WO1992008809A1

WO1992008809A1 - Assay device and method for antibody and antigen detection

Info

Publication number: WO1992008809A1
Application number: PCT/US1991/008399
Authority: WO
Inventors: Roger M. Clemmons
Original assignee: University Of Florida
Priority date: 1990-11-19
Filing date: 1991-11-19
Publication date: 1992-05-29
Also published as: AU9052491A

Abstract

An immunodiagnostic device for determining the presence of an antibody or antigen is provided which comprises a support member presenting a planar surface that comprises at least one delimited portion comprised of a material having a high surface area upon which a proteinaceous material can be immobilized in reactive form and, immobilized on said delimited portion, a proteinaceous material that binds the antibody or antigen. Also provided are immunoassay methods for detecting the presence of antibody and antigen in a fluid sample.

Description

ASSAY DEVICE AND METHOD FOR ANTIBODY AND ANTIGEN DETECTION

Background of the Invention

The present invention relates to a device and to a method that provide improved capability for the detection of agents such as antibodies and antigens.

Various immunoassays are known for determining the presence of an agent in a biological sample. In an enzyme-linked immunosorbent assay (ELISA) , for example, antigen adsorbed to a solid phase is contacted with a solution to be tested for the presence of antibody that binds the adsorbed antigen. The solid phase is then contacted with a ligand in the form of an enzyme linked to a molecule that is also recognized by the test antibody. A chromogen is thereafter introduced which generates a colored end-product in the presence of the enzyme portion of the ligand. The optical density of the solution, measured at the end of a defined period, is proportional to the amount of enzyme present and, hence, to the amount of test antibody.

The ELISA method is also useful for detecting a target antigen. In this case, a monoclonal antibody against a specific target antigen is adsorbed to the surface of a microtiter plate well made of a plastic material, such as polystyrene. Nonspecific binding is prevented by the use of a blocking agent, usually glycine or albumin. Subsequently a fluid suspected to contain the target antigen is contacted to the adsorbed monoclonal antibody, allowing the target antigen to interact and bind to the antibody. The surface is then washed to remove unbound antigen. Enzyme-conjugated antibody is then added to the well and allowed to bind to the previously bound target antigen. The enzyme-conjugated antibody may be monoclonal, against the same or another epitope of the target antigen, or polyclonal against a number of epitopes of the target antigen. The microtiter well is then washed free of the unbound enzyme-conjugated antibody. A substrate capable of producing a detectable end product in the presence of the enzyme system is then added to the well and incubated. The presence of the end product thereafter is determined visually or photometrically.

While the ELISA technique is advantageous in some respects, as noted above, numerous problems remain. For instance, because the immobilized antibody is typically bound to a bead or small particle, or coated on the surface of a microtiter plate well, long incubation and washing times are necessary. Even when several samples are run simultaneously, the procedure still requires many hours to complete. Additionally, strict protocol requirements for ELISA, such as the timing of the incuba¬ tion steps and measuring of reagents, tend to limit the use of ELISA to individuals with a relatively high degree of technical skill and training. Furthermore, expensive and relatively sophisticated equipment and laboratory facilities are required to conduct ELISA assays. (This last problem is also common to other known immunodiagnostic assays.)

Detection of the human immunodeficiency virus (HIV) , identified as the causal factor for acquired immune deficiency syndrome (AIDS) and pre-AIDS conditions, is of particular importance. HIV can in principle be detected by assaying for the presence of HIV antibodies or antigens. The presence of other pathogens, including the viruses causing hepatitis A, hepatitis B, hepatitis C and herpes, and the microorganisms causing chlamydia, gonorrhea and syphilis, can likewise be detected by assay for antibodies or antigens.

However, known methods for antibody and/or antigen detection have proven inadequate in certain respects. For example, known methods for HIV antibody detection in urine require substantial concentration, processing or both in order to provide levels of HIV antibody that are detectable by such methods. Thus, U.S. patent No. 4,865,966 discloses that conventional HIV diagnostic assays, such as the ELISA or Western Blot method, can be used to detect HIV antibodies in concentrated urine that has been concentrated at least 20 fold.

In addition to the foregoing disadvantages, conventional techniques also require invasive procedures to obtain blood or other suitable bodily fluids to use as test samples. Invasive sample gathering procedures greatly increase the costs incurred in obtaining and storing samples and in conducting the assays.

A need therefore exists for an immunodiagnostic device and method that avoid the above-described problems of time, technical skill, facility costs and invasive sample gathering. A device suitable to satisfy this need would additionally have to furnish accurate results, with a minimum of false negatives and false positives.

Summary of the Invention

It is therefore an object of the present invention to provide an immunodiagnostic device that gives sensitive and accurate results specific for the presence of an antibody or antigen in a period of minutes.

It is also an object of the present invention to provide a method of detecting antibodies or antigens which can be implemented simply, by contacting a fluid sample with a "dipstick"-style device and then developing a reaction product visible to the naked eye.

In accomplishing the foregoing objectives, an immunoassay device has been provided for detecting the presence of antibody or antigen in a fluid sample, comprising a support member presenting a planar surface that comprises at least one delimited portion comprised of a material having a high surface area upon which a proteinaceous material can be immobilized in reactive form and, immobilized on the delimited portion, a proteinaceous material that binds the antibody (in the case of antibody detection) or antigen (in the case of antigen detection) . Preferably, the material having a high surface area is nitrocellulose, nylon, polyamide, polycarbonate or cellulose acetate. In a preferred embodiment for antibody detection, the proteinaceous material comprises a nonrecombinant protein from a pathogenic microorganism. In a preferred embodiment for antigen detection, the immobilized proteinaceous material comprises a polyclonal antibody that binds an antigen from a pathogenic microorganism.

In a particular preferred immunoassay device, the support member has at least one recess into which a membrane made of a high-surface-area material is mechanically secured. Through-holes in the support member and the securing means allow the fluid to access and penetrate the membrane. In a particularly preferred embodiment, the support member presents a recess in its planar surface and the device comprises means for securing the material having a high surface area, said means being comprised of a retaining washer that (i) has a chamfered outer edge and a smooth surface which abuts the membrane and (ii) securingly fits into the recess.

In a further preferred embodiment, a plurality of proteinaceous materials from different pathogenic microorganisms (for antibody detection) or monoclonal or polyclonal antibodies that bind antigens from different pathogenic microorganisms (for antigen detection) are immobilized on different delimited portions of the surface of the device. In another preferred embodiment for antigen detection, the immobilized proteinaceous material comprises a polyclonal antibody that binds an antigen that is present during normal changes in states of health in humans, or a non-naturally occurring substance such as a drug.

In another preferred embodiment for antibody detection, the immunoassay device comprises a plurality of delimited portions, on one of which is immobilized a proteinaceous material that is recognized by an enzyme- conjugated anti-human immunoglobulin antibody such that, once said enzyme-conjugated anti-human immunoglobulin antibody is allowed to react with said immobilized protein, said enzyme is capable of catalyzing a detectable reaction involving a substrate, and on another of which is immobilized a proteinaceous material that is not recognized as human immunoglobulin by said enzyme- conjugated anti-human immunoglobulin antibody. These additional proteinaceous materials serve as positive and negative controls.

In accordance with another aspect of the present invention, an immunoassay method for antibodies is provided that comprises the sequential steps of bringing a fluid sample into contact with a proteinaceous material immobilized in reactive form on a delimited portion of a material having a high surface area; removing the proteinaceous material from contact with the fluid sample; contacting the proteinaceous material with a solution comprising at least one agent that recognizes human antibody or a human antibody-antigen complex, wherein the agent is conjugated to an enzyme such that the enzyme can catalyze a detectable reaction involving a substrate; removing the proteinaceous material from contact with the solution; bringing the proteinaceous material into contact with the substrate; and then determining whether the contact with the substrate has brought about a detectable reaction.

In preferred embodiments, the agent comprises an antibody that recognizes a human immunoglobulin, or an immune-complex binding material (e.g., RhC, Rheumatoid factor or Clq) that recognizes human immunoglobulin- antigen complexes.

In accordance with another aspect of the present invention, the conjugated agent is added to the fluid sample before bringing the sample into contact with the proteinaceous material. In accordance with a further aspect of the present invention, an immunoassay method for antigens is provided that comprises the sequential steps of diluting a fluid sample with a solution containing a first proteinaceous material that binds the antigen, wherein the first proteinaceous material is conjugated to an enzyme such that the enzyme can catalyze a detectable reaction involving a substrate; bringing the diluted fluid sample into contact with a second proteinaceous material that binds the antigen, wherein the second proteinaceous material is immobilized in reactive form on a delimited portion of a material having a high surface area, and wherein the second proteinaceous material binds at least one epitope on the antigen that is not bound by the first proteinaceous material; removing the second proteinaceous material from contact with the fluid sample; washing the second proteinaceous material; bringing the second proteinaceous material into contact with the substrate; and then determining whether the contact step has brought about the detectable reaction.

In accordance with yet another aspect of the present invention, there is provided an immunoassay method comprising the sequential steps of: diluting a fluid sample with a solution containing a first proteinaceous material that binds the antigen, wherein the first proteinaceous material is biotinylated; bringing the fluid sample into contact with a second proteinaceous material that binds the antigen, wherein the second proteinaceous material is immobilized in reactive form on a delimited portion of a material having a high surface area, and wherein the second proteinaceous material binds at least one epitope on the antigen that is not bound by the first proteinaceous material; removing the second proteinaceous material from contact with the fluid sample; washing the second proteinaceous material; bringing the second proteinaceous material into contact with a material that binds biotin, wherein the material is conjugated to an enzyme such that the enzyme can catalyze a reaction involving a substrate; washing the second proteinaceous material; bringing the second proteinaceous material into contact with the substrate; and then determining whether the contact step has brought about a detectable reaction.

In accordance with still another aspect of the present invention, there is provided an immunoassay method comprising the sequential steps of: diluting a fluid sample with a solution containing a first proteinaceous material that binds the antigen; bringing the fluid sample into contact with a second proteinaceous material that binds the antigen, wherein the second proteinaceous material is immobilized in reactive form on a delimited portion of a material having a high surface area, and wherein the second proteinaceous material binds to at least one epitope on the antigen that is not bound by the first proteinaceous material; removing the second proteinaceous material from contact with the fluid sample; washing the second proteinaceous material; bringing the second proteinaceous material into contact with a third proteinaceous material that binds the first proteinaceous material, wherein the third proteinaceous material is conjugated to an enzyme such that the enzyme can catalyze a reaction involving a substrate; washing the second proteinaceous material; bringing the second proteinaceous material into contact with a substrate for the enzyme; and then determining whether the contact step has brought about a detectable reaction.

Other objects, features and advantages of the present invention will become apparent to those skilled in the art from the following detailed description. It is to be understood, however, that the detailed description and specific examples, while indicating preferred embodiments of the present invention, are given by way of illustration and not limitation. Many changes and modifications within the scope of the present invention may be made without departing from the spirit thereof, and the invention includes all such modifications. Brief Description of the Drawing

The invention may be more readily understood by referring to the accompanying drawing by which

FIG. 1 is a line drawing of an immunoassay device according to the invention,

FIG. 2 is a line drawing of a preferred embodiment of an immunoassay device according to the invention, and

FIG. 3 is a line drawing of an immunoassay device according to the invention for use in a semiquantitative assay.

