WO1999045395A1 - Rapid confirmatory test for microbial infections - Google Patents

Abstract

A diagnostic test device contains a filter and detects antibodies having multiple antigen specificities from a blood sample for confirmatory testing of an infectious pathogen such as a microbe. The filter is an integral part of a strip and can be used for rapid strip-testing of whole blood and other particulate-containing solutions generally. Whole blood HIV tests are exemplified that are easy to carry out by untrained personnel and that can provide qualitative information about the subtype of an HIV. The multiple antigen testing capability is useful for confirmatory testing of other microbes such as syphilis and hepatitis C.

Description

Rapid Confirmatory Test for Microbial Infections

Field of the Invention

The present invention relates to diagnostic test devices for confirming microbial infection by detecting antibodies that react with at least two separate antigens related to the microbe.

Background of the Invention

Microbial infection commonly is diagnosed by detection of antibodies in blood of an individual patient, using an ELISA or agglutination-based test. Positive test results should be confirmed by a method that separately detects multiple antigens because of false negatives and false positives obtained when testing just one antigen or group of antigens to obtain one result. The most common confirmatory test is the tedious western blot assay. In a western blot, microbe lysate proteins are electrophoretically separated by size and incubated with blood to determine which of the microbial proteins react with antibodies in the blood. Western blot is slow. Rapid confirmation, although not possible now, would be very desirable from the viewpoint of treatment and patient notification, particularly when the microbial disease is life threatening such as AIDS.

Presently, the most important disease condition that requires such a rapid confirmatory test is HIV infection. Current technology for testing and confirmation of HIV exposure is reviewed here to exemplify the field of confirmatory testing for microbial disease generally.

To determine past or present infection with HIV, a blood sample is screened for the presence of antibodies targeted against HIV antigens, using an ELISA or agglutination test. A positive test result from the screen is confirmed by re-testing the blood sample by western blot or by competitive ELISA, which, when evaluated quantitatively, allow the differentiation of HIV types and, to some extent, subtypes. A western blot test is particularly useful for confirmatory testing because epitope information is obtained from the blood sample, and the more reliable result can guide an effective course of treatment.

A confirmatory HIV test based on detecting anti-HIV antibody in blood that binds antigen must include multiple antigens to pick up the presence of HIV-1 and HIV-2, as well as to detect various new strains of HIV having antigen that poorly cross reacts with previously identified strains. The HIV-1 and HIV-2 types share 40-60% amino acid homology among proteins coded for by their genes.

HIV-1 is subdivided further into several subtypes (from A to I) with an additional subtype O. Subtype O shows an amino acid homology of 55-70% with the other HIV-1 subtypes, and is regarded by some researchers as a new subgroup. Similarly, and to reflect the close relation of subtypes A-I, these are grouped together as subgroup M (for major). Unfortunately, often antibodies made against one HIV subtype do not react against another subtype. A screening test uses only one antigen and thus, can miss one or more subtypes.

The screening tests most often used are enzyme-linked immunosorbent assays (ELISA) and agglutination assays. The sensitivity of these methods has been increased by replacing whole-microbe lysate with recombinant proteins and peptides. Double-antigen assays are more sensitive in the early phase of seroconversion but have a lower crossreactivity with antibodies directed against variant HIVs. Consequently, subtypes other than B may be missed during early seroconversion. In particular, infections with subtype O strains may be missed even at later stages of infection.

The occurrence of these false-negatives from some anti-HIV subtype O specimens has prompted the modification of assays by inclusion of subtype O antigens. This is because HIV subtype O isolates are highly heterogeneous and there is low antibody cross-reactivity between HIV-1 subtype O sera and corresponding peptides from HIV-1 subgroup M or HIV-2 strains. Accordingly, confirmatory assays have been developed that detect exposure to microbes such as HIN, by incorporating multiple antigens in their test repertoire. A confirmatory test contains multiple antigens to detect differences due to microbe heterogeneity. The popular HIV western blot test, which contains proteins of an HIV strain, is the gold standard for investigating reactive results from ELISA screening. Although western blot can detect antibodies against isolates of the same HIV-1 subgroup (commercially available western blots are prepared with subtype B strains), this procedure may miss sera obtained during the early stages of seroconversion. HIV-1 western blot cannot reliably confirm HIV-2-positive sera, because about 20% of such specimens give false-negative results, especially when the antibody titer is low, as summarized by Gurtler in the Lancet, 348: 176 (1996). An HIV-1 subtype B western blot also misses subtype O infection in about 10% of the specimens. Thus, even this gold standard has serious problems.

Present immunoblot tests have not been modified to include subtype O-antigen, as has been done for ELISA since 1994. Accordingly, the risk of a false-negative immunoblot result remains. On the other hand, Gurtler has suggested that the easiest way to improve existing immunoblot strips would be to include highly immunogenic subtype-O-specific peptides (Gurtler et al. , J. Virol. Meth. 51:L 177-84 (1995).

Sometimes a western blot cannot deliver a final diagnosis of HIV infection when this test reacts only with HIV-core-derived bands (p24, p55), a pattern that is non-specific. The complexity in band patterns seen in western blot reflects mutations in HIV. For example, anti HIV-2 antibodies detected by an HIV-1 immunoblot show a common profile of p24,55, but antibodies with subtype O crossreactivity show strongest reactivity with p32 (integrase) and p51,66 (reverse transcriptase).

Another big problem with western blot confirmatory tests is their high cost. Partly in response to this problem, the World Health Organization has proposed using two or three different ELISAs, each based on a set of individually selected antigens, for confirmation of HIV exposure. That is, separate and different ELISA tests can be carried out separately to obtain results which are interpreted together to confirm the presence or absence of HIV exposure. Such use of multiple tests potentially could distinguish HIV-1 from HIV-2 infection, although approximately 30% of all specimens react with antigens of both HIV types.

Unfortunately, multiple ELISA tests as proposed, are carried out individually, with only one antigen at a time, or with a mixmre of antigens at a time. In order to obtain an accurate result, individual reactivity with each of three or more specific antigen tested typically must be determined. For example, an HIV confirmatory test should contain antigens having epitopes of at least two of the following HIV proteins: p24, gp41, gpl20 or gpl60. The multiple ELISA testing stratagems proposed for confirmation require tests be carried out separately on a sample, or with separate samples, as described, for example, by van der Groen et al., Bull. WHO 69: 747-752 (1991), Sato et al., Bull. WHO 72: 129-134 (1994), and Mortimer, Bull. WHO 70: 751-756 (1992).

Accordingly, an inexpensive and fast test device and method of its use are needed for simultaneous determination of antibodies against multiple antigens. Furthermore, a fast and simple confirmatory test that gives information relating to specific alternative alleles of an epitopic site of an antigen (i.e. specific alternative mutated forms of the same part of a protein) would be very useful for more accurate testing of a wide range of infectious disease agents. This is particularly important for microbes, wherein the organism mutates rapidly and presents shifting epitopic determinants, thereby complicating the task of accurate detection. Such test and method are desired not only for HIV testing but also for other diseases such as syphilis where patient compliance is a problem and a quick result is wanted before the patient leaves the clinical setting, perhaps never to be seen again.

