BACKGROUND OF THE INVENTION [02] Anti-retroviral therapies have led to a dramatic reduction in HIV-related illness and death in developed countries. However, 99% of deaths from Acquired Immune Deficiency Syndrome (AIDS) in 2002 occurred in low and middle income countries where 95% of persons with HIV/AIDS live, and only a small percentage of persons in these countries have access to anti-retroviral treatment (Weekly Epidemiological Record, No.

51/52,20 December 2002; New International Coalition Aims to Expand Global Access to HIV/AIDS treatment).

[03] Now that a number of agencies, including the recently-formed International HIV-1 Treatment Access Coalition (ITAC) served by the World Health Organization, have targeted the provision of anti-retroviral drugs (ARD) for developing countries, it becomes critical to develop methods that can evaluate viral burden (viral load) and the effectiveness of drug regimens, particularly because of the recent and significant concern of emerging viral resistance. Monitoring the progression of HIV-1 infection, predicting outcome, and measuring the viral response to ARD to guide treatment strategies is essential for managing the care of HIV-1 infected individuals and can have a major impact on morbidity and mortality.

[04] Because viral load monitoring cannot currently be supported in most resource- poor country laboratories, simple but effective methods must be devised. In addition, these methods must address issues related to limited laboratory capabilities including unstable electricity, poor instrument maintenance, non-calibrated pipettes, poor temperature control, etc. , and cost issues that severely limit availability of test kits. Current molecular methods to measure viral load are expensive and cannot be performed under these conditions.

[05] Currently, two strategies are available to measure HIV-1 response to anti- retroviral therapies: (1) HIV-1 RNA measurements by molecular methods, and (2) HIV-1 p24 antigen (p24 antigen) measurements by ELISA. RNA methods are well-established, used widely in industrialized nations, and are extremely effective in providing information to guide therapeutic management of patients. However, their limitations for use in developing countries are well known, and therefore, patient care in these countries cannot be effectively managed. It is certain that more simplified methods will be of immense benefit for monitoring HIV-1 viral load in countries where technical expertise and laboratory infrastructure are limited.

[06] The p24 antigen ELISA assay measures the viral capsid (core) HIV-1 p24 protein in blood resulting from virus replication. It is associated with periods of high levels of viremia during early and late infection, and fluctuates in concert with RNA levels. The sensitivity of the p24 antigen test in detecting HIV-1 infection as compared to the gold- standard RT-PCR is 99.9%, and the specificity following a procedure of neutralization to confirm its presence is nearly 100% (Schupbach et al, Int. J Afztifnicrob Agents, 16 (4): 441- 445 (2000) ). However, this method possesses a maximum sensitivity when testing serum of only about 10 pg/ml, making it ineffective in measuring low copy numbers of virus.

Nevertheless, the p24 antigen assay has been used extensively for monitoring HIV-1 disease progression, although it is used less widely now because of HIV-1 RNA testing.

[07] Several advances in technology may be exploited for the development of a simple and ultra-sensitive p24 antigen test that can parallel the utility of HIV-1 RNA monitoring.

First, a signal amplification method has been reported to increase the analytical sensitivity of the p24 antigen ELISA test, making it suitable to monitor HIV-1 infection (Schupbach et al.

AIDS 10 : 1085-1090 (1996) ). This method, tyramide signal amplification (TSA), incorporates a compound with the capacity to generate a large number of reactive intermediates that will subsequently bind avidin/HRP to produce an increased amount of color (higher sensitivity in the detection of low levels of p24 antigen). The sensitivity was reported to approach that of RNA testing (200-400 viral copies/ml, or 12-24 fg/ml of p24 antigen) making it suitable for monitoring viral levels in blood after anti-retroviral treatment (Schupbach et al., Int. R Antimicrob Agents, 16 (4): 441-445 (2000) ). However with highly active antiretroviral therapy (HAART), viral loads may decrease to <50 copies/mL, which is a level undetectable by the TSA test.

[08] Second, the use of paramagnetic microspheres in immunoassays has been shown to increase sensitivity over other solid support formats (Miltenyi et al. Cytometry 11: 231-238 (1990) ). Microspheres allow increased reaction kinetics due to their greater surface area compared to the conventional or any other solid support system or solid phase format.

Because of their spherical characteristics, there are none of the steric or diffusion limitations noted with other solid support system formats, thereby allowing a more dynamic and turbulent incubation step for immunological reactions to proceed. Also, the highly effective washing of the microspheres reduces non-specific protein binding that has an increased tendency to occur when molecules are randomly affixed to stationary surfaces. Because of their small size, microspheres are rapidly re-suspended in solution and separate easily by magnetocapture platforms. These qualities allow immunologic reactions to proceed more efficiently, thereby being more effective in detecting small quantities of analyte. However, microsphere technology has not been applied to the detection of HIV-1 p24 antigen.

[09] The instant invention addresses the critical needs discussed above. Using a new technology that incorporates signal amplification, a simpler and less expensive detection assay is provided that offers ultra-sensitive detection, semi-quantitative measurements, rapid turn-around time for results, and portability.

SUMMARY OF THE INVENTION [10] Accordingly, an object of the present invention is to provide a simple and highly sensitive method for use, for example, in the detection of low levels of biomolecules in a test sample. For example, proteins and peptides that are of diagnostic or scientific significance may be detected in a test sample. Such proteins include viral antigens and prions that may be present in blood, neuronal tissue or urine at very low levels, and biowarfare reagents such as ricin and botulinum toxin that may be present in an environmental sample.

[11] An additional object of the present invention is to provide a method that may be used in (1) the early diagnosis of HIV-1 infection, (2) the determination of viral load for treatment decisions, and (3) the monitoring of an individual's response to HIV-1 treatment, through the testing of any human body fluid such as plasma, serum, saliva, and whole blood for the presence of the p24 antigen.

[12] A further object of the present invention is to provide a kit comprising the elements needed to practice the methods described above.

[13] In one embodiment, the above-described objects of the present invention have been met by a method for detecting a biomolecule in a sample. The method comprises: (a) incubating a sample with a capture molecule that binds a selected biomolecule under conditions such that said selected biomolecule in said sample is bound by said capture molecule, wherein said capture molecule comprises a molecule that specifically binds to said selected biomolecule and said capture molecule is attached to a solid support, (b) incubating the resulting product of step (a) with a detector molecule that specifically binds said selected biomolecule under conditions such that said selected biomolecule bound by said capture molecule is also bound by said detector molecule, wherein said detector molecule comprises (i) a molecule that specifically binds to said biomolecule and (ii) at least one biotin moiety, (c) incubating the resulting product of step (b) with a linker molecule under conditions such that said linker molecule binds to a biotin moiety of said detector molecule, wherein said linker molecule comprises at least one avidin moiety or at least one streptavidin moiety, or a combination thereof, (d) incubating the resulting product of step (c) with an amplification reagent under conditions such that said amplification reagent binds to said linker molecule, wherein said amplification reagent comprises (i) a scaffold molecule and (ii) two or more biotin moieties, (e) incubating the resulting product of step (d) with a signal molecule under conditions such that said signal molecule binds to at least one biotin moiety of said amplification reagent, wherein said signal molecule comprises (i) a molecule that can be detected and (ii) at least one avidin moiety or at least one streptavidin moiety, or a combination thereof, and (f) detecting said signal molecule in the resulting product of step (e), thereby detecting a biomolecule in said sample.

[14] In a further embodiment, the above-described objects of the present invention have been met by a second method for detecting a biomolecule in a sample. The second method comprises: (a) incubating a sample with a capture molecule that binds a selected biomolecule under conditions such that said selected biomolecule in said sample is bound by said capture molecule, wherein said capture molecule comprises a molecule that specifically binds to said selected biomolecule and said capture molecule is attached to a solid support, (b) incubating the resulting product of step (a) with a detector molecule that specifically binds said selected biomolecule under conditions such that said selected biomolecule bound by said capture molecule is also bound by said detector molecule, wherein said detector molecule comprises (i) a molecule that specifically binds to said biomolecule and (ii) at least one avidin moiety or at least one streptavidin moiety, or a combination thereof, (c) incubating the resulting product of step (b) with an amplification reagent under conditions such that said amplification reagent binds to said avidin or said streptavidin, or both, of said detector molecule, wherein said amplification reagent comprises (i) a scaffold molecule and (ii) two or more biotin moieties, (d) incubating the resulting product of step (c) with a signal molecule under conditions such that said signal molecule binds to at least one biotin moiety of said amplification reagent, wherein said signal molecule comprises (i) a molecule that can be detected and (ii) at least one avidin moiety or at least one streptavidin moiety, or a combination thereof, and (e) detecting said signal molecule in the resulting product of (d), thereby detecting a biomolecule in said sample.

[15] In an additional embodiment, the above-described objects of the present invention have been met in a third method for detecting a biomolecule in a sample. The third method comprises: (A) incubating a sample with a capture molecule that binds a selected biomolecule under conditions such that said selected biomolecule in said sample is bound by said capture molecule, wherein said capture molecule comprises a molecule that specifically binds to said selected biomolecule and said capture molecule is attached to a solid support, (B) incubating the resulting product of step (A) with a detector-signal conjugate, said conjugate comprising (i) a detector molecule comprising (a) a molecule that specifically binds to said biomolecule and (b) at least one biotin moiety, (ii) a linker molecule bound to a biotin moiety of said detector molecule, wherein said linker molecule comprises at least one avidin moiety or at least one streptavidin moiety, or a combination thereof, (iii) an amplification reagent bound to an avidin moiety or a streptavidin moiety, or both, of said linker molecule, wherein said amplification reagent comprises (a) a scaffold molecule and (b) two or more biotin moieties, (iv) a signal molecule bound to a biotin moiety of said amplification reagent, wherein said signal molecule comprises (a) a molecule that can be detected and (b) at least one avidin moiety or at least one streptavidin moiety, or a combination thereof, and (C) detecting said signal molecule in the resulting product of step (B), thereby detecting a biomolecule in a sample.

[16j In a preferred embodiment, the above-described objects of the present invention have been met in a method for detecting HIV-1 p24 antigen in a sample. The method comprises: (a) incubating a sample with a capture antibody that specifically binds p24 antigen under conditions such that p24 antigen in said sample is bound by said capture antibody, wherein said capture antibody is attached to a solid support, (b) incubating the resulting product of step (a) with a detector antibody that specifically binds p24 antigen under conditions such that p24 antigen bound by said capture antibody is also bound by said detector antibody, wherein said detector antibody comprises at least one biotin moiety, (c) incubating the resulting product of step (b) with a linker molecule under conditions such that said linker molecule binds to a biotin moiety of said detector antibody, wherein said linker molecule comprises at least one avidin moiety or at least one streptavidin moiety, or a combination thereof, (d) incubating the resulting product of step (c) with an amplification reagent under conditions such that said amplification reagent binds to said linker molecule, wherein said amplification reagent comprises (i) a scaffold molecule and (ii) two or more biotin moieties, (e) incubating the resulting product of step (d) with a signal molecule under conditions such that said signal molecule binds to at least one biotin moiety of said amplification reagent, wherein said signal molecule comprises (i) a molecule that can be detected and (ii) at least one avidin moiety or at least one streptavidin moiety, or a combination thereof, and (f) detecting said signal molecule in the resulting product of step (e), thereby detecting HIV-1 p24 antigen in said sample.

[17] In a further embodiment, the above-described objects of the present invention have been met in a kit comprising the element necessary to practice the methods of the invention.

The kit comprises: (a) a capture molecule attached to a solid support, wherein said capture molecule comprises a molecule that specifically binds to pre-selected biomolecule, (b) a detector molecule, wherein said detector molecule comprises (i) a molecule that specifically binds to said pre-selected biomolecule and (ii) at least one biotin moiety, (c) an linker molecule, wherein said linker molecule comprises at least one avidin moiety or at least one streptavidin moiety, or a combination thereof, (d) an amplification reagent, wherein said amplification reagent comprises (i) a scaffold molecule and (ii) two or more biotin moieties, (e) a signal molecule, wherein said signal molecule comprises (i) a molecule that can be detected and (ii) at least one avidin moiety or at least one streptavidin moiety, or a combination thereof, and (f) a set of standards, wherein said standards allow an approximation of concentration of said pre-selected biomolecule in a sample.

[18] In still a further embodiment, the above-described objects of the present invention have been met in a second kit comprising the element necessary to practice the methods of the invention. The second kit comprises: (a) an anti-p24 antigen capture molecule attached to a solid support, wherein said capture molecule comprises a molecule that specifically binds to HIV-1 p24 antigen, (b) an anti-p24 antigen detector molecule, wherein said detector molecule comprises (i) a molecule that specifically binds to HIV-1 p24 antigen and (ii) at least one biotin moiety, (c) an linker molecule, wherein said linker molecule comprises at least one avidin moiety or at least one streptavidin moiety, or a combination thereof, (d) an amplification reagent, wherein said amplification reagent comprises (i) a scaffold molecule and (ii) two or more biotin moieties, (e) a signal molecule, wherein said signal molecule comprises (i) a molecule that can be detected and (ii) at least one avidin moiety or at least one streptavidin moiety, or a combination thereof, and (f) a set of standards, wherein said standards approximate a range of between about 25,000 and 500,000 copies of HIV-1 RNA.

[19] In still a further embodiment, the above-described objects of the present invention have been met in a third kit comprising the element necessary to practice the methods of the invention. The third kit comprises: (a) an anti-p24 antigen capture molecule attached to a solid support, wherein said capture molecule comprises a molecule that specifically binds to HIV-1 p24 antigen, (b) an anti-p24 antigen detector molecule, wherein said detector molecule comprises (i) a molecule that specifically binds to HIV-1 p24 antigen and (ii) at least one avidin moiety, (c) an amplification reagent, wherein said amplification reagent comprises (i) a scaffold molecule and (ii) two or more biotin moieties, (d) a signal molecule, wherein said signal molecule comprises (i) a molecule that can be detected and (ii) at least one avidin moiety or at least one streptavidin moiety, or a combination thereof, and (e) a set of standards, wherein said standards approximate a range of between about 25,000 and 500,000 copies of HIV-1 RNA.

BRIEF DESCRIPTION OF THE DRAWINGS [20] Figure 1 shows the four main steps of one embodiment of the invention: immunocapture, detection, amplification, and color production used to detect the HIV-1 p24 antigen (3). The immunocapture step uses a capture antibody (2) specific for the p24 antigen attached to a solid support (1) (microspheres) to immobilize free p24 antigen (3) from a test sample. The detection step comprises the addition of a different anti-p24 antibody that is biotinylated (4) (the detector molecule), followed by the addition of a linker molecule (5) (such as avidin or streptavidin) that binds to the biotinylated capture antibody. Preferably, attached to the linker molecule is a signal molecule that is used in the color production step.

The amplification step comprises addition of an amplification reagent (6) that binds to the linker molecule, where the amplification reagent comprises (i) a scaffold molecule and (ii) two or more biotin moieties. A signal molecule (7), comprising a detection molecule and an avidin moiety or a streptavidin moiety, is then added and allowed to bind a biotin moiety of the amplification reagent (6). In the color production step, the signal molecule is detected by the production of a visible color (8).

[21] Figure 2 shows a further embodiment of the invention where a re-amplification step is employed. After the addition of the amplification reagent (6) and the signal molecule (7), additional amplification reagent (8) and signal molecule (9) is added, followed by color production (10).

[22] Figure 3 shows an additional embodiment of the invention where an avidinylated detector antibody (4) is added. The avidinylated detector antibody is a combination of the biotinylated anti-p24 antigen detector antibody and avidin linker molecule shown in Figures 1 and 2.

[23] Figure 4 shows a further embodiment of the invention where the detector antibody is directly conjugated to the amplification reagent as a detector anti-p24 antigen bio-DNA conjugate (4).

[24] Figure 5 shows increased signal to noise ratios after the first cycle of bio-DNA/SA- HRP addition compared to an ELISA assay, for the detection of HIV-1 p24 antigen captured on paramagnetic microspheres.

[25] Figure 6 shows increased sensitivity using bio-DNA/avidin/SAHRP compared with ) an ELISA for the detection of HIV-1 p24 antigen.

[26] Figure 7 shows the results of signal to noise (S/N) ratio determinations from OD measurements of replicates of 2-3 experiments for the detection of HIV-1 p24 antigen.

Quantification of the HIV-1 infected culture supernatants had been previously performed by routine ELISA. The negative control was plasma diluted 9: 1 in viral lysis buffer (5% Triton-X, PBS). The cutoff for a positive sample is a S/N ratio > 2.0.

DETAILED DESCRIPTION OF THE INVENTION [27] Generally, the methods of the present invention comprise application and binding of a capture molecule to a solid support, incubation of the capture molecule-bound support with a test sample containing a biomolecule to be detected, addition of a detector molecule, addition of a linker molecule, addition of an amplification reagent, addition of a linker molecule-signal molecule conjugate and visual reading of the results (or reading with a simple instrument) at ambient temperatures.

[28] The technique uses substrate-based signal amplification methodologies that couple protein detection with several levels of amplification of a signal molecule, such as colorimetric signal amplification. Presently, there are no other products that use a colorimetric amplification technology in a rapid test format. Although the TSA method for HIV-1 p24 antigen ELISA is a signal amplification technology, it does not possess the procedural simplicity, robustness (room temperature performance and storage), or allow for the rapid (less than one hour) turnaround time for results. Further, it lacks sensitivity of detection past 0.5 pg/mL. Signal amplification methods are used for HIV-1 RNA detection, but these methods are very expensive, require significant laboratory infrastructure, and have long turnaround times.

[29] In one embodiment, the present invention relates to a method for detecting a biomolecule in a sample. The method is practiced in an assay system through the following steps: (a) incubating a sample with a capture molecule, where the capture molecule specifically binds a selected biomolecule that may be present in the sample and where the capture molecule is attached to a solid support, (b) adding a detector molecule that specifically binds the selected biomolecule, under conditions such that the selected biomolecule bound by said capture molecule is also bound by the detector molecule, where the detector molecule comprises (i) a molecule that specifically binds to the biomolecule and (ii) at least one biotin moiety, (c) adding a linker molecule under conditions such that the linker molecule binds to a biotin moiety of the detector molecule, where the linker molecule comprises at least one avidin moiety or at least one streptavidin moiety, or a combination of both, (d) adding an amplification reagent under conditions such that the amplification reagent binds to the linker molecule, where the amplification reagent comprises (i) a scaffold molecule and (ii) two or more biotin moieties, (e) adding a signal molecule under conditions such that the signal molecule binds to at least one biotin moiety of the amplification reagent, wherein the signal molecule comprises (i) a molecule that can be detected and (ii) at least one avidin moiety or at least one streptavidin moiety, or a combination of both, and (f) detecting the signal molecule. The detection of the signal molecule will reveal the presence of the selected biomolecule in the sample.

[30] An alternative method for detecting a biomolecule in a sample is also provided.

The alternative method comprises: (a) incubating a sample with a capture molecule, where the capture molecule specifically binds a selected biomolecule that may be present in the sample and where the capture molecule is attached to a solid support, (b) adding a detector molecule that specifically binds the selected biomolecule under conditions such that the selected biomolecule bound by the capture molecule is also bound by the detector molecule, where the detector molecule comprises (i) a molecule that specifically binds to the biomolecule and (ii) at least one avidin moiety or at least one streptavidin moiety, or a combination of both, (c) adding an amplification reagent under conditions such that the amplification reagent binds to the avidin or said streptavidin, or both, of the detector molecule, where the amplification reagent comprises (i) a scaffold molecule and (ii) two or more biotin moieties, (d) adding a signal molecule under conditions such that the signal molecule binds to at least one biotin moiety of the amplification reagent, where the signal molecule comprises (i) a molecule that can be detected and (ii) at least one avidin moiety or at least one streptavidin moiety, or a combination of both, and (e) detecting the signal molecule. The detection of the signal molecule will reveal the presence of the selected biomolecule in the sample.

[31] An further alternative method for detecting a biomolecule in a sample is provided.

The further alternative method comprises: (A) incubating a sample with a capture molecule, where the capture molecule specifically binds a selected biomolecule that may be present in the sample and where the capture molecule is attached to a solid support, (B) adding a detector-signal conjugate, where the conjugate comprises: (i) a detector molecule comprising (a) a molecule that specifically binds to the selected biomolecule and (b) at least one biotin moiety, (ii) a linker molecule bound to a biotin moiety of the detector molecule, wherein the linker molecule comprises at least one avidin moiety or at least one streptavidin moiety, or a combination of both, (iii) an amplification reagent bound to an avidin moiety or a streptavidin moiety, or both, of the linker molecule, wherein the amplification reagent comprises (a) a scaffold molecule and (b) two or more biotin moieties, (iv) a signal molecule bound to a biotin moiety of the amplification reagent, wherein the signal molecule comprises (a) a molecule that can be detected and (b) at least one avidin moiety or at least one streptavidin moiety, or a combination of both, and (C) detecting the signal molecule. The detection of the signal molecule will reveal the presence of the selected biomolecule in the sample.

[32] In a preferred embodiment, the present invention relates to a method for detecting the HIV-1 p24 antigen in a sample.

[33] This embodiment provides advantages in the detection of low levels of HIV-1 in a sample through (1) higher sensitivity than is attainable by current p24 antigen ELISA methods, (2) a cost-effective semi-quantitative serologic method that parallels viral load (RNA) measurements, (3) a simple procedure with visually read results, and (4) a format that allows performance in facilities where technical expertise is limited and which lack stable electricity or sufficient infrastructure.

[34] The HIV-1 p24 antigen assay allows early detection of HIV-1 infection by determining viral load before the expression of HIV-1 antibody occurs. Furthermore, it provides results that are available in less than 1 hour, as compared to RNA methods that require about 6 hours, and p24 antigen methods that require several hours. Such early and rapid determination of patient HIV-1 viral load allows immediate assessment of viral burden, and an easy method for detecting (1) early infection when screening blood units in facilities that cannot support RNA testing and (2) the occurrence of viral rebound when treatment regimens fail, thereby signaling the need for changes in therapy.

[35] The method of the preferred embodiment is practiced in an assay system through the following steps: (a) incubating a sample with a capture molecule, where the capture molecule specifically recognizes and binds to the HIV-1 p24 antigen that may be present in the sample and where the capture molecule is attached to a solid support, (b) adding a detector antibody that specifically binds p24 antigen under conditions such that p24 antigen bound by the capture antibody is also bound by the detector antibody, where the detector antibody comprises at least one biotin moiety, (c) adding a linker molecule under conditions such that the linker molecule binds to a biotin moiety of the detector antibody, where the linker molecule comprises at least one avidin moiety or at least one streptavidin moiety, or a combination of both, (d) adding an amplification reagent under conditions such that the amplification reagent binds to the linker molecule, where the amplification reagent comprises (i) a scaffold molecule and (ii) two or more biotin moieties, (e) adding a signal molecule under conditions such that the signal molecule binds to at least one biotin moiety of the amplification reagent, where the signal molecule comprises (i) a molecule that can be detected and (ii) at least one avidin moiety or at least one streptavidin moiety, or a combination of both, and (f) detecting the signal molecule. The detection of the signal molecule will reveal the presence of the p24 antigen in the sample.

Biomolecule [36] The specific biomolecules to be detected in the methods of the present invention are not particularly limited. The methods may be used to detect any biomolecule that may be present at low levels in a test sample. Exemplary molecules that may be detected using the methods of the present invention include polypeptides and peptides, glycoproteins, lipids, carbohydrates, nucleic acids, or any combination thereof [37] In particular, the methods of the present invention are useful in the detection and/or monitoring of biomolecules such as viral antigens and prions that may be present in a biological sample, and biowarfare reagents such as ricin and botulinum toxin that may be present in an environmental sample or a biological sample. Specific toxins include Staphylococcal enterotoxin B (SEB), Epsilon toxin of Clostridium perfringens and anthrax proteins.

[38] Included with the scope of the tenn"biomolecule"are synthetic compounds, both those having chemical characteristics of biomolecules and those that are not carbon based, such as synthetic toxins.

[39] More particularly, the methods of the present invention are useful in the detection and/or monitoring of the HIV-1 p24 antigen in a biological fluid.

Test sample [40] The test sample used in the methods of the present invention may be from any source that may contain a selected biomolecule of interest. When the methods are used for the detection of biomolecules from living organisms, such as in the detection of a viral antigen, a prion or a toxin, preferably the source of the test sample is a biological sample, such as plasma, serum, saliva, whole blood, semen, cerebrospinal fluid, urine, neuronal tissue or nasal material. Nasal material can include mucosal secretions from the anterior nasal passage or sinuses (secretions).

[41] When the methods are used for the detecting of the HIV-1 p24 antigen, the source of the test sample is any in which the HIV-1 p24 antigen may be found at a detectable level using the method. For example, plasma, serum, saliva, whole blood, semen, and cerebrospinal fluid may be used. Preferably, the sample is plasma or serum.

[42] When the methods are used for the detection of biomolecules in environmental sample, such as in the detection of toxins, the source of the test sample may be water, soil, air or any biological material.

