WO2010048503A1 - Live/dead viral load assay - Google Patents

Abstract

The present invention provides viral load assays, kits and methods able to distinguish between active and inactive viruses in a sample. Current diagnostic methods for analyzing the presence of viruses in a clinical sample, or the effects of an anti-viral treatment or a microbicide can only report the total amount of viruses and cannot distinguish between "live" and "dead" viruses. Using current viral load assays, the results would include dead viruses that are non-infective. Present methods are therefore not suitable for evaluating the effect of anti-viral therapies, such as highly active anti-retroviral therapy (HAART) or microbicides. In response, the present invention introduces a viral load assay that provides information on the levels of both live and dead viruses. Also provided are methods of amplifying viral detection after the live and dead viruses have been separated.

Description

LIVE/DEAD VIRAL LOAD ASSAY

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR

DEVELOPMENT

[0001] This invention was made with government support under Grant NIH - U01 Al 066709 awarded by the National Institutes of Health. The United States Government has certain rights in the invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application 61/108,255, filed October 24, 2008, and U.S. Provisional Application 61/169,640, filed April 15, 2009, which are incorporated herein in their entirety to the extent not inconsistent herewith.

BACKGROUND OF THE INVENTION

[0003] Viral load is the measure of the levels of a virus in a bodily fluid and the severity of a viral infection. Typically, viral load is calculated by estimating the amount of the virus in the body or a bodily fluid, such as the number of viral RNA copies per ml of blood. Detecting and measuring viral load is useful in diagnosing infections as well as in research settings to test the viricidal effects of compounds and chemical agents. Currently, there is great interest in developing anti-viral agents to combat disease and infections, such as influenza, AIDS, dengue fever, avian influenza and SARS. Improved methods of determining viral load would increase the ability to test the anti-viral effects of compounds and treatments in both in vitro and in vivo experiments. In particular, it would be greatly beneficial for such viral load assays and diagnostic kits to be able to distinguish between intact viruses (active or live viruses) and structurally broken viruses (inactive or dead viruses). [0004] For example, since 1996 the introduction of highly active anti-retroviral therapy (HAART) has led to a dramatic decline in AIDS-related morbidity and mortality in the United States. The lifespan of individuals infected with HIV has significantly increased. This dramatic effect, together with the hope that HIV could be eradicated, led clinicians and HIV treatment guideline committees to adopt an aggressive strategy in treating HIV, focusing primarily on the benefits of HAART (Ho, D. D., N Engl J Med. 1995; 333:450- 451 ). However, as current antiretroviral therapy is unable to eradicate HIV (Chun and Fauci, Proc Natl Acad Sci U S A. 1999; 96:10958-10961 ), and is associated with increased toxicities, HIV treatment guideline committees have recently adopted a less aggressive set of guidelines for treating HIV.

[0005] HAART often includes a cocktail of nucleoside reverse transcriptase inhibitors and protease inhibitors that prevent virus replication and assembly. While effective in reducing the progression of AIDS, significant side effects have been observed (Carr et al, Lancet. 1998; 351 :1881-1883; Holmberg et al, Lancet. 2002; 360:1747-1748). Because insufficient dosage may allow viruses to escape with drug resistance, most patients under HAART are overdosed. To date, all four classes of antiretrovirals (ARVs) and all 19 FDA-approved ARV treatments have been directly or indirectly associated with various degrees of side effects including death (Reisler et al, J Acquir Immune Defic Syndr. 2003; 34: 379-386). Common side effects include diabetes, atherosclerosis, dyslipidemia and lipodystrophy. Until there is a cure for AIDS, patients will have to be on HAART for life. Therefore, it is important to monitor the effect on HIV viral load in response to HAART in real time to optimize the dosage and to minimize adverse effects.

[0006] A viral load test is part of the standard of care in HIV treatment (HAART) in the developed world. However, current HIV viral load tests are costly, so that the majority of patients on HAART in the developing world are not tested. The consequences include treatment failure and non-adherence, resulting in drug-resistant viruses in the population. Children also need new viral load test as HIV antibodies are not a reliable indicator of HIV status in the first 12-18 months of life. A viral load assay, which can be done as early as 4-6 weeks after birth, is the only way to determine whether a baby is infected, but many babies are not tested in resource poor settings. Each year about 500,000 children contract HIV and 40-50% of them die within the first two years of life. An early viral load test can save many infants' lives. Therefore, a low cost viral load test is urgently needed.

[0007] Current HIV and other viral load assays are limited in that they are unable to differentiate active (intact, live) viruses from inactive (dead) viruses. For example, current HIV load assays detect viral RNA and will produce a positive viral load even in cases where the viruses are inactive. Once the therapy begins, many newly produced viruses are rendered non-infective. However, the current viral load assays cannot differentiate the inactive viruses from active viruses. As a result, these methods cannot accurately monitor the effect of the HAART in real time. Without real-time monitoring of the therapeutic response to HAART, drug dosages are largely based on indirect data (e.g., CD4 counts) and physicians' experience. Because insufficient dosage may allow viruses to escape with drug resistance, patients are often given overdosed medications. Therefore, it is important to monitor the effect of HAART in real time by determining both the "live" (active) and "dead" (inactive) HIV viral loads. Additionally, HIV and other viral load assays typically are high cost and are unsuitable for point-of-care use. Thus, it is important to determine the live (active) and dead (inactive) viral loads, especially for HIV, in a simple and cost effective manner.

[0008] Microbicides are compounds that deactivate, kill or reduce the infectivity of microbes, such as viruses or bacteria. Microbicides can be applied to surfaces, including surfaces on the human body, to prevent or reduce viral infection. For example, microbicides can be applied inside the vagina or rectum to protect against sexually transmitted infections (STIs) including HIV. They can be formulated as gels, creams, films, or suppositories. Microbicides in this context could work in at least four different ways: prevent the virus from entering human cells; enhance the body's normal defense mechanisms against the virus; inactivate the virus; and inhibit virus replication. For example, Carraguard®, Cyanoviran®, cellulose sulphate, and PRO 2000® provide a barrier to block virus entry. Acidform®, BufferGel® and Lactobacillus crispatus maintain an acidic pH to enhance the natural vaginal defense. C31G and Nonoxynol-9 (N-9) inactivate pathogens by stripping their outer membranes. Tenofovir (PMPA) acts by preventing replication of the virus after it has entered the cell. Presently, there are 23 microbicides against HIV in various stages of clinical development.

[0009] To test the effectiveness of a microbicide, it is similarly often necessary to measure the viral load of the targeted virus when mixed with the microbicide in a test tube as well as in bodily fluids. At present, the most sensitive method for detecting HIV viral load in vaginal secretions is based on nucleic acid amplification with Real-Time RT- PCR (Benki et al, J Acquir Immune Defic Syndr. 2008; 47:529-534). The current most commonly used HIV viral load analytical kit using nucleic acid amplification is the Amplicor HIV-1 Monitor® Test, v1.5 (Roche Diagnostics), having a lower detection limit of 50 copy/ml. This method has been used to evaluate the HIV-1 viral load reduction in human vaginal lavages after treatment with a microbicide. However, most samples treated with a viricidal microbicide gave equal or higher viral load results than the untreated negative controls. For testing, all viruses must be lysed (killed) to release RNA. Therefore, it is believed that a PCR-based method detects all viral RNAs, even those from inactive viruses. Therefore, this method does not provide useful information for immediate viricidal effect of a microbicide.

[0010] A second method for viral detection is not based on nucleic acid amplification. It is called nanoparticle-based biobarcode amplification method (Kim et al, Nanomed 2008; 3:293-303; Tang et al, J Acquir Immune Defic Syndr. 2007; 46:231-237). Its target molecule is the HIV core protein p24. Several steps are involved in this method: (i) capturing the target molecule by anti-p24 antibody linked to magnetic beads or coated on microtiter plates; (ii) reacting with gold beads linked with hundreds of biobarcode DNA; and (iii) releasing the biobarcode DNA for detection. This assay is the second most sensitive HIV detection method with a lower limit of 1 ,000 copy/ml. For testing, all viruses must be lysed (killed) to expose the core protein p24. Therefore, although these methods can detect the total HIV viral load for evaluating other types of anti-HIV treatment, they are also unsuitable for evaluating immediate effects of viricidal microbicides in women's vaginal secretions, because most viruses are killed upon contact with these microbicides. These methods are unable to distinguish between viruses inactivated by the microbicide and viruses inactivated by the detection process.

[0011] The inability of the viral load assay to differentiate between active (live) and inactive (dead) viruses impedes the evaluation of microbicide viricidal effects and other anti-viral treatments. This technological gap has impeded the progress of microbicide development and efficient treatment of viral diseases, particularly with regard to HIV. What is needed is a viral load assay able to differentiate between active and inactive viruses, particularly for the monitoring of anti-viral treatments and microbicide effects.

SUMMARY OF THE INVENTION

[0012] The present invention is able to determine the viral load of enveloped viruses (i.e., viruses having a viral envelope surrounding a protein or nucleic acid core), including RNA and DNA enveloped viruses. The present invention provides assays, diagnostic kits and methods for determining the levels of live and dead viruses in a fluid sample, preferably a bodily fluid taken from a patient. As used herein, "live" viruses refer to intact active viruses capable of infecting a cell and replicating. "Dead" viruses refer to inactive or deactivated viruses that have been structurally compromised or are just fragments of the whole virus. Active viruses have an intact viral envelope while the viral envelope of inactivated viruses are partially or completely removed or damaged, thereby exposing the internal core viral proteins and nucleic acids. As provided by the present invention, live viruses and dead viruses are detected and/or separated based on selectively capturing or binding the viral envelope and internal viral proteins or nucleic acids.