FIGS. 4a-b are line drawings in plan and side view of a preferred embodiment of an immunoassay device according to the invention,

FIG. 5 is an enlarged sectional view of a recess and well adapted for receiving a membrane used in the immunoassay device,

FIGS. 6a-b are plan and side views of a retaining washer adapted for insertion into a recess of the immunoassay device, FIG. 7 is an enlarged side view of the retaining washer, showing chamfered edges, and

FIG. 8 is an enlarged sectional view showing the disposal of a membrane and retaining washer within a recess of the immunoassay device. In the drawing, identical elements are numbered identically throughout.

Detailed Description of the Preferred Embodiments

An immunodiagnostic approach has been developed that avoids the above-described disadvantages of conventional technology for detecting the presence of antibody or antigen, and that includes a fast, simplified, noninvasive and accurate assay for the screening of diseases.

Examples of antibodies for which the present invention provides improved detection capability include antibodies specific to pathogenic microorganisms, such as viruses, bacteria and protozoa. Particular mention is made of antibodies specific to HIV-1, HIV-2, the hepatitis-A, hepatitis-B, hepatitis-C and hepatitis-D viruses, cytomegalovirus, Treponema pallidum (syphilis spirochete) , Neisserxa gonorrhoeae , Chlamydia trochomates , and Mycobacterium tuberculosis. Exemplary antigens for which the present invention provides improved detection capability include surface and intracellular antigens from pathogenic microorganisms, such as viruses, bacteria and protozoa. Particular examples include surface antigens from the microorganisms mentioned above.

Detection of antibodies specific to proteinaceous materials derived from sources other than pathogenic microorganisms is also within the scope of the present invention, as is detection of antigens derived from sources other than pathogenic microorganisms. In particular, it is contemplated that antigens detectable by the inventive immunoassay devices and methods encompass proteinaceous and other materials that are present during changes in states of health in humans. Examples of such materials include human chorionic gonadotropin, thyroidhormone, testosterone, progesterone and other hormones, and myoglobin. Other contemplated antigens include proteinaceous and non-proteinaceous materials indicative of the presence of abnormal or non- naturally occurring substances in the human body, such as: therapeutic drugs; drugs of abuse, including barbiturates, amphetamines, cocaine, cannabis, morphine and heroin; toxins, including botulinum toxin, endotoxin and tetanus toxin; and other organic compounds. In the latter cases, methods for producing antibodies that bind such substances are well ^~known to those skilled in the art. It is also contemplated that the inventive immunoassay device and method are applicable to non-human subjects, e.g., warm-blooded animals, for the detection of specific antigens in said subjects. The selection of the particular immunoassay (i.e., antigen vs. antibody assay) for determining the presence of a specific pathogen will depend on the nature of the pathogen. Infections that result in low levels of antigen and high levels of non-protective antibody are preferably detected using the antibody immunoassay method. In these cases, presence of the antibody indicates the presence of infection, since antibody levels are independent of antigen concentration. Thus, the antibody immunoassay is preferably employed to determine active infection with pathogens such as HIV-1, HIV-2 and hepatitis-C virus. The antibody immunoassay may also be used to detect exposure to certain infectious pathogens in the past, where the antibody produced is protective. If history of past illness is important, the antibody immunoassay is preferably employed. Examples include detection of hepatitis-A or hepatitis-B exposure.

Active infections in which antibodies produce immunity, however, cannot be detected by establishing the presence of antibody against the pathogen alone. In these cases, the antibodies are not present during the early active infection. In such cases, and moreover in cases in which the antibody is not produced where it may neutralize the infection, that is, in which local infections are not cleared by circulating antibodies, detection of the pathogen by determining the presence of disease antigens according to the present invention is the preferred immunoassay. Thus, the antigen immunoassay is preferably employed to determine active infection with pathogens such as hepatitis-A, hepatitis-B, gonorrhea, syphilis, chlamydia and cytomegalovirus.

Finally, in certain infections, such as infection with herpes virus, either immunoassay is appropriate. In infections with pathogens such as herpes virus, the presence of antigen confirms active infection, while the presence of antibody indicates the latent state of the infection. The present invention enables one to detect antigen in a wide range of fluids or fluidizable samples, i.e., samples that can be mixed with a carrier fluid. Antibodies can be detected in a sample comprised, for example, of urine, saliva, tears, feces, semen, a urogenital secretion or sweat, without mandatory resort to the invasive techniques conventionally required in the testing of blood and blood products like serum and plasma. Antigen can also be non-invasively detected in saliva, urine or other of the foregoing exemplary samples. Of course, serum, blood and plasma can also be assayed using the inventive method.

In particular, the present invention is useful for immunodiagnosis of voided urine and other nonconcentrated fluids. The term "nonconcentrated" here signifies the absence of pre-assay processing of the sample for the purpose of effecting a higher concentration of protein or other constituent in that fluid.

For example, the present invention enables detection of HIV antibodies in nonconcentrated fluids, including voided urine, in contrast to the method disclosed in U.S. patent No. 4,865,966. It has been discovered that the use of immobilized proteinaceous materials that are contacted, for a period of minutes, with the fluid sample, results in accumulation of antibodies or antigens at the site of immobilization of the proteinaceous material. Placement of an immunodiagnostic device according to the present invention in contact with a fluid sample thus provides an unexpected means to concentrate antibodies or antigens from the sample to such an extent as to allow detection that was previously not envisioned to be possible.

Immobilization of the proteinaceous material on the surface of an immunoassay device according to the invention is accomplished by techniques well known to those skilled in the art, such as western blotting, drying or application of vacuum. See, e.g., Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, at §10.8, John Wiley and Sons, New York (1987, 1990) , the contents of which are incorporated by reference. For antigen detection, use is preferably made of polyclonal antibodies or polyclonal antibody fragments that bind the antigen to be assayed. Monoclonal antibodies and fragments thereof can also be used as the immobilized proteinaceous material if desired.

Complex-binding agents can also be used as the immobilized proteinaceous material for antigen detection. Complex-binding agents are agents which selectively bind to antigen-antibody complexes, but do not bind to the antibody or antigen in isolation. These agents include RhC, Rheumatoid factor, and Clq (a subunit of the first complement component Cl) . RhC and Clq are characterized, and methods for their preparation are disclosed, in U.S. patents No. 4,783,525 and 4,595,654 respectively, the contents of each of which are hereby incorporated^' in their entireties by reference. It is contemplated that other agents that selectively bind to antibody-antigen complexes without binding to either in isolation are suitable for use as the immobilized proteinaceous material in the present invention.

It has been found that assay sensitivity is enhanced, to an extent unheralded in the art, by immobilizing the proteinaceous material on a support having a surface which, at least in delimited portions as described above, presents microscopic irregularities that increase the total surface area to which the proteinaceous material used is bound. Examples of materials having such an irregular surface include nylon, nitrocellulose, polyamide, polycarbonate and cellulose acetate. These materials are referred to as "high surface area materials."

In the context of the present invention, high surface area materials preferably are those materials having a binding capacity of about 50-500 μg protein/cm².

By increasing the amount of proteinaceous material immobilized on a delimited portion of such a material, it has further been discovered that detection of antibody or antigen is effected during a shorter exposure of the immobilized proteinaceous material to the test sample, even when the antigen is present in low titer, as is typically the case when voided urine or saliva is the test sample.

In an exemplary embodiment of an antibody assay according to the present invention, where the antibody to be assayed is anti-HIV antibody (anti-HIV-1 or anti-HIV- 2) and the test sample is urine, the amounts of immobilized proteinaceous material derived from HIV-1 and HIV-2 are about 0.50 to 3.0 μg and about 0.35 to 2.5 μg, respectively. Where the test sample is blood, the amounts are decreased to about 0.25 to 2.0 μg and about 0.20 to 1.5 μg, respectively.

In an exemplary embodiment of an antigen assay according to the present invention, where the antigen to be assayed is HBSAg, the immobilized proteinaceous material is an antibody that binds HBSAg, and the test sample is saliva or serum, the amount of immobilized antibody is about 0.1 to 10.0 μg.

The use of nonrecombinant proteins as the proteinaceous materials in an immunoassay device according to the present invention has been discovered to result in an additional, surprising increase in sensitivity and an unexpected decrease in the incubation time needed for detection of antibody in the sample assayed. In this context, "nonrecombinant" denotes a polypeptide obtained via expression of endogenous DNA or RNA, in contradistinction to expression of heterologous or recombinant DNA or RNA in a genetically-engineered host organism. It has been found that the increased sensitivity and shorter incubation time apparently is due to the fact that nonrecombinant proteinaceous materials have a higher affinity for antibody specific to them than do recombinant proteins.

The nonrecombinant proteins are prepared either by affinity purification or by HPLC preparation. In an exemplary embodiment of the invention for detection of HIV antibody, nonrecombinant HIV proteins such as gag or env can be used, as the sole proteinaceous material or in combination in the same assay. As indicated above, nonrecombinant gag (HIV core) and env (HIV envelope) proteins are those that are obtained from HIV virus per se, rather than by expression of isolated polynucleotides encoding these proteins in a transformed host.

In a preferred embodiment of the inventive immunoassay device adapted for use in the detection of HIV antibody, the planar surface of the device comprises a plurality of delimited portions, each comprised of a material having a high surface area upon which proteinaceous material can be immobilized in reactive form. The proteinaceous material immobilized thereon comprises (i) HIV lysate, and at least one of (ii) substantially homogeneous nonrecombinant HIV gag protein and (iii) substantially homogeneous nonrecombinant HIV env protein. These proteinaceous materials are immobilized on separate portions of the plurality.

For purposes of the present invention, proteins derived from only one component of a pathogenic microorganism, such as an HIV virus, are considered "substantially homogeneous." Thus, "substantially homogeneous gag protein" can contain one or more gag

(core) proteins, but no env (envelope) proteins or proteins from other HIV components. Other nonrecombinant

HIV proteins, such as the core proteins pl7, pl8, p24 and p55 and envelope proteins gp41, gpl20 and gpl60, likewise can be employed in this embodiment of the present invention.

In the preferred embodiment described above, the phrase "HIV lysate" refers to a preparation obtained from material produced when a cell culture undergoes lysis following infection from an HIV virus. The inoculant virus may be either HIV-1 or HIV-2 virus or some combination thereof. An HIV lysate thus contains a heterogeneous mixture of nonrecombinant HIV proteins, including both gag and env proteins. It is therefore distinguished from the substantially homogeneous gag protein and env protein materials which are immobilized on separate delimited portions of a support member according to an embodiment of the present invention

A suitable HIV lysate can be obtained by infecting a permissive human neoplastic T-cell line such as H9 with the HIV-1 virus and, after an appropriate incubation period, lysing the cells and subjecting the lysed material to purification through a sucrose density gradient. The selected fraction, at a density of about 1.16 g/ml, is the HIV lysate. See Popovic et al., Science 224:497-500 (1984), the contents of which are incorporated by reference. Alternatively, HIV lysate is commercially available from Organon Teknika and Diagnostic Biotechnology (pte) Ltd.