Summary of the Invention

The inventors have discovered a test device and method of its use that provides easy and quick testing of multiple antigens for confirmatory testing that obviates having to send a sample to a separate laboratory for a time consuming expensive confirmation. This device and method can test blood for infection by many different pathogens such as HIV, syphilis and hepatitis C. The test requires only a drop of blood, such as that obtainable from a fingerstick. The method and device allow, for the first time, rapid testing of patient populations or by other untrained personnel on a real time basis.

The inventors also have discovered an improvement to the diagnosis of microbial infection, the improvement comprising replacing at least one recombinant antigen of the microbe used in the test with two or more allelic peptides that cross react with an immunodominant region of a protein of the microbe. In one embodiment the allelic peptides are immobilized on a solid phase used in a heterogeneous format test device. In another embodiment the peptides are alternative alleles from the same spot of an immunodominant region of an envelope protein of a virus and the envelope protein is selected from the group consisting of HIV-1 gp 41, HIV-1 gp 120, HIV-2 gp 36, HIV-2 gp 120, hepatitis C envelope protein and syphilis envelope protein.

In one embodiment the invention provides a process for confirming infection by a specific microbe comprising the steps of: (a) providing a test device that contains a housing having an opening, an absorbent pad held by the housing, and a reagent layer in contact with the absorbent pad and aligned with the opening to receive an aqueous sample, the reagent layer comprising at least a first antigen and a second antigen of the microbe immobilized at separate regions of the reagent layer; (b) applying a blood sample to the opening of the test device; (c) applying at least one wash fluid to the test device; (d) allowing binding reactions to proceed in the reagent layer of the device; and (e) detecting two or more reactions in the reagent layer in response to the presence of antibody against at least two of the immobilized antigens.

In another embodiment, the invention is directed to a process for confirming infection by a specific microbe comprising the steps of: (a) providing a test device that contains a housing having an opening, an absorbent pad held by the housing, and a reagent layer in contact with the absorbent pad and aligned with the opening to receive an aqueous sample, the reagent layer comprising at least one immobilized polypeptide, the polypeptide comprising a first antigen and a second antigen of the microbe; (b) applying a blood sample to the opening of the test device; (c) applying at least one wash fluid to the test device; (d) allowing binding reactions to proceed in the reagent layer of the device; and (e) detecting two or more reactions in the reagent layer in response to the presence of antibody against the two antigens of the microbe.

In yet another embodiment, the invention is a microbial infection confirmatory test device that detects the presence of at least two antigens from a microbe, comprising: (a) a housing having an opening, (b) an absorbent pad held by the housing, and (c) a reagent layer in contact with the absorbent pad and in sample receiving relationship with the opening in the housing, comprising at least a first antigen and a second antigen of the microbe immobilized at separate regions of the reagent layer.

Other embodiments, objects and advantages will become apparent from the following detailed description taken in conjunction with the Figures.

Descriptions of the Figures

Figure 1 shows topside views of a visual test result from a device indicating alternative results, in accordance with an embodiment of the invention.

Figure 2 diagrams the top view of devices for an HIV-1 confirmatory test and for a multi-analyte test, in accordance with an embodiment of the invention.

Figure 3 is a sequence list of three representative peptides according to embodiments of the invention.

Detailed Description of the Preferred Embodiments

The inventors discovered how to improve reliability of diagnostic tests to provide "confirmation" of infection by a particular organism, or in some embodiments, a group of organisms. In each embodiment of the invention, at least two antigens are used simultaneously with the same sample. In one set of embodiments, the antigens are from different epitopic sites, that is, different proteins and/or different regions of a protein. Simultaneous tests of these sites provide added reliability that heretofore generally only was available in "confirmatory" laboratory tests such as Western blot tests of multiple antigens. In another set of embodiments, the antigens are peptides that represent expression of alleles (alternative phenotypic expressions) from the same immunodominant region of a protein from the infectious disease agent. That is, two or more alleles as used in the invention comprise sequences that code alternative peptides chosen from an epitopic site and which cross-react broadly with a wide range of native mutated forms of the organism. The inventors discovered that they could combine peptides that correspond to multiple alleles from just one epitopic site to achieve reliable test results that normally are only associated with a laboratory confirmatory test such as Western blot. This strategy can actually preempt new mutated forms by providing cross-reactivity to forms that have not been seen in nature, and is applicable to the design of vaccines as well. Co-pending application 60/104,686 entitled "Mixed peptides for early detection and therapy of viral infections" (Attorney docket No. 073294/0197) describes how to select polypeptide sequences that correspond with multiple alleles for this purpose and is specifically incorporated by reference in its entirety.

Confirmation test using different epitope regions

The invention successfully has confirmed HIV infection using antigens from different proteins/protein regions. Thus, in one embodiment for HIV testing, the device advantageously comprises, preferably in separate locations for separate detection, antigens that correspond to HIV proteins gpl20, gp41 and p24. In an advantageous method, a confirmed positive test result from this device is indicated when two of these three immobilized antigens create a colored signal in response to processing a blood test sample. In this same method, a confirmed negative test result is indicated by less than two of immobilized antigens creating a colored signal. In both cases, an optional procedural control line indicates to the user that the test procedure was carried out correctly.

Figure 1 shows 7 possible test results wherein the test procedure was carried out correctly, as indicated by development of a negative control line, wherein various combinations of immobilized antigen reacted with antibodies in blood samples. In this top view of a single test unit, the negative control line is at the top, and antigens are located from left to right as: p24; gp41 and gpl20. This figure shows three possible positive test results. Another alternative test result, gp41 with gpl20 is not shown. This device is considered a confirmation test device because it confirms whether a test sample has been exposed to any of a variety of HIV-1 strains. In other embodiments, peptides that cross- react with the same immunodominant region are used together to cover a range of strains of virus to more accurately detect (i.e. confirm) whether a sample has been infected. In this latter case, alternative peptides that cover the same epitopic region, preferably of an envelope protein of a microbe, are expressions of "alleles." Examples of such allelic expression in this context are peptides that correspond (share significant sequence identity) with the same immunodominant region, such as the immunodominant region of gp41. Specific examples of peptides that share significant sequence identity are Group M subtype D reactive peptides and Group O reactive peptides as disclosed in the co-pending patent applications. Further examples of peptides that share significant sequence identity may be found in co-pending application Nos. 60/104,686 filed 10/16/98, 60/111,995 filed 12/11/98, 60/104,685 filed 10/16/98, 09/238,635 filed 1/28/99 and 60/091,659 filed 7/02/98.