[0013] Assays, diagnostic kits and methods of the present invention provide a) a first substrate able to capture or bind the internal core proteins or nucleic acids of a targeted virus, and/or b) a second substrate able to capture or bind the viral envelope of the targeted virus. The first and second substrates with the captured viruses and virus fragments are then separated and the levels of viral envelope or internal viral proteins or nucleic acids bound to the substrates determined. The substrates may be any compound, solid support, cell or composition known in the art able to bind to the desired component of the targeted virus. In one embodiment, the substrates are beads (preferably magnetic beads), microtiter plates, bacteria, a dipstick, multi-capillary glass structure, or a filter paper strip. Preferably, the substrates comprise antibodies directed against a specific component of the viral envelope, a viral core protein or viral nucleic acid, such as antibodies that bind the HIV core protein p24 (anti-p24 antibodies) and antibodies that bind the HIV viral envelope gp120 antigen (anti-gp120 antibodies). Preferably, the antibodies and substrates directed against the viral envelope are directed against a protein or carbohydrate present on the surface of the viral envelope of the targeted virus. The substrates may be linked to additional moieties, including but not limited to magnetic beads, chemical labels, sugar binding lectins, and fluorescent tags, to enhance separation or detection of the bound substrates.

[0014] In one embodiment, the methods of the present invention comprise adding a first set of antibodies to a sample containing a mixture of intact and inactive viruses and detecting the levels of the bound antibodies, where the first set of antibodies are directed against the viral envelope of the targeted virus. A further embodiment comprises adding a second set of antibodies to the sample and detecting the levels of bound antibodies, where the second antibodies are directed against an internal viral protein or nucleic acid of the targeted virus.

[0015] Another embodiment of the present invention is an assay for determining the viral load of a sample containing a mixture of active and inactive viruses comprising a first substrate comprising a first set of antibodies, wherein the first set of antibodies is able to selectively bind a core protein or nucleic acid of a virus, or selectively bind a virus envelope. The assay optionally further comprises a second substrate comprising a second set of antibodies, wherein the second set of antibodies is able to selectively bind a core protein or nucleic acid of a virus or selectively bind a virus envelope with the proviso that if the first set of antibodies selectively binds a core protein or nucleic acid of a virus then the second set of antibodies selectively binds a virus envelope, and if the first set of antibodies selectively binds a virus envelope then the second set of antibodies selectively binds a core protein or nucleic acid of a virus. The assay also comprises a means for detecting the amount of core protein, nucleic acid or viral envelope bound to the first and second substrate. The means for detecting the amount of bound core proteins, nucleic acid or viral envelope can be any detection means known in the art including, but not limited to, polymerase-chain reaction (PCR), enzyme- linked immunosorbent assay (ELISA) and magnetic bead readers.

[0016] One embodiment of the invention is a method for detecting viral load in a sample containing a mixture of inactive viruses and active viruses comprising the steps of separating the inactive viruses in the sample from the active viruses; conducting a procedure to correct false positive data in the separated active viruses; and detecting and determining the viral count of the separated inactive viruses and the viral count of the separated active viruses. In a further embodiment, the separating step comprises: mixing a set of viral core antibodies or viral envelope antibodies with a sample, wherein the set of antibodies are linked to a solid support and are directed against one or more core proteins or nucleic acids of a virus or against a viral envelope; capturing the viruses or viral fragments having a core protein, nucleic acid or viral envelope able to bind to the set of antibodies; removing the solid support and purifying the captured viruses or viral fragments. In another further embodiment, the corrective procedure comprises mixing a corrective set of antibodies with a sample containing the separated active viruses, wherein the corrective set of antibodies are linked to a solid support and are directed against one or more core proteins of a virus. The inactive viruses or fragments thereof able to bind to the corrective set of antibodies are then captured and removed from the active viruses.

[0017] In one embodiment, the present invention provides a viral load assay capable of differentiating active from inactive viruses that is inexpensive and suitable for point-of- care testing. In one example, the viral load assay measures blood plasma HIV-1 RNA concentration. Untreated HIV-1 infection is characterized by high-level viral production and CD4 T-cell destruction, despite an often lengthy clinical latency period to the infection subsequently results in a significant net loss of CD4 T cells and, finally, AIDS. The level of steady-state viral load is a strong predictor of the rate of disease progression and, by itself or in combination with CD4 T-cell counts, has great prognostic value. As a result, current guidelines advocate the use of plasma viral load testing when considering highly active anti-retroviral therapy (HAART) initiation, monitoring response to therapy, and instituting a change in drug regimen based on the patient's response. To support the key clinical goal of achieving maximal viral load suppression, several sensitive viral load assays have previously been developed to quantify HIV-1 RNA. These include:

• Amplicor HIV-1 Monitor® v.15 Assays (RT-PCR) (Roche, Branchburg, NJ)

• Versant® HIV-1 RNA 3.0 Assay (bDNA) (Bayer, Tarrytown, NY)

• NucliSens® HIV-1 Assay (bioMerieux, Boxtel, Netherlands)

• LCx HIV RNA Quantitative Assay (Abbott Laboratories, Abbott Park, IL)

[0018] All these assays detect the RNA of HIV viruses, including RNA from inactive viruses produced during HAART. Consequently, they cannot provide useful information for immediate therapeutic response to HAART (Moore and Mermin, Lancet. 2008; 371 :1396-1397). Once on HAART, a long waiting period (often weeks or months) is needed for an accurate second viral load assay after the initial baseline. Indirect data, such as CD4 counts and physicians' experience, are frequently used for evaluating therapeutic response (Reynolds et al, AIDS. 2009; 23:697-700), but they are not sufficient. Also, these assays require sophisticated technology and high-priced equipment, such as real-time PCR. In contrast, the viral load assay and diagnostic kits of the present invention monitor HAART in real time thereby maximizing efficacy, lowering costs and minimizing side effects of the treatment. Such assays and kits can be made accessible to AIDS patients in resource poor settings, reduce health disparities, and can substantially improve the effectiveness of current AIDS treatment and the quality of life of millions of people living with HIV/AIDS.

[0019] The present invention provides assays, kits and methods for determining the viral load of a sample and for monitoring the viral load of a patient receiving treatment for a viral disease. One embodiment provides diagnostic kits for determining a viral load in a fluid sample, preferably a bodily fluid, comprising a means for collecting a fluid sample from a patient; a first and second substrate, where the first substrate is able to bind one or more core proteins or nucleic acids of a virus and the second substrate is able to bind a viral envelope, thereby binding inactive viruses to the first substrate and binding active viruses to the second substrate; and means for detecting the amount of inactive viruses bound to the first substrate and the amount of active viruses bound to the second substrate. The substrates can be any substrate known in the art able to bind the viral core proteins, nucleic acid and viral envelopes, particularly those containing or bound to antibodies directed against the core proteins, nucleic acids and viral envelopes. Preferably, the substrates, independently from one another, are selected from the group consisting of bacteria, magnetic beads, microtiter plates, dipsticks, multi-capillary glass structures and filter paper strips.

[0020] The kit optionally comprises a corrective set of antibodies wherein the corrective set of antibodies are linked to a solid support and are able to bind virus fragments containing partial viral envelopes. The corrective set of antibodies is contacted with viruses and virus fragments that bind to the second substrate, i.e. the substrate able to bind and capture the viral envelope. This removes inactive viruses and virus fragments containing a partial viral envelope from active viruses having a fully intact viral envelope.

[0021] Additionally, the diagnostic kits and assays optionally comprise a reagent comprising a detector antibody linked to either biotin linked magnetic beads or streptavidin/avidin linked magnetic beads. A detector antibody is an antibody able to bind to the target antigen (core protein, nucleic acid of the virus or viral envelope) while the target antigen has been bound and captured by the substrate. This allows the viral load to be detected using a handheld magnetic bead reader or magnetometer able to detect one or more magnetic beads. The kits may also contain one or more containers of a reagent comprising biotin linked magnetic beads; and one or more containers of a reagent comprising streptavidin/avidin linked magnetic beads. These additional reagents can be used to amplify the number of magnetic beads associated with each bound particle and improve detection. The kits and assays of the present invention may further comprising a reagent comprising a linker protein, wherein the first or second substrates additionally comprise a first linker antibody able to bind to said linker protein, and the magnetic beads linked to the detector antibody additionally comprise a second linker antibody also able to bind to the linker protein

[0022] The components of the kit can be contained in a housing, such as a plastic or metal housing, for practical storage and transport. The housing can include reference materials, such as requirements for the assay, recommended amounts and procedures for the size, age and sex of the patient, diagrams and instructions for use.

[0023] The viral diseases able to be detected and monitored by the present invention include but are not limited to influenza, HIV/AIDS, dengue fever, avian influenza and SARS. Preferably, the treatment is highly active anti-retroviral therapy (HAART). In one method, a first sample from the patient containing active and inactive viruses is contacted with a first and second substrate, where the first substrate is able to bind to one or more core proteins or nucleic acids of a virus and the second substrate is able to bind to a viral envelope, thereby binding inactive viruses to the first substrate and binding active viruses to the second substrate. Preferably the first substrate comprises antibodies able to bind to internal viral proteins and/or nucleic acids, and the second substrate comprises antibodies able to bind the viral envelope. The bound first and second substrates are separated and the amount of inactive viruses or fragments bound to the first substrate and the amount of active viruses bound to the second substrate are determined. In a further embodiment, a second sample from the patient is contacted with the first and second substrate where the second sample is taken at a point in time different than the first sample. The amount of inactive and active viruses or virus fragments from the second sample bound to the first and second substrates are compared with the first sample. Optionally for each sample, a procedure is conducted to remove inactive viruses bound to the second substrate to avoid false positives with regard to the active viral load determination. This procedure involves mixing a corrective set of antibodies with a sample containing the separated active viruses. The corrective set of antibodies are directed against one or more core proteins or nucleic acids of a virus and are able to capture and remove inactive viruses or fragments having an exposed core protein or nucleic acid able to bind to the corrective set of antibodies. [0024] One of the major functions of microbicides is to neutralize (kill) viruses, particularly infectious viruses such as HIV. To evaluate the potential efficacy of a viricidal microbicide, it is similarly necessary to develop a highly sensitive viral load assay that can differentiate active and inactive viruses. Therefore the methods of the present invention are also useful for evaluating the immediate effect on viral load reduction by microbicides in test tubes, ex vivo in vaginal lavage samples, and in vivo in women.

[0025] Current methods for evaluating the potential efficacy of a microbicide are based on large clinical trials, which are extremely costly and time consuming. So far, several lead microbicides, including N-9, Savvy, Ushercell, PRO 2000 and Carraguard, have all failed in large clinical trails, costing hundreds of millions of dollars and many years of extensive efforts. Because HIV spreads rapidly, especially among women (Quinn and Overbaugh, Science 2005; 308:1582-1583), and kills millions of people each year, methods that can accelerate and simplify the evaluation process of microbicides are urgently needed. The new live/dead viral load assay will allow researchers to vet quickly and economically microbicide candidates. Therefore, the new technology will significantly accelerate the development of anti-HIV microbicides.

[0026] Present commercially available HIV viral load assays are not suitable for evaluation of the viricidal effect of microbicides. Although it is known in theory that current methods based on amplification of viral RNA, such as the Real-Time RT-PCR kit (Amplicor HIV-1 Monitor®, Roche Diagnostics), cannot differentiate between active (infectious) and inactive (noninfectious) HIV (Chantry et al, Infect Dis Obstet Gynecol. 2006; 2006:95938), such methods have been repeatedly used to evaluate HIV inactivation, yielding questionable data (Giles and Mijch, Infect Dis Obstet Gynecol. 2005; 13: 237-240; Israel-Ballard et al, J Acquir Immune Defic Syndr. 2007; 45:318- 323). In one embodiment, the assays, kits and methods of the present invention includes separation of active and inactive viruses; false positive proofreading; and viral load detection of separated viruses and virus fragments. The results provide viral load information on both active and inactive viruses. It will be useful for evaluating viricidal effect of microbicides as well as anti-viral treatments. [0027] A further embodiment of the present invention provides a viral load test that is easy to use, simple, and low cost and can be used to test the effectiveness of microbicides and anti-viral treatments. The technology is based on a signal enhanced magnetic immunoassay and uses a dipstick, or another type of matrix or substrate, and a battery-powered magnetic bead reader. Magnetic immunoassay is a novel diagnostic assay using magnetic beads as an alternative label to conventional enzymes (ELISA), radioisotopes or fluorescence. It involves the specific binding of a magnetic labeled antibody to a target antigen. The presence of magnetic beads is then detected by a magnetic reader (magnetometer) which measures the magnetic field change induced by the beads. The signal measured by the magnetometer is proportional to the analyte (e.g., virus) quantity in the initial sample. A handheld, battery-powered magnetic bead reader provided herein is designed based on the non-linear magnetization technology. Its detection limit is 100 Dynabead M-280 beads (Invitrogen, Carisbad, CA). It is about 100-fold more sensitive than the standard ELISA. With a battery-powered instrument, the new test will be cheaper and more suitable for point-of-care testing.

[0028] One embodiment of the invention provides an assay comprising a first substrate able to selectively bind a core protein or nucleic acid of a virus or selectively bind a virus envelope. The substrate is preferably a dipstick, multi-capillary glass structure, filter paper strip, or other type of matrix able to be easily inserted into a container containing the sample. The assay further comprises a container containing a reagent comprising a detector antibody linked to either biotin linked magnetic beads or streptavidin/avidin linked magnetic beads. The detector antibody is able to bind to the same viral core protein, nucleic acid or envelope bound by the substrate. The assay further comprises one or more containers containing a reagent comprising additional biotin linked magnetic beads; and one or more containers containing a reagent comprising additional streptavidin/avidin linked magnetic beads. The containers can be any containers known in the art able to hold and store reagents. Preferably the containers are small enough to be included as part of a practical kit for field use and can be easily disposed after use. Optionally, the assay comprises a second substrate able to selectively bind a core protein or nucleic acid of a virus or selectively bind a virus envelope with the proviso that if the first substrate selectively binds a core protein or nucleic acid of a virus then the second substrate selectively binds a virus envelope, and if the first substrate selectively binds a virus envelope then the second substrate selectively binds a core protein or nucleic acid of a virus.

[0029] When the substrate, which can be either the first and/or second substrate, is contacted with the sample, the substrate binds the viral core protein, nucleic acid or envelope. The substrate is removed from the sample and added to the reagent containing the detector antibodies, which bind to the viral core protein, nucleic acid or envelope captured on the substrate. Each detector antibody is linked to a magnetic bead conjugated with biotin or streptavidin/avidin. The substrate can be removed and through alternating contact with the reagents containing the additional biotin linked and substravidin linked magnetic beads, magnetic beads will aggregate on the substrate. The magnetic beads are then detected using a handheld magnetic bead reader or magnetometer.

[0030] In a further embodiment, the substrate, which can be either the first and/or second substrate, is linked to a first antibody, which is able to bind to the target antigen (i.e., the viral core protein, nucleic acid or envelope), and a linker antibody which is able to bind to a linker protein. Each magnetic bead is linked to a detector antibody, which is able to bind to the captured target antigen, and a second linker antibody which is also able to bind to the linker protein. When the magnetic bead binds to the captured target antigen on the substrate, the linker protein is added and is able to cross-link the bound magnetic bead and the substrate.

[0031] In another embodiment, the present invention provides improved methods of amplifying viral detection using immunomagnetic signal amplification or biotin- streptavidin/avidin signal amplification. A sample containing a mixture of active and inactive viruses is contacted with a first and second substrate, where one of the first or second substrate is able to selectively bind a core protein or nucleic acid of a virus, and the other of the first or second substrate is able to selectively bind a virus envelope. The virus or virus fragment able to bind to the substrate is captured and subsequently bound to an antibody-linked magnetic bead conjugated with streptavidin/avidin, thereby forming a captured -virus conjugated magnetic bead complex. The substrate is then optionally disassociated from the virus or fragment thereof and removed from the captured-virus conjugated magnetic bead complex. The conjugated streptavidin/avidin is contacted with a plurality of biotinylated beads, thereby attaching the biotinylated beads to the magnetic bead complex. Additional streptavidin/avidin coated beads are bound to the attached biotinylated beads, thereby attaching the additional streptavidin/avidin beads to the magnetic bead complex. In a further embodiment, multiple rounds of biotinylated and streptavidin/avidin coated beads are added to the magnetic bead complex. The magnetic bead complex is then detected by means known in the art such as ELAST ELISA. In one embodiment, the biotinylated and streptavidin/avidin-coated beads are superparamagnetic beads and the magnetic bead complex is detected using a magnetometer.

BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 is an HIV diagram showing the viral envelope, core proteins, and internal RNA.

[0033] Figure 2 illustrates HIV assembly, budding, and maturation (as presented in Ohagen et al., Virology 1997; 228:112-114). Protease is critical for HIV to form a mature virus. With protease inhibitor, HIV viruses become immature, doughnut-shaped viruses.

[0034] Figure 3A shows normal mature HIV viruses without inhibitors, and Figure 3B shows HIV viruses treated with protease inhibitor KNI-272 resulting in doughnut-shaped immature viruses and disrupted viruses (as presented in Goto et al., J Med Virol. 2001 ; 63:203-209).

[0035] Figure 4A shows an electron micrograph of intact HIV-1 virions, and Figure 4B shows an electron micrograph of HIV viral cores without envelopes (as presented in Briggs et al., The EMBO Journal 2003; 22: 1707-1715). [0036] Figure 5 shows the comparison between two HIV-1 infection assays. Figure 5A illustrates HIV replicating in multiple rounds in PBMC before ELISA testing. Figure 5B illustrates luciferase- tagged HIV entering the target cell and replicating in only one round.

[0037] Figure 6 illustrates one method of separating active (live) and inactive (dead) viruses by bacteria. Inactive viruses bind to an antibody-coupled bacterium (bacterium A, e.g., Staphylococcus aureus) selective for a viral core protein or nucleic acid, and active viruses bind to an antibody-coupled bacterium (bacterium B) selective for the viral envelope.

[0038] Figure 7 illustrates one method of separating active (intact, live) and broken (dead) viruses using magnetic beads. Magnetic beads selective for a viral protein are added to a sample containing active viruses and virus fragments. The active viruses bind to the magnetic beads which are held in the test tube by a magnetic while the virus fragments remaining the sample are removed.

[0039] Figure 8 illustrates separation of active (intact, live) and broken (dead) viruses by antibody (Ab) coated plates. A sample containing active viruses and inactive viruses and virus fragments are added to a plate coated with either an antibody able to bind a viral core protein or nucleic acid (Anti-core Ab) or an antibody able to bind to a viral envelope (Anti-envelope Ab). The active viruses or inactive viruses and virus fragments are captured by the plates with the corresponding antibodies while the uncaptured viruses and virus fragments are washed away.

[0040] Figure 9 illustrates a corrective procedure to isolate inactive (dead) viruses with a partial envelope from active viruses having intact viral envelopes. Active viruses and any virus fragments containing a partial viral envelope are captured using an antibody coated plate, such as illustrated in Fig. 8. The active viruses and fragments with partial envelopes are added to a plate coated with an antibody able to bind a viral core protein or nucleic acid (Anti-core Ab). The fragments with partial envelopes are captured by the plate while the active viruses remain unbound and can be collected. [0041] Figure 10 illustrates direct detection of HIV-1 proteins by nanomagnetic particles. Microplates are coated with either active viruses or inactive viruses and virus fragments. Antibody linked magnetic beads able to bind to the viruses or virus fragments are added to the plate and are captured. The magnetic beads are then detected using a magnetic bead reader or magnetometer.

[0042] Figure 11 is a flow-chart describing operation of a new viral load assay of the present invention.

[0043] Figure 12 shows amplification of HIV viral load detection sensitivity by immunomagnetic signal-boosting. Active viruses bound by an antibody-linked bacterium are contacted with antibody linked streptavidin/avidin coated magnetic beads. The virus-streptavidin/avidin magnetic bead complex is harvested and contacted with biotin coated magnetic beads, which bind to the streptavidin/avidin coated magnetic beads. Additional alternating exposure to streptavidin/avidin coated magnetic beads and biotin coated magnetic beads results in a complex containing a greatly multiplied number of magnetic beads which are detected.

[0044] Figure 13 shows an example of magnetic bead aggregation by using biotin- streptavidin/avidin interaction including the approximate number of magnetic beads after each round of amplification.

[0045] Figure 14 shows detection of HIV viral load by an assay of the present invention utilizing magnetic beads (MB) amplified through multiple rounds of biotin and streptavidin/avidin exposure. A dipstick able to bind to a viral core protein is dipped into a sample containing viruses and virus fragments. The dipstick is then placed in a test tube containing a detector antibody linked to streptavidin/avidin magnetic beads followed by exposure to biotin magnetic beads. After multiple rounds of alternating exposure to streptavidin/avidin and biotin magnetic beads, the amount of magnetic beads on the dipstick are detected by a handheld magnetic bead reader or other type of magnetometer. [0046] Figure 15 illustrates a method to secure captured magnetic beads to a virus or virus fragment. A dipstick contains one antibody able to bind a target antigen, such as a protein, nucleic acid, particle or virus fragment of HIV, and a second antibody able to bind to a linker protein. The target HIV particle binds to the first antibody and is captured. A magnetic bead, similarly containing one antibody for the target antigen and a second antibody for the linker protein, binds to the captured HIV particle. The linker protein is added and is able to cross-link the bound magnetic bead and the dipstick.

[0047] Additional features and advantages of the present devices and methods may be obtained by reference to the following detailed description and accompanying drawings that set forth illustrative embodiments, in which the principles of the methods, devices and apparatuses are utilized.

DETAILED DESCRIPTION

[0048] The present invention is able to determine the viral load of enveloped viruses while distinguishing between live and dead viruses. As used herein, an "enveloped virus" is any virus containing a lipid bilayer surrounding an internal protein and nucleic acid core. Typically, the viral envelope contains additional proteins necessary for receptor binding and membrane fusion. Some virus strains, such as adenovirus or coxsackie virus, do not contain viral envelopes and would not be targeted by the present invention. Fig. 1 shows a diagram of HIV illustrating different components of the viral envelope and viral core. The viral envelope comprises a lipid membrane surrounding the viral core and may contain cellular proteins, such as MHC class Il proteins, and glycoproteins, such as gp 120. Proteins and nucleic acids within the viral core are not typically exposed unless the viral envelope has been compromised.

[0049] Not living organisms themselves, viruses must infect host cells to reproduce. Intact viruses capable of infection and reproduction in host cells are considered to be "live" or "active." If a virus fails to develop into a structurally mature virus (as shown in Figs. 2 and 3), or becomes damaged or disintegrated (as shown in Fig. 4), it loses the ability to infect and thus becomes a "dead" or "inactive" virus. [0050] A viral load assay of the present invention will have at least two steps: (i) separation of "live" and "dead" viruses and (ii) detection of the separated viruses. Preferably, the amount of each of the separated intact and inactive viruses is determined by the assay. The key difference between the present invention and current viral load assays is that the methods and assays of the present invention separate the active (intact) and inactive (broken) viruses from one another prior to determining the viral load. The methods and assays of the present invention are highly specific for detecting active and inactive HIV viruses. The sensitivity should be as high as the currently available methods or even better because the virus separation procedure also may concentrate the viruses. Thus, this new method can fill multiple technologic gaps of current HIV viral load assays.

[0051] A flow-chart of the steps in a viral load assay of the present invention is illustrated in Fig. 11 and shows separation of intact and partially intact viruses (which contain at least part of the viral envelope) from inactive (structurally broken) viruses. The viruses and virus fragments are separated through selective binding or capture by a substrate, such as antibody-linked bacteria, magnetic beads or coated microtiter plates, specific to the viral envelope or to a viral core protein or nucleic acid. Because partially broken viruses associated with fragmented envelopes may be co-purified with intact viruses, an additional corrective proofreading procedure may be performed. The partially intact viruses are further separated from the intact viruses and grouped with the broken viruses. The viral load for both the active virus (intact viruses) and inactive virus (broken and partially intact viruses) are detected and the viral load count for each group determined. The results provide information on both active and inactive viruses and are useful for evaluating the effects of antiviral treatments and microbicides.

[0052] One of the intended uses of the present invention is to test the effectiveness of microbicides used to neutralize (kill) viruses or reduce the number of active viruses in a bodily fluid able to cause an infection. A method or assay able to provide more accurate viral load information would increase the ability to identify potentially useful microbicides in an experimental setting as well as the effectiveness of the microbicide in bodilyfluids, particularly with infectious viruses such as HIV. [0053] The present invention also provides improved methods of viral detection using immunomagnetic signal amplification or biotin-streptavidin/avidin signal amplification. The intact and inactive viruses are separately captured using a first or second substrate as described above. The captured viruses or virus fragments are then captured with MBL/Ab-linked magnetic beads conjugated with streptavidin/avidin. The substrate is removed and the remaining conjugated magnetic beads with the captured virus or virus fragments are collected using a magnetic device. The conjugated magnetic beads are contacted with biotinylated nano-beads which bind to the conjugated streptavidin/avidin. Additional streptavidin/avidin coated beads are then bound to the attached biotinylated nano-beads. After multiple rounds, the initial conjugated magnetic beads will form a large aggregate of streptavidin/avidin and biotinylated beads, which can be detected using means ELAST ELISA.

[0054] Alternatively, the streptavidin/avidin-conjugated magnetic beads with the captured virus or virus fragment are contacted with alternating cycles of biotin and streptavidin/avidin-coated superparamagnetic beads forming a magnetic bead aggregate. The unbound free beads can be removed using differential centrifugation. The presence of the magnetic bead aggregate is then detected by a magnetic reader (magnetometer) which measures the magnetic field change induced by the aggregate with the captured virus.

[0055] These methods of detecting viruses can potentially eliminate the need for costly real-time PCR, and can be used with the other viral load assays and methods described herein. In particular, these viral detection methods can be used with methods of monitoring the viral load of a patient undergoing HAART. The resulting assay and method will be cost-effective, suitable for point-of-care testing, and can monitor the therapeutic response to HAART in real time. It can help clinicians to adjust drug combination and dosage. By maximizing efficacy, lowering cost, and minimizing side effects of HAART, this new method can reduce health disparity and improve the quality of lives of millions of people living with HIV/AIDS worldwide.

Example 1 - Real Time HAART Monitoring [0056] During HAART, the maturation of new viruses is suppressed, causing the production of structurally immature, doughnut-shaped viruses (see Figs. 2 and 3, Ohagen et al., Virology 1997; 228:112-114 and Goto et al, J Med Virol. 2001 ; 63:203- 209). Although these immature viruses have the same RNA as mature, active HIV, they are inactive viruses that cannot infect new cells. However, current HIV viral load assays cannot differentiate among these viruses. They detect them as if they were active viruses because they carry the same RNA. Therefore, for monitoring HAART in real time, it is necessary to develop a new method that can differentiate these non-infective viruses from fully mature, infective viruses.

[0057] It is possible to separate active HIV viruses from inactive ones. Because only broken viruses expose viral cores, they can be captured by anti-core antibodies, while intact viruses will not react with anti-core antibodies. As shown in Fig. 3b, the immature doughnut-shaped viruses have highly vulnerable envelopes and disintegrate to expose their core proteins (Goto et al, J Med Virol. 2001 ; 63:203-209). Disintegrated HIV viruses have either broken or lost their viral envelopes (see Fig. 4, Briggs et al., The EMBO Journal 2003; 22: 1707-1715), but their cores are relatively durable and contain detectable RNA (Warrilow et al, Retrovirology 2007; 4:77). The assay of the present invention has two procedures: (i) separation of intact and inactive viruses and (ii) viral load detection of each of the separated viruses.

[0058] The potential impact of this novel diagnostic assay will be in the field of HIV/AIDS health care. Worldwide, the number of people living with HIV/AIDS is about 35-40 million. Thanks to HAART, the lifespan of people living with HIV/AIDS has significantly extended. The HIV/AIDS community has been growing at a speed about 3- 5 million per year due to the rapid spreading of HIV and effectiveness of HAART. By the year 2025, the world population of people living with HIV/AIDS will reach 100 million in the absence of an HIV vaccine.

[0059] Because HAART has been associated with severe side effects including death, it is important to monitor the HAART dosage closely for all AIDS patients. However, current HIV viral load assays have multiple limitations: (i) inability to differentiate live from dead viruses; (ii) high cost; and (iii) unsuitability at point-of-care. Due to high host and complexity, such assays are not available in resource poor settings. As a result, the majority patients on HAART in the developing world are not monitored by any viral load assays. The new "live/dead" HIV viral load assay will fill these critical technology gaps. It will be inexpensive, suitable for point-of-care testing, and can monitor therapeutic response to HAART in real time. It can help physicians quickly and efficiently adjust the HAART drug combination and dosage.

Example 2 - Immuno-capture of HIV-1 by antibody-linked Staphylococcus

[0060] The major HIV-1 surface antigen is gp120, but it is highly clade-specific. To select a surface antigen that is universal among all HIV clades, the human host cell- derived antigen HLA-DR (class Il major histocompatibility complex, MHC) was chosen as the universal target. It is incorporated into all HIV-1 viral envelopes during viral production in the human host cells regardless of viral clades. Certain Staphylococcus aureus cells (Pansorbin; Calbiochem, San Diego, CA) express protein A that binds antibody (Ab) molecules with high affinity, especially rabbit antibodies. By immobilizing the mouse anti-HLA-DR IgG to four different surfaces: 96-well plate, goal-anti-mouse IgG-coupled agarose bead, and rabbit-anti-mouse Ab pretreated protein A-coupled agarose bead, and fixed S. aureus cells, HIV-1 immuno-capture was performed. Three viruses, HIVMN, HIV_Ada-M and HIV_Ba-L, were tested. The results showed that HLA-DR Ab- immobilized S. aureus had the highest HIV capture (77.7%). Bound virus was detected after treatment with 0.5% Triton X-100 and values are percentages of p24 detected comparing with total amount loaded 932.85 pg of p24/ml (Saarloos et al, J Virol. 1997; 71 :1640-1643).

[0061] The carbohydrate moiety of the HIV envelope gp120 includes high mannose N- linked glycan. The protein is largely covered by carbohydrates. HIV captures by targeting the gp120 carbohydrate and protein with the human mannose-binding lectin (MBL) and the human anti-gp120 mAb IgGI b12, respectively have been studied. The results showed that MBL-coated wells captured about 10% HIV viral particles, while lgG1b12 only captured 0.5%, which is only slightly higher than the negative control BSA-coated well (0.2%) (Ji et al, Current Protein and Peptide Science 2006; 7(4):317- 24). The captured HIV quantity was determined by p24 ELISA. The result indicates that carbohydrate on gp120 is a better target for viral capture than its protein, and ligand-immobilized on bacterial cells could perform better than microtiter wells (Saarloos et al, 1997).

Example 3 - Generating active and inactive viruses for evaluation

[0062] A simple way to evaluate diagnostic methods that can or cannot differentiate active and inactive HIV viruses is to use these methods to detect known amounts of both kinds of viruses. Two types of active HIV viruses are prepared in the laboratory (Fig. 5). First, active HIV-1_BaL is prepared by infection of PHA-stimulated human periphery blood monocytes. Second, luciferase-tagged HIV-1_∞nB is prepared by transfection of 293T cells. The ability to infect or gain entry to cultured U87-CD4-CCR5 cells by these HIV-1 viruses determines whether they are active or inactive. The ability to be detected by anti-p24 core protein antibody without using the lysis buffer determines whether the virus is intact or damaged. Infection by HIV-1 BaL is detected by p24-capture ELISA after lysis, while entry by luciferase-tagged HIV-1_COnB is detected by the luciferase assay.

[0063] To make inactivated viruses, one half of the active viruses are treated by a method that can disrupt the viral envelope and expose its core protein, but not produce any toxicity that may affect subsequent viral infection experiments. To select an appropriate virus inactivation method, the following procedures are performed.

[i± Heat treatment. The viral sample is exposed to flash-heat at 80-100⁰C hot water for 1-10 seconds. Preferably, the virus is exposed to the flash heat just long enough to break the viral envelope but not denature the p24 core protein. The purified p24 is used as a control.

QiX Alkaline or acid treatment. Extreme pH can disrupt the virus membrane and inactivate the virus. 1 M NaOH or HCI 1 :100 is added to the virus suspension to a final concentration of 10 mM. After 10 min at room temperature (20⁰C), the virus suspension is neutralized to pH 7.

(Hi) Lysis treatment. Mild detergent, such as Triton X-100, is used to make lysis solution in distilled water. To a virus solution, the detergent is added to a final concentration at 1 % and incubated for 10-30 min at 20⁰C. The viruses are lysed and detergent removed by dialysis against frequent changes of phosphate-buffered saline at 4°C. The molecular size cutoff of the dialysis membrane should be less than 10 kDa, so that the HIV-1 core protein p24 and RNA will not be lost.

(iv) Protease inhibitor. A common HAART agent, such as a protease inhibitor like Saquinvir or Ritonavir (Moravek, Brea, CA) is added to treat the HIV- infected cell culture. The untreated samples can be used as a control.

[0064] Based on the knowledge about HIV, all of the above methods should inactivate HIV viruses, but their ability to strip the viral envelope and expose the core protein p24 and RNA may vary. Since only envelope-damaged viruses will expose core proteins, by p24-capture ELISA with and without lysis buffer treatment, it can be determined whether the viral envelopes have been damaged. If the results between the two treatments are similar, the viral envelope has been stripped. If the p24 detection is significantly lower in samples without lysis buffer treatment than that with lysis buffer treatment, the viral envelope could be still largely intact.

[0065] Alternatively, Western blots can be performed on the purified "live" and "dead" viruses. The ratios of gp120 and gp41 to p24 of live versus dead viruses should be different. Therefore, Western blots can be used for evaluating "dead" viruses prepared by different methods and may provide insight into the nature of the dead viral particles. For example, the live viruses probed with antibodies to gp120, gp41 and p24 should show 3 bands at the appropriate molecular weights. In cases where preparation of the dead virus has removed the membrane or at least the gp120 and gp41 , only p24 will be present (or at least the intensity of p24 will be significantly more than the remaining gp120 and gp41 ). Alternatively, gp120, which is noncovalently associated with the viral envelope, may be removed with gp41 and p24 remaining.

[0066] If all of the viral inactivation methods can strip the HIV viral envelope to fully expose the viral core, the flash-heating treatment is preferable because it is the simplest and cleanest procedure. All of the inactivated viruses can be used for in vitro virus infection of cultured cells to confirm their inactive status. The active and inactive viruses in different amount combinations can be used for subsequent studies.

Example 4 - A new viral load measurement method that differentiates active and inactive viruses to evaluate antiviral treatment in real time

[0067] As discussed above, viral load assays in the market cannot distinguish active and inactive viruses and thus are not suitable for evaluating the immediate effects of HAART in real time. A new method is required to facilitate treatment monitoring. Active viruses can be separated from inactive ones by the methods and assays herein resulting in the true viral load. First, a system for separating active (intact) and inactive (structurally broken) viruses utilizing surface antigen-linked magnetic beads or coated microtiter plates is applied. Second, a proofreading system to correct possible false positive data is performed. Third, after the active and inactive viruses are separated, they are analyzed individually by currently sensitive detection methods.

[0068] To establish a new assay that can provide viral load information on both active and inactive viruses, the active and inactive viruses must first be separated. Selective capture technology can be used for this purpose. For example, an antibody, such as the anti-envelope (gp120) antibody, can be used to capture intact viruses, and a second antibody, such as the anti-core (p24) antibody, can be used to capture broken viruses (core proteins). The different antibodies are linked to magnetic beads, bacteria or coated to microtiter plates which are then separated.

[0069] As shown in Fig. 6 two virus-specific bacterial systems can be applied sequentially to capture and separate core-exposed, inactive viruses and intact, active viruses. One bacterial system (labeled Ab-coupled bacterium A in Fig. 6) is able to bind viral core proteins of inactive virus fragments, and the other bacterial system (labeled Ab-coupled bacterium B in Fig. 6) is able to bind the viral envelopes of active viruses. For example, the HLA-DR mouse lgG2a monoclonal Ab (clone L243, ATCC)-linked S. aureus (Saarloos et al, 1997) is highly effective for HIV capture and have been used to capture intact HIV. The anti-core (p24) (Polymun, Vienna, Austria) Ab-linked S. aureus can be used to capture broken viruses that expose core proteins.

[0070] The magnetic bead technology (Invitrogen, Carlsbad, CA) has been used to separate human immune cells and other types of viruses (Lien et al, Lab on a Chip 2007; 7:868-875). This method can be applied to separate intact and disintegrated HIV viruses. Because core antigens are inside the virus and are not exposed when the virus is intact, broken viruses are captured with an anti-core antibody and intact viruses captured with MBL or an anti-envelope antibody. As shown in Fig. 7, magnetic beads are first cross-linked with anti-p24 Ab or anti-HLA-DR Ab, or a mannose-binding lectin (e.g., ConA). The anti-p24 Ab-linked magnetic beads are added to a sample containing both active and inactive viruses prepared as described above. The mixture is incubated for 30 min with gentle agitation to facilitate binding. Next, a magnet is applied to trap the inactive viruses that expose core proteins. Intact viruses that have complete envelopes are removed and concentrated with magnetic beads that bind the viral envelope by either anti-HLA-DR Ab or ConA or both. The sample is washed to remove unbound substances. The inactive viruses are separated from active ones, both as purified fractions.

[0071] As an alternative, anti-envelope and anti-core antibody-coated microtitier plates are used for capturing intact and disintegrated HIV viruses, respectively. This method has been successfully used to capture HIV core protein p24 as a part of p24-ELISA. As shown in Fig. 8, the microtiter plates are coated with anti-envelope or anti-core antibody. The crude sample containing both intact and disintegrated viruses prepared as described above is added to an antibody coated plate. The plate is incubated for 30 min with gentle rotation to allow binding. The wells are washed gently with wash buffer to remove unbound fractions, and the intact or disintegrated viruses captured by the antibody-coated microtiter plates are purified. [0072] The virus capture procedure is important not just for isolation of intact or broken viruses, but also for enrichment of viral particles or viral core proteins to increase the sensitivity of the subsequent viral detection assays.

[0073] After the "live" and "dead" viruses are separately captured, the capturing efficiency of each virus can be assessed by measuring viral load directly in the captured fraction (the pellet) and indirectly by measuring the viral load reduction in the supernatant using p24-capture ELISA. For active viruses, infection assay can be performed for more accurate viral load assessment. Because after capture by antibody or MBL many previously active HIV viruses might be rendered inactive in the captured fraction after elution, the supernatant can be analyzed for viral infectivity. The results ware used to calculate viral load reduction for the capture of active viruses.

Example 5 - A Method for Data Proofreading

[0074] A diagnostic method may need to include steps that overcome possible false positive or false negative results. The likelihood to co-purify intact viruses is low with the use of anti-core antibodies for broken viruses, because the viral core is not exposed in intact viruses; however, the possibility exists for broken viruses to be co-purified with intact ones using anti-envelope antibodies. For example, when anti-envelope gp120 antibody-linked magnetic beads are used to isolate intact viruses, some broken viruses may be co-purified if they are associated with a partial envelope. To reduce the potential for false results it would be desirable to capture inactive viruses first and active viruses second within the same sample. Additionally, a method to proofread the detecting result is beneficial.

[0075] Fig. 9 illustrates a proofreading method for correcting possible false positive errors arising during intact virus isolation. First, along with active viruses, inactive viruses associated with a partial envelope are captured by the anti-envelope antibody coated beads or microtiter plates. After washing and gentle elution (such as 200 mM mannose), the captured fully enveloped and partially enveloped viruses are released. [0076] After elution, the viruses are reacted with anti-p24 antibody-coated beads or plates. This time, the viral core of the partially enveloped viruses is captured by the anti-core p24 antibody. With this data, the correct copy number of active viruses can be obtained by subtracting the copy number of partially enveloped viruses from that of the total enveloped viruses. After the intact, "live" viruses and broken "dead" viruses are captured by the above-mentioned methods, their infection capacity may be confirmed by in vitro HIV infection assays.

[0077] Captured HIV-1 BaL viruses can be used to infect PHA-stimulated human periphery blood monocytes and detected by p24-capture ELISA. The captured luciferase-tagged HIV-1_COnB viruses can be used to infect U87.CD4.CCR5 cells and detected by the Luciferase assay. The ability to infect cultured cells by these HIV-1 viruses determines whether they are active or inactive. The results are used to confirm each other and also to verify the infection capacity of the captured viruses.

Example 6 - Detection of Captured Viruses

Real-time RT-PCR

[0078] The currently most sensitive HIV viral load detection method on the market is the Real-time RT-PCR kit of Roche Diagnostics (AMPLICOR HIV-1 MONITOR® Test, v1.5). It is based on amplification of viral RNA. This method requires lysis of the virus to release its RNA. Since active viruses can be separated from the inactive ones according to the above-mentioned procedures, any sensitive and reliable detection methods, including the commercially available ones, can be used to detect the "live" and "dead" viruses individually. A straightforward method is to use the kit from Roche Diagnostics (AMPLICOR HIV-1 MONITOR® Test, v1.5) to test the purified active viruses and inactive viruses separately. This method is the most sensitive method for HIV viral load detection by far (low limit: 50 copy/ml). The potential drawback of this method, however, is that this method is only semi-quantitative, because the final result is based on nucleic acid amplification. The viral load data could be inflated due to signal amplification. Methods for direct measurement of viral particles, is achievable, could provide more precise information. Magnetic nanoparticle count

[0079] An alternative detection method is to use antibody-linked fluorescent magnetic particles to detect the number of viruses directly without amplification. As shown in Fig. 10, the procedures for capturing magnetic beads are as follows: (i) coat on separate microtiter plates with the purified intact and broken viruses; (ii) capture the anti-gp120 and anti-p24 antibodies-conjugated nanoparticles to these plates; (iii) release these particles and capture them with a magnet; (iv) measure these particles with various methods, including a flow-cytometer (for counting the number of particles), a magnetometer (for measuring the magnetic field strength), and/or a detection method with diaphragm or cantilever.

[0080] Existing optical methods, such as fluorescence and optical transmission, used for detecting the number of beads generally utilize two types of beads. They are paramagnetic and ferromagnetic (Spherotech, Lake Forest, IL). The paramagnetic beads by themselves are not magnetic, but react under the influence of a magnetic field.

Detection with paramagnetic beads - Diaphragm approach

[0081] By localizing the beads onto micromachined diaphragms, the diaphragms will deflect under the influence of a stationary magnetic field due to the induced force upon the beads. This force is proportional to the number of beads. The deflection can be monitored optically (reflected laser beam) or electronically (as part of a differential capacitor structure). The sensitivity of this method will be modeled using finite element calculations with the appropriate material parameters as input and will be compared to the existing methods.

Detection with paramagnetic beads - Cantilever Approach

[0082] Another potential method is to localize the beads onto an array of micromachined cantilevers and measure the change in resonance frequency as a function of the number of beads. This method is extremely sensitive and it might become possible to detect the presence of a single bead. Detection with ferromagnetic beads

[0083] Ferromagnetic beads retain some magnetism and it is possible to measure their magnetic field with a very sensitive magnetometer. One approach is the use of a microfabricated sensor using the 'giant magnetoresistive' effect. This sensor can be placed over the areas where the beads are localized and the output should be proportional to the number of beads collected.

[0084] Other methods might be identified and investigated as well. Without signal- amplification, such methods obtain viral count directly and thus can be more precise than the RT-PCR method. However, the size determination of the nano-particles and sensitivity of the detection methods can be challenging especially when the viral load is low.

Example 7 - Alternative methods for viral detection are needed

[0085] Current viral load assays based on real-time PCR are costly (e.g., $100/test with the Roche kit). The new assay will require two tests for both live and dead viral loads. This will double the expense, possibly making the new assay cost prohibitive. Therefore, it is important that HIV viral load be sensitively and cost-effectively detected without using real-time PCR. This process includes (i) improving the well developed ELAST ELISA with immunomagnetic viral capture and signal amplification; and (ii) developing a new method based on magnetic immunoassay enhanced with signal amplification.

Viral Detection Using ELISA

[0086] ELISA is a simple and convenient method for many clinical diagnoses, but the conventional ELISA is not sensitive enough for monitoring HIV viral load in patient samples. To overcome this problem, a new method called signal-amplification-boosted ELISA with biotinyl tyramide and heat-denatured plasmid has been developed (Schupbach, Rev. 2002; 4:83-92). Perkin Elmer Life Sciences (Boston, Mass) has developed a version called ELAST ELISA having enhanced detection sensitivity. With this method, a p24 ultrasensitive antigen assay (Up24) has been developed with a detecting range from 1 to 6,500 pg p24 (1 pg p24 equals about 8,000 HIV virions). However, it is still about 300-fold less sensitive than the current nucleic acid-based viral load assays. The present invention improves its sensitivity by targeted virus capture and signal amplification.

[0087] For use with the viral load assay of the present invention as shown in Fig. 12, the first step will be to separately capture the active and inactive HIV viruses with fixed bacterial cells or magnetic beads. Then, the captured viruses are used for a second round of capture by MBL/Ab-linked magnetic beads conjugated with streptavidin/avidin. After removing unbound beads by differential centrifugation (with sucrose or ficoll gradient) and wash, the captured beads will be separated from the bacteria with detergent and be harvested by a magnet device. At this point, a small number of magnetic beads can be proportionally amplified with the addition of excessive biotinylated nano-beads to allow biotin-streptavidin/avidin interaction. The bound beads can be harvested again by a magnet device or differential centrifugation. The unbound beads will be removed. A second round of streptavidin/avidin -coated beads will be added and to allow further biotin-streptavidin/avidin interaction. With multiple rounds of amplification, the initially trace amount of magnetic beads will be significantly increased and can be detectable with the ELAST ELISA detection system from Perkin Elmer. The last round streptavidin/avidin -coated beads can activate the biotinyl-tyramide reaction. With this system, the detection sensitivity of the present ELAST ELISA can be increased about 100 fold. Once the amplification method is finalized, a standard curve will be established with known amounts of viruses. Since the detection is based on the coated streptavidin/avidin on the magnetic beads provided in the second round virus capture, magnetic beads can be used for the initial virus capture if the bacterial cells are not used. The capture Ab-linked magnetic bead without streptavidin/avidin will not affect the downstream detection.

Biotin-Streptavidin/avidin Signal Amplification Magnetic Immunoassay

[0088] Magnetic immunoassay (MIA) is a novel type of diagnostic immunoassay utilizing magnetic beads as an alternative to conventional enzymes (ELISA), radioisotopes (RIA) or fluorescent immunoassays. This assay involves the specific binding of an antibody to its antigen, where a magnetic label is conjugated to the antibody. The presence of magnetic beads is then detected by a magnetic reader (magnetometer) which measures the magnetic field change induced by the beads. The signal measured by the magnetometer is proportional to the analyte (e.g., virus) quantity in the initial sample. As an example, a portable magnetic bead reader is now available from Magnisense (Paris, France) based on the non-linear magnetization technology (Nikitin et al, J. Appl. Phys. 2008; 103: 07A304). Its detection limit is 1000 MyOne beads (1 μm) (Invitrogen, Carlsbad, CA). If it is comparable to 1000 virion/ml, it would be more sensitive than the Up24 ELAST ELISA by about 8 fold, but it is still 40 times less sensitive than the real-time PCR method (25 virion/ml).

[0089] To improve the detection sensitivity, the harvested magnetic beads are amplified by several rounds of biotin-streptavidin/avidin interaction (Fig. 12). First, the virus-captured streptavidin/avidin-coated magnetic beads from MBL+/Ab-linked bacteria are harvested with a magnet device. These magnetic beads will be subject to amplification with biotin and streptavidin/avidin -coated superparamagnetic beads. This time, a magnet is not used for separation because the unbound beads are also magnetic. By adding excessive biotinylated magnetic beads (such as Biotin FeOdot 15, Genovis, Inc., Cambridge, MA) to the streptavidin/avidin -coated magnetic beads, they will form co-aggregates. The unbound free beads will be removed by differential centrifugation or by wash with a filtered cartridge that only allow free beads to pass through. After several rounds of amplification and wash, these beads will be read with the magnetic bead reader of Magnisense. Once the amplification method is finalized, a standard curve will be established with known amounts of viruses.

[0090] Ab-linked magnetic beads have been successfully used for capturing viruses (Lien et al, 2007). It is straightforward for these beads linked with Ab to detect the "live" and "dead" viruses that are already separately captured by bacteria or the first round of Ab-linked beads. Additionally, the protocol of ELAST ELISA has been well established by Perkin Elmer and the magnetic bead reader is commercially available by Magnisense. With targeted virus capture by magnetic beads and multiple rounds of signal amplification, viral load detection with 10-100-fold higher sensitivity than the present ELAST ELISA can be obtained. With the biotin-streptavidin/avidin signal amplification of magnetic beads, the sensitivity of the magnetic bead reading may also be increased for about 10-100 fold. Either method will be an inexpensive alternative to current viral load assays and can be selected for use in live/dead viral load assay of the present invention. The overall sensitivity should be as high as the real-time PCR by its cost should be 10 times less.

Example 8 - Standardized Viral Load Assays

[0091] The assays of the present invention require viral capture and detection. Because magnetic beads offer advantages for viral detection, it is preferable to use treated bacterial cells or other substrates for the virus capture and separation steps, where the bacterial cells or other substrates are treated to link to magnetic beads. A preferred assay protocol includes two differently treated bacterial cells for capturing intact and inactive viruses, and to detect them with MBL/Ab-linked streptavidin/avidin- coated superparamagnetic beads. The basic instruments for such an assay are simple and inexpensive. Table 1 presents an example of a live/dead viral load assay kit and protocols.

Table 1. Live/dead HIV viral load assay kit and protocols

For safety reasons, live HIV pseudovirus, not real virus, will be used as control. [0092] Current immuno-adsorbent S. aureus cells (Pansorbin; Calbiochem, San Diego, CA) are fixed with formalin with a short shelf life; however, lyophilization is performed to achieve longer shelf life. The bacteria are killed and fixed with isopropanol, and then they are coupled with appropriate antibodies and lyophilized with a Labconco lyophilizer. Likewise, Ab-coupled magnetic beads are also be lyophilized. Once lyophilization is achieved, the reagents will be stable for years.

[0093] One objective of the present invention is to develop a low cost viral load assay suitable for point-of-care testing, especially in resource poor settings. Currently, four high cost systems to measure HIV viral load are available from Abbott, Bayer, bioMerieax, and Roche, which are all based on nucleic acid amplification. The less expensive ELISA tests, including the heat-dissociated p24 antigen (HDAg) and ultrasensitive p24 (Up24), are about 3-4 orders of magnitude less sensitive. These tests have been tried in resource poor settings, but results have been unsatisfactory. In this assay, the immunoassay is modified using magnetic labeling and magnetic bead aggregation to drastically boost sensitivity. Unlike current viral load assays, the new test will not need high cost instruments, perishable reagents, and highly trained technicians. It will use a dipstick, or another type of applicator, substrate or matrix, a number of tubes with non-perishable reagents, magnetic beads, and a handheld, battery powered magnetic bead reader.

[0094] The magnetic signal magnetic beads bound to the captured viruses or virus fragments are amplified by nano-bead aggregation via multiple rounds of biotin- streptavidin/avidin interaction. In only four rounds, the magnetic signal of 1 magnetic bead can increase 313-fold (Fig. 13). This method is faster and more accurate than PCR because the size of a magnetic bead is standard and its number can be precisely counted. Each round of amplification is proportional to the number of the initially captured beads

[0095] For this HIV assay, the viral core target for capture is the HIV core protein p24 because it is the most abundant antigen in HIV (estimated 2000 copies/virus). To capture the viral protein, the first step is to obtain a viral sample, such as a blood or other fluid sample from a patient. Then, as shown in Fig. 14, to capture the HIV p24 protein, the sample is transferred to a test tube with a dipstick or other substrate coated with anti-p24 antibody and incubated for 1 h at room temperature with gentle agitation. To test and optimize the assay, samples containing known amounts of laboratory HIV-1 are used as source materials and heated to dissociate the p24 protein. The sample is diluted 1 :6 in 0.5% Triton X-100 and heated at 100⁰C for 5 min prior to transfer to the test tube.

[0096] Next, the captured viral core protein p24 on the dipstick is used to capture antibodies linked to magnetic beads conjugated with streptavidin/avidin. At this point, the small number of magnetic beads is proportionally amplified by transferring the dipstick to a new tube with excessive biotin-linked magnetic beads to allow biotin- streptavidin/avidin interaction. The biotin-magnetic beads conjugate with the streptavidin/avidin-magnetic beads on the dipstick. The unbound beads remain behind in the test tube while the dipstick is transferred to the next tube with excessive streptavidin/avidin -linked magnetic beads to allow further biotin-streptavidin/avidin interaction. With multiple rounds of amplification, the initial trace amount of magnetic beads is significantly increased and is readily detectable with a handheld magnetic bead reader or other type of magnetometer (Fig. 14).

[0097] There will be a size limitation for a dipstick to capture viral antigens and magnetic beads. The larger the magnetic-bead aggregate formed on the dipstick, or other substrate, the more fragile it becomes. A large aggregate on a dipstick can be easily broken off because the initial number of antibody-antigen linkages is limited. To overcome this problem, subsequent reinforcement linkages can be provided. For example (as shown in Fig. 15), the dipstick can be coated with two antibodies: one antibody is the anti-HIV core antibody for capturing the target antigen, and the other antibody is able to bind to a linker protein, which can be any protein that does not exist in the human bodily fluid, such as the bovine serum albumin (BSA). After the initial capture of the target antigen, the dipstick will be used to capture the first round magnetic beads, which also has one antibody able to bind to the target antigen and a second antibody able to bind to the linker protein. After capture of the magnetic beads, the unbound beads will be removed by washing and the linker (e.g., BSA) added. The additional antibodies on the dipstick and magnetic beads will bind to the linker protein and provide additional linkage between the captured beads and the dipstick. Likewise, a second or third round of linkers can be designed this way to reinforce the binding of captured beads and the sticks.

[0098] The antibody-conjugated dipsticks and beads can be lyophilized for storage at ambient temperature thereby reducing cost and improving ease of use of the test in the field. Additionally, the reagents will not be perishable, so that refrigeration will not be needed. Advantages of this new viral load test will include lower sample volume required, less sample preparation, no perishable reagents, no requirement for refrigeration or AC electric main power, lower instrument cost (especially compared to a real time PCR device), shorter result time (<2 h), higher accuracy, less requirement for technician training, substantially lower cost per test, and more suitable for point-of-care testing. The new test can also be readily scaled up for multi-sample testing and automation. While this viral load test may be optimal for HIV, it is similarly applicable for other viruses and testing for other viral diseases. Additionally, the material, size and porosity (contact area) of the dipstick, the type and size (100-1 ,000 nm) of magnetic beads, the size, shape, number and fluid volume of the dipping tubes, the type and amount of reagents, reaction time, methods of agitation and washes can be modified to adapt to the particular application. In addition to dipstick, other applicators, substrates and matrices can be used, including multi-capillary glass structure (photonic crystals) and porous filters with a syringe-type liquid handling system, and filter paper strips.

[0099] Having now fully described the present invention in some detail by way of illustration and examples for purposes of clarity of understanding, it will be obvious to one of ordinary skill in the art that the same can be performed by modifying or changing the invention within a wide and equivalent range of conditions, formulations and other parameters without affecting the scope of the invention or any specific embodiment thereof, and that such modifications or changes are intended to be encompassed within the scope of the appended claims. [0100] When a group of materials, compositions, components or compounds is disclosed herein, it is understood that all individual members of those groups and all subgroups thereof are disclosed separately. When a Markush group or other grouping is used herein, all individual members of the group and all combinations and subcombinations possible of the group are intended to be individually included in the disclosure. Every formulation or combination of components described or exemplified herein can be used to practice the invention, unless otherwise stated. Whenever a range is given in the specification, for example, a temperature range, a time range, or a composition range, all intermediate ranges and subranges, as well as all individual values included in the ranges given are intended to be included in the disclosure. Additionally, the end points in a given range are to be included within the range. In the disclosure and the claims, "and/or" means additionally or alternatively. Moreover, any use of a term in the singular also encompasses plural forms.