The foregoing embodiment of the invention represents a particular example of an immunoassay device for detecting the presence of a pathogenic microorganism. By replacing the HIV lysate with an appropriate corresponding heterogeneous mixture of pathogen proteins, and the gag and/or env proteins with one or more specific pathogen proteins for which detection of protein-specific antibody is desired, an immunoassay device adapted for detection of the presence of other pathogenic microorganisms can easily be provided.

The use of a specific, nonrecombinant antigen in addition to a lysate results in a diagnostic screening and confirmation test in the same assay, an additional advantage of the present invention over conventional diagnostic procedures. The present invention thus provides a more comprehensive and accurate test, while at the same time minimizing the occurrence of false positive results. Minimization of false positive results, particularly in the case of HIV diagnostic tests, is highly desirable. Indeed., in the case of HIV, such a minimization is required as a matter of public policy, due to the poor prognosis of patients diagnosed with AIDS. The incorporation of a secondary test in the present device and assay method also substantially reduces or eliminates the cost and effort associated with subsequent secondary testing on initial positive results, as required by the known assay methods.

An immunodiagnostic device according to the present invention can also include additional delimited portions, which preferably act as positive and negative control portions. A variety of positive control can be employed. In the case of antibody detection, a human IgG antibody is preferably immobilized on one or more of the additional delimited portions. These portions serve as positive controls, because the enzyme-conjugated antibody is expected always to bind with the hlgG, producing a detectable product upon contact with the substrate. Failure to produce a detectable product at this delimited portion thus indicates an inaccurate assay.

In the case of antigen detection, the positive control can be purely mechanical, in that the enzyme (usually bound to a protein) capable of catalyzing a reaction involving the substrate selected for use in the inventive method can be bound to the delimited portion, either directly or via a linker. Preferably the enzyme is the same enzyme used in the inventive method. In the alternative, if the preferred antigen immunoassay employs an additional step for detection of the antigen, such as using biotinylated antibodies to detect the antigen, then the biotinylated antibody can be bound to the delimited portion. Detection of the biotinylated material by an enzyme-labeled streptavidin or enzyme-labeled antibody against biotin would then be employed to provide a positive control.

The assayed antigen can also be immobilized on the delimited portion. In this case, the enzyme-conjugated proteinaceous material used in the assay method will bind thereto. However, this positive control is less preferable. If the assayed antigen is present in the fluid sample in high titer, it could bind all available enzyme-conjugated proteinaceous material added to the sample, leaving none to bind to the control. Hence, there would be an erroneous absence of signal from the positive control despite the large amount of antigen present.

Thus, this embodiment of the inventive device is effective over a more limited range of antigen titer.

As a negative control, a non-reactive protein, such as ovalbumin or bovine serum albumin, can be immobilized on an additional delimited portion. This portion serves as a negative control. In the case of antibody detection, no enzyme-conjugated antibody is expected to bind thereto, and thus no detectable product is expected to be produced. In the case of antigen detection, no antigen, and hence no complex of the antigen and the enzyme-conjugated proteinaceous material (or of the complex-binding agent and the antigen bound to the first proteinaceous material) , is expected to bind thereto. Likewise, no detectable product is expected to be produced. Generation of a detectable product at this delimited portion indicates an inaccurate assay. The present invention therefore provides even further assurance against false negatives and false positives. An immunodiagnostic device according to the present invention can detect antibody or antigen in a nonconcentrated fluid sample without the use of additional equipment to perform or read the results of such an assay. Additionally, an immunoassay device according to the present invention has the advantage that it yields results that are almost immediately ascertainable, because the presence of specific immunoglobulins or antigens can be evidenced by a darkening or coloring of the material upon which the proteinaceous material (e.g. , the nonrecombinant protein) is immobilized, due to generation of colored product by the enzymatic reaction.

In particular embodiments for the inventive antibody assay, the present invention provides for the detection of specific IgA immunoglobulins in urine, specific IgM or IgA immunoglobulins in serum and whole blood, and specific IgG immunoglobulins in urine, whole blood and serum. Specific immunoglobulins can be detected, using an immunoassay device according to the present invention, in a matter of minutes. For example, specific antibodies in nonconcentrated urine can be accurately detected in less than about 15 minutes, and in whole blood or serum in less than about 8 minutes. In particular embodiments for the inventive antigen assay, the present invention provides for the detection of specific surface antigens in saliva and serum. Specific antigens can be detected, using an immunoassay device according to the present invention, in a matter of minutes. For example, specific antigens in saliva or serum can be accurately detected in less than about 4 minutes.

An immunoassay device according to the invention can also be used to obtain semiquantitative results. For example, in an antibody assay according to the invention, differing but standardized amounts of proteinaceous material can be immobilized and an assay performed, as described above, to obtain an indication of the amount of antibody bound from the test sample. In an exemplary HIV diagnostic device, two or more standard amounts of homogeneous gragr protein can be immobilized and brought into contact with a fluid sample which contains an HIV- specific antibody. The intensity of the detectable reaction will differ at each location depending on the amount of antibody-binding protein immobilized therein. Comparison of the intensities with a positive control imparts semiquantitative information as to the amount of HIV-specific antibody in the assayed sample.

An immunodiagnostic device according to the invention can comprise a simple membrane bound to a support, such as a plastic dipstick. The proteinaceous material or materials are simply immobilized on distinct delimited portions of the membrane. Referring now to the drawing, in Figure 1 a membrane

1 comprised of a high surface area material is bonded to a plastic dipstick 3. On a delimited portion 5 of the surface of membrane 1 a proteinaceous material is immobilized.

Figure 2 illustrates a preferred embodiment of an immunodiagnostic device according to the invention incorporating specific proteinaceous materials and positive and negative controls. A plurality of delimited portions 5a-5c and 7a-7b are defined on the surface of membrane 1. A heterogeneous mixture of proteinaceous materials, such as a lysate, is immobilized on delimited portion 5a. Substantially homogeneous proteinaceous materials, such as homogeneous pathogen proteins, are immobilized on delimited portions 5b and 5c, with a different protein being immobilized on each delimited portion. A human immunoglobulin serving as a positive control is immobilized on delimited portion 7a, and a proteinaceous material that is not recognized as a human immunoglobulin is immobilized on delimited portion 7b, serving as a negative control.

Figure 3 illustrates a preferred embodiment for use in a semiquantitative immunoassay according to the invention, in which different amounts of the same proteinaceous material are immobilized on delimited portions 5d-5f defined on the surface of membrane 1.

A particularly preferred embodiment of the immunodiagnostic device is shown in Figs. 4-8. In this embodiment, a membrane is mechanically secured within a recess in the planar surface of the dipstick, and is accessible to the fluid sample on either side due to the presence of a through-hole in the dipstick. This embodiment results in superior product quality, low cost construction, ease of handling, protection of the membrane, and minimization of the fluid quantity needed to conduct the assay. The accuracy and reproduciblity of the assay results are also enhanced by use of the preferred embodiment. The preferred material for use in producing the foregoing particular embodiment is high density ABS plastic. ABS is a high strength plastic which does not absorb or adsorb liquids and is impervious to attack from other reagents used in the inventive assay. It is particularly important to use "virgin" plastic, i.e., plastic that has not been recycled. Recycled plastic typically contains mold release compounds and other contaminants, which could affect the assay and yield inaccurate results. The preferred color of the ABS is white, to accentuate the color change on the membranes produced by the assay results.

An immunoassay device according to the invention preferably is of a shape which is easy to hold. The device preferably also is adapted to fit snugly into a 10 millimeter ID test tube, of the type which is in common use in clinics and laboratories.

Figures 4a-b show a particularly preferred embodiment of an immunoassay device according to the invention, in the form of a dipstick. The dipstick 9 has three wells

11, each connected to a recess 13 via through hole 15 in recess floor 17 (shown enlarged in Fig. 5) . This embodiment is useful in a preferred assay according to the invention, which provides three results simultaneously, namely, a positive control, a negative control and a positive or negative test result. As shown in Fig. 8, in each recess 13 is disposed a membrane

19, which is then secured in place by means of a retaining washer 21. Each retaining washer 21 has a through hole 23 and chamfers 25 on its top and bottom edges (Figs. 6a-b, 7) .

The secured membranes 19 are located on the end of dipstick 9 distal from handle 27, which is also useful as a label area. The membranes 19 are located in recesses 13 as close together as possible (within constraints set by the strength of the ABS plastic) to minimize the amount of fluid necessary to completely submerge the membranes. The membrane material is preferably nitrocellulose, typically about 0.005 inches thick with a 0.45 micron pore size. This material is very brittle and subject to tearing, for example, if ultrasonic welding is employed to secure the membrane to the support member. The membrane is also highly adsorbent, and will wick and adsorb any fluid, including adhesives. Adsorption of contaminants such as adhesives can render the membrane incapable of performing its required function. Accordingly, membrane 19 preferably is secured by discrete mechanical means, such as the retaining washer 21 of the illustrated embodiment, which do not involve the use of membrane-contaminating substances for bonding purposes.

In the preferred embodiment, retaining washer 21, which secures membrane 19 in place, is also made of ABS plastic. The washer has an outer diameter slightly wider

(preferably about 0.001 inch wider) than recess 13, in which membrane 19 is disposed. Careful control of this dimension of retaining washer 21 is important, in order to allow a tight interference fit and thus to secure membrane 19 in place without bonding via adhesives or ultrasonic welding to hold the membrane is place.

Membrane 19 is cut into a disk approximately the diameter of recess 13. The thickness of retaining washer 21 is approximately equal to the depth of recess 13 minus the thickness of membrane 19 (e.g., about 0.005 inch less than the depth of recess 13) .

In assembling dipstick 9, membrane 19 is inserted into recess 13. Retaining washer 21 is then press-fit into recess 13, thus securing membrane 19 in place. Chamfers 25 prevent retaining washer 21 from cutting or otherwise damaging membrane 19 should the retaining washer be inserted into recess 13 at an angle. Although only one chamfer 25 is needed for this purpose, use of a chamfer on both the top and bottom outer edges of the retaining washer facilitates assembly of the dipstick, since it is then not necessary to orient the retaining washer. Typically, chamfer 25 is very small, e.g., about 0.005 inch.

It is necessary that the surface of retaining washer 21 in contact with membrane 19 be flat. Any burrs, ridges, or other obstructions will tend to act as blades and will cut the membrane, thus producing a tear which will render the membrane ineffective. On the other hand, if the retaining washer does not make adequate contact with the membrane, then the fluid sample will leak into the peripheral area of the membrane between the disk and the bottom of recess 13. Residual fluid sample in the peripheral area can result in an inaccurate assay. Thus, retaining washer 21 must be inserted into recess 13 with enough force to firmly hold the membrane in place and avoid creation of air pockets to trap fluid, but without so great a force that the membrane is ruptured. The membrane is then sandwiched in the recess in such a way that the center portion of the disk, between through holes 15 and 23, is open to allow the fluid sample to pass through the membrane unobstructed and interact with the proteinaceous material immobilized thereon.

In a preferred embodiment, well 11 is bevelled to allow a drop of fluid to rest therein while it is being drawn through the membrane. This embodiment facilitates the placement of the reagents required for various assay steps and minimizes the amount of fluid necessary for these steps.

The foregoing device can be employed in any method for detecting an agent (e.g., an antibody or antigen) using a membrane that is secured to a support member, especially membranes on which materials reactive with the assayed agent are immobilized. Those methods using membranes that are fragile and/or highly susceptible to contamination from adhesives are specifically contemplated.