Alleles in the context used herein means expressions of alternative mutated forms of a gene, and does not require that the gene be part of a chromosome.

Figure 2 shows a typical test orientation of immobilized antigen for an HIV test (left side of the figure) and for a multi-microbe test for HIV, hepatitis C and syphilis (right side of the figure.) The latter test exemplifies a multi-pathogen test that can be carried out. In the context of the claimed invention, such multi-pathogen test is considered a confirmation test because it confirms whether a test sample has been exposed to any of a group of pathogens.

Confirmation tests using different allelic forms from the same immunodominant epitope

The invention also successfully has been used to confirm HIV infection using alternative allelic forms of the same immunodominant region. An "immunodominant region" in the context used here, means a spot, located within a peptide sequence, or as a 3 -dimensional surface of a protein having greater immunoreactivity compared to the rest of the molecule. A favored immunodominant region which often is identified as a peptide sequence as well as a 3 dimensional surface is a cysteine loop. A skilled artisan can readily appreciate such a spot from publications of the particular target organism. For example, in the case of HIV, the gp41 immunodominant region and the V3 loop region are said to be immunodominant. Other organisms such as hepatitis C also have immunodominant regions that are found to give strong immunoreactivity, particularly within portion(s) of their envelope proteins.

Good results were obtained using alternative allelic forms of the gp41 immunodominant region of the HIV envelope protein. Specifically, the combined use of an HIV-1 Group M subgroup D reactive peptide with an HIV-1 Group O reactive peptide provided test results that equaled results obtainable with a Western blot method. In this case, a subgroup D peptide and a Group O peptide of the same immunodominant region are considered expressions of different alleles of that region.

An "antigen" is a molecule that reacts with an antibody. As used in the inventive device and method, the test antigen preferably contains one or more epitopes that are similar or the same as epitopes of the infectious disease agent. When testing for exposure to Human Immunodeficiency Virus (HIV) an advantageous antigen is a polypeptide that contains at least one segment that corresponds in sequence to that of a polypeptide produced by the virus. Exemplary sequences in this context are variations of conserved sequences that have been published by the Los Alamos National Laboratory, Los Alamos, New Mexico 87545 (HUMAN RETRO VIRUSES AND AIDS 1996). Acceptable peptides for use as a confirmatory HIV test are described in co-pending provisional application "Universal HIV- 1 Peptide Antigens," filed January 28, 1998 (attorney docket No. 073294/0161) and in co- pending U.S. utility application "Universal HIV-1 Peptide Antigens," filed March 2, 1998 (attorney docket No. 073294/0164), both of which are herein incorporated in their entireties by reference.

Most acceptable for HIV-1 testing are universal peptides described in the copending patent applications cited above and as further described here. These patent applications report the significance of amino acid sequence information between positions 567 to 617 from the HIV-1 gp41 protein that spans the immunodominant region. Preferred peptides are those that have sequences that are more closely related to sequences of the HIV-1 O strains than to the most likely sequence of M strains. To make this comparison, an M consensus sequence and an O consensus sequence published in the Los Alamos National Laboratory Data Base, are used as described in co-pending provisional application "Universal HIV-1 Peptide Antigens. "

More preferably, the peptide antigen is between about 25 and about 50 amino acids long. Even more preferably, the region of at least about 8 amino acids ranging from about 21 amino acids and about 29 amino acids from the position of the cysteine at the N-terminal side of the heptapeptide loop forms an alpha helix. Furthermore, the Hopp acrophilicity scale peptide profile should be about at least in 75% agreement with the profile of the classical HIV-1 M strain B subtype sequence, as determined by peptide analysis with "Peptide Companion Version 1.24 for Windows" software from Peptides International, Inc. Louisville, Kentucky 40299 U.S.A.

A universal peptide sequence useful for the HIV-1 test furthermore preferably should have a Janin accessibility scale peptide profile that is in about at least 80% agreement with the sequence profile of the classical HIV-1 M strain B subtype, as determined by this software. Also preferred is a sequence having a Hopp and Woods hydrophilicity scale peptide profile that is at least in 75 % agreement with the profile of the classical HIV-1 M strain subtype. Furthermore, the Kyte and Doolittle hydropathy scale profile of the peptide should be in at least 80% agreement with the profile of the classical HIV-1 M strain subtype (all determined by the Peptide Companion software.)

Acceptable antigens for use as a confirmatory device for syphilis testing are the 15.5 kDa, 17 kDa, 44.5 kDa (TmpA), and 47 kDa polypeptides, or portions thereof, made by the Treponema pallidum virus as described by Byrne et al. in J. Clin. Microbiol. 30: 115- 122 (1992). A positive confirmed syphilis test is when at least three of the four antigens reacts with a blood sample and produces three colored spots. In one embodiment, a confirmed negative syphilis test is when less than two of the four syphilis antigens react.

10 Acceptable antigens for use as a confirmatory device for hepatitis C testing include, for example, four different "HCV regions" known as core, NS3, NS4 and NS5, as discussed by Feucht et al. in J. Clin. Microbiol. 33:620-624 (1995). A confirmed positive test result can be determined by reactivity against two of these four protein antigens. Particularly preferred are two or more alternative epitopes that correspond to an immunological hot spot for envelope protein El or E2 from hepatitis C. Most preferred are alternative epitopes that correspond to the region of El that is coded for by nucleotide positions 1,023 to 1,053 of the genome and which is highly predictive of HCV type and subtype as reported by Bukh et al. , Proc. Natl. Acad. Sci. USA 90: 8234-38 (1993). An analogous hot spot region exists in hepatitis C envelope protein E2 as described by Okamoto et al., Virology 188: 331-341 (1992). The invention of using two or more alternative expressed alleles of the same site (that is, two or more amino acid sequences that correspond to the same region but that differ both in sequence and epitopic reactivity) is particularly useful for immunodominant regions of El and E2, which are known to differ greatly among samples in the wild. This embodiment of the invention solves the variability problem that forced previous workers to focus on other less desirable proteins such as non- structural proteins. The non-structural proteins generally exhibit less genetic variability and were more convenient to obtain reliably an antibody titer response. The invention, on the other hand, uses a novel strategy of employing multiple allelic forms of the same immunodominant region to obtain a reliable antibody titer response.

Antigens useful for confirmatory testing of exposure to other pathogens such as those responsible for lyme disease, toxoplasmosis, and other microorganisms such as rubella, mycoplasma, cytomegalo virus, herpes, HTLVI, HTLVII, Hepatitis B, and clamydia are known to the skilled artisan. Antigens also can be determined by cloning genes of a pathogen and testing each expressed gene or portions thereof for reactivity, as demonstrated by Feucht (Id.) using available reagents and methods. Such antigens are too numerous to list here but are contemplated by the inventors in the context of their invention. Chemically synthesized peptides and recombinant proteins can be immobilized

11 within devices as claimed by routine methods, such as spotting a water solution of the antigen onto a nitrocellulose membrane.

