated at room temperature for 30 minutes.

Finally, the conjugated gp41BSA-L was purified by dialysis against 5 mM sodium borate buffer having a pH of 9.0 using a dialysis membrane tubing with a molecular weight cut-off of 12,000 to 14,000, obtained from Spectrum Medical Industries, Inc., Los Angeles, CA. The buffer was changed 4 to 6 times during 24-48 hours of dialysis using a magnetic stir plate at 4 to 8°C. The purified conjugated gp41BSA-L was stored at 4-8°C. (iii) Heterobifunctional linkage of HIV-1 Envelope Glycoprotein 41 to Bovine Serum Albumin to Produce GP^-BSA.

The HIV-1 glycoprotein 41 (’gp41’) was linked to Bovine Serum Albumin ('BSA') in preparation for conjugation with the gold particles as follows. gp41was obtained from Ferring Diagnostics AB, Malmo, Sweden. Sephadex G-25 and G-10, Bovine Serum Albumin, hydroxylamine, EDTA, NaQ, Tris and dimethylformamide were obtained from Sigma, St Louis, MO.the sources identified previously. N-succinimidyl-S-acetylthioacetate and N-y-maleidobutyrloxy-succinimide were obtained from Calbiochem Novabiochem, Nottingham, England. Ultrogel AcA 34 was obtained from Spectrum Europe B.V., Breda, The Netherlands. SeroCard™ HIV 1/2 assays were obtained from Trinity Biotech, Dublin, Ireland. a) 5mg of peptide was dissolved in distilled water in a 12 x 75 mm glass test tube to a final concentration of lOmg/ml and an equal volume of 0.1 M borate buffer (pH 6.8) added. Two additions of 164 ul of 175uM/ml of N-succinimidyl-S-acetyl-thioacetate in dimethylformamide (DMF) were added to the peptide solution and incubated in the IE 950988 -20dark at room temperature for 30 minutes on each occasion. 230 ul of 0.5 M hydroxy lamine in 0.1 M Na^HPC^ , pH 7.0 was then added to the solution and incubated in the dark for 30 minutes at 3O-32°C.

The activated peptide was purified by applying the mixture to a 1 x 30 cm Sephadex G-10 gel permeation column equilibrated with 0.05 M Tris/ 0.145 M NaCl/ 2.5 mM EDTA, pH 7.0. Fractions were collected in 1.0 ml increments. lOul of each fraction was added to 190ul of a known anti-gp41 positive human serum sample, incubated at room temperature for 10 minutes followed by assaying on SeroCard™ HIV 1/2 rapid assay. Fractions giving negative test results were identified as containing activated peptide. These fractions were pooled, yielding activated gp41 peptide. b) BSA was activated as follows. 10 mg of BSA was added to a 12 x 75 mm glass test tube and dissolved in distilled water to a final concentration of 10 mg/ml. Two additions of 70 ul of 175uM/ml N-y-maleidobutyrloxy-succinimide in DMF were added to the solution, mixed and incubated in the dark at room temperature on each occasion for 30 minutes.

The activated BSA was purified by applying the mixture to a 2 x 25 cm Sephadex G-25 gel permeation column equilibrated with 0.05M Tris, pH 7.2.

Fractions were collected in increments of 1.0 ml. Protein containing fractions were identified using measurement of file optical density of each fraction at a wavelength of 280nm and pooled to yield activated BSA. c) Activated peptide and activated Bovine Serum Albumin were linked by combining them in varying molar ratios (typically 1:1 to 1:50, BSA:peptide) in a 12 x 75 mm glass test tube for 4 to 24 hours in the dark at room temperature.

This material was purified by applying the mixture to a 2 x 100 cm Ultrogel AcA-34 gel permeation column equilibrated with 0.05 M Tris, pH 7.2. The purified conjugated gp4 rBSA was stored at 2-8°C.

IE 950988 -21(iv) Conjugation of Envelope Glycoprotein 41IBovme Serum Albumin or Envelope Glycoprotein 41/Bovine Serum Albumin-Lactosvl with the Gold Particles to Produce first Substance-Label Both gp4,BSA and gp4lBSA-L, as prepared by the methods disclosed in sections (AKiia) or (A)(iib) above, respectively, are suitable for conjugation with the gold particles and were used to produce first substance-labeL Initially, the minimal amount of gp41BSA or gp4IBSA-L needed to fully stabilize the suspension of gold particles, prepared in step (A](i) above, was determined as follows. milliliter aliquots of gold solution were dispensed into 12 x 75 mm glass test tubes. The pH was adjusted to between 7.2 and 7.5 using 40 μΙ of 0.25 M sodium borate having a pH of 7.25. 10 to 100 μΙ containing known quantities of gp41BSA or gp41BSA-L were then added to the tost tubes. The resultant solutions were mixed and incubated at room temperature for 5 minutes. Finally, 100 μΙ of 10% sodium chloride solution were added to each test tube.