Methods according to the present invention provide highly accurate and sensitive immunodiagnostic processes for detecting antibodies or antigens in fluid samples. The fluid sample does not require concentration or processing. The sensitivity and specificity achieved with immunoassays according to the present invention to date has been equal to that of conventional ELISA and Western blot. In this context, the term "sensitivity" refers to the ability of the assay to detect the presence of antibodies or antigens when they are present in a sample, and the term "specificity" refers to the ability of the assay to distinguish the target antibody or antigen from other antibodies.

Table 1 presents data showing the sensitivity and specificity of an exemplary HIV antibody assay according to the invention. The sample size was 137, with an incidence of HIV positivity of 33%.

Statistical Analysis of a rapid HIV antibody assay n = 137; incidence of HIV positivity = 33%

In a particular embodiment, first-morning urine samples are assayed for antibodies according to the method of the invention. Use of first-morning urine is preferred in order further to enhance accuracy; however, voided urine which is not a first morning sample may be used to achieve positive results visible to the naked eye.

In another preferred embodiment of the present invention, up to about 1.0% each of glycine and glutamine, preferably about 0.5% glycine and about 0.5% glutamine, and optionally up to about 1.0% lactose, is added to the urine sample to be assayed. The advantages of this embodiment include a surprising, approximately two-fold increase in the overall sensitivity of the inventive assay, in addition to prevention of denaturation of the proteins from the sample during cryopreservation and prevention of non-specific binding of interfering proteins to the membrane. The effects of glycine and glutamine are additive but independent of each other. Enhancement of specific binding begins at a concentration of about 0.25% glycine or glutamine; however, above 1% there appears to be a reduction of specific binding. The first step in detecting an antibody in the fluid sample according to a method of the invention involves contacting the sample with an immobilized proteinaceous material for which it is specific. The sample is contacted with the proteinaceous material for a specific period of time. In the exemplary embodiment of detection of anti-HIV antibodies, the sample is contacted with the

HIV proteinaceous material for at least about 15 minutes

(for urine) or at least about 1 minute (for blood or serum) . In another preferred embodiment of the present invention, vigorous agitation is applied during the contacting step in order to accelerate performance of the assay. In this embodiment, the same results that would otherwise be obtained by longer periods of incubation are achieved with unexpected rapidity. Thus in the previous exemplary HIV assay, the required contact time for blood would be about 1 minute, with a total assay time of about 10 minutes. For urine, the contact and total times would be about 5 and 14 minutes, respectively. The next step in a method according to the present invention involves the removal of the immobilized proteinaceous material from contact with the fluid sample. The proteinaceous material is then brought into contact with at least one agent that recognizes human antibody or an antibody-antigen complex per se, said agent being conjugated to an enzyme which catalyzes a detectable reaction involving a substrate. Binding of the conjugated agent to the selected antibody, bound to the immobilized proteinaceous material, or to the complex formed by the antibody and the immobilized proteinaceous material, is effected by incubation in a buffer solution containing the conjugated agent. Subsequent interaction of the enzyme, conjugated to the bound agent, with a substrate for the enzyme results in production of a detectable output, as described below.

Alternatively, the agent can be added to the fluid test sample before bringing the sample into contact with the immobilized proteinaceous material. As applied to the exemplary HIV assay according to the invention, the contact and total assay times for blood would be about 1 and 7 minutes, respectively, while for urine the times would be about 15-30 and 21-36 minutes, respectively.

Table 2 summarizes the typical times required for carrying out antibody immunoassays according to the method of the invention.

Table 2

All times in minutes

Suitable agents for use in an antibody assay method according to the present invention include agents which bind to human antibodies, such as enzyme-conjugated anti- human IgA, IgG or IgM. Agents which selectively bind to the antigen-antibody complex, but do not bind to the antibody or antigen in isolation, are also suitable. The latter agents, referred to as "complex-binding agents," include enzyme-conjugated RhC and enzyme-conjugated Clq (a subunit of the first complement component Cl) . RhC and Clq are characterized, and methods for their preparation are disclosed, in U.S. patents No. 4,783,525 and 4,595,654 respectively, the contents of each of which are hereby incorporated in their entireties by reference. It is contemplated that other agents that selectively bind to antibody-antigen complexes without binding to either in isolation are suitable for use in the present invention.

Care must be taken in conjugating the complex-binding agent with the selected enzyme. Preferably the enzyme is oxidized, in particular using the periodate method, and subsequently linked to the complex-binding agent. Such treatment of the enzyme, rather than the complex-binding agent, is especially preferred when the complex-binding agent is RhC, as it has been found that treatment of RhC in certain media, such as strongly ionic buffers, destroys its activity. While the reason for this loss of activity is not known with certainty, it is believed that RhC is an ionic dimer, and as such is susceptible to structural disruption and loss of functional groups upon treatment with ionic buffers. This problem does not arise when the enzyme, rather than the complex-binding agent, is treated. The conjugation of the enzyme and the complex-binding agent can also be effected using the one- step glutaraldehyde method. This method may be preferable in certain circumstances, but the efficiency of the method is low.

In a preferred embodiment of an antibody assay according to the present invention, the complex-binding agent (e.g., RhC) is biotinylated, whereby enzyme conjugated streptavidin increases the amount of enzyme per unit of antibody conjugated. Biotinylation can be carried out employing methods known to those skilled in the art.

A wide variety of enzymes can be used in practicing an antibody assay method of the present invention. Preferred enzymes include alkaline phosphatase, horseradish peroxidase, and glucose oxidase. Suitable substrates for the previously mentioned enzymes include nitroblue tetrazolium and diaminobenzadine. Also, β- galactosidase can be used with a suitable substrate. The next step in an antibody assay according to the invention is removal of the immobilized proteinaceous material(s) from contact with the solution containing the remaining unbound agent and into contact with the substrate. In a preferred embodiment, the removal of the proteinaceous material from the fluid sample is accomplished by washing with an isotonic buffer solution, such as phosphate buffered saline (PBS) or tris buffered saline (TBS) . The final step of the antibody assay according to the invention is determination whether the contact with the substrate, effected in the previous step, results in production of a detectable output. Interaction of the conjugated enzyme with the substrate, as a result of the aforesaid contact, to produce a detectable output indicates the presence of antibody in the fluid sample. Absence of a detectable output indicates that no conjugated enzyme was present to interact with the substrate, and thus no antibody was present with which the enzyme-conjugated agent could bind.

In a preferred embodiment of an antibody assay of the present invention, the detectable product is visible and distinguishable to the naked eye; that is, the use of equipment to detect a specific antibody is unnecessary, as has been noted above. However, equipment may be used to read the device, so as to either increase the sensitivity or more easily quantitate the result.

Antigen assay methods according to the invention are similar to the foregoing antibody assays. The first step in detecting an antigen in the fluid sample in an embodiment of the inventive method involves diluting a fluid sample suspected to contain an antigen with a solution containing a first proteinaceous material that binds the antigen. Preferably, the first proteinaceous material is a monoclonal antibody or fragment thereof.

In a first preferred embodiment of the inventive immunoassay method, the first proteinaceous material is conjugated to an enzyme, such that the enzyme can catalyze a detectable reaction involving a substrate. When the fluid sample is diluted with the solution containing the conjugated first proteinaceous material, the proteinaceous material binds to any of the assayed antigen that is present in the fluid sample, forming a complex.

Known enzyme conjugation methods as described above can also be employed to produce the conjugated first proteinaceous material. Preferably the enzyme is oxidized, in particular using the periodate method, and subsequently linked to the first proteinaceous material. The above-mentioned enzymes and substrates are also suitable for use in antigen assays according to the invention.

Addition of glycine and/or glutamine to the fluid sample, as described above in the context of the inventive antibody assays, confers similar advantages in an antigen assay according to the invention. The next step of the inventive antigen assay method involves bringing the diluted fluid sample into contact with a second proteinaceous material that also binds the antigen to be assayed, said second proteinaceous material being immobilized in reactive form on a delimited portion of a high-surface-area material as described previously. The second proteinaceous material preferably is a polyclonal antibody or fragment thereof, but can also be a monoclonal antibody or fragment thereof or a complex- binding agent. If a monoclonal antibody or fragment is used, it should bind to an epitope other than that which the first _, proteinaceous material binds. In the embodiment in which a complex-binding agent is used, the agent recognizes the complex of antigen and first proteinaceous material (e.g., antigen-antibody) . As a result of the foregoing contacting step, a higher-order complex is formed that also includes the second proteinaceous material. Thus, in one preferred embodiment, the total complex is a polyclonal-antigen- monoclonal complex. In another preferred embodiment, the total complex is formed between the complex-binding agent, e.g., RhC, and the complex including the antigen and the first proteinaceous material.

The fluid sample is contacted with the second proteinaceous material for a specific incubation time. In the exemplary embodiment for detection of HBSAg in saliva or serum, the fluid sample is contacted with the second proteinaceous material reactive with HBSAg for at least about 1 minute. In another preferred embodiment of an antigen assay according to the present invention, vigorous agitation is applied during the contacting step in order to accelerate performance of the assay. In this embodiment, the same results that would otherwise be obtained by longer periods of incubation are achieved with unexpected rapidity. In an exemplary HBSAg urine assay, the required contact time would be about 5 minutes, with a total assay time of about 8 minutes. Subsequent to the contacting of the second proteinaceous material with the fluid sample, the second proteinaceous material is removed from contact therewith. The material is then washed, preferably with an isotonic buffer solution such as phosphate buffered saline (PBS) or tris buffered saline (TBS) . Washing is essential in order to prevent unbound enzyme-conjugated first proteinaceous material from subsequently coming into contact with the substrate, thus resulting in an inaccurate assay. In the next step, the second proteinaceous material is brought into contact with the enzyme substrate. Any antigen-comprising complex (e.g., polyclonal-antigen- monoclonal, or RhC-antibody/antigen) that is present will come into contact with the enzyme substrate, and thus bring about a detectable reaction.

The final step of the antigen assay according to the first preferred embodiment of the invention is determining whether the contact with the substrate, effected in the previous step, results in production of a detectable output. Interaction of the enzyme, conjugated to the first proteinaceous material, with the substrate, as a result of the aforesaid contact, to produce a detectable output indicates the presence of antigen in the fluid sample. Absence of a detectable output indicates that no complex was present to interact with the substrate, and thus that no antigen was present in the fluid sample. As with the inventive antibody assays, the detectable product of the inventive antigen assay preferably is visible and distinguishable to the naked eye.

In a particularly preferred embodiment, the sensitivity of the inventive assay is increased through enzyme amplification. This technique is described, for example, in Clinica Chimica Acta, 148 (1985) 119-124, and in J^". Immunol . Methods, 76 (1985) 389-393, and also in U.S. Patent No. 4,769,321 (to Self, Sept. 6, 1988), the disclosure of which is hereby incorporated in its entirety by reference. The enzyme amplification technique couples a first enzymatic reaction with a redox cycling enzyme system, such that a product of the first enzymatic reaction is subsequently cycled between oxidized and reduced form. Both stages of the assay are accompanied by the formation of colored product, with multiple molecules of the colored product being produced during the cycling stage. The technique thus yields much a higher rate of absorbance change than that obtainable in conventional, non-cycling assays.