[43] The biological sample may be used directly isolated from a subject. Alternatively, the sample may be frozen to preserve it prior to use in the assay, or stored refrigerated for about two weeks. The sample may also be concentrated by a molecular sizing column, filtration, or centrifugation in order to increase the sensitivity of the assay.

[44] The sample may also be fractionated, for example to remove molecules that may interfere with the detection of the selected biomolecule; supplemented, for example to add factors, such as blocking reagents (e. g. BSA, casein, Triton X-100, polymers, or nucleic acids) that increase the ability of the capture molecule and the detector molecule to recognize and/or bind the selected biomolecule; and subjected to column purification, filtration, centrifugation, dialysis, lysis, and/or enzymatic digestion. The sample may also be processed by heating or boiling it, or through acidification or neutralization. For example, the sample could be heated to at least 95°C for 3 to 8 minutes, preferably 100°C for 5 minutes. The sample may also be processed through a combination of treatments, such as heating an acidified sample.

Support [45] The solid support may be any structure that provides a support for the capture molecule. Preferably, the solid support is a membrane, paramagnetic or latex microsphere, or microtiter well. When the solid support is a material such as a paramagnetic or latex microsphere, the solid support may be contained in an open container, such as a multi-well tissue culture dish, or in a sealed container, such as a screw-top tube, both of which are commonly used in laboratories. The skilled artisan will understand that the circumstances under which the methods of the current invention are performed will govern which solid supports are most preferred and whether a container is used.

[46] In a preferred embodiment, the solid support is a paramagnetic microsphere which contains surface modifications for efficient conjugation of the capture molecule. Preferably, the paramagnetic microsphere is composed of Fe203 or Fe304 particles coated with a polymer (polystyrene) shell. Preferably, the size of the paramagnetic microsphere is about 50 nm to about 4.5 um, more preferably about 1-3 um. Preferably, the paramagnetic microsphere is coated with Protein A, Protein G, carboxylate or amine modifications to aid in the attachment of capture molecule to the surface of the microspheres. Exemplary paramagnetic microspheres include those available from Dynal, Inc. (catalogue # 142. 03) ; Miltenyi Biotec Inc. (catalogue # 130-048-102); Sigma Corp. (catalogue # CLB4); and Bionor Inc (catalogue &num in-hiv-conf03).

Capture Molecule and Detector Molecule [47] The methods of the present invention may be adapted for the detection of any biomolecule by simply altering the capture molecule and the biomolecule-binding portion of the detector molecule used in the method (e. g. , the capture antibody attached to the solid support and the biotinylated detector antibody) such that the capture molecule and the biomolecule-binding portion of the detector molecules utilized specifically recognize and bind the biomolecule for which the method is being used.

[48] The capture molecule and the biomolecule-binding portion of the detector molecule may recognized and bind the same or different portions or epitopes of the biomolecule under investigation. Preferably, the capture molecule and the biomolecule-binding portion of the detector molecule recognize and bind different portions or epitopes of the biomolecule.

[49] The specific molecules used as the capture molecule and the biomolecule-binding portion of the detector molecules used in the methods of the present invention are not particularly limited. Molecules useful as the capture molecule and the biomolecule-binding portion of the detector molecules include monoclonal, polyclonal, or phage derived antibodies, antibody fragments, haptens, nucleic acids, nucleic acid aptamers, protein A, protein G, folate, folate binding proteins, maleimide and sulfhydryl reactive. groups both those commercially available and those that may be produced for use with the methods of the present invention.

[50] Preferably, the capture molecule and the biomolecule-binding portion of the detector molecules are monoclonal, polyclonal, or phage derived antibodies, or antibody fragments. More preferably, the capture molecule and the biomolecule-binding portion of the detector molecules are monoclonal antibodies.

[51] Any method for generating monoclonal antibodies, for example by in vitro generation with phage display technology and in vivo generation by immunizing animals, such as mice, can be used in the present invention. These methods include the immunological methods described by Kohler and Milstein (Nature 256,495-497 (1975) ) and Campbell ("Monoclonal Antibody Technology, The Production and Characterization of Rodent and Human Hybridomas"in Burdon et al. , Eds. , Laboratory Techniques in Biochemistry and Molecular Biology, Volume 13, Elsevier Science Publishers, Amsterdam (1995) ); as well as by the recombinant DNA method described by Huse et al. (Science 246, 1275-1281 (1989) ). Standard recombinant DNA techniques are described in Sambrook et al.

("Molecular Cloning, "Second Edition, Cold Spring Harbor Laboratory Press (1987) ) and Ausubel ("Current Protocols in Molecular Biology, "Green Publishing Associates/Wiley- Interscience, New York (1990) ). Each of these publications, and the methods disclosed therein, is incorporated herein by reference.

[52] Other binding molecules include functional antibody equivalents that have binding characteristics that are comparable to those of the antibodies, and include, for example, chimerized, humanized, and single-chain antibodies as well as fragments thereof may also be used. Methods of producing such functional equivalents are disclosed in PCT Application WO 93/21319, European Patent Application No. 239, 400 ; PCT Application WO 89/09622; European Patent Application 338, 745 ; and European Patent Application EP 332,424. Each of these publications, and the methods disclosed therein, is incorporated herein by reference [53] The capture molecule and the biomolecule-binding portion of the detector molecule are not limited to intact antibodies, but encompass other binding molecules such as antibody fragments and recombinant fusion proteins comprising an antibody fragment.

[54] As used herein, "antibody fragments"include any portion of an antibody that retains the ability to bind to the epitope recognized by the full length antibody, generally termed "epitope-binding fragments. "Examples of antibody fragments preferably include, but are not limited to, Fab, Fab', and F (ab') 2, Fd, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv) and fragments comprising either a VL or VH domain. Epitope- binding fragments, including single-chain antibodies, may comprise the variable region (s) alone or in combination with the entirety or a portion of the following: hinge region, CH1, CH2, and CH3 domains.

[55] The antibodies and binding molecules of the present invention should have a high affinity for the selected biomolecule under investigation. Preferably, when antibodies are used as the capture molecule and in the detector molecules, they will have an affinity of 10-6- 10-l°/M, more preferably they will have an affinity of at least 10-8/M, most preferably they will have an affinity of at least 10-9/M.

[56] Exemplary capture molecules for use in the detection of the HIV-1 p24 antigen in a test sample include the anti-p24 antigen monoclonal antibodies 13B6 and 13G4 produced by Dr. George Lewis at the University of Maryland, or others that are commercially available from Dako Corp. (anti-HIV-1 p24 Val-1, catalogue # M857), Immunodiagnostics Corp.

(Human p24 polyclonal antibody IgG), and Perkin-Elmer Life Sciences, Inc. (HIV-1 anti- p24 rabbit polyclonal antibody).

[57] Quantities of the capture molecule to be attached to the solid support will be determined empirically by checkerboard titration with different quantities of biomolecule that would be expected to mimic quantities in a test sample. Generally, the quantity of the biomolecule in the test sample is expected to be in the microgram to milligram range. An unknown concentration of the biomolecule (e. g. , HIV-1 p24 antigen) in a test sample will be added at specified volumes, and this will influence the sensitivity of the test. If large volumes of the test sample (e. g. , 200-400 uL) are used, modification of the rapid test format may be needed to allow for the larger sample volumes. Generally, however, the concentration of the capture molecule will be about 2 to about 5 micrograms per mL.

[58] The capture molecule can be attached to a solid support by routine methods that have been described for attachment of biomolecules to plastic or other solid support systems (e. g. , membranes or microspheres). Examples of such methods may be found in U. S. Patent No. 4,045, 384 and U. S. Patent No. 4,046, 723, both of which are incorporated herein by reference.

[59] Conjugation should be optimized to ensure stabilization of the colloidal suspension, maintenance of the binding ability of the capture molecule, and minimization of surface interaction with other molecules present in the test reaction.

[60] Attachment of the capture molecule to membranes may be performed by direct addition in PBS, or other buffers of defined pH, followed by drying in a convection oven.

[61] In practicing the methods of the present invention, a test sample suspected of containing the selected biomolecule under investigation is applied to the support containing the capture molecule. Depending on the identity of the support, the support may be contained within a culture device of some type. When the support is a membrane, for example, a swallow glass dish slightly bigger that the length and width of the membrane may be used. When the support is a microsphere, the microspheres may be contained in a tube, such as a polypropylene or polystyrene microfuge or screw-top tube. The identity of the container is not critical, but it should be constructed of a material to which the reagents used in the methods of the present invention do not adhere.

[62] The quantity of test sample used is not critical, but should be an amount that can be easily handled and that has a concentration of biomolecule that is detectable within the limits of the methods of the present invention. The test sample should also be sufficient to adequately cover the support, and may be diluted if needed in this regard. For example, the quantity of the test sample may be between 5 uL and 2 mL. Preferably, the quantity of the test sample is be between 5 uL and 1 mL. Most preferably, the quantity of the test sample may be between 5 uL and 200 uL.

[63] While the concentration of the biomolecule in the test sample is not critical, it should be within the detection limits of the methods of the present invention. The skilled artisan will understand that the concentration may vary depending on the volume of the test sample, and thus it is difficult to provide a concentration range over which a biomolecule may be detected. However, biomolecules present at concentrations in the femtogram (10-15) range, can be detected using the methods of the present invention. Therefore, preferably a test sample used in the methods contains between about 1 x 10-6 g and about 1 x 10-15 g of the biomolecule, more preferably between about 1 x 10-6 g and about 1 x 10-riz g of the biomolecule, most preferably between about 1 x 10~6 g and about 1 x 10-9 g of the biomolecule.

[64] The limits of detection of the methods of the present invention are a function of a number of factors based on the identity of the biomolecule of interest. However, the method can generally be used to detect biomolecule, such as proteins, at levels as low as 1,3, 5,7, 10,20, 30,40, 50,60, 70,80, 90,100, 150,200, 250,300, 350,400, 450,500, 750,1000, 1250,1500, 2000 or 3000 fg/ml of test sample.

[65] The length of time during which the capture molecule-bearing support is incubated with the test sample is not critical. Preferably, the incubation proceeds from between about 10 minutes and about 60 minutes. More preferably, the incubation proceeds from between about 10 minutes and about 30 minutes. Most preferably, the incubation proceeds from between about 10 minutes and about 15 minutes.

[66] The temperature at which each of the incubation steps of the methods is performed is also not critical. Preferably, the temperature at which the incubations occur is between about 18°C and about 37°C. More preferably, the incubation temperature is between about 18°C and about 30°C. Most preferably, the incubation temperature is at ambient temperature (20°C).

[67] While each of the incubation steps of the present invention can take place in a fixed, stationary position, it is preferable that the incubation steps occur under gentle agitation, rocking or shaking. Such movement ensures proper mixing and exposure of all of the elements used in the method. For example, when paramagnetic microspheres are used as the support, the test sample and reagents can be combined in a microtiter well or sealed tube, and mixed on a magnetocapture platform, such as the Bionor Platform (Bionor Corp, Norway). The Bionor Platform is small (20 cm x 30 cm), easily carried, and can be connected to a 12V car battery for portability.

Biotinylation of Detector Molecules [68] Following the incubation of the capture molecule-bearing support and the test sample, a detector molecule is added to the assay system under conditions that allow the detector molecule to recognize and bind the selected biomolecule that is bound by the / capture molecule, which in turn, is attached to the solid support.

[69] The detector molecule is comprised of two parts, (i) a molecule that specifically binds to the selected biomolecule, and (ii) at least one biotin moiety.

[70] As described above, the portion of the detector molecule that specifically binds to the selected biomolecule is not critical and includes monoclonal, polyclonal, or phage derived antibodies, antibody fragments, haptens, nucleic acids, nucleic acid aptamers, protein A, protein G, folate, folate binding proteins, maleimide and sulfhydryl reactive groups, both those commercially available and those that may be produced for use with the methods of the present invention. Preferably, the biomolecule-binding portion of the detector molecule is a monoclonal, polyclonal, or phage derived antibody, or antibody fragment. More preferably, the biomolecule-binding portion of the detector molecule is a monoclonal antibody.

[71] The biotinylation of the detector molecule may be by routine methods (Altin et al.

Anal Biochem. 224: 382-389 (1995), incorporated herein by reference) or through the use of a commercial biotinylation kit, such as EZ-NHS-LC-biotin (Pierce Biotechnology Inc., Rockford, IL, USA). Preferably, at least 1-4 biotin molecules are incorporated per nucleotide of the detector molecule when a polynucleotide is used. More preferably, at least 3-4 biotin molecules are incorporated per detector molecule.

[72] Biotinylated detector molecules may be purified by dialysis against PBS using a molecular weight exclusion of 10,000 kD. The extent of detector molecule biotinylation may be determined in the final preparation using an avidin-HABA reagent to determine the molar ratio of biotin to detector molecule (Green N. M. Biochem J. 94: 32c-24c (1965), incorporated herein by reference).' [73] After the biotinylated detector molecule has been prepared, it is added to the assay system. The amount of biotinylated detector molecule added to the assay system depends on the quantity of the test sample used, and the surface area of the support. Thus the skilled artisan would understand that the concentration of biotinylated detector molecule added to the assay system will depend on the quantities of other elements already added. However, it is preferable that a quantity of biotinylated detector molecule between 0.1 and 1 times the amount of the capture molecule attached to the solid support be added. More preferably, a quantity of biotinylated detector molecule between 0.5 and 1 times the amount of the capture molecule attached to the solid support. Most preferably, a quantity of biotinylated detector molecule between 0.1 and 0.25 times the amount of the capture molecule attached to the solid support.

[74] The length of time during which the detector molecule is incubated in the assay system is not critical. Preferably, the incubation proceeds from between about 10 minutes and about 60 minutes. More preferably, the incubation proceeds from between about 10 minutes and about 30 minutes. Most preferably, the incubation proceeds from between about 10 minutes and about 15 minutes.

Linker Molecule [75] The linker molecule used in the methods of the present invention serves as a bridge between the biotinylated detector molecule and the amplification reagent. The linker molecule is preferably avidin or streptavidin, or polymerized avidin or streptavidin.

[76] In preferred embodiments, the linker molecule is conjugated to a signal molecule.

Preferred signal molecules are discussed below. In a more preferred embodiment, the linker molecule is avidin conjugated to horseradish peroxidase (HRP) (avidin-HRP) or streptavidin-HRP. Such a linker molecule-signal molecule conjugate allows the same reagent to be added before and after the addition of the amplification reagent, thus reducing the number of different reagents needed to practice the methods. However, the skilled artisan will understand that an unconjugated linker molecule may be preferred under some circumstances to form the bridge between the biotinylated detector molecule and the amplification reagent.

[77] When avidin-HRP is used as the linker molecule-signal molecule conjugate, the formation of a bridge between the biotinylated detector molecule and the amplification reagent is dependent on the molar ratio of avidin-HRP to biotin. To obtain the highest sensitivity and the lowest non-specific background, due to the potential of avidin-HRP to bind to other reagents or the solid support, the amount of avidin-HRP may be varied, starting with a molar excess and subsequently lowering the amount, in the manner reported by Saito et al. , 1999 (Saito et al. Clin. Chem. 45 (5): 665-669 (1999); see also methods of determining the concentration of avidin-HRP to biotin as described by Niemeyer et al. Nuc. Acid Res.

27 (23): 4553-4561 (1999); both references are incorporated herein by reference). Assessment may also be performed using different concentrations of the amplification reagent in a matrix design. The exact cause of any background from avidin-HRP may be assessed by eliminating one reagent at a time, and noting unexpected signal.

[78] The source of avidin-HRP is not particularly limited, and may be commercially obtained such as through Sigma-Aldrich Corp. or Pierce Biotechnology Inc.

[79] The length of time during which the linker molecule is incubated in the assay system is not critical. Preferably, the incubation proceeds from between about 10 minutes and about 60 minutes. More preferably, the incubation proceeds from between about 10 minutes and about 30 minutes. Most preferably, the incubation proceeds from between about 10 minutes and about 15 minutes.

Amplification [80] If the concentration of the biomolecule in the test sample is great enough, and a linker molecule-signal molecule conjugate is utilized, the amplification/re-amplification step may be bypassed and the signal production step may be performed (as described below) after incubation of the linker molecule-signal molecule conjugate in the assay system.

[81] However, an important feature of the invention is the ability to easily and quickly detect the presence of very small amounts of a biomolecule in a test sample. The speed and ease of the method are based in part on the use of the amplification/re-amplification step.

[82] The amplification reagent is comprised of two elements, (i) a scaffold molecule and (ii) two or more biotin moieties.

[83] The scaffold molecule serves as a platform for the attachment of multiple biotin moieties, to which multiple signal molecules may attached, thus increasing the number of signal molecules per biomolecule in the test sample. The identity of the scaffold molecule employed is not critical. Preferred examples of the scaffold molecule include synthetic or natural, organic or inorganic polymers of nucleic acids, such as DNA and RNA, proteins, carbohydrates, and lipids, or any combination thereof.

[84] When a polynucleotide is used as the amplification reagent, the polynucleotide may be a linear polynucleotide, such as a linear DNA (bio-DNA) or RNA molecule, or a circular polynucleotide, such as a circular DNA or RNA molecule. Preferably, the scaffold molecule is a DNA polynucleotide or a RNA polynucleotide.

[85] An example of a DNA polynucleotide that may be used as the scaffold molecule is a 500 bp sequence from bacteriophage lambda with a GC content of 30-50%. The 500 bp bacteriophage lambda DNA is obtained by PCR amplification of lambda DNA with the following primers: 5'primer: 5'-GATGAGTTCGTGTCCGTACAACTGG-3' (SEQ ID NO : 1) 3'primer : 5'-GGTTATCGAAATCAGCCACAGCG-3' (SEQ ID NO : 2).

(see, Kuehnelt, D. M. , et al. Quantitative PCR of bacteriophage lambda DNA using a second- generation thermocycler. PCR Methods & Applications. 3: 369-371 (1994), incorporated herein by reference in its entirety).

[86] The size of the scaffold is also not critical, but should be large enough to serve as a platform for multiple biotin moieties. In a preferred example, the scaffold molecule contains between about 20 and 100 biotin moieties, more preferably between about 50 and 100 biotin moieties, and most preferably between about 75 and 100 biotin moieties.

[87] When a DNA polynucleotide is used as the scaffold molecule, the length of the polynucleotide is preferably between about 80 nucleotides and about 500 nucleotides.

[88] Each of the biotin moiety on the amplification reagent is capable of binding to the immobilized linker molecule. Each biotin moieties is also capable of binding to subsequently added signal molecules (in the form of a linker molecule-signal molecule conjugate). While the skilled artisan will understand how to biotinylate molecules such as antibodies and polynucleotides, reference is made to Altin et al. (Anal Biochem. 224: 382- 389 (1995) ), which is hereby incorporated by reference. Biotinylation kits are also available from commercial sources, such as the PCR Biotinylation Kit (KPL, Inc. , Gaithersburg, MD).

[89] The quantity of amplification reagent added to the assay system will be dependent on the quantity of the reagents added to the assay system in the previous steps. In general however, the quantity of amplification reagent added is such that the amount of biotin on the amplification reagent is at least 3 fold excess over the amount of linker molecule (avidin) previously added. Since each avidin is a tetrameric molecule capable of binding 4 biotin, in a preferred example, the quantity of amplification reagent added to the assay system is between about 3x and 12x fold excess, more preferably between about 3x and 9x fold excess, and most preferably between about 3x and 6x fold excess.

[90] The amount of the amplification reagent used in assay methods can severely affect background (Constantine et al. Ultra-low detection of HIV-1 p24 antigen using immuno- PCR. Abstract, XIV It7ter. AIDS Conf 2002, Barcelona, Spain, July 7-12,2002), resulting in a low signal to noise ratio. Assessment of the background may be performed using serial dilutions of the amplification reagent at concentrations of 1 pg/ml to 1 ng/ml, followed by assessment of the highest signal to noise ratio. Subsequently, modifications to this concentration may be performed after optimization of all other reagents, if necessary, such as performed by Niemeyer et al. (1999), incorporated herein by reference. Equally important for optimal amplification and signal/noise is the molar ratio of biotin to the scaffold molecule, and this may be assessed empirically, as mentioned above.

[91] The length of time during which the amplification reagent is incubated in the assay system is not critical. Preferably, the incubation proceeds from between about 10 minutes and about 60 minutes. More preferably, the incubation proceeds from between about 10 minutes and about 30 minutes. Most preferably, the incubation proceeds from between about 10 minutes and about 15 minutes.

Re-amplification [92] A second amplification step (re-amplification) can be performed to boost the sensitivity of the method. This re-amplification includes addition of more linker molecule and with subsequent addition of more amplification reagent. The re-amplification will produce a signal proportionate to the amount of biomolecule in the test sample. The amplification/re-amplification can be repeated as many times as desired to increase the sensitivity of the method. Preferably, the methods of the present invention repeat the amplification step 2,3, 4,5, 6,7, 8,9, or 10 or more times.

Attachment of Detector Molecule to the Amplification Reagent [93] Reducing the number of reagents that have the potential to non-specifically bind to other reagents or to the solid support may be helpful. Thus, in each embodiment of the present invention, one or more of the reagents can be combined into one conjugate.

[94] For example, (i) the portion of the detector molecule that binds to the biomolecule and (ii) the linker molecule can be joined prior to addition to the assay system. Thus, rather then using a biotinylated detector molecule and an avidin or streptavidin linker molecule, the detector molecule may be avidinylated. An example of such a conjugate would be a monoclonal antibody with at least one avidin moiety.

[95] Another example is a conjugate comprising (i) the portion of the detector molecule that binds to the biomolecule and (ii) the amplification reagent. Such a conjugate could be formed using the chemical methods of Hendrickson et al. (Nuc. Acid Res. 23 (3): 522-529 (1995), incorporated herein by reference). The amplification reagent may be conjugated to the portion of the detector molecule that binds to the biomolecule by other methods described in the literature. For example, a standard procedure is one in which a linker molecule (e. g., Sulfo-SMCC) is attached to both a detector antibody and an amplification reagent (Hendrickson et al. , 1995).

[96] As briefly mentioned above, another means by which the number of reagents may be reduced is to use a signal molecule-conjugated linker molecule for both (1) linking the biotinylated detector molecule to the amplification reagent, and (2) binding signal molecules to the amplification reagent. The use of such a linker molecule-signal molecule conjugate also serves to increase the amount of signal molecule in the assay system by linking a signal molecule to every linker molecule (e. g. , avidin) used. However, the skilled artisan will understand that there may be some applications where it is preferable to used an unconjugated linker molecule to link the biotinylated detector molecule to the amplification reagent.

[97] A further means by which the number of reagents may be reduced would be to combine the following components: (1) the detector molecule, (2) the amplification reagent, and (3) the signal molecule. Other combinations will be apparent to the skilled artisan.

Signal Molecule [98] The signal molecule of the present invention comprises (i) a molecule that can be detected and (ii) at least one avidin moiety. The identity of the molecule that can be detected is not critical, but should be one that can be conjugated to an avidin moiety.

[99] Preferably, the signal molecule produces a colorimetric product that is visible to the unaided eye. However, to increase sensitivity (if needed), the signal molecule can also be a fluorometric or chemiluminescent compound. More preferably, the signal molecule is one that produces a color change upon either precipitation (e. g. , colloidal gold) or interaction with a substrate (i. e. , enzyme substrate) such that detection equipment is not be necessary.

[100] A signal molecule may also be used that allows a quantitative or semi-quantitative determination of the amount of the biomolecule (e. g. , p24 antigen) in a test sample; i. e., fluorescence. However, a colorimetric, visually read, endpoint can be made semi- quantitative. For example, a color guide can be provided that grades the intensity of the color produced (e. g. , 1+-4+), and can be used to compare the test result with a standardized guide representing different quantities of analyte.

[101] In a preferred embodiment, the portion of the signal molecule that can be detected is horseradish peroxidase (HRP), or similar enzyme/substrate system that is commonly used in ELISA or Western blot systems, colloidal gold, alkaline phosphatase, glucose oxidase, or B- galactosidase. The signal molecule may also be a fluorescent molecule, including Alexa fluors (e. g. , 350 and 546), Texas Red, Cy fluors (e. g. Cy 2 and Cy 3), DAPI, fluorescein, and tetramethylrhodamine.

[102] In a more preferred embodiment, the portion of the signal molecule that can be detected is HRP. Upon addition of an HRP substrate, oxidation of the substrate occurs and a color change results, thus providing a visually-readable assay signal for the presence of the biomolecule. Many different substrates may be used for color development when HRP is employed, such as ABTS, chemiluminescent (luminol), alkaline phosphatase, 4CN, TMB, AEC/DAB, and OPD. Other substrates, such as NBT/BCID, Fast Red, INT dye, PNPP, ONPG, x-gal, and IPTG may be used with other signal molecules.

[103] The quantity of the signal molecule added to the assay system will depend on the quantity of the reagents added in the previous steps. In general however, the quantity of signal molecule added is such that there is at least a ten-fold molecular excess of substrate.

In a preferred example, the quantity of signal molecule added to the assay system is between about 10 and 100 fold excess, more preferably between about 50 and 100 fold excess, and most preferably between about 50 and 75 fold excess.

[104] The length of time during which the signal molecule is incubated in the assay system is not critical. Preferably, the incubation proceeds from between about 10 minutes and about 60 minutes. More preferably, the incubation proceeds from between about 10 minutes and about 30 minutes. Most preferably, the incubation proceeds from between about 10 minutes and about 15 minutes.

[105] The detection of the signal molecule may be performed during or after incubation of the signal molecule in the assay system. If a substrate is required to be added to the assay system in order to detect the signal molecule, the instructions accompanying the substrate will be followed.

Blocking Solutions [106] A variety of commercially available solutions may be used as blocking reagents in the method described herein including BSA, heterogeneous nucleic acid, prionex (or any commercially available blocking reagent), casein, gelatin, collagen, PVP, PVA, Tween 20, and Triton X-100. In each method of the present invention, blocking solution is added as the diluents for each reagent in the assay. They are removed by aspiration after specific binding of reagents to their targets has occurred.

Washing Solution [107] Between the addition of reagents in the methods of the present invention, the assay system is preferably subjected to washing to reduce the incidence of non-specific binding.

[108] While the number of wash cycles and soak times is empirically determined, in general either water or a low or high molarity salt solution with a detergent such as Tween 20, Triton X-100, or NP-40 may be used as the washing solution. 1-3 washes, each lasting 5-10 minutes may be performed, after incubation of each of the reagents used in the methods. Preferably, washing takes place between each incubation step, e. g. , after addition of the capture molecule to the solid support, after addition of the test sample, after addition of the detector molecule, after addition of the linker molecule, after addition of the amplification reagent, and after addition of the linker molecule-signal molecule conjugate.

[109] Preferably, the washing solution is PBS with 0.05% Tween 20, and the assay system is subjected to 2-3 washes between incubation steps, each wash lasting about 5 minutes.

[110] In a preferred embodiment, the incubation and washing steps are performed using a platform that gently rocks a microtiter well containing the mixture of sample and reagents.

For example, when paramagnetic microspheres are used as the support, the test sample and reagents can be combined in a microtiter well and mixed on a magnetocapture platform, such as the Bionor Platform (Bionor Corp, Norway). The Bionor Platform is small (20 cm x 30 cm), easily carried, and can be connected to a 12V car battery for portability. It also contains a pump for aspiration of wash fluid and a reading lamp.

[111] When a membrane is used, the test sample, membrane, and other reagents can be combined sequentially in a microtiter well and gently rocked on the Bionor platform.

Kit [112] The present invention also includes a kit that may be used to detect a biomolecule in a test sample.

[113] In one embodiment, the present invention relates to a kit for detecting a biomolecule in a sample comprising: (a) a capture molecule attached to a solid support, wherein said capture molecule comprises a molecule that specifically binds to pre-selected biomolecule, (b) a detector molecule, wherein said detector molecule comprises (i) a molecule that specifically binds to said pre-selected biomolecule and (ii) at least one biotin moiety, (c) an linker molecule, wherein said linker molecule comprises at least one avidin moiety or at least one streptavidin moiety, or a combination thereof, (d) an amplification reagent, wherein said amplification reagent comprises (i) a scaffold molecule and (ii) two or more biotin moieties, (e) a signal molecule, wherein said signal molecule comprises (i) a molecule that can be detected and (ii) at least one avidin moiety or at least one streptavidin moiety, or a combination thereof, and (f) a set of standards, wherein said standards allow an approximation of concentration of said pre-selected biomolecule in a sample.

[114] In another embodiment, the present invention relates to a kit for detecting a biomolecule in a sample comprising: (a) an anti-p24 antigen capture molecule attached to a solid support, wherein said capture molecule comprises a molecule that specifically binds to HIV-1 p24 antigen.

(b) an anti-p24 antigen detector molecule, wherein said detector molecule comprises (i) a molecule that specifically binds to HIV-1 p24 antigen and (ii) at least one biotin moiety, (c) an linker molecule, wherein said linker molecule comprises at least one avidin moiety or at least one streptavidin moiety, or a combination thereof, (d) an amplification reagent, wherein said amplification reagent comprises (i) a scaffold molecule and (ii) two or more biotin moieties, (e) a signal molecule, wherein said signal molecule comprises (i) a molecule that can be detected and (ii) at least one avidin moiety or at least one streptavidin moiety, or a combination thereof, and (f) a set of standards, wherein said standards approximate a range of between about 25,000 and 500,000 copies of HIV-1 RNA.

[115] In yet another embodiment, the present invention relates to a kit for detecting a biomolecule in a sample comprising: (a) an anti-p24 capture molecule attached to a solid support, wherein said capture molecule comprises a molecule that specifically binds to HIV-1 p24 antigen, (b) an anti-p24 detector molecule, wherein said detector molecule comprises (i) a molecule that specifically binds to HIV-1 p24 antigen and (ii) at least one avidin moiety, (c) an amplification reagent, wherein said amplification reagent comprises (i) a scaffold molecule and (ii) two or more biotin moieties, (d) a signal molecule, wherein said signal molecule comprises (i) a molecule that can be detected and (ii) at least one avidin moiety or at least one streptavidin moiety, or a combination thereof, and (e) a set of standards, wherein said standards approximate a range of between about 25,000 and 500,000 copies of HIV-1.

[116] The identity and properties of each of the reagents used in the kits are the same as those defined above for the methods of the present invention.

[117] With regard to the standards used in the kits, calibration may be performed to correlate color development with viral RNA copy number to allow a semi-quantitative measurement of the p24 antigen. This may be accomplished by using a sample containing 50,000 copies of viral RNA (corresponding to 3 pg of p24 antigen) (US Aidsinfonet. org, Fact Sheet 411,2002) and selecting a sample dilution that results in a moderate color development. For samples with unknown quantities of p24 antigen, such sample dilution may result in a stronger or weaker color depending on viral loads above and below 50,000 copies of viral RNA, respectively. In a test assay kit, a standard solution containing 50,000 copies of viral RNA as a control may be included, allowing comparison of color development of the test sample to that of the standard. For samples with a resultant color less than that of the standard, the viral load can be considered to be less than 50,000 copies of viral RNA. For more exact estimates, repeat testing of the sample using lower levels of standards will allow a more exact estimate of copy number (e. g. , standards of 5,000 and 25,000 copies of viral RNA). Conversely, unknown samples producing a stronger color may be re-tested with standards in the range of 100,000 and 500,000 copies of viral RNA. If color development of the unknown is at a maximum, further dilution of the sample with a correction for the dilution would be necessary to estimate copy number. The comparison of color development of the test sample with that of the standards will allow results to be expressed as a range (e. g. , between 25,000 and 50,000 copies of viral RNA or between 100,000 and 500,000 copies of viral RNA). A standard curve will be established to define the linear range of the visual assay. A set of standards, wherein the standards approximate a range of between about 25,000 and 500,000 copies of HIV-1 RNA, which corresponds to a range between about 1.5 to 30 pg of HIV-1 p24 antigen, is a preferred set of standards for use with the kits of the present invention.

[118] The kits of the present invention may also include written instructions for use of the kit. Kits may also contain all of the components and apparati needed to practice the methods of the present invention. For example, the kits may also contain all necessary containers (such as dilution tubes or microfuge screw-top tubes), solid supports such as microwell plates or slides, microspheres, pre-measured reagents and solutions (such as all reagents used in the method, washing solutions, blocking solutions, and substrates for the molecule that can be detected).

[119] The kits may also include other components to aid in the detection of the selected molecule. The identity of these other components will be obvious to the skilled artisan based on the identity of the molecule that is the subject of the detection.

EXAMPLES [120] The broad scope of this invention is best understood with reference to the following examples, which are not intended to limit the invention to specific embodiments.

Example 1 [121] Magnetic beads were coated with 0.1 ug/ml p24 antigen and then incubated with human anti-p24 antibody for 30 min at room temperature (RT) followed by 2 washes with PBS and 0.05% Tween 20. Biotinylated anti-human detector antibody was added at 1 or 2 ug/ml for 30 min at RT followed by 2 washes. Avidin-HRP was added at 1 ug/ml for 30 min at RT followed by 3 washes. HRP substrate was added to one set of beads to determine the baseline optical density.

[122] In the parallel set of reactions, 100 pg/ml of a 500-bp biotinylated lambda DNA sequence was added for 30 min at RT after addition of the avidin-HRP, followed by 3 washes. The 500 bp bacteriophage lambda DNA is obtained by PCR amplification of lambda DNA with the following primers: 5'primer: 5'-GATGAGTTCGTGTCCGTACAACTGG-3' (SEQ ID NO : 1) 3'primer: 5'-GGTTATCGAAATCAGCCACAGCG-3' (SEQ ID NO : 2).