After an additional 5 minutes the change in color of colloidal gold suspension was recorded. Retention of the reddish color indicated stabilization of the gold particles by the corresponding quantity of the peptide-BSA conjugate. Loss of the reddish color and appearance of a purple or blue color indicated aggregation of the gold particles by the high salt concentration of sodium chloride and, therefore, insufficient quantity of protecting peptideBSA conjugate. The minimum quantity of peptide-BSA conjugate which retained the reddish color of the gold particles was considered as the stabilizing concentration and was used for future gold-peptide BSA conjugate preparation. Using this method, it was found that 25 μρ of gp4lBSA, or alternately 25 fjg of gp41BSA-L, gave optimal stabilization of 30 nm gold particles to be linked with the above peptide-BSA molecules.

After the above determination was made, 70 ml of the suspension of 30 nm gold particles was dispensed into a clean glass Erlenmeyer flask. The pH of the suspension was adjusted to between 7.2 and 7.5 using 0.25 M sodium borate having a pH of 7.25.

Next, 1750 μρ of gp4,BSA or gp4]BSA-L was dissolved in 1.0 ml of 10 mM of sodium borate buffer and was added to the suspension of gold particles. The resultant diluted suspension was mixed gently and incubated at room temperature for 5 minutes. -224. 9 5 ft Λ Λ Q 950988 After incubation, 1.4 ml of 10% sterile Bovine Serum Albumin, obtained by dissolving 10 gm of BSA from Sigma, St Louis, MO in 100 ml of distilled water and filtering the solution with a 0.2 μ filter, was added to the diluted suspension and mixed wel The diluted suspension was incubated at room temperature for 30 minutes.

The gold particle bound/gp41BSA or gold particle bountf/gp«,BSA-L was separated from the unbound gp41BSA or unbound gp41BSA-L, respectively, by centrifuging the diluted suspension at 28,000xg for 45 minutes at 8°C. The supernatant was carefully aspirated. The pellet was resuspended using 0.1% polyethylene glycol (PEG) 20K in 0.01 M borate having a pH of 9.0. in a volume equal to that aspirated. The centrifugation, aspiration and resuspension steps were repeated at least two time more for a total of 3 or 4 times. The final resuspended pellet contained the gold particle bound/gp4lBSA or gold particle bound/gp«,BSA-L, that is, the first substance-iabeL It was stored at 4°C in 0.1% PEG 20K in 0.01 M borate having a pH of 9.0 at 25 to 35 times the starting concentration of gold particle suspension, that is, 2.8 to 2.0 mt respectively for the above conjugation starting with 70 ml of gold. (v) Impregnation of First Substance-Label onto the First Matrix As used hereinafter, gp41Au refers to either gold particle hound/gp41BSA or gold particle boundfgp41BSA-L, prepared and purified by the methods disclosed above. The first matrix used was derived from glass fiber sheets, 28.5 x 21.5 cm and 0.4 mm thick available from Lydall Manning Co., Troy, NY, though other sources are known to those with skill in the art.

A diluent, Gold Conjugate Dilution Buffer 1, was prepared by containing 50mM Tris, 1.0% BSA, 0.5% PEG 15-20K, 0.3% PVP 30K, 2.5% sucrose and 0.9% T-20. The solution had a pH of 7.3 and was filtered using a 0.45// pore size filter. 40 ml of a diluted suspension of gp41Au was prepared by mixing 3.33 ml of gp4iAu suspension with 36.67 ml of diluent yielding a titre of 1:12.

Alternatively, the following diluent may be used: A diluent, Gold Conjugate dilution buffer 2, was prepared by combining 40 mM Tris, 1% BGG, 0.5% PEG 15-20K, 0.3% PVP 3OK, 3% sucrose and 0,9% T-20. The solution was filtered using a 0.45 μ pore size filter. 40 ml of a diluted suspension of gp4IAu was prepared by mixing 3.33 ml of gp4iAu suspension with 36.67 ml of diluent yielding a titre of 1:12.

The resultant diluted suspension was gently mixed ana incuoatea at room temperature for 5-10 minutes. One glass fiber sheet was placed in a suitable plastic tray of dimensions slightly longer than the first matrix sheet. 40 ml of diluted suspension was then uniformly distributed over the entire area of the first matrix by rapidly dispensing the suspension on the matrix followed by immediately tilting the plastic tray carrying the matrix in the direction of the unwetted portion of the matrix. Uniform distribution of the gold suspension was detected visually through uniform reddish staining of the entire matrix.

The diluted suspension-covered first matrix was allowed to stand for 10-15 minutes at room temperature. Then, the diluted suspension-covered first matrix was transferred onto a wire mesh tray where it was dried under -23vacuum at ambient temperature for 1.5 to 10 hours, completing production of a first substance-label-impregnated first matrix. The finished impregnated first matrix was stored in a desiccated container pending incorporation into the finished test strip.