[0101] One of ordinary skill in the art will appreciate that starting materials, reagents, purification methods, materials, substrates, device elements, analytical methods, assay methods, mixtures and combinations of components other than those specifically exemplified can be employed in the practice of the invention without resort to undue experimentation. All art-known functional equivalents, of any such materials and methods are intended to be included in this invention. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein.

[0102] All publications referred to herein are incorporated herein to the extent not inconsistent herewith. Some references provided herein are incorporated by reference to provide details of additional uses of the invention. All patents and publications mentioned in the specification are indicative of the levels of skill of those skilled in the art to which the invention pertains. References cited herein are incorporated by reference herein in their entirety to indicate the state of the art as of their filing date and it is intended that this information can be employed herein, if needed, to exclude specific embodiments that are in the prior art. For example, when a compound is claimed, it should be understood that compounds known in the art including the compounds disclosed in the references disclosed herein are not intended to be included in the claim.

Claims

1. A method for detecting a viral load in a sample containing a mixture of inactive (broken) viruses and active (intact) viruses, said method comprising the steps of: a. separating the inactive viruses from the active viruses; b. conducting a procedure to correct false positive data in the separated active viruses; and c. detecting and determining the viral count of the separated inactive viruses and the viral count of the separated active viruses.

2. The method of claim 1 wherein the inactive and active viruses are HIV.

3. The method of claim 1 wherein the sample is a bodily fluid.

4. The method of claim 1 where said separating comprises the steps of: a. mixing a set of viral core antibodies with said sample, wherein the set of viral core antibodies are linked to a first substrate and are directed against one or more core proteins or nucleic acids of a virus; b. capturing inactive viruses or fragments thereof having an exposed core protein or nucleic acid able to bind to the set of viral core antibodies; c. removing said first substrate and purifying the captured viruses or fragments thereof.

5. The method of claim 4 wherein the first substrate is one or more bacteria, magnetic beads or microtiter plates.

6. The method of claim 4 wherein the set of viral core antibodies comprises anti-p24 antibodies.

7. The method of claim 1 where said separating comprises the steps of: a. mixing a set of envelope antibodies with said sample, wherein the set of envelope antibodies are linked to a second substrate and are directed against a viral envelope; b. capturing active viruses having an viral envelope able to bind to the set of envelope antibodies; c. removing said second substrate and purifying the captured viruses.

8. The methods of claim 7 wherein the second substrate is one or more bacteria, magnetic beads or microtiter plates.

9. The methods of claim 7 wherein the set of envelope antibodies comprises anti- gp120 antibodies.

10. The method of claim 7 where said separating further comprises the steps of: a. mixing a set of viral core antibodies with said sample, wherein the set of viral core antibodies are linked to a first substrate and are directed against one or more core proteins or nucleic acids of a virus; b. capturing inactive viruses or fragments thereof having an exposed core protein or nucleic acid able to bind to the set of viral core antibodies; c. removing said first substrate and purifying the captured viruses or fragments thereof.

11. The method of claim 7 wherein said step of conducting a procedure to correct false positive data comprises: a. mixing a corrective set of antibodies with a sample containing separated active viruses, wherein the corrective set of antibodies are linked to a solid support and are directed against one or more core proteins of a virus; and b. capturing and removing inactive viruses or fragments thereof having a core protein able to bind to the corrective set of antibodies.

12. An assay for determining a viral load in a sample containing a mixture of inactive viruses and active viruses comprising: a. a first substrate comprising a first set of antibodies, wherein said first set of antibodies is able to selectively bind a core protein or nucleic acid of a virus or selectively bind a virus envelope; b. a second substrate comprising a second set of antibodies, wherein said second set of antibodies is able to selectively bind a core protein or nucleic acid of a virus or selectively bind a virus envelope with the proviso that if the first set of antibodies selectively binds a core protein or nucleic acid of a virus then the second set of antibodies selectively binds a virus envelope, and if the first set of antibodies selectively binds a virus envelope then the second set of antibodies selectively binds a core protein or nucleic acid of a virus; and c. a means for detecting the amount of core protein, nucleic acid or viral envelope bound to the first and second substrate.

13. The assay of claim 12 wherein the first substrate is one or more bacteria, magnetic beads or microtiter plates.

14. The assay of claim 12 wherein the second substrate is one or more bacteria, magnetic beads or microtiter plates.

15. The assay of claim 12 wherein the core protein of a virus is a core protein of HIV.

16. The assay of claim 12 wherein the virus envelope is from HIV.

17. The assay of claim 12 wherein said first or second substrate is a dipstick, multi- capillary glass structure or filter paper strip.

18. The assay of claim 17 further comprising a reagent comprising a detector antibody linked to either biotin linked magnetic beads or streptavidin/avidin linked magnetic beads.

19. The assay of claim 18 further comprising: a) a reagent comprising biotin linked magnetic beads; and b) a reagent comprising streptavidin/avidin linked magnetic beads.

20. The assay of claim 19 further comprising a magnetic bead reader or magnetometer able to detect one or more magnetic beads.

21. The assay of claim 18 further comprising a reagent comprising a linker protein, wherein said first or second substrate further comprises a first linker antibody able to bind to said linker protein, and said magnetic beads comprise a second linker antibody able to bind to said linker protein.

22. A method of monitoring the viral load of a patient receiving treatment for a viral disease comprising the steps of: a. contacting a first sample from the patient containing active and inactive viruses with a first and second substrate, where the first substrate is directed against one or more core proteins or nucleic acids of a virus and the second substrate is directed against a viral envelope, thereby binding inactive viruses to the first substrate and binding active viruses to the second substrate; b. conducting a procedure to remove inactive viruses bound to the second substrate; and c. detecting and determining the amount of inactive viruses bound to the first substrate and the amount of active viruses bound to the second substrate.

23. The method of claim 22 wherein said viral disease is selected from the group consisting of influenza, HIV/AIDS, dengue fever, avian influenza and SARS.

24. The method of claim 22 wherein said treatment is highly active anti-retroviral therapy (HAART).

25. The method of claim 22 further comprising the steps of contacting a second sample from the patient with the first and second substrate, wherein the second sample is taken at a point in time separate from the first sample; and comparing the amount of bound active and inactive viruses from the second sample to the first sample.

26. A method of amplifying viral load detection of a sample containing a mixture of inactive viruses and active viruses comprising the steps of: a. contacting said sample with a first and second substrate, where one of the first or second substrate is able to selectively bind a core protein or nucleic acid of a virus, and the other of the first or second substrate is able to selectively bind a virus envelope; b. capturing a virus or fragment thereof having a core protein, nucleic acid or viral envelope able to bind to said first substrate; c. binding the captured virus or fragment thereof with an antibody-linked magnetic bead conjugated with streptavidin/avidin, thereby forming a magnetic bead complex; d. disassociating the first substrate from the virus or fragment thereof and removing the first substrate from the magnetic bead complex; e. binding the conjugated streptavidin/avidin with a plurality of biotinylated beads, thereby attaching the biotinylated beads to the magnetic bead complex; f. contacting additional streptavidin/avidin beads with the attached biotinylated beads, thereby attaching the additional streptavidin/avidin beads to the magnetic bead complex.

27. The method of claim 26 further comprising detecting the magnetic bead complex using ELAST ELISA.

28. The method of claim 26 wherein the biotinylated and streptavidin/avidin-coated beads are superparamagnetic beads.

29. The method of claim 28 further comprising detecting the magnetic bead complex using a magnetic bead reader or magnetometer.

30. A diagnostic kit for determining a viral load in a sample comprising: a means for collecting a sample from a patient; a first and second substrate, where the first substrate is able to bind one or more core proteins or nucleic acids of a virus and the second substrate is able to bind a viral envelope, thereby binding inactive viruses to the first substrate and binding active viruses to the second substrate; a means for detecting the amount of inactive viruses bound to the first substrate and the amount of active viruses bound to the second substrate; and a housing able to contain components of said kit.

31. The diagnostic kit of claim 30 wherein said first and second substrate, independently from one another, are selected from the group consisting of bacteria, magnetic beads, microtiter plates, dipsticks, multi-capillary glass structures and filter paper strips.

32. The diagnostic kit of claim 31 wherein said first substrate comprises an antibody able to selectively bind a core protein or nucleic acid of a virus, and said second substrate comprises an antibody able to selectively bind a virus envelope.

33. The diagnostic kit of claim 30 further comprising a reagent comprising a detector antibody linked to either biotin linked magnetic beads or streptavidin/avidin linked magnetic beads.

34. The diagnostic kit of claim 33 further comprising one or more containers containing a reagent comprising biotin linked magnetic beads; and one or more containers containing a reagent comprising streptavidin/avidin linked magnetic beads.

35. The diagnostic kit of claim 34 wherein said means for detecting comprises a handheld magnetic bead reader or magnetometer able to detect one or more magnetic beads.

36. The diagnostic kit of claim 33 further comprising a reagent comprising a linker protein, wherein said first or second substrate further comprises a first linker antibody able to bind to said linker protein, and said magnetic beads comprise a second linker antibody able to bind to said linker protein

37. The diagnostic kit of claim 30 further comprising a corrective set of antibodies wherein the corrective set of antibodies are linked to a solid support and are able to bind virus fragments containing partial viral envelopes.

38. The diagnostic kit of claim 30 wherein the core proteins, nucleic acids and viral envelope are from HIV.