(see, Kuehnelt, D. M. , et al. Quantitative PCR of bacteriophage lambda DNA using a second- generation thermocycler. PCR Methods & Applications. 3: 369-371 (1994), incorporated herein by reference in its entirety).

[123] Avidin-HRP was added a second time followed by the enzyme substrate for optical density (OD) determination. The results are shown in Figure 2.

[124] As shown in Figure 5, after one cycle of bio-DNA/avidin-HRP amplification, the S/N (signal to noise) ratio increased from 40.7 to 798 when using 2 ug/ml of biotinylated anti-human Ab and from 20.2 to 116 when using 1 ug/ml of secondary detector Ab. In this experiment, there was an increase in S/N values ranging from 5. 7 x to 19. 6 x over the routine ELISA method.

Example 2 [125] Microtiter well plates were coated with anti-p24 Ab (5 ug/ml) 13B6 and 13G4 (Dr.

George Lewis at the University of Maryland). Lysed HIV-1 culture supernatant was added at 570 pg/ml, 57 pg/ml, or 570 fg/ml in separate coated dishes for 2 hr at 37°C followed by 4 washes with PBS and 0.05% Tween 20. Biotinylated human anti-p24 Ab was added for 1 hr at 37°C followed by 4 washes. Avidin-HRP was added at 10 ng/ml for 30 min at 37°C followed by 4 washes. Substrate was added to determine baseline OD. In a parallel set of reactions, a biotinylated 500-bp segment of bacteriophage lambda DNA was added at 100 ng/ml for 30 min at 37°C after the avidin-HRP, followed by 4 washes. The 500 bp bacteriophage lambda DNA is obtained by PCR amplification of lambda DNA with the following primers: 5'primer: 5'-GATGAGTTCGTGTCCGTACAACTGG-3' (SEQ ID NO : 1) 3'primer: 5'-GGTTATCGAAATCAGCCACAGCG-3' (SEQ ID NO : 2).

(see, Kuehnelt, D. M. , et al. Quantitative PCR of bacteriophage lambda DNA using a second- generation thermocycler. PCR Methods & Applications. 3: 369-371 (1994), incorporated herein by reference in its entirety).

[126] Avidin-HRP was added a second time followed by substrate for OD determination.

The results are shown in Figure 6. After 1 cycle of bio-DNA/avidin-HRP amplification, the S/N ratio increased from 15. 3 to 22.0 for 570 pg/ml p24 antigen, from 2.8 to 4.3 for 57 pg/ml p24 antigen, and from 1.0 to 1.75 for 570 fg/ml p24 antigen. In this experiment, there was a one log increase in sensitivity of detection of p24 antigen over the routine ELISA.

Example 3 [127] Equal volumes (100 uL) of biotinylated anti-p24 antibody (NEN; Boston, MA, catalogue # IP801) and various concentrations of HIV-1 infected human lymphocyte culture supernatant produced by the Institute of Human Virology, University of Maryland (diluted in viral lysis buffer: 0.5% Triton-X in PBS) were incubated in 1.5 mL Eppendorf Microtubes (Brinkman/Eppendorf Corp. ; Westbury, NY) for 1 hour at RT on the Bionor Testing Station.

100 uL of 6-7 x 108 magnetic (2.8 um diameter) Dynal beads/mL, coated with anti-HIV-1 p24 monoclonal antibodies 13B6 and 13G4 (University of Maryland, Baltimore, MD), and 15 uL of a 1: 10 dilution of blocking buffer (1: 2 DNA Blocking Reagent; Roche Diagnostics Corp.; Indianapolis, IN and Stabilcoat: Surmodics Corp.; Eden Prairie, MN) (Barletta et al., 2004) were added and incubated for 1 hr, RT on the Bionor Testing Station. Aliquots (100 uL) of this mixture of 13B6 and 13G4 antibody coated magnetic beads, bound to HIV-1 p24 antigen and to NEN biotinylated anti-HIV-1 antibody were then aliquoted into individual microwells of an ELISA plate, captured by the Prolinx bar magnetic separator, and washed 2X with PBST (PBS with 0.05% Tween 20). 60 uL of Streptavidin-HRP (0.2 ug/mL) (KPL ; Gaithersburg, MD) were then added to the captured magnetic beads and incubated for 30 min, RT, on a plate agitator. Magnetic beads were then captured by the magnetic separator, washed 2X with PBST and 60 uL of TMB (KPL; Gaithersburg, MD) was added for 30 min at RT for color development. Solution was aspirated from the beads, placed in another microplate well, and 60 uL of 2N HC1 was added for colorimetric detection at 450 nm.

Samples were considered positive if the signal to noise ratio (S/N) was > 2.0. Negative controls were either plasma or serum diluted 9: 1 with 5% Triton-X, PBS.

[128] Figure 7 shows the level of sensitivity of detection (75 pg/mL HIV-1 p24 antigen; S/N = 2.2) which was consistently attained in replicates of multiple experiments. A sensitivity of detection of 7.5 pg/mL HIV-1 p24 antigen was attained, although inconsistently, by this method (25% of experiments, data not shown).

[129] Additional experiments have been conducted to examine the amplification potential of three types of biotinylated polymers: a 500 bp (25% biotinylated) DNA; a 3000 mw (50% biotinylated) dextran: BioDex3; and a 70,000 mw (450% biotinylated) dextran: (Biodex70.

Preliminary studies have shown that the S/N for Cycle 1 (no amplification) at 150 pg/mL HIV-1 p24 antigen increased from a range 2.2-2. 5 to a range of 3.0-3. 2 when using Biodex3 at 10 ug/mL (S/N = 3.0) and Biodex70 at 1 and 5 ug/mL (S/N = 3.1 and 3.2, respectively) for the first amplification step (data not shown). The background control was also increased which interfered with the potential to increase sensitivity of detection of HIV- 1 p24 antigen to concentrations lower than 150 pg/mL.

Example 4-Method for the Detection of a Biomolecule in a Test Sample [130] A. Sanzple Collection, Processing and Storage : [131] This method may be used with biological samples such as any human body fluid (e. g. , whole blood, plasma, serum, saliva, neuronal tissues, and urine) or environmental samples such as water or soil.

[132] All specimens used in this assay may be processed on the same day as collected or stored frozen at-20°C or below until tested. Clear, non-hemolyzed plasma or serum specimens should be used whenever possible.

[133] B. Test Protocol : [134] Note: All steps require optimization depending on the antigen or antibody.

1. A solid support (e. g. , microwells, microbeads, or membranes) is coated with a capture antibody (5-10 ug/mL) for 8-16 hrs at room temperature (RT). The capture antibody solution is removed (such as by aspiration), and the solid support is washed 1-4X using 0.05% detergent in PBS (PBST).

2. A blocking reagent (such as 1: 2 DNA Blocking Reagent; Roche Diagnostics Corp.; Indianapolis, IN and Stabilcoat: Surmodics Corp.; Eden Prairie, MN) is added to the solid support for a 1 hr incubation at RT, followed by removal of the blocking reagent and washing 1-4X using PBST.

3. The test sample (10-100 uL) is added to the solid support for a 10 min to 1 hr incubation at RT, followed by removal of the test sample and washing 1-4X using PBST.

4. A detector antibody (2-5 ug/mL) is added to the solid support for a 10 min to 1 hr incubation at RT, followed by removal of the detector antibody and washing 1-4X using PBST.

5. A linker molecule (2-5 ug/mL) is added to the solid support for a 10 min to 1 hr incubation at RT, followed by removal of the linker molecule and washing 1-4X using PBST.

6. An amplification reagent (10-100 uL) is added to the solid support for a 10 min to 1 hr incubation at RT, followed by removal of the amplification reagent and washing 1- 4X using PBST.

7. Steps 5. and 6. may be repeated 2-3 times.

8. A signal-generating molecule (10-100 uL) is added to the solid support for a 10 min to 1 hr incubation at RT, followed by removal of the signal molecule and washing 1-4X using PBST.

9. A substrate of the signal-generating molecule (10-100 uL) is added for 10 min to 1 hr at RT.

10. The colored end product resulting from step 9. is compared to a set of standards and quantified.

[135] While the invention has been described in detail and with reference to specific embodiments thereof, it will be apparent to one of skill in the art that various changes and modifications can be made therein without departing from the spirit and scope thereof.