(B) PREPARATION AND IMMOBILIZATION OF SECOND SUBSTANCE The next steps involved the preparation of the second substance and immobilization of second substance onto the second matrix. The second substance comprised gp«,BSA or gp4,BSA-L prepared according to the methods described above. This second substance was diluted in phosphate buffered saline having a pH of 7.4 to a concentration of between 14 mg/mL The second matrix comprised nitrocellulose membrane having a pore size of between about 6 and 15//. Suitable nitrocellulose membrane is available from Schleicher & SchuelL Keene, NH or Gelman Sciences, Ann Arbor, Ml, among other sources known to those with skill in tha art The second substance was immobilized onto the second matrix by microprocessor-controlled airbrush spraying. A microprocessor-controlled airbrush was used because it gave repeatable results. A narrow, approximately 1-2 mm wide, strip of second substance was centrally deposited approximately 10-13 mm from the edge of a 25 mm wide by 50 meters long nitrocellulose membrane roL The immobQized second matrix was dried at room temperature for 20-60 minutes and then dried under vacuum for 1.5 to 2.0 hours.

(C) ASSEMBLY OF DEVICE A working device was assembled as follows. Liquid absorber pad strips 18 x 295 mm, and sample absorber pad strips 20 x 295 mm, were cut from cellulose absorber sheets supplied by Gelman Sciences, Ann Arbor, Mi. Other sources of cellulose absorber sheets are known to those with skill in the art, including Whatman Specialty Products, Fairfield, NJ and Schleicher & Schuell, Inc., Keene, NH.

First matrix containing first substance-label were cut into 8 x 295 mm strips. Second matrix containing immobilized second substance were cut into 30 cm long strips. Vinyl plastic backing with adhesive 59 x 290 mm long was obtained from G&L Precision Die Cutting, Inc., Campbell, CA.

A second matrix strip was affixed to the vinyl plastic backing 19 mm from the bottom edge of the backing. A liquid absorber pad strip was laid on top of the backing overlapping 1 to 2 mm of the top edge of the second matrix strip. Next, a first matrix strip was laid on the backing overlapping about 1 mm of the bottom edge of the second matrix strip. Finally, a sample absorber pad strip was laid at the bottom of the backing such that the sample absorber pad touched the first matrix strip.

The backing was turned over and placed on a flat surface. Uniform pressure was applied to the backing to affix the strips. Next, the backing with the affixed strips were cut into 8 mm sections perpendicular to the long axes of the strips. Finally, the sections were placed in a suitable plastic housing completing the assembly.

Referring now to Figures 4, there is shown an assembled test device 400 according to one aspect of the present invention. The test device 400 has a plastic housing 410 containing a test strip 420. The test strip 420 comprises a sample application end 422 and a sample collection end 424. At the sample application end 422 of the test strip 420 is a sample absorber pad 430 having a sample application port 432. Adjacent the sample absorber 950988 -24pad 430 is a first substance 440. A test window 450 in housing 410 reveals the second matrix 460 and immobilized second substance 462, thereupon. At the sample collection end 424 is a liquid absorber pad 470.

Example 2: Method of Use of Device for the Detection of Antibody to HIV-1 glycoprotein 41 In accordance with another aspect of the present invention, there is provided a method of using a test device for the detection or quantification of antibody to AIDS virus as follows. A test device was made according to Example 1 above. Referring now to Figure 4, four drops, equal to about 200 fA, of sample fluid obtained from either human serum or human plasma and containing analyte antibody to HIV-1 envelope glycoprotein 41 virus was applied to the sample port 432 over the sample absorber pad 430 using a plastic disposable pipette.

Alternatively, in order to minimise the sample volume required, one drop or 50 μΐ of sample, either human serum, plasma or whole blood may be added to the device at the sample absorber pad followed by 3 drops of a wash solution manufactured according to the following composition: mM Sodium Borate, 1.0% PEG 15 - 20k and 0.05% Sodium Azide pH 7.75.

Sample moved through the sample absorber pad 430 encountering first substance-label upon entering the first matrix 440. At least some of the analyte antibody in the sample was bound to first substance-label by one of the at least two epitope binding sites present on the analyte antibody, thereby, forming analyte antibody/first substance-label complexes (antibody to HIV-1 envelope glycoprotein 41 virusigpJAu complexes). At least some of these complexes moved though the first matrix into the second matrix 460 where they encountered immobilized second substance 462. At least some of the complexes that encountered immobilized second substance was bound to the immobilized second substance by one of the at least two binding sites present on the analyte antibody, thereby, forming immobilized second substance/analyte antibody/first substance-label complexes (immobilized BP«i/antibody to HIV-1 envelope glycoprotein 41 virus/gp41/Au complexes). Unbound sample components and unbound analyte antibody/first substance-label complexes continued to flow past the immobilized second substance towards the liquid absorber pad 470 at the sample collection end 424 of the test device.

The gold particles immobilized to the second matrix through the second substance turned the location of the second substance a visually detectable reddish color that was detectably more color saturated than the rest of the second matrix visible through the test window 450. A greater intensity of reddish color at the location of the second substance indicated the presence of a greater amount of antibody to HIV-1 gp41 in the sample. The test was completed in less than about 3-5 minutes after application of the sample fluid to the sample application port 422. 9509ββ -25The specificity of the device according to one aspect of the present invention was tested using a sample of serum or plasma that did not contain antibody to HIV-1 gp«v The sample was applied to a test device 400 at the sample application port 432. After allowing the sample fluid 5 minutes to traverse the device from sample application pad 430 to liquid absorber pad 470, no characteristic reddish color was present at the location of the second substance, beyond that present essentially throughout the second matrix.