In a second embodiment of the antigen immunoassay method according to the invention, the first proteinaceous material is biotinylated rather than being conjugated to an enzyme. After the formation of the complex of the antigen with the first and second proteinaceous materials, the complex is contacted with enzyme-conjugated streptavidin, and subsequently washed.

In a further modification of the second embodiment of the inventive antigen assay method, an enzyme- conjugated proteinaceous material that binds biotin, e.g. , an anti-biotin polyclonal or monoclonal antibody or fragment thereof, is contacted with the complex comprising the antigen and the first and second proteinaceous materials. The amount of enzyme per unit of proteinaceous material conjugated again is increased.

In a third embodiment of the antigen immunoassay method according to the invention, the first proteinaceous material is not conjugated to an enzyme. Upon formation of the complex including the antigen and the first and second proteinaceous materials, the complex is contacted with a third enzyme-labeled proteinaceous material, e.g., an antibody, that binds the first proteinaceous material. A washing step follows. The resulting complex is then contacted with the enzyme substrate, and the presence or absence of a detectable output from the enzymatic reaction is determined in the same manner as above. Table 3 summarizes the typical times required for carrying out antigen assays according to the invention.

initial incubation time/total time All times in minutes

The following examples are provided as non-limiting illustrative embodiments of the present invention. In the examples, the following buffers and reagents are used:

a) Tris Buffered Saline (TBS, loading buffer) : 20 mM Tris-HCl, 0.5 M NaCl, pH 7.5.

To 2.54 g Tris-HCl, 0.472 g Tris and 29.22 g NaCl is added a quantity of deionized water sufficient to make 1 L buffer solution. b) Barbital Buffer (BB, washing buffer) : To a solution of 20 iriM sodium barbital (4.12 g/L) and 1 mM MgCl (0.5 ml 2M MgCl/L) are added 2% (v/v) acetic acid, to adjust the pH of the solution to 9.6.

c) Non-Fat Dry Milk, Caseinate, Glycine,

Glutamine, Barbital Buffer (MCGGBB: blocking buffer)

To a solution of 20 mM Barbital Buffer at pH 9.6 are added 5% non-fat dry milk (5 g/100 ml) , 5% sodium caseinate (5 g/100 ml), 2.5% glycine (2.5 g/100 ml) and

2.5% glutamine (2.5 g/100 ml) .

d) Glycerol/thimerosal solution (G/T stabilizer) To a solution of 5% glycerol (5 ml/100 ml deionized

H₂0) is added a solution of 0.01% thimerosal (10 mg/100 ml) .

e) Non-Fat Dry Milk, Caseinate, NP40, Barbital Buffer (MCNBB dilution buffer)

The buffer is prepared by combining in solution 20 mM BB at pH 9.6, 5% non-fat dry milk (5 g/100 ml), 3% sodium casienate (3 g/100 ml) and 0.1% NP40 (0.1 ml/100 ml) .

f) Non-Fat Dry Milk, Caseinate, NP40, Barbital Buffer (MCNBB-U, urine dilution buffer)

The buffer is prepared by combining in 80 ml of 20 mM BB (at pH 9.6) with 5% sodium casienate (5 g/100 ml) under heat and with stirring. After the casienate dissolves, 24% non-fat dry milk (24 g/100 ml), and 0.7% NP40 (0.7 ml/100 ml), are added. The mixture it topped off with BB to yield 100 ml of buffer. The MCNBB-U can be stored at 4°C.

g) Substrate buffer

A 100 ml substrate buffer is prepared by combining 0.393 M 2-amino-2-methylpropanol (3.5 g/100 ml) with 0.5 mM MgCl₂ (10.2 mg/100 ml), and adjusting the pH to 9.8. To this buffer are added 50 mg nitroblue tetrazolium and 25 mg 5-bromo-4-chloro-3-indyl phosphate dissolved in 5 ml N,N-dimethylformamide, to form the substrate solution.

h) Rabbit-derived, anti-hlgG antibody conjugated with alkaline phosphatase, diluted 1:1000 in MCNBB, is used as the conjugated agent.

i) Non-Fat Dry Milk, Caseinate, Glycine, Glutamine, NP40, Barbital Buffer (MCGGNBB dilution buffer)

The buffer is prepared by combining in solution 20 mM BB at pH 9.6, 5% non-fat dry milk (5 g/100 ml), 3% sodium caseinate (3 g/100 ml), 0.5% glycine (0.5 g/100 ml), 0.5% glutamine (0.5 g/100 ml) and 0.1% NP40 (0.1 ml/100 ml) .

j) Amplified substrate solution

A 100 ml amplified substrate buffer is prepared by combining 2-amino-2-methylpropanol (3.12 gm/100 ml) with 5 ml of Mg/Zn/HEDTA solution (1.72 gm magnesium acetate, 1.15 gm zinc sulfate and 3.04 gm HEDTA/ 100 ml H₂0) , adjusting the final solution to pH 9.8 with 3 N hydrochloric acid. To this buffer are added 81.76 mg nitroblue tetrazolium (NET) , 43.36 mg 5-bromo-4-chloro-3- indyl phosphate (BCIP, dissolved in 5 ml N,N- dimethylformamide) , 10 IU 3-α-hydroxysteroid dehydrogenase, 0.02 mol NADP, 4 mg diaphorase and 10 μ ol glycochenodeoxycholic acid to form the final volume of amplified substrate.

Example 1: comparison with planar surface (microtiter plate)

Varying concentrations of HIV-2 viral lysate were bound to the surface of a nitrocellulose membrane (a "high surface area material") and to the flat surface of a polystyrene microtiter plate. For the membrane test, the HIV lysate was bound to the nitrocellulose in 50 mM TBS at pH 7.5. The membrane was washed 3X with BB at pH 9.6 and blocked overnight with a solution of non-fat dry milk, sodium caseinate, glutamine and glycine in TBS at pH 9.6. For the microtiter plate test, the lysate was bound to the plate surface using carbonate buffer at pH 9.6. The plate was washed in TBS at pH 7.5 and blocked overnight with a solution of non-fat dry milk, sodium caseinate, glutamine and glycine in TBS at pH 7.5. All subsequent steps were carried out identically, with the exception of substituting the TBS of the microtiter plate system for the BB of the membrane system. Incubation times for the membrane system were 15 minutes with serum and conjugated agent, and 5 minutes for the substrate. All assessments were performed visually.

The titer of an HIV-2-positive serum sample was determined with both the microtiter and membrane methods. The HIV-2-positive serum was diluted 1:50, 1:100, 1:200, 1:500, 1:1000, 1:2000 and 1:5000.

Test results are shown in Tables 4 and 5. Utilizing the microtiter method, Table 4 shows that the strongest reaction was noted when 0.5 μg of HIV-2 viral lysate was bound. The serum titer was determined to be 1:2000 at the point at which a slight color change was seen. At a concentration of either 1.0 μg or 0.25 μg of HIV-2 lysate, the serum titer was 1:1000. This indicates that the sensitivity of the microtiter method is dependent on the concentration of the HIV-2 lysate bound. Moreover, there is an inhibition of the response when the optimal concentration is exceeded.

In contrast, a dose response curve could be demonstrated with the membrane method throughout the range of serum dilutions used. The serum titer when HIV- 2 lysate was bound to the membrane was greater than 1:5000 for all concentrations used. However, as shown in Table 5, at 0.1 μg of HIV-2 lysate, the result was slightly over background. This suggests that there was also a concentration-dependent effect on the results obtained with the membrane system. On the other hand, within the range of HIV-2 lysate utilized, the process was not saturated. No inhibition of the reaction was seen in the range of HIV-2 lysate used. Thus, depending on the concentration of HIV-2 lysate used on the membrane or microtiter surface, the immunoassay using the membrane according to the invention was about 2.5 to 10 times more sensitive than the assay using the microtiter plate.

Table 4

Simple planar surface (microtiter plate of smooth polystyrene)

1.0 0.5 0.25 0.1 μg

HIV-2 viral lysate concentration, μg

+ = strong reaction - = weak reaction 0 = no reaction Table 5

High surface area material (nitrocellulose membrane) agent: RhC enzyme: alkaline phosphatase

Serum Titer

1:1000

1:5000

1.0 0.5 0.25 0.1 μg

HIV-2 viral lysate concentration, μg

Darkness scale indicating strength of reaction:

8 = very heavy black

7 = black

5 = visible gray

1 = weak reaction, trace barely visible

0 = no visible reaction

Example 2: preparation of HIV immunoassay device An HIV immunoassay device according to the invention comprises an extrusion molded ABS plastic dipstick and a nitrocellulose membrane (0.45μm) which are sonically bonded together. HIV antigens are loaded onto delimited portions of the nitrocellulose membrane of the immunoassay device. HIV lysates (HIV-1 and HIV-2) are diluted 1:12 with TBS. 0.5 μg of HIV-1 and 0.31 μg of HIV-2 (1 μg and 0.72 μg for use in a urine assay, respectively) are loaded in a 3 μl volume. The lysates are applied to separate delimited portions of the nitrocellulose surface layer of the immunoassay device and allowed to dry by sitting for 45 minutes. The device is then washed 3X with 3 ml BB. HIV-1 gag protein is next diluted 1:12 with TBS. 0.5 μg of gag protein (1 μg for use in a urine assay) is loaded in a 3 μl volume. The protein is applied to a separate delimited portion of the nitrocellulose surface layer of the immunoassay device and allowed to dry by sitting for 45 minutes. The device is then washed 3X with 3 ml BB.

In a similar manner, HIV-1 env protein is next diluted 1:12 with TBS. 0.5 μg of gag protein (1 μg for use in a urine assay) is loaded in a 3 μl volume. The protein is applied to a separate delimited portion of the nitrocellulose surface layer of the immunoassay device and allowed to dry by sitting for 45 minutes. The device is then washed 3X with 3 ml BB. The negative and positive controls are finally applied. The negative control (ovalbumin, 0.2 g/100 ml) is applied to the device in the same manner, using the same quantity of protein. The positive control (human IgG) is finally as follows: hlgG stock solution is diluted in TBS to a concentration of 50 ng/3 μl. Then 50 ng of the hlgG is applied to a separate delimited portion of the nitrocellulose surface layer and allowed to dry by sitting for 45 minutes, after which time the device is washed 3X with 3 ml BB. After immobilization of the proteinaceous materials on the membrane, the device is placed in a 15 ml screw- capped tube, to which is added 5 ml MCGGBB blocking buffer. The tube containing the device is then placed on a rocker and incubated with rocking for 16 hours at room temperature, removed from the blocker, rinsed 3X in BB, and placed in a tube containing 5 ml G/T for five minutes. Excess fluid is removed by shaking, and the device is allowed to dry at room temperature in a desiccator under vacuum until dry. Finally, the device is placed in a sealable bag (e.g., a zip-lock bag) and stored at 4°C until use. Example 3: Serum and whole blood test

Serum is prepared for assay as follows: A serum sample obtained by normal laboratory procedures is heated at 56°C for 30 minutes, after which 20 μl of thimerosal- TBS solution per ml of serum are added. The sample can then be stored refrigerated or frozen. Before use in the assay, the serum sample is diluted 1:100 in MCNBB.

In the assay, 1 drop (50 μl) of the diluted sample is placed on each delimited region of the immunoassay device. After an incubation period of 1 minute, excess sample is removed by shaking, and the device is rinsed by dipping up and down 5X in 3 ml BB in a test tube. The rinsing procedure is then repeated. Next, 1 drop of the dilute (1:1000) conjugated agent is placed on each delimited region. After an incubation period of 2 minutes, excess agent is removed by shaking, and the device is rinsed as before. Subsequently, 1 drop (50 μl) of substrate is placed on each delimited region. The device is incubated 2-5 minutes, and the results of the reaction are determined visually.

A similar procedure is used to assay whole blood, except that the blood is not heated or stored, but used directly after being obtained from the patient.

Example 4: Urine test Urine is prepared for assay as follows: A serum sample obtained by normal procedures is heated at 56°C for 30 minutes. Next, 2.7 ml of the heated sample is pipetted into a test tube, and 300 μl of a booster reagent containing 5% glycine, 4% glutamine and 0.1 thimerosal in BB is added. The sample can then be cryopreserved for storage, if required.

Before use in the assay, the urine sample is thawed if necessary and brought to room temperature. 500 μl of MCNBB-U are added to the sample with thorough mixing. The treated sample is transferred to a test tube. Next, the immunoassay device (double antigen loaded) is placed in the test tube to a depth sufficient to cover the delimited regions of the membrane, and incubated for a period of 15 minutes. During the incubation period, the device is dipped up and down 5X initially, and then every 5 minutes. Alternatively, the device can be vigorously agitated throughout a 5 minute period. The device is then removed from the test tube, and excess sample is removed by shaking. The remainder of the procedure is as for the assay of blood or serum, above.

Example 5: Addition of conjugated agent to sample RhC is conjugated to alkaline phosphatase by the glutaraldehyde method. The conjugated RhC is then added to urine at a final concentration of 1 mg/ml (protein concentration) . The sample mixture is incubated with the immunoassay device for 30 minutes with agitation, washed twice with TBS, and then incubated with the substrate solution. Results show a positive reaction at the delimited portions of the membrane on which HIV-1 and HIV-2 proteins and hlgG are immobilized.

Example 6: semiquantitative method Human IgG was placed in a number of portions of the device according to the invention in various concentrations. The device was then processed and developed in the normal manner. The minimum detectable hlgG concentration was at 5 ng/spot, and an increasing "dot" intensity was observed at 10, 25 and 50 ng/spot. Above 50 ng/spot, the intensity of the "dot" did not appear to change. Thus, by comparing the intensity of the dot appearing in HIV portion to that appearing in the positive control portion of the device, a semi- quantitative result can be obtained. The result can be further refined by providing hlgG portions having concentrations of 10 and 25 ng/spot. Results indicate whether the sample contains <10 ng, 10-25 ng or >25 ng.

Example 7: preparation of HBSAg (hepatitis B surface antigen) immunoassay device An HBSAg immunoassay device according to the invention comprises an extrusion molded ABS plastic dipstick and a plurality of nitrocellulose membrane (0.45μm pore size) which are held in membrane chambers with ABS retaining washers, as described above. Horse- derived, anti-human HBSAg polyclonal antibodies (0.1-10 μg, affinity purified and ad/ay specific) are loaded onto one of the nitrocellulose membranes of the immunoassay device. Horse-anti-HBSAg (10 mg/ml) are diluted 1:50 with TBS. One μg of the antibody is loaded in a 5 μl volume. The antibody is applied to a nitrocellulose membrane of the immunoassay device and allowed to dry by sitting for 45 minutes. The device is then washed 3X with 3 ml of BB.

The negative and positive controls are applied. The negative control (ovalbumin, 0.2 g/100 ml) is applied to a separate membrane of the device in the same manner, using the same quantity of protein. The positive control (alkaline phosphatase-conjugated, mouse-monoclonal anti- HBSAg ad/ay, 5 ng/5 μl) is applied as follows: stock solutions of alkaline phosphatase-conjugated, mouse- monoclonal anti-HBSAg subtype ad and ay (1 mg/ml) is diluted 1:1000 with TES to a concentration of 5 ng/5 μl. Then, 5 ng of alkaline phosphatase-conjugated, mouse- monoclonal anti-HBSAg subtype (ad/ay) is applied to a separate nitrocellulose membrane. The negative and positive controls are allowed to dry by sitting for 45 minutes, after which time the device is washed 3X with 3 ml BB.

After immobilization of the proteinaceous materials on the membranes, the device is placed in a 15 ml screw- capped tube, to which is added 5 ml MCGGBB blocking buffer. The tube containing the device is then placed on a rocker and incubated with rocking for 16 hours at room temperature, removed from the blocker, rinsed 3X in BB, and placed in a tube containing 5 ml G/T for five minutes. Excess fluid is removed by shaking, and the device is allowed to dry at room temperature in a desiccator under vacuum until dry. Finally, the device is place in a sealable bag (e.g., a zip-lock bag) and stored at 4°C until use.

Example 8: Saliva test for HBSAg

Saliva is obtained using normal laboratoryprocedures which may include the stimulation of salivation using dilute solutions of acetic acid (vinegar) . Saliva is collected in a sterile container (e.g., urine cup) and 1 ml is transferred to a screw-capped plastic, cryogenic tube and may be refrigerated or frozen. Before use in the assay, the saliva sample is diluted 1:1 in MCGGNBB containing 0.1 μg/ml of alkaline phosphatase-conjugated, mouse monoclonal anti-HBSAg antibody (subtype ad/ay) . In the assay, 1 drop (50 μl) of the diluted sample is placed upon each delimited region of the immunoassay device. After an incubation period of 1 minute, excess sample is removed by shaking, and the device is rinsed by dipping up and down 5X in 3 ml BB in a test tube. Next, 1 drop (50 μl) of the amplified substrate solution is placed upon each delimited region. The device is incubated 2-5 minutes, and the results of the reaction are determined visually.

The amplified substrate takes advantage of the optimal pH for the action of alkaline phosphatase conjugated with the antigen detecting antibody and uses the optimal pH necessary to ensure that the "redox" enzymes react with their substrate in the proper direction. Utilizing BCIP to act as a trap for inorganic phosphorus (Pj) allows the specific directing of the "redox" enzyme pathways. The formation of the colored insoluble product from NET is enhanced since NADP converted to NADH by alkaline phosphatase enters the "redox" cycle as a cofactor allowing further formazan to form by the action of diaphorase. The cycle of NAD⁺ to NADH by the action of 3-α_-hydroxysteroid dehydrogenase allows the NADH to recycle. As such, for each NADH molecule formed from alkaline phosphatase, 20-200 molecules of for azan may be produced by the "redox" cycling enzyme system. While some NBT may be converted to formazan by alkaline phosphatase, the overall reaction is enhanced 100 fold over substrates for alkaline phosphatase not utilizing the enhancing "redox" enzyme system. Since the substrate and enzymes are only activated by the action of alkaline phosphatase upon NBT and NADP, background levels are unaffected. However, the sensitivity of the method is improved, since few alkaline phosphatase molecules are required to produce a visible product.

The overall reaction can be demonstrated below, showing the enzymatic pathways involved in formazan formation initiated by alkaline phosphatase and enhanced by a "redox" cycle.

Purple-Black Colored Product

A similar procedure can be used where the mouse monoclonal anti-HBSAg antibody is not conjugated to an enzyme. In this instance, a second 1 minute incubation with an alkaline phosphatase-conjugated anti-mouse IgG antibody is interjected, with an additional washing step (dipping device up and down 5X in 3 ml BB) , before the addition of the amplified substrate solution. Example 9: Serum and whole blood test

Serum is prepared for assay as follows: A serum sample obtained by normal laboratory procedures is heated at 56 °C for 30 minutes, after which 20 μl of thimerosal- TBS solution per ml of serum are added. The sample can then be stored refrigerated or frozen. Before use in the assay, the serum sample is diluted 1:10 in MCNBB containing 1:100 mouse anti-HBSAg (10 μg/ml) .

In the assay, 1 drop (50 μl) of the diluted sample is placed on each delimited region of the immunoassay device. After an incubation period of 1 minute, excess sample is removed by shaking, and the device is rinsed by dipping up and down 5X in 3 ml BB in a test tube. The rinsing procedure is then repeated. Subsequently, 1 drop (50 μl) of substrate is placed on each delimited region. The device is incubated 2-5 minutes, and the results of the reaction are determined visually.

Example 10: Urine test

Urine is prepared for assay as follows: A serum sample obtained by normal procedures is heated at 56°C for 30 minutes. Next, 2.7 ml of the heated sample is pipetted into a test tube, and 300 μl of a booster reagent containing 5% glycine, 4% glutamine and 0.1 thimerosal in BB is added. The sample can then be cryopreserved for storage, if reguired.

Before use in the assay, the urine sample is thawed if necessary and brought to room temperature. 500 μl of MCNBB-U (containing in addition 50 μg/ml of mouse anti- HBSAg) are added to the sample with thorough mixing. The treated sample is transferred to a test tube. Next, the immunoassay device is placed in the test tube to a depth sufficient to cover the membranes, and incubated for a period of 15 minutes. During the incubation period, the device is dipped up and down 5X initially, and then 5X every 5 minutes. Alternatively, the device can be vigorously agitated throughout a 5 minute period. The device is then removed from the test tube, and excess sample is removed by shaking. The remainder of the procedure is as for the assay of blood or serum, above.

Example 11: RhC method

100 μg of RhC is immobilized on a delimited portion of high surface area material. The selected first proteinaceous material, conjugated to the selected enzyme, is added to the fluid sample. Subsequently, the immunoassay device is incubated in the diluted fluid sample for 30 minutes with agitation. The device is then washed twice with BB, and then incubated with the selected substrate solution. Results show a positive reaction at the delimited portion when either horse anti- HBSAg or mouse anti-HBSAg are mixed with samples containing HBSAg (ad/ay) .

Example 12: developmental results

A series of experiments were laid out to provide an example of the application of the technology in detecting HBSAg in biologic fluids, utilizing a horse-derived anti- HBSAg (ad/ay) polyclonal antibody and a mouse-derived anti-HBSAg monoclonal antibody to capture purified HBSAg (mixture of subtypes ad and ay) upon a nitrocellulose membrane (high surface area material) . Detection of the antibody-antigen-antibody complexes was performed using alkaline phosphatase-conjugated, goat-derived anti- antibody (either against horse or mouse antibody, depending upon which material was not bound to the membrane) .

Exp. 1: To determine the concentration of horse or mouse antibodies which could be detected using goat-anti- horse or -anti-mouse antibodies, respectively, the antibodies were bound to membranes using 5 μl loading volumes of 1:10, 1:100, 1:500, 1:1000, 1:2000, 1:5000, -46-

1:10000, and 1:20000 dilutions of the stock antibodies (10 mg/ml for horse antibody and 1 mg/ml for mouse antibody). Detection was achieved using a 1:1000 dilution of the respective goat-anti-antibodies. The results suggested that horse anti-HBSAg antibodies were most visible at 1:10 dilutions, while mouse anti-HBSAg antibodies were best utilized at 1:100 dilutions.

Exp. 2: To determine the concentrations of antigens detectable with the respective horse or mouse antibodies, a mixture of HBSAg subtypes ad/ay were bound to the membranes in the device, then contacted with horse anti- HBSAg antibodies (1:10) followed by goat-anti-horse antibodies (1:1000) . 25 ng of HBSAg ad/ay was detectable above background. Using mouse anti-HBSAg antibodies (1:100), 50 ng of HBSAg ad/ay was detectable.

Exp. 3: To determine which antibody (polyclonal or monoclonal) function best in the method when bound to the membrane, devices were loaded with horse or mouse anti- HBSAg antibodies. HBSAg ad/ay was then incubated for 3 minutes with each respective antibody and subsequently washed away. Then, the complementary mouse or horse anti-HBSAg were incubated with the device for 3 minutes and the device was washed. Subsequently anti-mouse or anti-horse antibodies conjugated with alkaline phosphatase were incubated, respectively, for three minutes. Following a final wash, the device was incubated with substrate solution. The results were similar in each case.

To further clarify this point, HBSAg ad/ay was diluted in horse anti-HBSAg or mouse anti-HBSAg and then applied to devices with corresponding mouse anti-HBSAg or horse anti-HBSAg, respectively. The devices were incubated for 3 minutes and washed. Subsequently, each device was incubated with enzyme-labeled goat anti-mouse or anti-horse antibodies, washed and developed. The results of this experiment indicated that binding of HBSAg:mouse anti-HBSAg to polyclonal antibodies upon the membrane was identical to the results obtained with sequential addition of the components. On the other hand, markedly less binding occurred between HBSAg:horse anti-HBSAg complexes and membrane bound monoclonal antibodies. Thus, the embodiment in which polyclonal antibodies are bound to the membrane and the antigen is reacted with monoclonal antibodies in solution is indicated as preferred.

Exp. 4: In order to provide evidence that the method could be used to detect HBSAg in biological fluids, two known positive serum samples and one known positive saliva sample were analyzed with the device according to the preferred embodiment. One negative serum sample and one negative saliva sample were also run, in parallel. Both positive serum samples and positive saliva sample were positive, while the negative samples showed no detectable reaction.

Claims

What is claimed is:

1. An immunoassay device for detecting the presence the presence of an analyte selected from the group consisting antibodies and antigens in a fluid sample, comprising

(a) a support member presenting a planar surface that comprises at least one delimited portion comprised of a material having a high surface area upon which a proteinaceous material can be immobilized in reactive form and, immobilized on said delimited portion,

(b) a proteinaceous material that binds said analyte.

2. An immunoassay device as claimed in claim 1, wherein said material having a high surface area is nitrocellulose, nylon, polyamide, polycarbonate, or cellulose acetate.

3. An immunoassay device as claimed in claim 2, wherein said material has a protein-binding capacity of about 50-500 μg/cm².

4. An immunoassay device as claimed in claim 1, wherein said planar surface comprises a plurality of delimited portions, upon each of which is immobilized a proteinaceous material.

5. An immunoassay device as claimed in claim 4, wherein each said proteinaceous material immobilized on said plurality of delimited portions is independently the same or different.

6. An immunoassay device as claimed in claim 5, wherein at least one proteinaceous material immobilized on a portion of said plurality is recognized by an enzyme-conjugated anti-human immunoglobulin antibody such that, once said enzyme-conjugated anti-human immunoglobulin antibody is allowed to react with said immobilized protein, said enzyme is capable of catalyzing a detectable reaction involving a substrate, and wherein at least one additional proteinaceous material immobilized on a different portion of said plurality is not recognized as human immunoglobulin by said enzyme- conjugated anti-human immunoglobulin antibody.

7. An immunoassay device as claimed in claim 6, wherein said proteinaceous material that is recognized is IgG and said proteinaceous material that is not recognized is ovalbumin or bovine serum albumin.

8. An immunoassay device as claimed in claim 1, wherein said analyte is an antibody and wherein said proteinaceous material comprises nonrecombinant protein from a pathogenic microorganism.

9. An immunoassay device as claimed in claim 8, wherein said pathogenic microorganism is a virus, a bacterium or a protozoan.

10. An immunoassay device as claimed in claim 8, wherein said pathogenic microorganism is an HIV virus.

11. An immunoassay device as claimed in claim 8, wherein said planar surface comprises a plurality of delimited portions, each comprised of a material having a high surface area upon which said proteinaceous material can be immobilized in reactive form, and wherein on one portion of said plurality is immobilized a heterogeneous mixture of proteins obtained from said pathogenic microorganism, and on a different portion of said plurality is immobilized a substantially homogeneous nonrecombinant protein obtained from said pathogenic microorganism.

12. An immunoassay device as claimed in claim 11, wherein said heterogeneous mixture of pathogen proteins is HIV lysate, and wherein said nonrecombinant protein is HIV gag protein or HIV env protein.

13. An immunoassay device as claimed in claim 12, wherein said HIV lysate comprises HIV-1 lysate or HIV-2 lysate.

14. An immunoassay device as claimed in claim 11, further comprising at least one proteinaceous material, immobilized on a different portion of said plurality, that is recognized by an enzyme-conjugated anti-human immunoglobulin antibody such that, once said enzyme- conjugated anti-human immunoglobulin antibody is allowed to react with said immobilized protein, said enzyme is capable of catalyzing a detectable reaction involving a substrate, and at least one proteinaceous material, immobilized on another portion of said plurality, that is not recognized as human immunoglobulin by said enzyme- conjugated anti-human immunoglobulin antibody.

15. An immunoassay device as claimed in claim 14, wherein said proteinaceous material that is recognized is hlgG and said proteinaceous material that is not recognized is ovalbumin or bovine serum albumin.

16. An immunoassay device as claimed in claim 5, wherein said agent is an antibody and wherein said device comprises a plurality of nonrecombinant proteins from different pathogenic microorganisms, each immobilized on separate delimited portions of said plurality.

17. An immunoassay device as claimed in claim 4, wherein different quantities of the same proteinaceous material are immobilized on at least two of said plurality of delimited portions.

18. An immunoassay method for detecting the presence of antibody in a fluid sample, comprising the sequential steps of

(a) bringing a fluid sample into contact with a proteinaceous material that binds said antibody, wherein said proteinaceous material is immobilized in reactive form on a delimited portion of a material having a high surface area;

(b) removing said proteinaceous material from contact with said fluid sample;

(c) contacting saidproteinaceousmaterial with a solution comprising at least one agent that recognizes human antibody or a human antibody-antigen complex, wherein said agent is conjugated to an enzyme such that said enzyme can catalyze a detectable reaction involving a substrate;

(d) removing said proteinaceous material from contact with said solution;

(e) bringing said proteinaceous material into contact with said substrate; and then

(f) determining whether said contact in step (e) has brought about said detectable reaction.

19. An immunoassay method according to claim 18, wherein in step (a) said proteinaceous material is in contact with said fluid sample for a period of about 1 to 30 minutes.

20. An immunoassay method as claimed in claim 18, wherein said agent comprises an antibody that recognizes human immunoglobulin.

21. An immunoassay method as claimed in claim 20, wherein said agent comprises is selected from the group consisting of anti-hlgM antibody, anti-hlgA antibody and anti-hlgG antibody.

22. An immunoassay method as claimed in claim 18, wherein said agent is RhC or Clq.

23. An immunoassay method as claimed in claim 22, wherein said agent is biotinylated.

24. An immunoassay method as claimed in claim 18, wherein said enzyme is alkaline phosphatase, horseradish peroxidase, glucose oxidase or jS-galactosidase.

25. An immunoassay method as claimed in claim 18, wherein said fluid sample is selected from the group consisting of urine, saliva, tears, feces, semen, a urogenital secretion, and sweat.

26. An immunoassay method as claimed in claim 25, wherein said fluid sample is a urine sample that is combined with a solution of up to about 1.0% glycine and about 1% glutamine.

27. An immunoassay method as claimed in claim 26, wherein said solution further comprises up to about 1.0% lactose.

28. An immunoassay method as claimed in claim 27, wherein said urine sample is combined with a solution of about 0.5% glycine, about 0.5% glutamine, and about 1.0% lactose.

29. An immunoassay method as claimed in claim 18, wherein said fluid sample is selected from the group consisting of blood, serum, and plasma.

30. An immunoassay method as claimed in claim 18, wherein said proteinaceous material comprises nonrecombinant protein from a pathogenic microorganism.

31. An immunoassay method as claimed in claim 30, wherein said pathogenic microorganism is a virus, a bacterium or a protozoan.

32. An immunoassay method as claimed in claim 31, wherein said pathogenic microorganism is HIV.

33. An immunoassay method as claimed in claim 18, wherein in step (a) said proteinaceous material is in contact with said fluid sample for a period of about 1 to 5 minutes, and wherein step (a) is accompanied by agitation.

34. An immunoassay method as claimed in claim 18, wherein step (d) is accomplished by washing with an isotonic buffer solution.

35. An immunoassay method as claimed in claim 18, wherein step (a) comprises bringing a plurality of nonrecombinant proteins from different pathogenic microorganisms, each immobilized in reactive form on a different delimited portion of a material having a high surface area, into contact with said fluid sample.

36. An immunoassay method as claimed in claim 18, wherein step (a) comprises bringing a plurality of different amounts of the same proteinaceous material, each immobilized in reactive form on a different delimited portion of a material having a high surface area, into contact with said fluid sample.

37. An immunoassay method as claimed in claim 18, wherein step (a) comprises bringing said fluid sample into contact with each of

(i) a heterogeneous mixture of proteins obtained from a pathogenic microorganism, and

(ii) a substantially homogeneous nonrecombinant protein obtained from said pathogenic microorganism, said protein mixture and said nonrecombinant protein being immobilized in reactive form on separate delimited portions of a material having a high surface area.

38. An immunoassay method as claimed in claim 37, wherein said protein mixture is HIV lysate, and wherein said nonrecombinant protein is an HIV gag protein or an HIV env protein.

39. An immunoassay method as claimed in claim 38, wherein said HIV lysate is HIV-1 lysate or HIV-2 lysate.

40. An immunoassay method for detecting the presence of antibody in a fluid sample, comprising the sequential steps of

(a) providing a fluid sample into which has been added an agent that recognizes a human antibody- antigen complex, wherein said agent is conjugated to an enzyme such that said enzyme can catalyze a detectable reaction involving a substrate, said agent being present in said fluid sample in a detectable amount; (b) bringing at least part of said fluid sample into contact with a proteinaceous material that binds said antibody, wherein said proteinaceous material is immobilized in reactive form on a delimited portion of a material having a high surface area; (c) removing said proteinaceous material from contact with said fluid sample;

(d) bringing said proteinaceous material into contact with said substrate; and

(e) determining whether said contact in step (d) brought about said detectable reaction.

41. An immunoassay method according to claim 40, wherein in step (b) said proteinaceous material is in contact with said fluid sample for a period of about 1 to 30 minutes.

42. An immunoassay method as claimed in claim 40, wherein said agent is RhC or Clq.

43. An immunoassay method as claimed in claim 40, wherein said agent is biotinylated.

44. An immunoassay method as claimed in claim 40, wherein said enzyme is alkaline phosphatase, horseradish peroxidase, glucose oxidase or jS-galactosidase.

45. An immunoassay method as claimed in claim 40, wherein said fluid sample is selected from the group consisting of urine, saliva, tears, feces, semen, a urogenital secretion, and sweat.

46. An immunoassay method as claimed in claim 45, wherein said fluid sample is a urine sample that is combined with a solution consisting of up to about 1.0% glycine or glutamine.

47. An -immunoassay method as claimed in claim 46, wherein said solution further comprises up to about 1.0% lactose.

48. An immunoassay method as claimed in claim 47, wherein said urine sample is combined with a solution consisting of about 0.5% glycine, about 0.5% glutamine, and about 1.0% lactose.

49. An immunoassay method as claimed in claim 40, wherein said fluid sample is selected from the group consisting of blood, serum, and plasma.

50. An immunoassay method as claimed in claim 40, wherein said proteinaceous material comprises nonrecombinant protein from a pathogenic microorganism.

51. An immunoassay method as claimed in claim 50, wherein said pathogenic microorganism is a virus, a bacterium or a protozoan.

52. An immunoassay method as claimed in claim 51, wherein said pathogenic microorganism is HIV.

53. An immunoassay method as claimed in claim 40, wherein in step (b) said proteinaceous material is in contact with said fluid sample for a period of about 1 to 5 minutes and wherein step (b) is accompanied by agitation.

54. An immunoassay method as claimed in claim 40, wherein step (d) is accomplished by washing with an isotonic buffer solution.

55. An immunoassay method as claimed in claim 40, wherein step (b) comprises bringing at least part of said fluid sample into contact with a plurality of nonrecombinant proteins from different pathogenic microorganisms, each immobilized in reactive form on separate delimited portions of said material having a high surface area.

56. An immunoassay method as claimed in claim 40, wherein step (b) comprises bringing at least part of said fluid sample into contact with a plurality of different amounts of the same proteinaceous material, each immobilized in reactive form on separate delimited portions of said material having a high surface area.

57. An immunoassay method as claimed in claim 40, wherein step (b) comprises bringing at least part of said fluid sample into contact with each of (i) a heterogeneous mixture of proteins obtained from a pathogen, and (ii) at least one substantially homogeneous nonrecombinant protein obtained from said pathogen, said proteinaceous materials being immobilized in reactive form on separate delimited portions of a material having a high surface area.

58. An immunoassay method according to claim 57, wherein said protein mixture is HIV lysate, and wherein said nonrecombinant protein is an HIV gag protein or an HIV env protein.

59. An immunoassay method as claimed in claim 57, wherein said HIV lysate is HIV-1 lysate or HIV-2 lysate.

60. An immunoassay device as claimed in claim 1, wherein said analyte is an antigen and said proteinaceous material is an antibody or antibody fragment that binds said antigen.

61. An immunoassay device as claimed in claim 60, wherein said proteinaceous material is a polyclonal antibody or polyclonal antibody fragment.

62. An immunoassay device as claimed in claim 1, wherein said analyte is an antigen which is a surface or intracellular antigen from a pathogenic microorganism.

63. An immunoassay device as claimed in claim 62, wherein said pathogenic microorganism is a virus, a bacterium or a protozoan.

64. An immunoassay device as claimed in claim 62, wherein said pathogenic microorganism is HIV-1, HIV-2, hepatitis-A virus, hepatitis-B virus, hepatitis-C virus, hepatitis-D virus, cytomegalovirus, Treponema pallidum, Neisseria gonorrhoeae, Chlamydia trochomates or Mycobacterium tuberculosis . -se¬

es. An immunoassay device as claimed in claim 1, wherein said analyte is an antigen which is a proteinaceous or non-proteinaceous material that is present during a change in state of health in a human patien .

66. An immunoassay device as claimed in claim 65, wherein said antigen is a hormone or myoglobin.

67. An immunoassay device as claimed in claim 66, wherein said antigen is human chorionic gonadotropin, thyroid hormone, testosterone or progesterone.

68. An immunoassay device as claimed in claim 1, wherein said analyte is an antigen which is a therapeutic drug, a drug of abuse or a toxin.

69. An immunoassay device as claimed in claim 5, wherein said analyte is an antigen and wherein said device comprises a plurality of proteinaceous materials that bind to different antigens, wherein each said proteinaceous material is immobilized on a separate delimited portion of said plurality thereof.

70. An immunoassay device as claimed in claim 5, wherein said plurality of delimited portions comprises a portion on which a positive control is immobilized and a different portion on which a negative control is immobilized.

71. An immunoassay device as claimed in claim 1, wherein said material having a high surface area is mechanically secured within at least one recess in said planar surface of said support member.

72. An immunoassay device as claimed in claim 71, wherein said recess has a floor provided with a through hole that communicates with the opposing surface of said support member.

73. An immunoassay device as claimed in claim 72, wherein said opposing surface is recessed around said through hole.

74. An immunoassay device as claimed in claim l, wherein said material having a high surface area is secured to said support member without bonding therebetween.

75. An immunoassay device as claimed in claim 1, wherein said support member presents a recess in said planar surface and said device comprises means for securing said material having a high surface area, said means being comprised of a retaining washer that (i) has a chamfered outer edge and a smooth surface which abuts said membrane and (ii) securingly fits into said recess.

76. An immunoassay device for detecting the presence of an analyte in a fluid sample, comprising:

(a) a support member presenting a planar surface,

(b) a membrane upon which a material reactive with said agent can be immobilized in reactive form and, immobilized thereon,

(c) a material reactive with said analyte, wherein said support member presents a recess in said planar surface and said device comprises means for securing said membrane, said means being comprised of a retaining washer that (i) has a chamfered outer edge and a smooth surface which abuts said membrane and (ii) securingly fits into said recess.

77. An immunoassay method for detecting the presence of an antigen in a fluid sample, comprising the sequential steps of (a) diluting a fluid sample with a solution containing a first proteinaceous material that binds said antigen, wherein said first proteinaceous material is conjugated to an enzyme such that the enzyme can catalyze a detectable reaction involving a substrate;

(b) bringing said fluid sample into contact with a second proteinaceous material that binds said antigen, wherein said second proteinaceous material is immobilized in reactive form on a delimited portion of a material having a high surface area, and wherein said second proteinaceous material binds at least one epitope on said antigen that is not bound by said first proteinaceous material binds;

(c) removing said secondproteinaceous material from contact with said fluid sample;

(d) washing said secondproteinaceousmaterial;

(e) bringing said secondproteinaceousmaterial into contact with said substrate; and then

78. An immunoassay method according to claim 77, wherein said first proteinaceous material is a monoclonal antibody or monoclonal antibody fragment.

79. An immunoassay method according to claim 77, wherein said second proteinaceous material is a polyclonal antibody or polyclonal antibody fragment.

80. An immunoassay method according to claim 77, wherein said fluid sample is serum, saliva or whole blood and in step (a) said first proteinaceous material is in contact with said fluid sample for a period of about 1 minute.

81. An immunoassay method according to claim 77, wherein said fluid sample is urine and in step (a) said first proteinaceous material is in contact with said fluid sample for a period of about 15 minutes.

82. An immunoassay method as claimed in claim 77, wherein said first proteinaceous material is biotinylated.

83. An immunoassay method as claimed in claim 77, wherein said enzyme is alkaline phosphatase, horseradish peroxidase, glucose oxidase or jS-galactosidase.

84. An immunoassay method as claimed in claim 77, wherein said fluid sample is selected from the group consisting of saliva, serum, whole blood, plasma, urine, tears, feces, semen, a urogenital secretion, and sweat.

85. An immunoassay method as claimed in claim 84, wherein said fluid sample is a urine sample that is combined with a solution of up to about 1.0% glycine and about 1% glutamine.

86. An immunoassay method as claimed in claim 85, wherein said solution further comprises up to about 1.0% lactose.

87. An immunoassay method as claimed in claim 86, wherein said urine sample is combined with a solution of about 0.5% glycine, about 0.5% glutamine, and about 1.0% lactose.

88. An immunoassay method as claimed in claim 77, wherein said antigen is a surface or intracellular antigen from a pathogenic microorganism.

89. An immunoassay method as claimed in claim 88, wherein said pathogenic microorganism is HIV-1, HIV-2, hepatitis-A virus, hepatitis-B virus, hepatitis-C virus, hepatitis-D virus, cytomegalovirus, Treponema pallidum, Neiεseria gonorrhoeae, Chlamydia trochomates or Mycobacterium tuberculosis.

90. An immunoassay method as claimed in claim 77, wherein said antigen is a proteinaceous or non- proteinaceous material that is present during a change in state of health in a human patient.

91. An immunoassay method as claimed in claim 90, wherein said antigen is a hormone or myoglobin.

92. An immunoassay method as claimed in claim 91, wherein said antigen is human chorionic gonadotropin, thyroid hormone, testosterone or progesterone.

93. An immunoassay method as claimed in claim 77, wherein said antigen is a therapeutic drug, a drug of abuse or a toxin.

94. An immunoassay method as claimed in claim 77, wherein said fluid sample is serum, saliva or whole blood and in step (a) said first proteinaceous material is in contact with said fluid sample for a period of about 1 minute, and wherein step (a) is accompanied by agitation.

95. An immunoassay method as claimed in claim 77, wherein said fluid sample is urine and in step (a) said first proteinaceous material is in contact with said fluid sample for a period of about 5 minutes, and wherein step (a) is accompanied by agitation.

96. An immunoassay method as claimed in claim 77, wherein step (d) is accomplished by washing with an isotonic buffer solution.

97. An immunoassay method as claimed in claim 77, wherein in step (a) said solution comprises a plurality of enzyme-conjugated proteinaceous materials that bind different antigens.

98. An immunoassay method as claimed in claim 77, wherein said substrate is an amplified enzyme substrate.

99. An immunoassay method for detecting the presence of an antigen in a fluid sample, comprising the sequential steps of

(a) diluting a fluid sample with a solution containing a first proteinaceous material that binds said antigen, wherein said first proteinaceous material is biotinylated;

(b) bringing said fluid sample into contact with a second proteinaceous material that binds said antigen, wherein said second proteinaceous material is immobilized in reactive form on a delimited portion of a material having a high surface area, and wherein said second proteinaceous material binds at least one epitope on said antigen that is not bound by said first proteinaceous material binds; (c) removing said secondproteinaceousmaterial from contact with said fluid sample;

(d) washing said secondproteinaceousmaterial;

(e) bringing said secondproteinaceousmaterial into contact with a material that binds biotin, wherein said material is conjugated to an enzyme such that said enzyme can catalyze a reaction involving a substrate;

(f) washingsaidsecondproteinaceousmaterial;

(g) bringing said secondproteinaceousmaterial into contact with said substrate; and then (h) determining whether said contact in step

(g) has brought about said detectable reaction.

100. An immunoassay method for detecting the presence of an antigen in a fluid sample, comprising the sequential steps of (a) diluting a fluid sample with a solution containing a irst proteinaceous material that binds said antigen;

(c) removingsaid secondproteinaceousmaterial from contact with said fluid sample;

(d) washing said secondproteinaceousmaterial; (e) bringing said secondproteinaceousmaterial into contact with a third proteinaceous material that binds said first proteinaceous material, wherein said third proteinaceous material is conjugated to an enzyme such that said enzyme can catalyze a reaction involving a substrate;

(f) washing said secondproteinaceous material;

(g) bringingsaid secondproteinaceousmaterial into contact with a substrate for said enzyme; and then

(h) determining whether said contact in step (g) has brought about said detectable reaction.