Allelic test antigen peptides in accordance with one embodiment of the invention can be used as replacements for one or more protein antigens (either formed by natural synthesis, or formed recombinantly) to improve a test assay. In a particular embodiment, the invention is an improvement to existing heterogeneous tests for microbial infection. As described in the above-cited co-pending applications, large protein antigens generally are disfavored for use in such binding assays due to non-specific binding of protein. Furthermore, multiple types of proteins often are combined in the same (or in parallel) tests to cover a wider range of epitope variations of the microbe, which may be, for example an RNA virus which mutates rapidly and is hard to detect with just one species of protein.

In one embodiment of the invention, two, three, or in some cases more allelic peptides are used simultaneously to cover a wide range of possible epitope variations. In this context, one or more recombinant antigen proteins from the organism that are used in the test are replaced by two or more allelic peptides that cross-react with an immunodominant region of the organism. In an advantageous embodiment, an immunodominant region is selected, peptides that represent alleles of that region are selected and prepared according to principles enumerated in the referenced co-pending applications, and two, three or more of such allelic peptides replace at least one recombinant protein used in the heterogeneous test. Such heterogeneous tests are well known under the names El A, ELISA, ARIS, FIA, SLFIA and the like. An example of this embodiment is provided as Example 2.

Test devices in accordance with one embodiment of the invention have a housing comprised of a water impermeable material in which other test components such as an absorbent pad, reagent layer, filter and a reagent used to obtain a test result are held. The housing has at least one opening to admit a fluid sample although additional openings may be desired. In some cases, a wick may exist at least partly outside the housing, or the filter may exist at least partly outside the housing. In an advantageous device configuration all mechanical parts are completely held within the housing. Also acceptable in some

12 embodiments is a housing having at least one light transmissive portion to allow detection of a signal either visually, or by instrumentation.

Most acceptable is a housing that comes apart during use so that the user can remove the filter to expose the reagent layer for application of a reagent and/or wash fluid. In this embodiment a sleeve that holds the filter is removably attached to the housing such that contact of the filter is favored over contact of the sleeve with the surface of the reagent layer. Preferably the sleeve is attached to the housing by a bayonet mount. After a sample is applied, and an optional wash solution added, the sleeve is removed and further optional reagent solution and a wash solution are added directly to the reagent layer. In some applications a sample is added to the device and further processing is carried out at a separate location or after storage of the device for a few hours. In these situations, the sleeve remains attached to the housing to prevent or delay the release of moisture from the device until the later processing steps are carried out. The housing also may contain a cover to protect the opening and further guard against the release of moisture.

Multiple housings can be incorporated into a multi-test unit to allow high volume testing. The latter embodiment is acceptable for infectious disease testing of blood samples at blood banks. Especially acceptable in this context is a 96 well multiple-test device that can be used with micropipettor devices. In one embodiment 8 (or 12) test devices that correspond in size to a column (or row) of a microtiter plate are used in applications where intermediate numbers of samples are processed. The invention has particular advantages when used in a multi-test format over other methods such as microtiter plate enzyme linked immunosorbent assays. For example, the invention obviates waste disposal required by these other methods and allows optical perception of several test spots, (including one or more calibrators) in one well, and is particularly useful for confirmatory tests. In an alternative embodiment, each well has a separate antigen and groups of wells are used for a single confirmatory test.

The housing and other parts of the test device may be constructed from well-known materials in accordance with well-known methods of the prior art. Material suitable for the invention should not interfere with the production of a detectable signal and should have a

13 reasonable inherent strength, or strength can be provided by means of a supplemental support. Natural, synthetic, or namrally occurring materials that are synthetically modified, can be used including, but not limited to: cellulose materials such as paper, cellulose, and cellulose derivatives such as cellulose acetate and nitrocellulose; fiberglass; cloth, both naturally occurring (e.g., cotton) and synthetic (e.g., nylon); porous gels such as silica gel, agarose, dextran, and gelatin; porous fibrous matrixes; starch based materials, such as Sephadex(r) brand cross-linked dextran chains; ceramic materials; films of polyvinyl chloride and combinations of polyvinyl chloride-silica; and the like. See, for example, U.S. Patent Nos. 5,075,078, 5,552,276, 4,912,034 and 5,212,065, which are incorporated herein by reference in their entireties. Constructions other than those described in these patents readily will be apparent to the skilled artisan.

The test device most preferably incorporates the features of (1) positioning parts with a positioning "sleeve" to allow even fluid flow between the parts without interference by the sleeve itself, (2) arranging parts to minimize transverse flow, (3) using friction-held parts and water swellable parts to allow fluid to more evenly flow through junctions between the parts, and (4) using a dispersing layer downstream of the filter to help disperse fluid more evenly to the reagent layer. Each of these four strategies improves test performance and should be considered independently for a particular test device configuration. The physical assembly of components from known materials within the housing generally will be understood to a skilled artisan but for clarity, some details are provided here in the form of definitions of some terms used in the claims.

A "blood sample" as termed herein means whole blood obtained by, for example, a finger prick or venipuncture either with or without an anti-coagulant added; a plasma sample; serum sample; or a partly processed or diluted derivative of any of these such as centrifuged blood.

A "whole blood" sample is a blood sample that contains red blood cells, white blood cells and other components such as chylomicrons and lipoprotein.

A "filter" removes more than half of particles that are suspended within a test sample and which are larger than the rated sieving size of the filter material. The "rated

14 sieving size" of a material is determined by and provided from a manufacturer of the material and typically is stated in microns. For example, a glass fiber having a porosity of ten microns should remove red blood cells from a blood sample because such cells have a dimension that is greater than 10 microns. In some cases the rated sieving size of the material may be close to or even larger than one or more dimensions of the particle that is to be held back by the cell filter. Depending on the filter thickness, retardation of particle movement and not simply holding back the particles will suffice for a given test device. How well a given filter material works is best determined empirically, by applying test samples to devices made using the material.

When used for test samples that contain red cells the filter preferably removes at least 90% of the red cells. The filter may comprise one or more materials in physical contact. In practice, the filter should have significant depth to allow entrapment of cells that typically comprise about one half of whole blood volume. When used for testing of saliva samples, the filter preferably retards cells and other debris that may exist in such test specimens. In an advantageous embodiment the filter comprises two glass fiber pads mechanically held together. The filter also may comprise a first portion that traps or slows the movement of cells and a second portion downstream from the first portion. In this case the second portion serves to disperse filtrate to an adjacent material such as a reagent layer.

A "filtrate" of a blood sample or whole blood sample is a portion of the sample that is passed through a filter or component of a filter. For example, a filtrate of a whole blood sample will have had most of the originally present red cells removed by the filter. In some cases the filter may remove blood cells or blood molecules by virtue of binding between the filter surface(s) and the blood cells or blood molecules. Preferably, the filtrate from a filter will lack more than 90% of the originally applied red cells by virtue of the sieving action of one or more portions of the filter.

An "absorbent pad" imbibes water generally and serves as a final repository for much or most of the liquid that enters the housing of a test device. Examples of an absorbent pad include paper, ethyl cellulose, porous plastic, sintered glass, and other polymers that accept water such as polyvinyl pyrrolidone and acrylamide copolymers. An

15 absorbent pad may consist of more than one piece. An acceptable absorbent pad is comprised of ethyl cellulose and is held in place by the device housing, wherein a surface of the pad contacts a reagent layer.

An "antibody" is IgG, IgE, IgM, IgA and the like and may be obtained from blood, a blood product or saliva. Although the use of saliva samples is not discussed in great detail it is understood that the invention is used for the detection of analyte from saliva as well as from blood. Advantages of the invention, such as the operation of the filter extend to saliva samples as well as to blood samples.

An "opening in the housing" may be a hole, aperture or simply a region that accepts a fluid sample. In some cases, the housing itself may be partly water permeable and partly water impermeable. In these instances the water permeable portion(s) act as an "opening" in context of the invention. More than one opening may be used in the test device.

The phrase "passes fluid" has its regular meaning that is well known to the skilled artisan. The relative rate of passing fluid can be determined by a number of techniques known to the artisan, including for example, measuring how much time is required for the material to become completely wetted upon contact with water. An advantageous material for trapping cells in the filter is glass fiber and an advantageous material for a dispersant layer in contact with this glass fiber typically is cellulose because cellulose can pass water at a lower rate compared to glass fiber.

The second portion or "dispersing" portion of a filter, when used may provide a lower flow rate of sample and of wash fluid compared to the cell trapping material in the filter. In addition to providing a more reproducible test result, the low flow rate provided by the dispersing portion allows more time for chemical substances to react before these substances contact the reagent layer. Increasing the reaction time, particularly for binding reactions, allows greater test sensitivity. Acceptable materials for use in the filter to trap cells include Gelman Cytosep glass fiber filter membrane and Pall Corporation blood separation membrane. Acceptable for a "dispersing" portion of a filter, if one is used, are cellulose-based materials such as filter paper.

16 A "reagent layer" is an absorbent or group of absorbents that contains at least one reagent, contacts the filter and receives sample from the filter by virtae of this contact. An advantageous reagent layer is a membrane that comprises the immobilized test reactants such as antibody or antigen more advantageously, this layer comprises at least three antigens that are immobilized on three separate portions of the layer.

Typically, an absorbent pad that contacts the reagent layer accepts and draws in fluid from the reagent layer. Although the reagent layer preferably comprises an immobilized test reactant, the test reactant may become immobilized during the test itself by for example, precipitation or by binding to the reagent layer during the test procedure. The reagent layer may assume any of a variety of shapes. An advantageous shape is a thin layer between the filter and the absorbent pad, although another shape such as a plug may be used, depending on the features of the other components. Fluid preferably moves traverse to the major surfaces of a reagent layer, although some device configurations may utilize lateral flow along the longest axis of a reagent layer. In an advantageous embodiment for testing of HIN antibody in a blood sample, the reagent layer is a nitrocellulose membrane with HIV antigen(s) bound to it. The reagent layer, however, need not be a "layer" per se, but may consist of a pad, rod or other shaped absorbent.

A "sleeve" is a water impermeable solid surface that holds the filter. The sleeve may be a portion of the test device housing or may be a separate part that is separately attached to the housing. In the latter case, the sleeve preferably is reversibly attached to the housing by a bayonet mount. An advantageous construction in this context is to place the filter into the sleeve and to assemble the housing from two portions, an upper housing portion and a lower housing portion. The lower portion holds the absorbent pad and a reagent layer. The two housing portions are snap-fit together and the sleeve is attached by a bayonet mount. During fitting of the sleeve, friction between the sleeve and the filter allows the filter to push onto the reagent layer. "Friction" in this context means that the filter is held in place by mechanical pressure of the filter against the walls of the sleeve. In some embodiments, the mechanical pressure is augmented by an adhesive that holds the filter to the sleeve surface. When using friction fit to attach the filter to the sleeve, the

17 filter preferably has a depth that is at least one-tenth its diameter. To maintain friction of the filter within the sleeve and also to maintain good contact between the filter and a reagent layer, it is acceptable that the filter (particularly the dispersant portion, if included), or, (in some cases), the sleeve surface, to swell upon wetting.

The term "swell" in the context of a part or part surface used in the test device means that the part increases its size or that the surface increases its thickness upon wetting. Acceptable in this context is cellulose, although the use of many alternate materials is readily appreciated by a skilled artisan.

An "optical change" in the context of determining the presence or absence of a test analyte means an absorbance, reflectance, fluorescence, phosphorescence, or chemiluminescence change produced within the test device. This change may be determined by eye or by an instrument. One acceptable embodiment employs gold particles to produce a reflectance change that is visually perceived. In an advantageous embodiment the filter is separated from the reagent layer by removal of the sleeve, which exposes a portion of the reagent layer where an additional reagent may be added and a signal is developed from the presence of gold particles.

A "bayonet mount" has the usual meaning in the art and refers to a fastening means whereby a first part firmly attaches to a second part by overlapping indentations along a circular surface that fits into a depression of the second part. After inserting one part into the other, either part is rotated to firmly seal the two together.

An advantageous device configuration comprises an absorbent pad, reagent layer and a two-part filter within a single housing. Acceptable materials for the housing include water impermeable plastics such as polystyrene, polypropylene, polyvinyl chloride and the like. Acceptable materials for the filtering portion of the filter, the dispersing portion of the filter, the reagent layer and the absorbent pad are glass fiber, ethyl cellulose, nitrocellulose and ethyl cellulose respectively. When nitrocellulose is used for the reagent layer, however, the material of the filter should be chosen for its ability to premix the test sample and any test reagent that may be present in the filter. Two or more materials can be physically combined to make up a filter, reagent layer or absorbent pad. Two materials are

18 acceptable for the filter, an upper filtering material and a lower dispersal material. Most acceptable is a dispersal layer that passes fluid at a slower flow rate compared to passage of fluid through an upper filtering material. The use of a dispersal layer is superior over the use of a single part filter. A skilled artisan will readily appreciate that one or more portions of the device summarized above can be replaced by a single piece of a suitable material. In this context, many absorbent, porous or capillary possessing materials through which a solution containing the analyte can be transported by a wicking action are suitable for construction of the test device and are known to the skilled artisan.

According to one embodiment of the test device, the reagent layer is in fluid contact with both the filter and absorbent pad 90 (i.e. it may contact both directly or via an intervening layer(s)). Preferably, the reagent layer protrudes beyond the sleeve to the absorbent pad as shown in this figure. When using a reagent layer in this manner, it is acceptable that the filter, one of the filter portions, or the sleeve be removable to allow direct optical detection of a signal that forms below the filter. Most acceptable in this context is a reagent layer comprised of a clear polycarbonate porous membrane in which at least two antigens immobilized on polystyrene microparticles have been spotted. According to this embodiment, an optical signal develops at the bottom of the polycarbonate filter and can be seen by inspecting the bottom of the polycarbonate filter after removal of the filter. Also acceptable is to place a colloidal gold detection reagent in the filter in a dried form so that the detection reagent may become resuspended during application of a test sample.

In some embodiments, an optional wick may be used to bring a fluid sample into the container. In these cases, a portion of the wick that is exposed to the outside of the container is contacted with the sample, which then wicks into the container. The wick may itself have filtering properties and can replace the filter.

A 96 well multiple test format also is envisaged for the test device. A 96 well cassette consists of an upper housing and lower housing as described in copending application U.S. No. 08/933,943 entitled Diagnostic Test Devices with Improved Fluid Movement and Resistance to Interferences, filed September 19, 1997. Each well position of the upper housing contains a filter and dispersant layer. Each well position of the lower

19 housing contains reagent layer and absorbent pad. In an advantageous embodiment the lower housing contains a continuous sheet of absorbent layer and a continuous absorbent pad.

During use, a single test device is obtained and sample and wash fluids are added directly to either the filter or the reagent layer of the device. An additional wick also can be used to bring a fluid sample into the device, if desired. Alternately, if desired, a sample can be introduced into the device by leaving a portion of the filter exposed to the outside, where it can be used to directly absorb sample fluid and then bring this fluid, or a filtrate into the housing.

After adding the fluid sample, an aqueous wash solution is applied. The wash solution preferably contains a non-ionic detergent such as Tween 20 (r) and may also contain a protein such as bovine serum albumin.

Most acceptably, after adding a wash solution, the filter is removed and then a reagent solution is added directly to the reagent layer. An advantageous reagent solution in this context contains a detection agent such as colloidal gold coated with protein A. The detection agent is optionally washed with a wash solution.

For the acceptable embodiment whereby the filter is removed, it is most acceptable that the sleeve be attached to the device reversibly by, for example a bayonet mount. According to this embodiment, contact between the filter and a reagent layer is established upon attaching the sleeve that holds the filter to the device housing. This contact is broken when the sleeve is removed after washing a sample into the reagent layer. In a further embodiment, the sleeve remains in place and the opening at the top of the sleeve is covered after addition of sample (and optional wash). The cover (and optionally the sleeve) can be removed later for further processing if needed.

Reagents needed for operation of the test device may be placed in the filter, reagent layer, dispersant (if used), and/or in an aqueous fluid that is applied to the device. For convenience in manufacturing, a reagent such as gold sol can be prepared and used as a liquid phase reagent and added during operation of the device. For optimum convenience

20 to the test user, however, it is acceptable to place all reagents except for the wash reagents within the device in a dried form.

In one embodiment, a detection agent such as colloidal gold is placed in an optional lower portion ("dispersant") of the filter during manufactare of the device. In this case, a user would apply a blood sample to the device, followed by a wash, removal of the filter, and then another optional wash.

Adding sample and wash solution starts sequential and automatic reactions in the test device. These reactions include two or more binding reaction(s), an optional separation step and an optional enzymatic reaction step according to the test format employed. Many types of ligand antigens are useful for the test device and are contemplated for the invention.

One advantageous format is a binding assay in which at least two, but more preferably three or more antigens are immobilized. In this format the test substance in the test sample, such as, for example, an anti-HIV antibody or other anti-microbe antibody, has at least one epitope, or, in the advantageous case of an antibody analyte as for HIV testing, a binding site that participates in a binding reaction with a specific binding member immobilized in the reaction layer. In such a binding assay two molecules that bind specifically (i.e. with an association constant of at least 100,000 under typical assay conditions) form the basis of concentrating a signal producing substance to form an optical signal. An advantageous signal producing substance is colloidal gold. Molecules that act as "binding partners" with an analyte in a test sample typically are antibodies specific to the analyte, but, as the skilled artisan will readily appreciate, other molecules that bind specifically to the analyte molecule also can be used.

The confirmation assay typically uses multiple immobilized antigens selected from antigens that are diagnostic of the pathogen. These antigens can be selected from lists of antigens that are known to react with and indicate, for example, the presence of antibodies against a particular microbe.

After adding one or more fluids to the test device and then allowing one or more reactions to proceed, an optical change(s) in the reagent layer is determined. When used in

21 a binding assay format to form this optical change, the test device may contain a chemical label to generate the signal or the user may add this label. The label generally is any substance which is attached to a specific binding member and which is capable of producing a signal that is detectable visually or by an instrument. An acceptable label is further described in US patent application No. 08/577,108 (CHILDS et al. , entitled PARTICLE ASSISTED IMMUNO ASSAY) which is herein incorporated in its entirety by reference.

The above-summarized procedure can be modified by the use of an aqueous "pre- treatment" solution that is applied to the device before test fluid application. The pre- treatment solution comprises a protein and a detergent. The protein may be, for example, bovine serum albumin, human serum albumin, casein, non-fat milk product or gelatin. The protein is preferably at a concentration of from 0.05% to 10% and more preferably between 0.2% and 2% (wgt/vol). Most acceptable is a 1 % solution of bovine serum albumin. The detergent is an amphoteric molecule or composition such as triton X-100, Tween 20, Tween 80, NP-40, zwitter ionic detergents and the like. The detergent is preferably at a concentration above its critical micelle concentration and typically should be at a concentration of between 0.05% and 3 % (wgt/vol). The use of Tween-20 detergent is most acceptable at a concentration of between 0.2% and 2% . The pre-treatment solution should have a pH in the range of 5.0 to 10.0 and preferably contains a buffering component such as sodium phosphate or Tris-Cl at a concentration of between lmM and 1M and more preferably between lOmM and 200mM. Most acceptable is a 50mM concentration of Tris- Cl at pH 8.0.

The acceptable embodiment whereby all solutions are added to a single horizontal location at the top of the device (either before or after separating the top and bottom halves) is particularly suited for automated instrumentation. The inventors realized that the filter, reagent pad and absorbent can be sized to allow their manufactare into a 96 well microtiter plate. Furthermore, 8 (or 12) devices can be manufactured into a vertical (or horizontal) strip of multiple test devices that can be assembled to become part of a microtiter plate. Such microtiter plate embodiments can be processed by existing microtiter plate

22 instruments. In these embodiments the user can add sample, wash and/or reagent solutions by hand or by automated instrumentation.

When a multiple sample device such as a 96 well microtiter plate is used for the invention, preferably one antigen is immobilized in each well. For example, of the 8 wells in a vertical strip of multiple test devices, four at one end separately may contain immobilized peptides having epitopes from gpl60, gp41, p24 and pl20 proteins. The remaining four wells can have another set of these same proteins, to allow a confirmatory test of two separate blood samples.

A skilled artisan will readily appreciate that a device of the present invention can be packaged with other components such as an instruction pamphlet, a wash reagent that may be either a dried material that is redissolved in water or buffer, or already present as a fluid, and optionally one or more reagents to add to the device such as gold particles treated with a binding partner for a binding reaction. Accordingly, the invention allows the production and use of kits for infectious disease testing from blood.

The present invention will now be illustrated by the following examples, which are not intended to be limiting in any way.

EXAMPLE ONE

In this example, filter holders were constructed with filters composed of the following components in sequential order from top to bottom: a blood separation filter; a backup blood separation filter; and a dispersant layer. The dispersant layer was situated in direct contact with a reagent layer in which three polypeptide members of binding pairs, namely HIV antigen p24, gp41 and gpl20, were immobilized as shown in Figure 1. The blood separation filters and the dispersant layer were chemically treated by exposure to a solution of buffer, detergent, protein and anticoagulant.

The preparation of the reagents used is generally known to the skilled artisan and is described in co-pending US patent application nos. 08/577,108 (Childs et al. , entitled PARTICLE ASSISTED IMMUNOASSAY), 08/577,630 (Childs et al., entitled STRIP TEST FOR HIV) and 08/912,580 (Childs et al. , entitled DIAGNOSTIC TEST DEVICES

23 WITH IMPROVED FLUID MOVEMENT AND RESISTANCE TO INTERFERENCES) which are herein incorporated in their entireties by reference.

In the test procedure, two drops of pre-treatment solution are added to the filter of the device. Then one drop of heparinized blood is added to the filter. Three drops of pre- treatment solution are added to wash the blood from the top of the filter (the primary filter) into the middle (backup filter) region, the lower (dispersant layer) region and the reagent layer.

The test fluid preparation filter holder is then removed from the detection device, which contains the reagent layer and an absorbent pad. After removing the filter holder, the reagent layer of the test device is exposed. Exposing the reagent layer allows for the addition of wash fluid. Two drops of wash buffer are added to the reagent layer on which HIV antigens gpl20, gp41 and p24 have been immobilized as three separate dots, along with a procedural control line of anti-human goat Fab'₂, as shown in Figure 1. Then two drops of colloidal gold labeled protein A are added, followed by two drops of wash buffer. The presence of anti-HIV antibody in the sample is detected as color formation from the deposition of colloidal gold within less than five minutes from the time of applying the blood sample. A reaction with an immobilized antigen is determined as the formation of color at the site of immobilization of that antigen.

A total of 51 known HIV-1 positive and 129 known HIV negative specimens, as determined by a western blot method, were tested according to this protocol. A positive result was determined as the formation of two or more colored dots. A negative result was determined as the formation of one or no colored dots. A correctly performed test was determined by the formation of the control line. The test results were 100 percent accurate. All 51 positive samples gave a strong positive result reacting with p24, gp41 and gpl20 antigens to produce colored spots. All 129 negative samples gave a negative result, and did not react with any of the three test antigens.

Example Two

24 In this example two allelic peptides for an immunodominant region of gp41 envelope protein of HIV-1 and a third peptide that cross-reacts with gp36 envelope protein of HIV-2 were incorporated into an enzyme-based microtiter plate test according to an embodiment of the invention. The heterogeneous test format chosen is an improvement of an immobilized antigen format wherein one or more recombinant proteins of the HIV microbe are replaced with allelic peptides that correspond to an immunodominant region of the microbe. By replacing recombinant protein(s) with two or more allelic peptides, nonspecific binding from the protein is alleviated and a greater concentration of antigenic binding sites, per unit of mass takes place.

Microtiter plates were coated with a mixture of three peptides having sequences SEQ ID NO: 1 (peptide 1), SEQ ID NO: 2 (peptide 2) and SEQ ID NO: 3 (peptide 3) as follows. Each peptide was diluted into carbonate buffer pH 9.6. One hundred fifty microliters of solution that contained 200ng of peptide 1 , 300ng of peptide 2 and 400ng of peptide 3 in combination totaling 900ng were placed into each well of a polystyrene plate. The plate was incubated at 37°C for 2 hours. After incubation, the plate was washed 5X with PBS [pH 7.4] 1 % Tween-20 on an automatic washer. The plate was blotted on a paper towel several times and then 350 microliters of Milk Blocking Solution (2% milk) were added to each well and incubated at 37°C for 2 hours. After incubation each well was washed 5X with PBS [pH 7.4] 1 % Tween-20 and blotted on a paper towel several times. The plate was stored at -20°C with desiccant until used.

The plate was used to test for HIV exposure from blood samples as follows. Ten microliters of sample were added to 90 microliters of proprietary sample diluent [2% milk in PBS containing 0.5% NaCl] to each well and mixed thoroughly by pipetting up and down. The plate was covered with plastic sealer and incubated at 37°C for 30 minutes. After incubation, the plate was washed 5X with PBS [pH 7.4] l %Tween-20 and blotted on a paper towel several times. One hundred microliters of 1 :40,000 conjugate (IgG F(ab)'₂) diluted in PBS [pH 7.4] were added to each well. The plate was covered with plastic sealer

25 and incubated at 37°C for 30 minutes. After incubation, wells were washed 5X with PBS [pH 7.4] 1 % Tween-20 on an automatic washer and the plate was blotted on a paper towel several times. One hundred microliters of TMB Peroxidase Subtrate were added to each well. The plate was covered with plastic sealer and incubated at room temperatare for 30 minutes. After incubation 100 microliters of Stop Reagent (IN H₂SO₄) were added to each well. Absorbance of each well at 450nm was determined on an automatic reader and used to determine the presence or absence of binding between sample antibody and immobilized peptide.

The test was carried out with 31 blood samples obtained from the Ivory Coast of Africa. The results are shown in Table 1 and indicate that the allelic peptide ELISA test results agree with a known test reference.

Example Three

In this example the two allelic peptides for an immunodominant region of gp41 envelope protein of HIV-1 and a third peptide that cross-reacts with gp36 envelope protein of HIV-2 that are described in Example Two were incorporated into the test device described in Example One and tested with the same 31 blood samples as used for Example Two. The results obtained are shown in Table 1 and indicate that the allelic peptide rapid test results agree with a known test reference.

26 Table 1

ELISA Rapid Test Reference Test

Sample ID Peptide Test Peptide Test: BioRad for HIV- Results HIV 1-O/2 1. Gene Lab 2.2 on * for HIV-2

772 0.051 N/N

1007 0.360 N/N

980 0.272 N/N

1009 0.060 N/N

1313 0.103 N/N

1320 0.084 N/N

1074 0.004 N/N

775 0.043 N/N

792 0.166 N/N Indeterminate

793 0.110 ST/N Indeterminate

798 0.043 N/N

876 2.669 4+/N HIV-1 positive

889 0.046 N/N

947 0.074 N/N

951 0.059 N/N

953 0.220 N/N

966 0.101 N/N Indeterminate

979 0.440 N/N Indeterminate

1046 0.293 N/N

1048 0.757 N/N

1069 ND ND

1083 0.420 N/N

1111 2.538 N/4+ HIV-2 positive

1118 0.098 N/N

1120 0.063 N/N

1122 0.070 N/N

1273 0.097 N/N

1276 0.094 N/N

1285 >3.0 N/4+ HIV-2 positive

1307 0.095 N/N

1308 0.135 N/N

N = negative; ST = very weakly reactive;

27 Although the present invention has been described with reference to certain acceptable embodiments, modifications or changes may be made therein by those skilled in this art without departing from the scope or spirit of the present invention, as defined by the appended claims. Further, although the inventors have described certain embodiments, designs, materials, etc. as preferred, it is understood that such preferred embodiments merely represent the preferred embodiments that have resulted from the inventors' work to date and that many other acceptable embodiments can be discovered and employed through practice of the invention and routine trial and error, and that some of such other embodiments ultimately may be more preferred than those specifically described.

Each of the documents cited herein is explicitly incorporated by reference in its entirety.

In the appended claims, the articles "a" and "an" shall mean "at least one" unless otherwise indicated.

28

Claims

What is claimed is:

1. A process for confirming infection by a specific microbe comprising the steps of:

(a) providing a test device that contains a housing having an opening, an absorbent pad held by the housing, and a reagent layer in contact with the absorbent pad and aligned with the opening to receive an aqueous sample, the reagent layer comprising at least a first antigen and a second antigen of the microbe immobilized at separate regions of the reagent layer;

(b) applying a blood sample to the opening of the test device;

(c) applying at least one wash fluid to the test device;

(d) allowing binding reactions to proceed in the reagent layer of the device; and

(e) detecting two or more reactions in the reagent layer in response to the presence of antibody against at least two of the immobilized antigens.

2. The process of claim 1, wherein the microbe test device further comprises a filter in fluid communication with the reagent layer and aligned between the reagent layer and the opening to receive an aqueous sample into the filter to make filtrate, and the blood sample is whole blood.

3. The process of claim 1 , wherein the microbe is HIV and each of the first and second antigens is an immunogenic polypeptide less than 100 amino acids long and contains a different epitope selected from the group consisting of HIV- 1 gp 120, HIV-1 gp 41, HIV- 1 gp 160, HIV-1 p24, HIV-2 gp 105, HIV-2 gp 36 and HIV-2 p24.

29

4. The process of claim 1, wherein the microbe is a virus and the two antigens comprise alternative allelic expression of the same spot of an immunodominant region of an envelope protein of the virus.

5. The process of claim 4, wherein the virus is HIV, the immunodominant region is the V3 loop of gp 120 or the immunodominant region of gp 41, and the two antigens correspond to expression of an HIV-1 Group O strain and an HIV-1 Group M Subgroup D strain respectively.

6. The process of claim 5, wherein at least one of the antigens is a modified form of a wild-type sequence having at least one less hydrophobic amino acid compared to the wild type.

7 The process of claim 5, wherein at least one of the antigens is a modified form of a wild-type sequence having greater secondary structure compared to the wild type.

8. A process for confirming infection by a specific microbe comprising the steps of:

(a) providing a test device that contains a housing having an opening, an absorbent pad held by the housing, and a reagent layer in contact with the absorbent pad and aligned with the opening to receive an aqueous sample, the reagent layer comprising at least one immobilized polypeptide, the polypeptide comprising a first antigen and a second antigen of the microbe;

(b) applying a blood sample to the opening of the test device;

(c) applying at least one wash fluid to the test device;

(d) allowing binding reactions to proceed in the reagent layer of the device; and

30 (e) detecting two or more reactions in the reagent layer in response to the presence of antibody against the two antigens of the microbe.

9. The process of claim 8, wherein the peptide has greater secondary structure compared to the wild-type sequences.

10. The process of claim 8, wherein the peptide has at least 2 fewer hydrophobic acid residues compared to the wild-type sequences.

11. The process of claim 8, wherein the microbe is HIV, one of the two antigens corresponds to HIV-1 Group O and the other antigen corresponds to HIV-1 Group M Subgroup D and both antigens are allelic expressions of an envelope region selected from the group consisting of the V-3 loop and the immunodominant region of gp 41.

12. The process of claim 8, wherein the microbe is HIV and the two antigens are selected from the group consisting of HIV-2 gp 105, HIV-2 gp 36, HIV-2 p24, HIV-1 gp 120, HIV-1 gp 41, HIV-1 gp 160 and HIV-1 p24.

13. The process of claim 1, wherein the microbe is syphilis and the antigens are selected from the group consisting of p47, TmpA, pl7 and p 15.

14. The process of claim 1, wherein the reagent layer comprises at least 3 immunogenic polypeptide antigens containing different epitopes that react with antibody against syphilis microbe.

15. The process of claim 1, wherein the microbe is hepatitis C and the antigens are selected from the group consisting of an HCV core protein antigen, NS3, NS4 and NS5.

31

16. A microbial infection confirmatory test device that detects the presence of at least two antigens from a microbe, comprising:

(a) a housing having an opening,

(b) an absorbent pad held by the housing, and

(c) a reagent layer in contact with the absorbent pad and in sample receiving relationship with the opening in the housing, comprising at least a first antigen and a second antigen of the microbe immobilized in the reagent layer.

17. The test device of claim 16, wherein the peptide antigens are expressions of alternative alleles from the same spot of an immunodominant region of an envelope protein of the virus.

18. The test device of claim 17, wherein the envelope protein is selected from the group consisting of HIV-1 gp 41, HIV-1 gp 120, HIV-2 gp 36, HIV-2 gp 120, hepatitis C envelope protein and syphilis envelope protein.

19. The test device of claim 16, wherein the peptide antigens contain alternative epitopes selected from the group consisting of syphilis p 47, syphilis TmpA, syphilis pl7, syphilis p 15, hepatitis C NS3, hepatitis C NS4, hepatitis C NS5, hepatitis C envelope protein El, hepatitis C envelope protein E2, HIV-1 gp 120, HIV-1 gp 41, HIV-1 gp 160, HIV-1 p24, HIV-2 gp 105, HIV-2 gp 36 and HIV-2 p24.

20. The test device of claim 16, wherein the reagent layer comprises at least 3 peptide antigens that react with antibody against the microbe.

21. The test device of claim 16, wherein the first antigen and the second antigen of the microbe are immobilized in the reagent layer at separate locations.

32

22. In a microbe infection test device that utilizes at least one recombinant antigen of the microbe to detect the presence of antibody against the antigen, an improvement comprising replacing a recombinant antigen of the test device with two or more allelic peptides that cross react with an immunodominant region of a protein of the microbe.

33