The sensitivity of the device for the detection of antibody to HIV-1 gp41 according to one aspect of the present invention was tested by comparing the results of 242 samples evaluated using both the present device and a commercially available test. Comparing the results against an indirect, Elisa test employing alkaline phosphatase antihuman IgG conjugate, as is well known to those with skill in the art, sixty five (65) samples were identified by 1Q both assays as negative and one hundred seventy seven (177) specimens were identified by both assays as positive for HIV-1 related antibodies. Comparing the results obtained with the present device for a Mixed-Titer Performance Panel, PRB 202 from Boston Biomedica, Inc., Bridgewater, MA, twenty-five (25) samples yielded a specificity of 100%, and a sensitivity of 96% based on two FDA licensed Western Blot procedures or 100% when correlated with a third FDA licensed Western Blot. Further, the device for the detection of antibody to HIV-1 gp,, according to one aspect of the present invention correctly identified the seroconversion sample from a Seroconversion Panel J, PRB 910 supplied by Boston Biomedica, Inc. in complete agreement with other FOA approved HIV-1 antibody Elisa procedures.

Example 3: Device and Method of Use for the Detection of Antibody to HIV-2 glycoprotein 36 Analyte antibody to HIV-2 gtycoprote'm 36 was detected using a device and method analogous to the embodiment described above in Example 1 and Example 2 above for the detection of immunoglobulin to HIV-1 glycoprotein 41. A summary of the construction of the device is as follows: (A) PREPARATION AND IMPREGNATION OF FIRST SUBSTANCE-LABEL ® Preparation of the Gold Particles Gold particles of the proper size ware produced as described under Example 1(AXi), above. -26(ii) Covalent Linkage of HIV-2 Envelope Glycoprotein 36 to Bovine Serum Albumin to Produce GP-BSA Using Glutaraldehyde The first substance used comprised synthetically produced HIV-2 peptide encoded by the highly conserved region of the virus envelope gene. The peptide contained amino acids 585-603, had a molecular weight of 2412 and was obtained from Ferring Diagnostics AB, Malnio, Sweden. It is referred to in this Example as gp».

Using an analogous method to the method described under Example KAHiia), 3 mg of gpH were added to 6.0 mg BSA dissolved in 1.0 ml PBS, pH 72. 25 μΙ 1% glutaraldehyde was then added to the solution and the solution was stirred at room temperature for 3.5 hours. Next, 25 μΙ of freshly prepared sodium cyanoborohydride solution, having a concentration of 20 mg/ml distilled water, were added to the solution and stirring continued for minutes at room temperature. Finally, the resultant gpw BSA conjugate was purified on a 18 x 1.6 cm Sephadex G-25 column equilibrated with PBS. Fractions were collected, evaluated for presence of immunoreactive conjugate, pooled and stored as described above. (iii) Conjugation of Envelope Glycoprotein 36/Bovine Serum Albumin with the Gold Particles to Produce First Substance-Label Conjugation of gpx Bovine Serum Albumin (gp^BSA) to the gold particles was achieved in analogy to the procedure outlined under Example 1(A)(iii), above. Briefly, after determination of the stabilizing concentration of gpwBSA as being equal to 15 μρ per milliliter of the 30 nm gold particles, 3750 μρ gpx in 5 ml 10 mmolar sodium borate, pH 7.3 were added to 250 ml of gold particles also at pH 7.3. The resultant solution was mixed and incubated for ten (10) minutes at room temperature.

Next, five (5) ml of 0.2 μ filtered 10% BSA/ml distilled water was added and the solution incubated at ambient temperature for one (1) hour. Separation of gold particle bound from unbound gp^BSA was accomplished by a centrifugation/aspiration/resuspension cycle repeated three times. Finally, the purified gpxBSAAu conjugate was reconstituted in 10 ml 0.1% PEG 20K in 0.01 M borate pH 9.0 to a concentration of 2.5 mg gold particles per milliliter suspension. The solution was stored at 4°C. 9509ο β -27fw) Impregnation of First Substance-Label onto the First Matrix To prepare gp^BSAAu conjugate for impregnation of glass fiber sheets, in a method analogous to that of Example l(A)ftv), the concentrated suspension of gp^BSAAu was diluted 1:12 fold (3.33 ml conjugate were added to 36.67 ml diluent) with the 0.45 μ filtered Gold Conjugate Dilution Buffer 1 containing 1% BSA, 0.5% PEG 15-20 K, 0.3% PVP 30 K, 2.5% Sucrose and 0.9% T-20 in 50 mmolar Tris buffer pH 7.3. Application of diluted conjugate to glass fiber sheet, drying and storage of impregnated first matrix was same as outlined above.

(B) PREPARATION AND IMMOBILIZATION OF SECOND SUBSTANCE The preparation and immobilization of the second substance, gpxBSA, was performed in a method analogous to that of Example 1(B), above.

(C) ASSEMBLY OF DEVICE A working device for the detection and quantification of unmunoglobulin to HIV-2 envelope glycoprotein 36 was assembled manufactured in a method analogous to that of Example 1(C), above.

Example 4: Manufacture of Device for the Co-Detection of Antibody 15 to HIV-1 alvconrotein 41 and HIV-2 glycoprotein 36 According to one aspect of the present invention, a test device for co-detection of HIV-1 glycoprotein 41 and HIV-2 glycoprotein 36 was constructed using processes analogous to those given in Examples 1 and 3, above. The device comprised first matrix impregnated with first substances gp4,BSAAu and gp^BSAAu, and second matrix containing immobilized second substances gp41BSA and gp^BSA. 40 ml of first substance was prepared by addition of 3.33 ml of gp41BSAAu and 3.33 ml of gp^BSAAu stock suspension to 33.34 ml Gold Conjugate Dilution Buffer 1. The first substance was mixed and impregnated on glass fiber sheets. The sheets were dried by vacuum and stored under desiccation until used.

Second substances gp41BSA and gp^BSA were diluted in PBS, pH 7.4, to a concentration of 2.2 and 1.6 mg per milliliter, respectively. The second substances were sprayed onto a 12 μ pore size nitrocellulose membrane using airbrush #1 to apply gp41BSA as a narrow 1-2 mm line 10 mm from bottom edge of second matrix, and airbrush #2 to apply gpxBSA as a narrow line 15 mm from bottom edge of second matrix. The second matrix was dried at room temperature for approxbnately 30 minutes, and then under vacuum for between approximately 1.5 to 2.0 hours and stored under desiccation until used. 09 g -28Example 5: Method of Manufacturing Control Substance for Use with the Test Device of Examples 1, 3 and 4 According to one aspect of the present invention, a control substance comprising a monoclonal antibody to BSA was manufactured for use with the test devices described in the above Example 1, 3 and 4. The control substance captured all gold-labelled, synthetic peptide-BSA conjugates (first substance), that had not reacted with second substance, by specific immunological reaction with epitopes present on the BSA moiety. The reddish color of the captured conjugates served as a visually identifiable marker indicating the viability of the test device and the completion of the test. The control substance was manufactured as follows.

Partially purified. Ammonium Sulfate (A.S.) precipitated and dialyzed, monoclonal Anti BSA antibody was obtained from Beckman Instruments, Inc. Carlsbad, CA, USA. The monoclonal antibody was dissolved in 10 mM of sodium phosphate buffer having a pH 7.4,145 mM sodium chloride (PBS) to obtain a concentration of 1-2 mg,ml. The dissolved monoclonal antibody was immobilized onto the second matrix about 19 mm from the bottom edge of the matrix for the devices of Examples 1, 3 and 4, employing the same airbrush microprocessor unit as for the spraying of gp41BSA in Example 1(B), above.

Competitive inhibition by Bovine Serum Albumin contamination will inhibit, partially or completely, the limited capacity of the immobilized Anti BSA antibody to bind the gp41BSAAu or gp^BSAAu. Therefore, m order to permit immunological recognition of the BSA moiety linked to the gp41BSAAu or gpnBSAAu conjugate by the immobilized Anti BSA antibody, the Gold Conjugate Dilution Buffer used in,the preparation of the immobilization solution had to be substantially completely free of any Bovine Serum Albumin contamination.

The Gold Conjugate Dilution Buffer used previously for dilution of gp41BSAAu or gp^BSAAu in Example 1 and Example 3, above, respectively, contained 1% BSA to stabilize the gold conjugate in the impregnating solution. Therefore, this buffer was replaced by 0.2% Human Serum Albumin in otherwise unchanged Gold Conjugate Dilution Buffer.

Second substances gp41BSA, gpKBSA prepared as in Example 4 were applied on second matrix as line 1 and line 2,10 mm and 15 mm, respectively, from the bottom edge of the 25 mm wide nitrocellulose membrane strip, together with partially purified monoclonal Anti BSA antibody at 1-2 mg,ml PBS, as line 3, 19 mm from the bottom edge of the membrane. A set up of three airbrushes were employed for uniform, consistent application of all three reagents. The sensitized second matrix was dried at room temperature and by vacuum as discussed previously, stored in desiccated container.

The present invention can be embodied in other specific forms without departing from its spirit or essential characteristics. The described embodiments are to be considered in all respects only as illustrative and not restrictive. The scope of the invention was, therefore, indicated by the appended claims rather than the foregoing description. All changes which come within the meaning and range of equivalency of the claims are to be embraced within their scope.

Claims (55)

1. A device for detecting the presence or amount of analyte antibody in a sample, comprising: a first zone that comprises a first matrix and a first substance that includes an epitope, said epitope being capable of binding to said antibody, the first substance having a label thereupon; and a second zone spatially separate from the first zone and in fluid communication with the first zone, said second zone comprising a second matrix and a second substance that includes an epitope substantially identical to the epitope on the first substance, the second substance immobilized on the second matrix; wherein the first substance is capable of moving from the first zone to the second zone after application of the sample to the first zone.

2. The device of Claim 1, wherein the epitope on the first substance is from Human Immunodeficiency Virus (HtV).

3. The device of Claim 2, wherein the first substance and the second substance each comprise at least one substance selected from the group consisting of HIV-1, HIV-2, HIV-1 glycoprotein 41, HIV-2 glycoprotein 36, and a fragment, derivative or combination of any of the foregoing.

4. The device of Claim 1, wherein the first matrix and the second matrix form a unitary matrix.

5. The device of Claim 1, wherein the sample is selected from the group consisting of a fluid, blood, serum, plasma, urine, sputum, cell culture supernatant, cell culture suspension, tissue culture supernatant, tissue culture suspension, cerebrospinal fluid, bone marrow, aqueous fluid, lacrimal fluid, saliva, pus, seminal fluid, amniotic fluid, sweat, mammary gland secretion, lymph, intraarticular fluid, pericardial fluid, peritoneal fluid and a combination thereof.

6. The device of Claim 1, wherein the sample is selected from the group consisting of serum and plasma.

7. The device of Claim 1, wherein the enalyte antibody is at least one substance selected from the group consisting of IgA, IgD, IgE, IgG, IgM, a fragment of any of the foregoing and a combination of any of the foregoing.

8. The device of Claim 1, wherein the analyte antibody is either IgG or IgM.

9. The device of Claim 1, wherein either the first matrix or the second matrix comprises nitrocellulose, polystyrene, polyvinyl, polycarbonate, nylon, cellulose acetate, glass fiber and filter paper.

10. The device of Claim 1, wherein both the first matrix and the second matrix comprise nitrocellulose.

11. The device of Claim 1, wherein the first zone further comprises a sample application zone and a separation zone in fluid communication with each other, wherein sample application zone is adapted for application of the sample, and wherein the separation zone separates the sample application zone from the second zone. -3012. The device of Claim 1, wherein the second zone further comprises a detection zone and a collection zone in fluid communication with each other, wherein the second substance is immobilized upon the second matrix in the detection zone, and wherein the collection zone is adapted to collect excess fluid.

12. 13. The device of Claim 1, wherein the second zone further comprises a control substance 5 immobilized thereupon.

13. 14. The device of Claim 13, wherein the control substance comprises at least one substance selected from the group consisting of a polyclonal antibody of human origin, a polyclonal antibody of non-human origin, a monoclonal antibody and a combination of any of the foregoing.

14. 15. The device of Claim 13, wherein the control substance comprises monoclonal antibody to 10 Bovine Serum Albumin.

15. 16. The device of Claim 1, wherein the label comprises at least one substance selected from the group consisting of an enzyme, a radioactive substance, a florescent compound, a chromogen, colored latex, gold particles, a peroxidase, a textile dye, alkaline phosphatase, σ-glucosidase, jS-gatactosidase, glucose oxidase, urease, and a fragment or combination of any of the foregoing. 15

16. 17. The device of Claim 1, wherein the label comprises gold.

17. 18. The device of Claim 1, further comprising a control zone having a control matrix and a control substance immobilized to the control matrix, wherein the control matrix is in fluid communication with the first zone, and wherein the control substance is capable binding the first substance, thereby immobilizing the first substance to the control matrix.

18. 20 19. A device for detecting the presence or amount of analyte antibody to each of two antigens, antigen-1 and antigen-2, in a sample, comprising: a first zone that comprises a first matrix, an antigen-1 first substance and an antigen-2 first substance, the antigen-1 first substance including an epitope from antigen-1, and the antigen-2 first substance including an epitope from antigen-2, each of the first substances having a label thereupon; 25 and a second zone spatially separate from the first zone and in fluid communication with the first zone, said second zone comprising a second matrix, an antigen-1 second substance and an antigen-2 second substance, the antigen-1 second substance including an epitope from antigen-1 that is substantially identical to the epitope from antigen-1 on the antigen-1 first substance, the antigen-2 30 second substance including an epitope from antigen-2 that is substantially identical to the epitope from antigen-2 on the antigen-2 first substance an epitope, each of the second substances being immobilized on the second matrix; wherein each of the first substances is capable of moving from the first zone to the second zone after application of the sample to the first zone. 35 20. The device of Claim 19, wherein antigen-1 is HIV-1 and antigen-2 is HIV-2. -3121. The device of Claim 20, wherein each first substance and each second substance comprise at least one substance selected from the group consisting of HIV-1 glycoprotein 41, HIV-2 glycoprotein 36, and a fragment, derivative or combination of any of the foregoing.

19. 22. The device of Claim 19, wherein the second zone further comprises a control substance immobilized thereupon.

20. 23. A device for detecting the presence or amount of a polyvalent analyte in a sample comprising: a first zone that comprises a first matrix and a first substance, the first substance having a label thereupon; and a second zona that comprises a second matrix and a second substance, the second substance immobilized to the second matrix; wherein the second zone is spatially separate from the first zone and in fluid communication with the first zone; wherein the first substance is in relation to the First matrix such that the first substance is capable of moving from the first zone to the second zone after application of the sample to the first zone; wherein a first epitope binding site and a second epitope binding site on the analyte antibody are capable, respectively, of sequential binding to a first epitope on the first substance and a second epitope on the second substance, such that, after the completion of both bindings, both epitope binding sites are simultaneously occupied; wherein the first epitope on the first substance and the second epitope on the second substance are substantially identical.

21. 24. The device of Claim 23, wherein the first matrix and the second matrix form a unitary matrix.

22. 25. The device of Claim 23, wherein the sample comprises two or more antigenically different polyvalent analytes, and wherein the first zone further comprises at least one first substance corresponding to each polyvalent analytes for a total of at least two first substances; each first substance being in relation to the first matrix such that each first substance is capable of moving from the first zone to the second zone after application the sample to the first zone, each first substance having an epitope capable of binding to at least one first epitope binding site on a corresponding polyvalent analyte, and each first substance having a label thereupon; wherein the epitope of each first substance is substantially non-identical with at least one epitope of one other first substance.

23. 26. The device of Claim 25, wherein the second zone further comprises at least one second substance corresponding to each polyvalent analyte; each second substance being immobilized upon the second matrix, and each second substance being capable of binding to at least one second epitope binding site on a corresponding polyvalent analyte.

24. 27. The device of Claim 23, wherein the sample comprises two or more antigenically different polyvalent analytes, and wherein the second zone further comprises at least one second substance corresponding to each polyvalent analyte; each second substance being immobilized upon the second matrix, and each second -32950088 substance being capable of binding to at least one second epitope binding site on a corresponding polyvalent analyte.

25. 28. The device of Claim 27, wherein the first zone further comprises at least one first substance corresponding to each polyvalent analyte; each first substance being in relation to the first matrix such that each first substance is capable of moving from the first zone to the second zone after application of the sample to the first zone, each first substance being capable of binding to at least one first epitope binding site on a corresponding polyvalent analyte, and each first substance having a label thereupon, wherein the epitope of each first substance is substantially non-identical with at least one epitope of one other first substance.

26. 29. The device of Cleim 23, wherein the semple is selected from the group consisting of a fluid, blood, serum, plasma, urine, sputum, cell culture supernatant, cell culture suspension, tissue culture supernatant, tissue culture suspension, cerebrospinal fluid, bona marrow, aqueous fluid, lacrimal fluid, saliva, pus, seminal fluid, amniotic fluid, sweat, mammary gland secretion, lymph, intraarticular fluid, pericardial fluid, peritoneal fluid and a combination thereof.

27. 30. The device of Claim 23, wherein the polyvalent analyte is at least one substance selected from the group consisting of immunoglobulin, IgA, IgD, IgE, IgG, IgM, a peptide, a receptor, a hormone, a fragment thereof and a combination thereof.

28. 31. The device of Clabn 23, wherein the first matrix or the second matrix comprises nitrocellulose, polystyrene, polyvinyl, polycarbonate, nylon, cellulose acetate, glass fiber and filter paper.

29. 32. The device of Claim 23, wherein the first zone further comprises a sample application zone and a separation zone, wherein sample is applied to the sample application zone, and wherein the separation zone separates the sample application zone from the second zone.

30. 33. The device of Claim 23, wherein the second zone further comprises a detection zone and a collection zone, wherein the second substance is immobilized upon the second matrix in the detection zone, and wherein the collection zone is adapted to collect excess fluid.

31. 34. The device of Claim 23, wherein the first substance comprises at least one naturally occurring or synthetically produced substance selected from the group consisting of a peptide, a glycoprotein, a hormone, a hormone receptor, an enzyme, a virus, a bacteria, a fragment thereof and a combination thereof.

32. 35. The device of Claim 23, wherein the label comprises at feast one substance selected from the group consisting of an enzyme, a radioactive substance, a florescent compound, a chromogen, colored latex, gold particles, a peroxidase, a textile dye, alkaline phosphatase, σ-glucosidase, /?-galactosidase, glucose oxidase, urease, a fragment thereof and a combination thereof.

33. 36. The device of Claim 23, wherein the second substance comprises at least one naturally occurring or synthetically produced peptide or a fragment thereof.

34. 37. The device of Claim 23, further comprising a control zone having a control matrix and a control substance immobilized to the control matrix, wherein the control matrix is in fluid communication with the -33first zone, and wherein the control substance is capable binding the first substance, thereby immobilizing the first substance to the control matrix.

35. 38. The device of Claim 23, wherein the second zone comprises a control substance.

36. 39. The device of Claim 38, wherein the control substance comprises at least one substance 5 selected from the group consisting of a polyclonal antibody of human origin, a polyclonal antibody of non-human origin, a monoclonal antibody and a combination of any of the foregoing.

37. 40. A device according to any of Claims 1,19 or 23, for use in detecting the presence or amount of analyte antibody to Human Immunodeficiency Virus m a sample.

38. 41. A method for detecting the presence or amount of analyte antibody to Human 10 Immunodeficiency Virus in a sample, said method comprising the steps of: a) providing a device according to Claon 1; b) applying the sample to the first zone, said antibody m said sample comprising first and second epitope binding sites; c) binding the first epitope binding site on the analyte antibody to the first epitope on the first 15 substance, thereby forming an analyte antibody/first substance complex; d) allowing the analyte antibody/fhrst substance to migrate from the first zone to the second zone; e) allowing the second epitope binding site on the analyte antibody of the first substance/analyte antibody to bind to the second epitope on the immobilized second substance in the 20 second zone, thereby forming immobilized first substance/analyte antibody/second substance; and f) detecting the label upon the first substance of the immobilized first substance/analyte antibody/second substance, wherein detection of the label is an indication of presence or amount of the analyte antibody.

39. 42. The method of Claim 20, wherein the application step, step b), is selected from the group 25 consisting of dipping a portion of the first matrix in the sample, spotting the sample on the first matrix and a combination thereof.

40. 43. The method of Claim 41, wherein the detecting step, step f), comprises visually identifying a color change on the second zone.

41. 44. The method of Claim 41, further comprising the step of filtering the sample between the 30 providing step, step a), and the applying step, step b).

42. 45. A method for detecting the presence or amount of a polyvalent analyte in a sample comprising the steps of: a) providing a device according to Claim 24; b) applying the sample to the first zone; -34950988 c) allowing the first epitope binding site on the analyte antibody to bind to the first epitope on the first substance and the analyte antibody/first substance to migrate from the first zone to the second zone; d) allowing the second epitope binding site on the analyte antibody of the first substancelanalyte antibody to bind to the second epitope on the immobilized second substance in the second zone, thereby forming immobilized First substancelanalyte antibody/second substance; and e) detecting the label upon the first substance of the immobilized first substancelanalyte antibody/second substance, wherein detection of the label is an indication of presence or amount of the analyte antibody.

43. 46. The method of Claim 45, wherein the application step, step b), is selected from the group consisting of dipping a portion of the first matrix in the sample, spotting the sample on the first matrix and a combination thereof.

44. 47. The method of Claim 45, wherein the detecting step, step e), comprises visually identifying a color change on the second zone.

45. 48. The method of Claim 45, further comprising the step of filtering the sample between the providing step, step a), and the applying step, step b).

46. 49. A device for detecting the presence or amount of analyte antibody in a sample, the device being substantially as described herein with reference to the Examples and as illustrated in the accompanying drawings .

47. 50. A device for detecting the presence or amount of analyte antibody to each of two antigens, antigen-1 and antigen-2, in a sample, the device being substantially as described herein with reference to the Examples and as illustrated in the accompanying drawings. -35o c n q q 8 950988

48. 51. A device for detecting the presence or amount of a polyvalent analyte in a sample, the device being substantially as described herein with reference to the Examples and as illustrated in the accompanying 5 drawings.

49. 52. A device according to any of Claims 1 to 40 for use in detecting the presence or amount of analyte antibody to Human Immunodeficiency Virus in a sample, in which a HIV-1 Envelope Glycoprotein 41 is linked to 10 Bovine Serum Albumin to produce GP^'-BSA.

50. 53. A device as claimed in Claim 52 in which the HIV-1 Envelope Glycoprotein 41 is linked to Bovine Serum Albumin to produce GP 41 -BSA using a Heterobifunctional linkage. 15

51. 54. A device as claimed in Claim 52 or 53 in which the HIV-1 Envelope Glycoprotein 41 is linked to Bovine Serum Albumin to produce GP 41 -BSA in accordance with the method described herein with reference to Example l(iii). 20

52. 55. A device according to any of Claims 1 to 40 or 49 to 54 for use in detecting the presence or amount of analyte antibody to Human Immunodeficiency Virus in a 95 0 9 fl 8 -35sample, in which impregnation of the first substance label onto the first matrix is carried out using a diluent comprising a Gold Conjugate dilution buffer which is prepared by combining 40 mM Tris, 1% BGG, 0.5% PEG 15-20K, 0.3% PVP 30K, 3% sucrose and 0.9% T-20, the solution being filtered using a 0.45 μ pore size filter, a diluted suspension of gp 41 Au being prepared by mixing 8.3 ml of gp 41 Au suspension with 91.7 ml of diluent for yielding a titre of 1:12.

53. 56. A method for detecting the presence or amount of analyte antibody to Human Immunodeficiency Virus in a sample, the method comprising the steps of providing a device as claimed in any of Claims 1 to 40 or 49 to 55, the method being substantially as described herein with reference to the Examples.

54. 57. A method for detecting the presence or amount of polyvalent analyte in a sample, the method comprising the steps of providing a device as claimed in any of Claims 1 to 40 or 49 to 55, the method being substantially as described herein with reference to the Examples .

55. 58. A method for detecting the presence or amount of analyte antibody to Human Immunodeficiency Virus in a sample, the method comprising the steps of 950988 -37a) providing a device as claimed in any of Claims 1 to 40 or 49 to 55, b) applying the sample to the first zone, said antibody in said sample comprising first and second epitope binding sites; c) binding the first epitope binding site on the analyte antibody to the first epitope on the first substance, thereby forming an analyte antibody/first substance complex; d) allowing the analyte antibody/first substance to migrate from the first zone to the second zone; e) allowing the second epitope binding site on the analyte antibody of the first substance/analyte antibody to bind to the second epitope on the immobilized second substance in the second zone, thereby forming immobilized first substance/analyte antibody/second substance; and f) detecting the label upon the first substance of the immobilized first substance/analyte antibody/second substance, wherein detection of the label is an indication of presence or amount of the analyte antibody, wherein one drop or 50 μΐ of sample, either human serum, plasma or whole blood may be added to the device at the sample absorber pad followed by three drops of a wash solution manufactured according to the following composition: