WO2010011775A2 - Detection and quantification of antigens - Google Patents

Detection and quantification of antigens Download PDF

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Publication number
WO2010011775A2
WO2010011775A2 PCT/US2009/051440 US2009051440W WO2010011775A2 WO 2010011775 A2 WO2010011775 A2 WO 2010011775A2 US 2009051440 W US2009051440 W US 2009051440W WO 2010011775 A2 WO2010011775 A2 WO 2010011775A2
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WO
WIPO (PCT)
Prior art keywords
seq
antibody
antigen
hiv
binding
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PCT/US2009/051440
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French (fr)
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WO2010011775A3 (en
Inventor
Jean-Charles Grivel
Silvia S. Chen
Angelique Biancotto
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The Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services
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Publication of WO2010011775A2 publication Critical patent/WO2010011775A2/en
Publication of WO2010011775A3 publication Critical patent/WO2010011775A3/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G01N33/56988—HIV or HTLV
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/15—Retroviridae, e.g. bovine leukaemia virus, feline leukaemia virus, feline leukaemia virus, human T-cell leukaemia-lymphoma virus
    • G01N2333/155—Lentiviridae, e.g. visna-maedi virus, equine infectious virus, FIV, SIV
    • G01N2333/16—HIV-1, HIV-2
    • G01N2333/161—HIV-1, HIV-2 gag-pol, e.g. p55, p24/25, p17/18, p.7, p6, p66/68, p51/52, p31/34, p32, p40
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/10—Detection of antigens from microorganism in sample from host

Definitions

  • This invention relates generally to assays for the detection of the presence of an analyte in a biological sample. More specifically, the present invention relates to an immunoassay for the detection of a HIV antigen, such as a HIV-I GAG polypeptide, in a biological sample.
  • a HIV antigen such as a HIV-I GAG polypeptide
  • the first antibody binds to an epitope of the p24 polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO.7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:
  • the volume of the biological can be varied in the methods and immunosystems of the present invention.
  • the biological sample is between about 20 ⁇ l and about 50 ⁇ l in volume.
  • the methods comprise the step detecting the presence of a second antigen in the biological sample wherein the second antigen is different from the p24 polypeptide.
  • Figure 1 depicts a typical p24 standard curve obtained using an assay described herein.
  • the graph represents the individual points a titration curve run in quadruplicate. Above each data point the CV of the data is indicated. The coefficient of determination is calculated to fit a 4 parameter logistic regression. Details are described in Example 3.
  • Figure 2 depicts inter-assay variation using an assay described herein.
  • the figure represents the inter-assay variation calculated for four assays run in triplicates at different concentrations. For each concentration, the data points (12 per concentration) are plotted together with the median, the interquartile box, and the range of the data. The number above each data point represents the CV of the results at the corresponding concentration. Details are described in Example 3.
  • Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, e.g., an ⁇ carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs may have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid.
  • Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions similarly to a naturally occurring amino acid.
  • Such antibodies can be produced using various techniques known in the art. For example in vitro methods involve use of recombinant libraries of human antibody fragments displayed on bacteriophage ⁇ e.g., McCafferty et al, 1990, Nature 348:552-554; Hoogenboom & Winter, 1991, J MoI Biol 227:381; and Marks et al, 1991, JMoI Biol 222:581), yeast cells (Boder and Wittrup, 1997, Nat Biotechnol 15:553-557), or ribosomes (Hanes and Pluckthun, 1997, Proc Natl Acad Sd USA 94:4937-4942).
  • biological fluid refers to a fluid from a host and includes, without limitations, whole blood, serum, plasma, urine, tears, mucus, nasal swab, sweat, milk, ascites fluid, saliva, oral fluid, semen, seminal fluid, vaginal secretions, breast milk, stool, sputum, gastric fluid, cerebrospinal fluid, pleural fluid, synovial fluid, interstitial fluid, lymph fluid, amniotic fluid, peritoneal fluid, fetal fluid, fluid from ulcers and other surface eruptions, blisters, and abscesses, and extracts of tissues including biopsies of normal, malignant, and suspect tissues or any other constituents of the body which may contain an analyte or antigen of interest.
  • fluorescence or “fluorescence signal” or “reporter signal” mean a signal measured in a fluorimeter or similar device.
  • the signal may be given as fluorescent unit.
  • BLAST and BLAST 2.0 are used, with the parameters described herein, to determine percent sequence identity for the nucleic acids and proteins of the invention.
  • Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information.
  • the phrase "providing a biological sample” means to obtain a biological sample for use in, systems, kits, and methods described in this invention. Most often, this will be done by removing a sample of cells from a host, but can also be accomplished by using previously isolated cells (e.g., isolated by another person, at another time, and/or for another purpose), or by performing the methods of the invention in vivo. Archival tissues, having treatment or outcome history, will be particularly useful.
  • the term “seroconversion” refers to the development of detectable specific antibodies to microorganisms, e.g., bacteria, viruses, in the blood serum of a subject as a result of infection or immunization. Prior to seroconversion, blood tests are seronegative for the antibody; after seroconversion, blood tests are seropositive for the antibody.
  • a specified antibody binds to a particular antigen at least two times the background and more typically more than 10 to 100 times background. Specific binding to an antibody under such conditions requires an antibody that is selected for its specificity for a particular antigen.
  • solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein (see, e.g., Harlow & Lane, Antibodies, A Laboratory Manual (1988) for a description of immunoassay formats and conditions that can be used to determine specific immunoreacti vity) .
  • tissue biopsy refers to an amount of tissue removed from a subject for diagnostic analysis. In a patient with cancer, tissue may be removed from a tumor, allowing the analysis of cells within the tumor. In a patient afflicted with AIDS, tissue may be removed, e.g., from a lymph node.
  • tissue biopsy can refer to any type of biopsy, such as needle biopsy, fine needle biopsy, surgical biopsy, etc.
  • the present invention provides compositions, systems, kits, and methods comprising two binding partners and a biological sample.
  • the two binding partners are antibodies.
  • One binding partner referred to herein as a capture binding partner
  • the other binding partner referred to herein as a detection binding partner
  • Both binding partners bind to the same analyte, such as a HIV antigen, albeit to different or non- overlapping epitopes of the analyte.
  • various combinations of binding partners can be used in the kits, systems, and methods described herein.
  • the methods of the present invention are not limited to detection of any particular HIV protein, including any form of Gag, Pol, Env, Nef, Vif, Rev, Vpr, Tat or VpuA, but are useful to also detect non-HIV antigens.
  • a GAG polypeptide is a polypeptide having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence shown in SEQ ID NO:2.
  • a GAG polypeptide has the amino aid sequence of SEQ ID NO:2.
  • the analyte of interest is captured through binding to a capture binding partner.
  • the capture binding partner is an antibody specifically binding to the analyte of interest, i.e., the analyte to be detected in a biological sample.
  • Other useful capture binding partners are molecules to which the analyte of interest has an affinity, i.e., to which it binds.
  • a detecting binding partner e.g., a detecting antibody, binds to the captured analyte. It will be appreciated that an antibody identified herein which is useful as a capture antibody is also useful as a detecting antibody, and vice versa. Binding of a capture antibody will not interfere with the binding of a detecting antibody.
  • Antibodies useful to practice methods of the present invention can be monoclonal antibodies, polyclonal antibodies, or fragments thereof.
  • a preferred antibody is a monoclonal antibody.
  • a first antibody is an antibody binding to an epitope of a p24 polypeptide comprising the amino acid sequence OfNH 2 -PIVQNIQGQMVHQAISPRTL- COOH (SEQ ID NO:4).
  • Preferred antibodies binding thereto are 111/182, 112/021, and 112/047 or fragments thereof (Niedrig et al, 1991, J Virol 65:4529-4533) ( Figure 5).
  • a first antibody is an antibody binding to an epitope of a p24 polypeptide comprising the amino acid sequence OfNH 2 -SEGATPQDLNTMLNTVG-COOH (SEQ ID NO: 8).
  • a preferred antibody binding thereto is a polyclonal antibody or a fragment thereof (Truong et al, 1997, J Med Virol 51(3):145-51) ( Figure 5).
  • a first antibody is an antibody binding to an epitope of a p24 polypeptide comprising the amino acid sequence OfNH 2 -DLNTMLNTVG-COOH (SEQ ID NO:12).
  • a preferred antibody binding thereto is 1 11/052 or a fragment thereof (Niedrig et al, 1991, J Virol 65:4529-4533) ( Figure 5).
  • a first antibody is an antibody binding to an epitope of a p24 polypeptide comprising the amino acid sequence of NH 2 - DLNTMLNTVGGHQAAMQMLKETINEEAAEWDR-COOH (SEQ ID NO: 13).
  • a preferred antibody binding thereto is a polyclonal antibody or a fragment thereof (Pialoux et al, 2001, AIDS 15(10): 1239-49) ( Figure 5).
  • a first antibody is an antibody binding to an epitope of a p24 polypeptide comprising the amino acid sequence of NH 2 -LKETINEE AAEWDRVHP V-
  • a first antibody is an antibody binding to an epitope of a p24 polypeptide comprising the amino acid sequence OfNH 2 -ETINEEAAEWD-COOH (SEQ ID NO: 16).
  • Preferred antibodies binding thereto are 113/038 and 111/073 or a fragment thereof (Niedrig et al, 1991, J Virol 65:4529-4533) ( Figure 5).
  • a first antibody is an antibody binding to an epitope of a p24 polypeptide comprising the amino acid sequence OfNH 2 -EAAEWDRVHP-COOH (SEQ ID NO:18).
  • Preferred antibodies binding thereto are l-B-7, 3-B-7, 6-D-12, 6-E-7, 8-D-5, or a fragment thereof (Niedrig et al, 1988, J Gen Virol 69:2109-2114; Niedrig et al, 1989, J Virol 63:3525-3528) ( Figure 5).
  • a first antibody is an antibody binding to an epitope of a p24 polypeptide comprising the amino acid sequence OfNH 2 -QGPKEPFRDYVDRFY-COOH (SEQ ID NO:37).
  • a preferred antibody binding thereto is VlO or a fragment thereof (Matsuo et al, 1992, J Gen Virol 73:2445-2450) ( Figure 5).
  • a first antibody is an antibody binding to an epitope of a p24 polypeptide comprising the amino acid sequence of NH 2 -
  • a first antibody is an antibody binding to an epitope of a p24 polypeptide comprising the amino acid sequence of NH 2 -
  • Quantum dots are free floating and can be attached to a variety of molecules via metal coordinating functional groups. These groups include but are not limited to thiol, amine, nitrile, phosphine, phosphine oxide, phosphonic acid, carboxylic acids or other ligands. By bonding appropriate molecules to the surface, the quantum dots can be dispersed or dissolved in nearly any solvent or incorporated into a variety of inorganic and organic films. Quantum dots (QDs) can be coupled to streptavidin directly through a maleimide ester coupling reaction or to antibodies through a maleimide-thiol coupling reaction.
  • Such supports include natural polysaccharides such as cellulose, modified celluloses, agarose, cross-linked dextrans, amino-modified cross-linked dextrans, guar gums, modified guar gums, xanthan gums, locust bean gums and hydrogels.
  • Other examples include cross-linked synthetic hydrophilic polymers such as polyacrylamide, polyacrylates, polyvinyl alcohol (PVA) and modified polyethylene glycols.
  • polymeric structures are those made of inorganic solids, including clay minerals such as kaolinite, bentonite, talc, and mica; metal oxides such as alumina, titanium dioxide, and zinc oxide; insoluble inorganic salts such as silica gel, hydroxyapatite, and calcium phosphate gel; metals such as gold, silver, platinum, and copper; and semiconductor compounds such as GaAs, GaP, and ZnS.
  • inorganic solids including clay minerals such as kaolinite, bentonite, talc, and mica; metal oxides such as alumina, titanium dioxide, and zinc oxide; insoluble inorganic salts such as silica gel, hydroxyapatite, and calcium phosphate gel; metals such as gold, silver, platinum, and copper; and semiconductor compounds such as GaAs, GaP, and ZnS.
  • the material is not limited thereto.
  • the polymeric structure may be used in combination of two or more thereof.
  • Ferromagnetic fluorescent particles for use in the present invention include SPHEROTM Fluorescent Carboxyl Ferromagnetic Particles, such as Fluorescent Yellow Carboxyl Ferromagnetic (2.0-2.9 ⁇ m; Spherotech, Inc., catalog # FCFM-2052-2), Fluorescent Yellow Carboxyl Ferromagnetic (4.0-4.9 ⁇ m; Spherotech, Inc., catalog # FCFM-4052-2), and Fluorescent Nile Red Carboxyl Ferromagnetic (4.0-4.9 ⁇ m; Spherotech, Inc., catalog # FCFM4056-2).
  • SPHEROTM Fluorescent Carboxyl Ferromagnetic Particles such as Fluorescent Yellow Carboxyl Ferromagnetic (2.0-2.9 ⁇ m; Spherotech, Inc., catalog # FCFM-2052-2), Fluorescent Yellow Carboxyl Ferromagnetic (4.0-4.9 ⁇ m; Spherotech, Inc., catalog # FCFM-4052-2), and Fluorescent Nile Red Carboxyl Ferromagnetic
  • Each bead set can be coated with a reagent specific to a particular assay, e.g., with a capture binding partner, preferably, a first antibody, as described herein.
  • the capture binding partner then allows the capture and detection of an analyte of interest from a sample, e.g., a GAG polypeptide.
  • lasers excite the internal dyes that identify each microsphere particle, and also any reporter dye captured during the assay.
  • a reporter dye e.g., is a fluorescent moiety, as described herein, attached to a second antibody.
  • the assay as described herein can be converted into a high throughput assay.
  • the biological sample is a sample of genital tract cells and/or genital tract secretions from a woman.
  • a sample may be obtained by, e.g., pap smear, cervicovaginal lavage, as a cytobrush, as a biopsied cervix tissue, or as an endocervical curettage.
  • Pap smear or genital tract cell and secretion collection methods such as cervicovaginal lavage (CVL), offer clinicians and investigators a simple noninvasive method of sampling cells and mucus secreted by the uterus, cervix and vaginal tissues in a state readily amenable to in vitro studies and methods described herein.
  • CVL sampling is a routine gynecological procedure.
  • a preferred biological sample is obtained from a human. Another preferred biological sample is obtained from subject having or suspected of having a lentivirus infection. Another preferred biological sample is obtained from subject having or suspected of having AIDS. Another preferred biological sample is obtained from subject treated with an antiviral agent.
  • Biological samples may be prepared for use in the methods described herein. Sample preparation includes steps necessary to prepare a raw sample for analysis.
  • a biological sample comprises a buffer.
  • the buffer may be mixed with the biological sample. While any suitable buffer can be used, the preferable buffer has low fluorescence background, is inert to the detectably labeled particle, and can maintain the working pH.
  • the buffer concentration can be any suitable concentration, such as in the range from about 1 to about 200 mM. Any buffer system may be used as long as it provides for solubility, function, and detection of the analyte of interest.
  • the buffer is selected from the group consisting of phosphate, glycine, acetate, citrate, acidulate, carbonate/bicarbonate, imidazole, triethanolamine, glycine amide, borate, MES, Bis-Tris, ADA, aces, PIPES, MOPSO, Bis-Tris Propane, BES, MOPS, TES, HEPES, DIPSO, MOBS, TAPSO, Trizma, HEPPSO, POPSO, TEA, EPPS, Tricine, Gly-Gly, Bicine, HEPBS, TAPS, AMPD, TABS, AMPSO, CHES, CAPSO, AMP, CAPS, and CABS.
  • phosphate glycine, acetate, citrate, acidulate, carbonate/bicarbonate, imidazole, triethanolamine, glycine amide, borate, MES, Bis-Tris, ADA, aces, PIPES, MOPSO, Bis-Tris
  • an analyte of the present invention may include without limitations, drugs, metabolites, co-factors, hormones, cytokines, cell- surface-receptors, antibodies, antigens, enzymes or other proteins whose expression is characteristic of a disease, disorder, or pathological condition.
  • the method of detecting an analyte in a biological sample has a dynamic range of between about 0.2 pg/ml and about 20,000 pg/ml.
  • a microtiter plate contains (i) a first sample having about 0.2 pg/ml of an analyte of interest in one well, (ii) a second sample having about 2 pg/ml of an analyte of interest in a second well, a third sample having about 20 pg/ml of an analyte of interest in third well, a fourth sample having about 200 pg/ml of an analyte of interest in a fourth well, a fifth sample having about 2,000 pg/ml of an analyte of interest in fifth well, and a sixth sample having about 20,000 pg/ml of an analyte of interest in a sixth well, an assay having a dynamic range of five
  • Accuracy may be determined by standard methods, e.g., using a series of standards of concentrations that span the dynamic range and constructing a standard curve (see Figure 1 and Examples herein). Standard measures of fit of the resulting standard curve may be used as a measure of accuracy, e.g., an R 2 greater than about 0.7, 0.75, 0.8, 0.85, 0.9, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98, 0.99, or 0.995. In a preferred embodiment of the present invention, the accuracy of the assay described herein is greater than 0.99, preferably greater than 0.995, more preferably greater than 0.9975.
  • methods and assays of the present invention are characterized by having a low detection limit of less than about 100 pg/ml, less than about 90 pg/ml, less than about 80 pg/ml, less than about 70 pg/ml, less than about 60 pg/ml, less than about 50 pg/ml, less than about 40 pg/ml, less than about 30 pg/ml, less than about 20 pg/ml, less than about 10 pg/ml, less than about 9 pg/ml, less than about 8 pg/ml, less than about 7 pg/ml, less than about 6 pg/ml, less than about 5 pg/ml, less than about 4 pg/ml, less than about 3 pg/ml, less than about 2 pg/ml, less than about 1 pg/ml, less than about 0.9 pg/ml, less than about 0.8 pg
  • the "detecting" of an analyte includes detecting the analyte directly or indirectly. In the case of indirect detection, a label that has been attached to the analyte, may be detected.
  • the method for detecting an analyte in a biological sample using an assay having a dynamic range of between about 0.2 and about 20,000 pg/ml and a coefficient of variation of less than 10% comprises the steps of (a) binding a capture binding partner specifically binding an analyte of interest to a solid support, (b) contacting the capture binding partner with a biological sample suspected of containing the analyte of interest for a time sufficient to allow binding of the analyte of interest to the capture binding partner, thereby forming a capture binding partner/analyte complex, (c) contacting the capture binding partner/analyte complex with a detecting binding partner specifically binding the analyte of interest for a time sufficient to allow binding of the detecting
  • a preferred method for detecting an antigen in a biological sample using an assay having a dynamic range of between about 0.2 and about 20,000 pg/ml and a coefficient of variation of less than 10% comprises the steps of (a) binding a first antibody specifically binding an antigen to a solid support, (b) contacting the first antibody with a biological sample suspected of containing the antigen for a time sufficient to allow binding of the antigen to the first antibody, thereby forming a first antibody/antigen complex, (c) contacting the first antibody/antigen complex with a second antibody specifically binding the antigen for a time sufficient to allow binding of the second antibody to the antigen of the first antibody/antigen complex, thereby forming a first antibody/antigen/second antibody complex, wherein the second antibody produces a detectable signal, and (d) detecting the detectable signal.
  • the first antibody and the second antibody bind to different binding sites, e.g., epitopes on the antigen.
  • this method comprises an assay, in which binding partner pairs, e.g. antibodies, to the same analyte, e.g., same antigen, are used.
  • binding partner pairs e.g. antibodies
  • the invention also encompasses binding partner pairs, e.g., antibodies, wherein both antibodies are specific to the same analyte, e.g., the same antigen, and wherein at least one member of the pair comprises a label as described herein.
  • a method for detecting a HIV-I antigen in a biological sample using an immunoassay having a dynamic range of between about 0.2 and about 20,000 pg/ml and a coefficient of variation of less than 10% is provided.
  • this method comprises the steps of (a) binding a first antibody specifically binding a HIV-I antigen to a solid support, (b) contacting the first antibody with a biological sample suspected of containing the HIV-I antigen for a time sufficient to allow binding of the HIV-I antigen to the first antibody, thereby forming a first antibody/HIV- 1 antigen complex, (c) contacting the first antibody/HIV- 1 antigen complex with a second antibody specifically binding the HIV-I antigen for a time sufficient to allow binding of the second antibody to the HIV-I antigen of the first antibody/HIV- 1 antigen complex, thereby forming a first antibody/HIV- 1 antigen/second antibody complex, wherein the second antibody produces a detectable signal, and (d) detecting the detectable signal.
  • the first antibody and the second antibody bind to different epitopes of the HIV-I antigen. Thereby the HIV-I antigen in the biological sample is detected.
  • a capture binding partner may be attached to a solid support.
  • a preferred solid support is a microtiter plate or a plurality of microspheres, e.g., paramagnetic beads.
  • the invention provides a capture binding partner for an analyte of interest, e.g., an antigen, such as a HIV-I antigen, attached to a microtiter plate.
  • the invention provides a capture binding partner for an analyte of interest, e.g., an antigen, such as a HIV-I antigen, attached to a plurality of microspheres.
  • the invention provides a binding partner for an analyte of interest, e.g., an antigen, such as a HIV-I antigen, attached to paramagnetic beads.
  • an analyte of interest e.g., an antigen, such as a HIV-I antigen
  • Any suitable capture binding partner that is specific for the analyte to be captured may be used.
  • a preferred capture binding partner is an antibody, more preferred, a monoclonal antibody. Attachment of an antibody to a solid support may be accomplished through a variety of methods known in the art. Binding moieties, such as an antibody, may be coupled to a solid support using reversible or non-reversible interactions.
  • a non-reversible or covalent attachment in which the orientation of the capture binding partner is such that capturing of the analyte of interest is optimized, is particularly useful.
  • a capture binding partner is covalently attached to a solid support in an oriented way.
  • a capture binding partner is covalently attached to a microtiter plate in an oriented way.
  • a capture binding partner is covalently attached to a plurality of microspheres in an oriented way.
  • a capture binding partner is covalently attached to paramagnetic beads in an oriented way.
  • Non-reversible or covalent interactions may be made using a support that includes at least one reactive functional group, such as a hydroxyl, carboxyl, sulfhydryl, or amino group that chemically binds to the binding moiety, optionally through a spacer group.
  • Suitable functional groups include N-hydroxysuccinimide esters, sulfonyl esters, iodoacetyl groups, aldehydes, epoxy, imidazolyl carbamates, and cyanogen bromide and other halogen- activated supports.
  • Such functional groups can be provided to a support by a variety of known techniques.
  • the method of detecting an analyte in a biological sample comprises the step of contacting the capture binding partner, such as a first antibody, with a biological sample suspected of containing an analyte of interest, such as a HIV-I antigen, for a time and under conditions sufficient to allow binding of the analyte of interest, such as the HIV-I antigen, to the capture binding partner, such as the first antibody, thereby forming a capture binding partner/analyte complex, such as a first antibody/HIV- 1 antigen complex.
  • the capture binding partner such as a first antibody
  • the biological sample suspected of containing the analyte of interest is added to a container (or vessel, plastic tube, etc.), containing a suspension comprising the capture binding partner bound to the solid support. Thereby the biological sample is contacted with the capture binding partner, such as a first antibody.
  • the first antibody is an anti-HIV-1 p24 antibody, preferably monoclonal antibody 4F6 or a fragment thereof.
  • the biological sample contacted to the capture binding partner such as the first antibody
  • the biological sample contacted to the capture binding partner is about 10 ⁇ l, about 20 ⁇ l, about 30 ⁇ l, about 40 ⁇ l, about 50 ⁇ l, about 100 ⁇ l, or about 200 ⁇ l.
  • the volume of the biological sample contacted to the capture binding partner, such as the first antibody is less than 10 ⁇ l. In some embodiments of the present invention, the volume of the biological sample contacted to the capture binding partner, such as the first antibody, is less than 20 ⁇ l.
  • the volume of the biological sample contacted to the capture binding partner, such as the first antibody is between about 10 ⁇ l and about 200 ⁇ l, preferably between about 20 ⁇ l and about 100 ⁇ l, more preferably between about 20 ⁇ l and about 50 ⁇ l.
  • the time allowed for binding of the detecting binding partner, such as a second antibody, to the analyte, such as a HIV-I antigen, of the capture binding partner/analyte complex, such as the first antibody/HIV- 1 antigen complex will vary depending on the conditions. It will be apparent that shorter binding times are desirable in some settings, especially in a clinical setting.
  • the time allowed for binding of the detecting binding partner, such as the second antibody to the analyte of interest, such as the HIV-I antigen is less than about 20 minutes. In some embodiments, the time allowed for binding of the detecting binding partner, such as the second antibody to the analyte of interest, such as the HIV-I antigen, is less than about 10 minutes.
  • Unbound label may then optionally be washed away.
  • the detecting antibody such as the second antibody to the analyte of interest, such as the HIV-I antigen
  • unbound label and detecting binding partner that may have bound nonspecifically, are washed away leaving substantially bound to the solid support only capture binding partner/analyte/detecting binding partner complexes, such as first antibody/HIV- 1 antigen/second antibody complexes. In other embodiments, no washing is performed. Other variations will be apparent to one of skill in the art. 4. Detecting The Detectable Signal
  • Imaging systems typically comprise a standard 50-55 mm fl.4 photographic lens coupled to a camera. Such systems can be used to image an entire multiwell plate. Further, a detectable signal can also be detected using a charge coupled device (CCD) as described in US 2003/0078737. Suitable CCD cameras are available from Alpha- Innotech (San Leandro, CA), Stratagene (La Jolla, CA), BioRad (Richmond, CA), and Beckman-Coulter (Fullerton, CA). [0272] Another manner of detecting the analyte, such as a HIV-I antigen, is to utilize a conjugate comprising a third antibody attached to a signal -generating compound.
  • CCD charge coupled device
  • the conjugate may comprise, for example, an antigen or anti-antibody capable of binding to the bound second antibody (e.g., anti-mouse antibody or an anti- human antibody) attached to a signal- generating compound capable of generating a detectable signal. Detection of the signal, thus, indicates presence of the complexes and, thus, presence of the analyte, such as the HIV-I antigen in the sample. The signal generated is actually proportional to the amount of antigen present in the sample.
  • variation of the plurality of solid supports allows changing the dynamic range of the assays described herein.
  • the changing of the dynamic range of the assay is performed in the context of the Luminex technology.
  • using the Luminex technology one detects the fluorescence signal(s) on a single bead.
  • the more fluorescent detector antibody is bound per bead the more sensitive the assay will be.
  • the more analyte bound to each bead the more sensitive the assay will be.
  • the analyte is abundant, this typically is not a problem, because every bead will have a high number of analyte bound.
  • the limiting quantity of the analyte is shared by all the beads, each of which can only bind a small amount of the analyte, and therefore decreases the fluorescence signal(s) detected on each bead. If less beads are present, then they each individual bead will bind more of the limiting amount of the analyte, and therefore produce a stronger fluorescent signal(s). In contrast thereto, a bulk assay will not have such limitations, and also will not have such a flexibility.
  • an antigen of interest is detected and quantified using a detecting binding partner, such as a second antibody, which may be labeled with a fluorochrome.
  • a detecting binding partner such as a second antibody, which may be labeled with a fluorochrome.
  • Methods described herein can also be adapted to be read by conventional fluorescence plate readers.
  • compositions and methods are used to detect a lentivirus antigen, such as a HIV-I antigen
  • a lentivirus antigen such as a HIV-I antigen
  • the detection of an amount or concentration of the lentivirus antigen in a biological sample is used for diagnosis, prognosis, and/or treatment selection of a lentivirus infection, such as AIDS.
  • a lentivirus antigen such as a HIV-I antigen
  • the presence of any lentivirus antigen at all in a biological sample from a subject is an indication of a disease or pathological state, and the threshold is essentially zero.
  • HIV antibody screening of the blood may no longer fulfill the criterion of being the most effective strategy and method in determining the HIV status of an individual. Assays for the early detection of HIV exposure and/or infection are needed, especially in a resource-poor setting.
  • a method of detecting exposure to HIV in a subject comprises the steps of (a) evaluating a biological sample from a subject for the presence of HIV and (b) determining whether the subject is HIV-positive or HIV-negative, wherein the presence of HIV in the biological sample indicates the subject is HIV-positive, and the absence of HIV in the sample indicates the subject is HIV-negative.
  • the step of evaluating the biological sample for the presence of HIV is performed using any of the assay methods described herein.
  • the method of detecting exposure to HIV in a subject comprises the step of providing a biological sample from the subject.
  • analyte levels e.g., HIV antigen levels
  • An analyte e.g., a HIV antigen
  • tl a first time
  • t2 a second time
  • t2 a second time
  • the levels of the HIV antigen determined can be used to determine whether to recommend that the subject should continue or discontinue anti-viral therapy or whether a different antiviral therapy than the one used fro the first treatment of the subject should be recommended. For example, if the quantification of the HIV antigen indicates progression of the viral infection, either continuing the anti-viral therapy, increasing the dose of the anti- viral agent and/or recommend another anti-viral therapy should be considered. Likewise, if quantification of the HIV-I antigen indicates regression of the viral infection or no viral infection, the viral therapy may be discontinued or the dose of the viral agent may be lowered.
  • kits and systems are also provided by the present invention.
  • such kits and systems may include any or all of the following compositions described herein: assay reagents, buffers, a first antibody, a second antibody, a biological sample, a calibration standard, a HIV antigen, a HIV-I antigen or polypeptide, a HIV-I GAG polypeptide, a HIV-I p24 polypeptide, a solid support, a capture binding protein, a capture binding protein bound to a solid support, a first antibody, a first antibody bound to a solid support, a detecting binding partner, a second antibody, a label, a labeled second antibody, or any other compound or composition described herein.
  • references to particular buffers, media, reagents, cells, culture conditions and the like, or to some subclass of the same, is not intended to be limiting, but should be read to include all such related materials that one of ordinary skill in the art would recognize as being of interest or value in the particular context in which they are presented. For example, it is often possible to substitute one buffer system or culture medium for another, such that a different but known way is used to achieve the same goals as those to which the use of a suggested method, material or composition is directed.
  • Table 6 shows U.S. FDA-Licensed Anti-HIV-1 Confirmatory Tests on seroconversion panel AD (same samples as in Table 2)
  • SEQ ID NO:3 p24 polypeptide sequence 1 PIVQNIQGQM VHQAISPRTL NAWVKVVEEK AFSPEVIPMF SALSEGATPQ DLNTMLNTVG
  • SEQ ID NO: 18 p24 epitope sequence EAAEWDRVHP
  • SEQ ID NO:20 p24 epitope sequence
  • SEQ ID NO:34 p24 epitope sequence
  • SEQ ID NO:48 p24 epitope sequence

Abstract

The present invention provides compositions, kits, systems, and methods for detecting antigens in a biological sample, preferably HIV antigens and in particular a HIV-1 p24 antigen. A preferred p24 immuosystem has a wide dynamic range together with a high sensitivity. A preferred assay of the present invention uses a solid support coupled to a high affinity first antibody directed against an antigen together with a labeled second antibody binding to the same antigen.

Description

DETECTION AND QUANTIFICATION OF ANTIGENS
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional Application No. 61/082,937, filed on July 23, 2008, the entire content of which is hereby incorporated here in by reference for all purposes.
FIELD OF THE INVENTION
[0002] This invention relates generally to assays for the detection of the presence of an analyte in a biological sample. More specifically, the present invention relates to an immunoassay for the detection of a HIV antigen, such as a HIV-I GAG polypeptide, in a biological sample. Compositions, kits, systems, and methods described herein, provide a wide dynamic range for detection and quantification of such antigens.
BACKGROUND OF THE INVENTION
[0003] Acquired Immunodeficiency Syndrome (AIDS) is an infectious and incurable disease transmitted through sexual contact with individual infected with the Human Immunodeficiency Virus type-1 (HIV-I) or by exposure to HIV-I contaminated blood or blood products. HIV-I includes the formerly named viruses Human T-cell Lymphotrophic Virus Type III (HTLV III), Lymphadenopathy Associated Virus (LAV), and AIDS Associated Retrovirus (ARV). HIV-I is a retrovirus related to a group of cytopathic retroviruses, lentiviruses, on the basis of morphologic features, genomic organization, and nucleotide sequence (Gonda et al. , 1985, Science 277: 177- 179; Stephan et al. , 1986, Science 231 :589-594; Korber, B. (ed.) et al, Human Retroviruses and AIDS. A Compilation and Analysis of Nucleic Acid and Amino Acid Sequences. Published by Theoretical Biology and Biophysics, Los Alamos National Laboratory, Los Alamos, N. Mex.; Reviewed in, Schochetman, G. and George, J. R., (1994) AIDS Testing. Springer- Verlag, N.Y., Berlin, Heidelberg). HIV-I is an enveloped virus containing several structural proteins. Of particular relevance, the core of the virus is formed by condensation of cleavage products from a highly processed gag-pol polyprotein precursor (Prl80gag-pol), which is cleaved into a pol precursor polypeptide and a gag precursor polypeptide (Pr55gag). Subsequently, the core precursor Pr55gag is cleaved into pi 7 (myristylated gag protein), p24 (major structural protein), p7 (nucleic acid binding protein), and p9 (proline-rich protein). The envelope contains two structural proteins, gpl20 (envelope glycoprotein) and gp41 (transmembrane protein), which are products of the cleavage of the envelope polyprotein precursor, gplόO.
[0004] Early diagnosis and monitoring of HIV-I infection in patients are paramount for the initiation and the evaluation of antiviral therapies. The most common markers of HIV-I infection are antibodies against viral structural proteins. In the United States, screening of blood and blood products by testing for the absence or presence of antibodies against HIV-I antigens or the absence or presence of HIV-I antigens is mandated. Some assays measure viral nucleic acid with molecular techniques that can detect a single viral genome (Maldarelli et al , 2007, PLoS Pathog 3 :e46; Palmer et al , 2003, J Clin Microbiol 41 :4531 -6). These molecular assays require the isolation of nucleic acid often from large sample volumes. The numerous stages involved and the instrumentation required for the extraction, the amplification, and the detection of the nucleic acid, often at a prohibitive cost, precludes their use in resource-poor settings (reviewed in Fiscus et al, 2006, PLoS Med 3: e417) as well as in experimental research. Similarly, in HIV experimental research, due to the large number of samples measured, it is often difficult to accommodate the costs of the detection of HIV-I nucleic acids.
[0005] Within several weeks of infection with HIV, individuals generally enter a clinical phase characterized by extensive viremia and acute symptoms. During this period, prior to seroconversion, HIV-I p24 core antigen can be detected transiently in serum or plasma specimens (antigenemia) (e.g., Kessler et al, 1987, JAMA 258:1196-1199; Phair, 1987,
JAMA 258: pl218; Allain et al, 1986, The Lancet ii:1233-1236). After seroconversion, the core protein apparently is complexed with antibodies in circulating immune complexes, making core protein detection difficult and requiring immune complex disruption techniques (Schύpbach et al, 1996, AIDS 10:1085-1090; Kageyama, et al, 1988, J Virol Methods 22: 125-131 ; Mathiesen ef α/., 1988, J Virol Methods 22: 143-148; Steindl et al, 1988, J Immunol Methods 217:143-151). After the initial viremic phase and throughout the remainder of the disease, the virus generally establishes a steady state level (reviewed in Coffin, 1995, Science 267: 483-489).
[0006] In resource-limited settings, the early diagnostic of HIV-I infection relies widely on the measurement of the HIV-I major core protein p24 (Fiscus et al., 2007, J Infect Dis 195:416-24), a method also embraced in experimental research. This method requires a smaller sample volume and is less laborious than its molecular counterparts. However, it remains nonetheless expensive and is often crippled by a small dynamic range, which necessitates the measurement of several sample dilutions to fall within the dynamic range of the assay. Accurate analysis of ELISAs often necessitates the measurement of several sample dilutions in such a way that one or several dilutions fall within the dynamic range of the assay.
[0007] Because of these limitations, commercial p24 ELISA to date fall into two groups: (i) ultra sensitive assays that often use an amplification technique to lower their limit of detection (Fiscus et al, 2007 , J Infect Dis 195:416-24; Respess et a!., 2005, J Clin Microbiol 43:506-8; Sutthent et al, 2003, J Clin Microbiol 41 :1016-22; Tehe et al, 2006, J Clin Virol 37:199-205) and (ii) less sensitive assays that allow the measurement of samples with high concentrations of antigen. The ultra sensitive p24 assays allow the detection of low antigen concentrations, in the range of 0.2 to 100 pg/ml (Boni et al, 1997, AIDS 11 :F47-52) while less sensitive assays allow detection above 1 ng/ml (e.g., NCI kit, VKP, Cell Biolabs). No p24 ELISA kit that has an inherent wide dynamic range together with a high sensitivity is available.
[0008] The HIV research and diagnostic community is in dire need of a reliable, sensitive and low-cost assay to measure viral replication in different experimental and medical settings. The compositions, systems, kits, and methods described herein satisfy the need of the HIV medico-scientific community as they have a unique and exquisite dynamic range and can be provided at a lower cost than presently available.
BRIEF SUMMARY OF THE INVENTION
[0009] The present invention provides economical and efficient methods and immunosystems for detecting antigens in a biological sample employing an assay that combines a wide dynamic range of detection with high sensitivity. [0010] Accordingly, in one aspect, the invention provides methods for detecting an antigen in a biological sample using an immunoassay having a dynamic range of between about 0.2 and about 20,000 pg/ml and a coefficient of variation of less than 10%. In some embodiments, the methods comprise the steps of (a) binding a first antibody specifically binding an antigen to a solid support, (b) contacting the first antibody with a biological sample suspected of containing the antigen for a time and under conditions sufficient to allow binding of the antigen to the first antibody, thereby forming a first antibody/antigen complex, (c) contacting the first antibody/antigen complex with a second antibody specifically binding the antigen for a time and under conditions sufficient to allow binding of the second antibody to the antigen of the first antibody/antigen complex, thereby forming a first antibody/ antigen/second antibody complex, wherein the second antibody produces a detectable signal, and (d) detecting the detectable signal and thereby detecting the antigen in the biological sample. The first antibody and the second antibody bind to different epitopes of the antigen.
[0011] Various antigens can be detected using compositions and methods of the present invention. A preferred antigen is a HIV-I antigen. In some embodiments, the HIV-I antigen is a GAG polypeptide. [0012] In some embodiments, the GAG polypeptide is a p24 polypeptide. In some embodiments, the p24 polypeptide has an amino acid sequence which is at least 90% identical to the amino acid sequence of SEQ ID NO:1. In some embodiments, the p24 polypeptide has the amino acid sequence of SEQ ID NO:1.
[0013] Various first antibodies can be used in the methods and immunosystems of the present invention. In some embodiments, the first antibody is a monoclonal antibody or a fragment thereof. Where the antigen is a p24 polypeptide, a preferred monoclonal antibody or fragment thereof is one specifically binding to the p24 polypeptide. In some embodiments, the first antibody binds to an epitope of the p24 polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO.7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, and SEQ ID NO:50.
[0014] In some embodiments where the antigen is a p24 polypeptide, the first antibody is selected from the group of antibodies consisting of 4F6, KC57, 111/182, 112/021, 112/047, ID8F6, F5-2, CB-13/5, 3D3, CD-4/1, 15F8C7, 111/052, 91-5, 47-2, 714/01, 1109/01, IG5C8, 14D4E11, 113/038, 111/073, l-E-4, l-E-9, 2-E-4, 2-H-4, 8-D-2, 8-H-7, 8-G-9, 10-E-7, 10-G- 9, l l-C-5, C5123, l-B-7, 3-B-7, 6-D-12, 6-E-7, 8-D-5, FFl, 113/072, 25.3, 13-102-100, RL4.72.1, 406/01, 38:9.6k, EB1A9, EF7, 30:3E5, LH-104-E, 1B2C12, LH-104-K, LH-104- A, 1A7, 1.17.3, 1F6, 23A5G5, 23A5G4, 3D10G6, F5-4, MO9.42.2, MO9.50.2, V-IO, V107, LH-104-C, 12-B-4, 9A4C4, 11D11F2, 1 IClOBlO, CD12B4, BE3, L14, 110/015, 108/03, 32:32K, C5200, FH2, 13B5, 106/01, LH-104-B, LH-104-B, and fragments thereof. A preferred first antibody is 4F6.
[0015] Various second antibodies can be used in the methods and immunosystems of the present invention. In some embodiments, the second antibody is a monoclonal antibody or a fragment thereof. [0016] Where the antigen is a p24 polypeptide, a preferred monoclonal antibody or fragment thereof is one specifically binding to the p24 polypeptide. In some embodiments, the second antibody binds to an epitope of the p24 polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, and SEQ ID NO:50. The second antibody binds to an epitope non-overlapping with the epitope to which the first antibody binds.
[0017] In some embodiments, the second antibody is selected from the group of antibodies consisting of 4F6, KC57, 111/182, 112/021, 112/047, ID8F6, F5-2, CB-13/5, 3D3, CD-4/1, 15F8C7, 111/052, 91-5, 47-2, 714/01, 1109/01, IG5C8, 14D4E11, 113/038, 111/073, l-E-4, l-E-9, 2-E-4, 2-H-4, 8-D-2, 8-H-7, 8-G-9, 10-E-7, 10-G-9, l l-C-5, C5123, l-B-7, 3-B-7, 6- D-12, 6-E-7, 8-D-5, FFl, 113/072, 25.3, 13-102-100, RL4.72.1, 406/01, 38:9.6k, EB1A9, EF7, 30:3E5, LH-104-E, 1B2C12, LH-104-K, LH-104-A, 1A7, 1.17.3, 1F6, 23A5G5, 23A5G4, 3D10G6, F5-4, MO9.42.2, MO9.50.2, V-10, V107, LH-104-C, 12-B-4, 9A4C4, 11D11F2, 11C1OB1O, CD12B4, BE3, L14, 110/015, 108/03, 32:32K, C5200, FH2, 13B5, 106/01, LH-104-B, LH-104-B, and fragments thereof. [0018] In some embodiments, the second antibody is KC57.
[0019] In a preferred embodiment, the first antibody is 4F6 and the second antibody is KC57.
[0020] The second antibody may comprise various detectable labels. A preferred label is a fluorochrome. A preferred second antibody is a second antibody labeled with a fluorochrome. In some embodiments, the fluorochrome is phycoerythrin.
[0021] In some embodiments, the methods comprise measuring a fluorescence signal produced by the second antibody.
[0022] In some embodiments, the solid support comprises a plurality of solid supports and step (d) comprises measuring a fluorescence signal for each individual member of the plurality of solid supports having bound the first antibody/antigen/second antibody complex.
[0023] In one aspect the antigen detection assay relies on using beads coupled to a high affinity monoclonal antibody against an antigen together with a non-overlapping monoclonal antibody coupled to a label, preferably a fluorochrome, such as phycoerythrine. [0024] Various solid supports can be used in the methods and immunosystems of the present invention. In some embodiments, the solid support is selected from the group consisting of a microsphere, a bead, a particle, a fiber, a monolith, a membrane, a plastic strip, and a microtiter plate.
[0025] In some embodiments, the solid support is a bead. In some embodiments, the solid support is a microsphere. In some embodiments, the solid support is carboxymethylated. In some embodiments the solid support is a Luminex bead. In some embodiments, the solid support is magnetic, preferably a magnetic bead. In some embodiments, the solid support is magnetic and fluorescent, preferably a magnetic and fluorescent bead. In some embodiments, the solid support is ferromagnetic, preferably a ferromagnetic bead. In some embodiments, the solid support is ferromagnetic and fluorescent, preferably a ferromagnetic and fluorescent bead.
[0026] Various biological samples can be analyzed using the methods and immunosystems of the present invention. In some embodiments, the biological sample is selected from the group consisting of serum, blood, lymph, and plasma. In some embodiments, the biological sample is obtained from a human. In some embodiments, the human is a patient having or suspected of having AIDS. In some embodiments, the biological sample is obtained from a patient treated with an anti-viral drug.
[0027] The volume of the biological can be varied in the methods and immunosystems of the present invention. In some embodiments, the biological sample is between about 20 μl and about 50 μl in volume.
[0028] In some embodiments, the methods comprise using a multiplex assay format and assaying for a second antigen present in the biological sample.
[0029] In some embodiments where the biological sample is obtained from a subject having or suspected of having AIDS, the methods further comprise the step of detecting a HIV-I antibody in the biological sample.
[0030] In some embodiments, the methods comprise the step detecting the presence of a second antigen in the biological sample wherein the second antigen is different from the p24 polypeptide.
[0031] In some embodiments, step (a) comprises binding of the first antibody to an activated solid support. In some embodiments, the methods comprise a washing step after step (a).
[0032] In some embodiments, the methods further comprise the step of determining the amount of the antigen. In some embodiments, the plurality of solid supports comprises at least 10 members and the methods further comprise measuring the signal from each of the at least 10 members.
[0033] In some embodiments, the methods further comprise the step of determining the amount of the solid support having bound the first antibody.
[0034] In some embodiments, the methods further comprise the step of adding the solid support having bound the first antibody to a multi-well container. [0035] In some embodiments, the methods further comprises the step of lysing the biological sample.
[0036] In some embodiments, step (d) comprises resuspending the solid support having bound the first antibody/antigen/second antibody complex in an assay buffer.
[0037] In some embodiments, the method is adapted to a high throughput method. [0038] In another aspect of the present invention an immunosystem for detecting an antigen in a biological sample is provided. The immunosystem has a dynamic range of between about 0.2 pg/ml and about 20 ng/ml and a coefficient of variation of less than 10%. In a preferred embodiment, the immunosystem comprises (i) a first antibody reactive to an antigen, wherein the first antibody is bound to a solid support, (ii) a biological sample containing or suspected of containing the antigen, and (iii) a second antibody reactive to the antigen, wherein the second antibody binds to a different epitope of the antigen than the first antibody.
[0039] In some embodiments, the second antibody comprises a label. [0040] Embodiments of the methods described herein may also be used as embodiments of the immunosystems.
[0041] Further embodiments of the immunosystem and methods are as described herein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0042] Figure 1 depicts a typical p24 standard curve obtained using an assay described herein. The graph represents the individual points a titration curve run in quadruplicate. Above each data point the CV of the data is indicated. The coefficient of determination is calculated to fit a 4 parameter logistic regression. Details are described in Example 3.
[0043] Figure 2 depicts inter-assay variation using an assay described herein. The figure represents the inter-assay variation calculated for four assays run in triplicates at different concentrations. For each concentration, the data points (12 per concentration) are plotted together with the median, the interquartile box, and the range of the data. The number above each data point represents the CV of the results at the corresponding concentration. Details are described in Example 3.
[0044] Figure 3 depicts the linearity of the detection of 4.98 μm magnetic beads binding a phycoerithrin-labeled antibody. Details are described in Example 6.
[0045] Figure 4 depicts a typical standard curve using HIV-I p24 polypeptide as an antigen. Details are described in Example 6.
[0046] Figures 5 A and 5B depict the amino acid sequence of a p24 polypeptide for HIV-I strain HXB2 (SEQ ID NO:3) and a p24 antibody epitope map (SEQ ID NOS:4-50) (HIV Molecular Immunology Database). The names of the monoclonal antibodies (MAbs) and the location of well characterized linear binding sites of 21 amino acids or less are indicated relative to the protein sequence of the HXB2 strain. This map is meant to provide the relative location of epitopes on a given protein. Above each linear binding site, the MAb name is given followed by the species in parentheses (h, human; p, non-human primate; m, mouse; o, others). DETAILED DESCRIPTION OF THE INVENTION
I. DEFINITIONS
[0047] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. The following references provide one of skill with a general definition of many of the terms used in this invention: Singleton et al. , Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them unless specified otherwise. [0048] It must be noted that, as used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a compound," includes mixture of compounds, reference to "an antigen" includes mixtures of antigens, reference to "a pharmaceutical carrier" includes mixtures of two or more such carriers, and the like. [0049] The headings used herein are for organizational purposes only and are not meant to be used to limit the scope of the description or the claims. As used throughout this application, the word "may" is used in a permissive sense (i.e., meaning having the potential to), rather than the mandatory sense (i.e., meaning must). Similarly, the words "include", "including", and "includes" mean including, but not limited to. [0050] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein. Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated-range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.
[0051] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e. g. "such as") provided herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.
[0052] As used herein, the term "about" refers to a range of values of plus or minus 10% of a specified value. For example, the phrase "about 200" includes plus or minus 10% of 200, or from 180 to 220.
[0053] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function similarly to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, γ- carboxyglutamate, and O-phosphoserine. Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, e.g., an α carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs may have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions similarly to a naturally occurring amino acid.
[0054] Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical
Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.
[0055] As used herein, the term "analyte" means a substance, compound, or composition whose presence, absence, amount, or concentration in a sample or specimen is to be detected or determined. Within the context of the current patent, a preferred "analyte" is an "antigen." [0056] As used herein, the term "antibody" refers to a protein functionally defined as a binding protein (a molecule able to bind to a specific epitope on an antigen) and structurally defined as comprising an amino acid sequence that is recognized by one of skill as being derived from the framework region of an immunoglobulin encoding gene. It includes whole antibody, functional fragments, modifications or derivatives of the antibody. It can also be a genetically manipulated product, or chimeric antibody, such as a humanized antibody. Antibodies can be a polyclonal mixture or monoclonal. Antibodies can be intact immunoglobulins derived from natural sources or from recombinant sources and can be immunoreactive portions of intact immunoglobulins, i.e., fragments with antigen-binding capability. Antibodies may exist in a variety of forms including, for example, rlgG, Fv, Fab, Fab', F(ab)2, as well as in single chains. Single-chain antibodies, in which genes for a heavy chain and a light chain are combined into a single coding sequence, may also be used. The term also refers to recombinant single chain Fv fragments (scFv). The term antibody also includes bivalent or bispecifϊc molecules, diabodies, triabodies, and tetrabodies. Bivalent and bispecifϊc molecules are described in, e.g., Kostelny et al. (1992, J Immunol 148:1547), Pack and Pluckthun (1992, Biochemistry 31 : 1579), Hollinger et al. (1993, supra), Gruber et al. (1994, J Immunol (152 (11):5368-74), Zhu et al. (1997, Protein Sci 6:781), Hu et al. (1996, Cancer Res. 56:3055), Adams et al. (1993, Cancer Res. 53:4026), and McCartney et al. (1995, Protein Eng. 8:301). [0057] An antibody immunologically reactive with a particular antigen can be generated by recombinant methods such as selection of libraries of recombinant antibodies in phage or similar vectors, see, e.g., Huse et al. (1989, Science 246:1275-1281); Ward et al. (1989, Nature 341 :544-546); and Vaughan et al. (1996, Nature Biotech. 14:309-314), or by immunizing an animal with the antigen or with DNA encoding the antigen. [0058] Typically, an immunoglobulin has a heavy and light chain. Each heavy and light chain contains a constant region and a variable region, (the regions are made up by the assembly of immunoglobulin-like domains). Light and heavy chain variable regions contain four "framework" regions interrupted by three hypervariable regions, also called "complementarity-determining regions" or "CDRs." The extent of the framework regions and CDRs have been defined. The sequences of the framework regions of different light or heavy chains are relatively conserved within a species. The framework region of an antibody, that is the combined framework regions of the constituent light and heavy chains, serves to position and align the CDRs in space. [0059] For purposes of the present invention, a "fragment" or "antibody fragment" is defined as a subunit of an antibody, preferably, a monoclonal antibody, which reacts in the same manner, functionally, as the full antibody with respect to binding properties. An "antibody fragment" comprises a portion of an antibody such as F(ab)2, F(ab')2, Fab, Fv, sFv, and the like. Regardless of structure, an antibody fragment binds to the same antigen that is recognized by the intact antibody. The term "antibody fragment" also includes any synthetic or genetically engineered protein that acts like an antibody by binding to a specific antigen to form a complex. For example, antibody fragments include isolated fragments consisting of the variable regions, such as the "Fv" fragments consisting of the variable regions of the heavy and light chains, recombinant single chain polypeptide molecules in which light and heavy variable regions are connected by a peptide linker ("scFv proteins"), and minimal recognition (CDR) units consisting of the amino acid residues that constitute the hypervariable region.
[0060] The phrase "single chain Fv" or "scFv" refers to an antibody in which the variable domains of the heavy chain and of the light chain of a traditional two-chain antibody have been joined to form one chain. Typically, a linker peptide is inserted between the two chains to allow for proper folding and creation of an active binding site.
[0061] A "chimeric antibody" is an immunoglobulin molecule in which (a) the constant region, or a portion thereof, is altered, replaced or exchanged so that the antigen binding site (variable region) is linked to a constant region of a different or altered class, effector function and/or species, or an entirely different molecule which confers new properties to the chimeric antibody, e.g., an enzyme, toxin, hormone, growth factor, drug, etc.; or (b) the variable region, or a portion thereof, is altered, replaced or exchanged with a variable region having a different or altered antigen specificity. [0062] A "humanized antibody" is an immunoglobulin molecule which contains minimal sequence derived from non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the framework (FR) regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin (Jones et al, 1986, Nature 321:522-525; Riechmann et al, 1988, Nature 332:323-329; and Presta, 1992, Curr O. Struct Biol 2:593-596). Humanization can be essentially performed following the method of Winter and co-workers (Jones et al, 1986, Nature 321 :522-525; Riechmann et al, 1988, Nature 332:323-327; Verhoeyen et al, 1988, Science 239:1534-1536), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such humanized antibodies are chimeric antibodies (U.S. Patent No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. [0063] The term "fully human antibody" refers to an immunoglobulin comprising human variable regions in addition to human framework and constant regions. Such antibodies can be produced using various techniques known in the art. For example in vitro methods involve use of recombinant libraries of human antibody fragments displayed on bacteriophage {e.g., McCafferty et al, 1990, Nature 348:552-554; Hoogenboom & Winter, 1991, J MoI Biol 227:381; and Marks et al, 1991, JMoI Biol 222:581), yeast cells (Boder and Wittrup, 1997, Nat Biotechnol 15:553-557), or ribosomes (Hanes and Pluckthun, 1997, Proc Natl Acad Sd USA 94:4937-4942). Similarly, human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, e.g., in U.S. Patent Nos. 6,150,584, 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the following scientific publications: {e.g., Jakobavits, 1988, Adv Drug Deliv Rev. 31:33-42; Marks et al, 1992, Bio/Technology 10:779-783; Lonberg et al, 1994, Nature 368:856-859; Morrison, 1994, Nature 368:812-13; Fishwild et al, 1996, Nature Biotechnology 14:845-51; Neuberger, 1996, Nature Biotechnology 14:826; Lonberg & Huszar, 1995, Intern. Rev. Immunol. 13:65-93). Alternatively, human monoclonal antibodies can be obtained by immunizing SCID-hu mice and using their B cells to produce monoclonal antibodies following techniques know in the art, including, but not limited to, cell fusion and viral transformation.
[0064] As used herein, the phrase "antibody reactive to analyte" means that the antibody has an area on its surface or in a cavity which specifically binds to a particular analyte, i.e., it has a binding affinity (usually expressed as Ka) for the analyte.
[0065] As used herein, the term "antigen" refers to a substance that prompts the generation of antibodies and can cause an immune response. Antigens are usually proteins or polysaccharides. This includes parts (coats, capsules, cell walls, capsids, fiagella, and toxins) of bacteria, viruses, and other microorganisms. The term encompasses all substances that can be recognized by the adaptive and innate immune system and by an antibody or antibody fragment in vitro.
[0066] As used herein, the terms, "anti-viral agent" or anti-viral drug" refer to an agent that inhibits viral proliferation and is known to those of skill in the art. Anti -viral agents are used against infections or disease states caused by a virus, including, without limitation, a lentivirus, a hepatitis A virus, a hepatitis B virus, and a hepatitis C virus.
[0067] As used herein, the term "biological fluid" refers to a fluid from a host and includes, without limitations, whole blood, serum, plasma, urine, tears, mucus, nasal swab, sweat, milk, ascites fluid, saliva, oral fluid, semen, seminal fluid, vaginal secretions, breast milk, stool, sputum, gastric fluid, cerebrospinal fluid, pleural fluid, synovial fluid, interstitial fluid, lymph fluid, amniotic fluid, peritoneal fluid, fetal fluid, fluid from ulcers and other surface eruptions, blisters, and abscesses, and extracts of tissues including biopsies of normal, malignant, and suspect tissues or any other constituents of the body which may contain an analyte or antigen of interest.
[0068] As used herein, the term "biological sample" refers to any sample obtained from a living or dead organism. The term "biological sample" refers to a sample of biological tissue or fluid that contains nucleic acids or polypeptides, e.g., a HIV nucleic acid or a HIV polypeptide. Such samples are typically from humans, but include tissues isolated from non- human primates (e.g., monkeys) or rodents (e.g., mice, and rats) and other mammals. Numerous types of biological samples can be used in the present invention, including, but not limited to, sections of tissues such as biopsy and autopsy samples, frozen sections taken for histological purposes, and biological fluids, etc. Biological samples also include explants and primary and/or transformed cell cultures derived from patient tissues. A "biological sample" also refers to a cell or population of cells or a quantity of tissue or fluid from an animal. Most often, the sample has been removed from an organism, but the term "biological sample" can also refer to cells or tissue analyzed in vivo, i.e., without removal from the organism. The definition also includes samples that have been manipulated in any way after their procurement, such as by treatment with reagents, washed, or enrichment for certain cell populations, such as CD4+ T lymphocytes, glial cells, macrophages, tumor cells, peripheral blood mononuclear cells (PBMC), and the like. The term "biological sample" encompasses a clinical sample, and also includes cells in culture, cell supernatants, cell extracts, tissue samples, organs, bone marrow, and the like. [0069] As used herein, the term "calibration material" refers to any standard or reference material containing a known amount of an analyte to be measured.
[0070] As used herein, the term "capture binding partner" refers to a molecule able to bind to a specific epitope on an antigen. A capture binding partner, typically, does not comprise a label. Within the context of the current patent, a preferred capture binding partner is an antibody.
[0071] As used herein, the term "coefficient of determination" refers to the square of the coefficient of correlation and denotes how much of the observed data is explained by the model to which they are fitted. For example, an R2 of 0.6 means that the model fitted explains 60% of the variance of the data. [0072] As used herein, the term "coefficient of variation" or "CV" or "%CV" means standard variation divided by average multiplied by 100.
[0073] "Conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical or associated, e.g., naturally contiguous, sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode most proteins. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to another of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are "silent variations," which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes silent variations of the nucleic acid. One of skill will recognize that in certain contexts each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, often silent variations of a nucleic acid which encodes a polypeptide is implicit in a described sequence with respect to the expression product, but not with respect to actual probe sequences. As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologues, and alleles of the invention. Typically, conservative substitutions for one another: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins (1984))
[0074] As used herein, the term "conjugation" refers to any process wherein two compounds or compositions are linked together to form a conjugate, in particular and within the context of the present invention, for example, an antibody/antigen complex.
[0075] As used herein, the phrase "correlating an amount" means comparing an amount of a substance, analyte or antigen (such as a HIV polypeptide) that has been determined in one sample to an amount of the same substance, analyte or antigen determined in another sample. The amount of the same substance, analyte or antigen determined in another sample may be specific for a given compound or agent contacted with the sample or a disease state.
[0076] As used herein, the terms "detecting binding partner" or "detection binding partner" refer to a molecule able to bind to a specific epitope on an antigen. A detecting binding partner, typically, comprises a label. Within the context of the current patent, a preferred detecting binding partner is an antibody.
[0077] Synonyms for the phrase "determining an amount" are contemplated within the scope of the present invention and include, but are not limited to, detecting, measuring, testing, or determining the presence, absence, amount, or concentration of an analyte or antigen, such as a HIV polypeptide, by any quantitative or qualitative assay.
[0078] As used herein, the term "dynamic range" means the range of analyte concentrations to be measured. Dynamic range refers to the range of analyte concentrations that can be detected or quantified by the method of the present invention without the need for dilution or other treatment of a sample to alter the concentration of the analyte in a sample. Such treatment does not preclude preparing a sample for use in an assay.
[0079] As used herein, the terms "epitope" or "antigenic determinant" refer to a site on an antigen to which an antibody binds. Epitopes can be formed both from contiguous amino acids or noncontiguous (discontinuous) amino acids juxtaposed by tertiary folding of a protein. Other epitopes are formed by quaternary structures, e.g., by the assembly of several polypeptides. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. An epitope typically includes at least 3, and more usually, at least 5 or at least 8-10 amino acids in a unique spatial conformation. Methods of determining spatial conformation of epitopes include, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance. See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, Glenn E. Morris, Ed (1996).
[0080] As used herein, the terms "fluorescence" or "fluorescence signal" or "reporter signal" mean a signal measured in a fluorimeter or similar device. The signal may be given as fluorescent unit.
[0081] As used herein, the term "fluorescent moiety," refers to one or more fluorescent entities whose total fluorescence is such that the moiety may be detected in a fluorimeter or similar devices, including, but not limited to, a single molecule detector. A fluorescent moiety may comprise a single entity (e.g., a Quantum Dot or fluorescent molecule) or a plurality of entities (e.g., a plurality of fluorescent molecules). It will be appreciated that when "moiety," as that term is used herein, refers to a group of fluorescent entities (e.g., a plurality of fluorescent dye molecules), each individual entity may be attached to a detecting binding partner separately or the entities may be attached together, as long as the entities as a group provide sufficient fluorescence to be detected.
[0082] The terms "identical" or percent "identity," in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., about 60% identity, preferably 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region, when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection (see, e.g., Altschul et al., 1977, Nucl Acids Res 25:3389-3402 and Altschul et al, 1990, J MoI Biol 215:403-410). Such sequences are then said to be "substantially identical." This definition also refers to, or may be applied to, the compliment of a test sequence. The definition also includes sequences that have deletions and/or additions, as well as those that have substitutions, as well as naturally occurring, e.g., polymorphic or allelic variants, and man-made variants. As described below, the preferred algorithms can account for gaps and the like. Preferably, identity exists over a region that is at least about 25 amino acids or nucleotides in length, or more preferably over a region that is 50-100 amino acids or nucleotides in length. [0083] For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Preferably, default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
[0084] A "comparison window", as used herein, includes reference to a segment of one of the number of contiguous positions selected from the group consisting typically of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. MoI. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat 'I. Acad. ScL USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by manual alignment and visual inspection (see, e.g., Current Protocols in Molecular Biology (Ausubel et al., eds. 1995 supplement)).
[0085] Preferred examples of algorithms that are suitable for determining percent sequence identity and sequence similarity include the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al, \911, Nucl Acids Res 25:3389-3402 and Altschul et al, 1990, J MoI Biol 215:403-410). BLAST and BLAST 2.0 are used, with the parameters described herein, to determine percent sequence identity for the nucleic acids and proteins of the invention. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive- valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, e.g., for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always < 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative- scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11 , an expectation (E) of 10, M=5, N=-4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. ScL USA 89:10915 (1989)) alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and a comparison of both strands. [0086] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, 1993, Proc Nat 'I Acad Sci USA 90:5873-5787). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01 , and most preferably less than about 0.001. Log values may be large negative numbers, e.g., 5, 10, 20, 30, 40, 40, 70, 90, 110, 150, 170, etc.
[0087] An indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the antibodies raised against the polypeptide encoded by the second nucleic acid, as described below. Thus, a polypeptide is typically substantially identical to a second polypeptide, e.g., where the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions, as described below. Yet another indication that two nucleic acid sequences are substantially identical is that the same primers can be used to amplify the sequences. [0088] The terms "isolated," "purified," or "biologically pure" refer to material that is substantially or essentially free from components that normally accompany it as found in its native state. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. A protein or nucleic acid that is the predominant species present in a preparation is substantially purified. In particular, an isolated nucleic acid is separated from some open reading frames that naturally flank the gene and encode proteins other than protein encoded by the gene. The term "purified" in some embodiments denotes that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. Preferably, it means that the nucleic acid or protein is at least 85% pure, more preferably at least 95% pure, and most preferably at least 99% pure. "Purify" or "purification" in other embodiments means removing at least one contaminant from the composition to be purified. In this sense, purification does not require that the purified compound be homogenous, e.g., 100% pure.
[0089] As used herein, the term "label" refers to a composition detectable by spectroscopic, fluorimetric, photochemical, biochemical, immunochemical, chemical, or other physical means. For example, useful labels include fluorescent moieties, electron-dense reagents, enzymes (e.g., as commonly used in an ELISA), biotin, digoxigenin, or haptens and proteins or other entities which can be made detectable, e.g., by incorporating a radiolabel into a peptide or used to detect antibodies specifically reactive with an antigen. The radioisotope may be, for example, 3H, 14C, 32P, 35S, or 125I. The labels may be incorporated into the antibodies at any position. Any method known in the art for conjugating the antibody to a label may be employed, including those methods described by Hunter et al. (1962, Nature, 144:945); David et al. (1974, Biochemistry, 13:1014); Pain et al. (1981, J. Immunol. Meth., 40:219); and Nygren (1982, J Histochem. and Cytochem., 30:407). The lifetime of radiolabeled peptides or radiolabeled antibody compositions may be extended by the addition of substances that stabilize the radiolabeled peptide or antibody and protect it from degradation. Any substance or combination of substances that stabilizes the radiolabeled peptide or antibody may be used including those substances disclosed in US Patent No. 5,961,955.
[0090] The term "lentivirus" as used herein, refers to human immunodeficiency virus- 1 (HIV-I), human immunodeficiency virus-2 (HIV-2), simian immunodeficiency virus (SIV), and feline immunodeficiency virus (FIV). [0091] As used herein, the term "limit of detection" refers to the lowest concentration at which one can identify an analyte of interest in a sample.
[0092] As used herein, the term "lysing" refers to the breakage of a cell membrane, plasma membrane, and viral particles sufficient to release at least some intracellular or viral content, such as polypeptides and nucleic acids. Lysing can be performed by mechanical, chemical, or osmotic mechanisms that compromise the integrity of a cell or virus particle.
[0093] As used herein, the terms "monitoring" or "monitoring a response to a therapeutic agent" refer to use of the present invention to determine a disease state of a subject after a therapeutic agent has been administered. An index value of the subject may be compared to an index value for the same subject that was determined before initiation of use of the therapeutic agent.
[0094] The terms "polypeptide," "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues joined together by a peptide bond. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers, those containing modified residues, and non-naturally occurring amino acid polymer. It may be appreciated that polypeptides can contain amino acids other than the 20 amino acids commonly referred to as the 20 naturally occurring amino acids. Also, polypeptides can include one or more amino acids, including the terminal amino acids, which are modified by any means known in the art (whether naturally or non-naturally). Examples of polypeptide modifications include e.g., by glycosylation, or other-post-translational modification. Modifications which may be present in polypeptides of the present invention, include, but are not limited to, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a polynucleotide or polynucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamate, formylation, gamma-carboxylation, glycation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer- RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination.
[0095] The phrase "providing a biological sample" means to obtain a biological sample for use in, systems, kits, and methods described in this invention. Most often, this will be done by removing a sample of cells from a host, but can also be accomplished by using previously isolated cells (e.g., isolated by another person, at another time, and/or for another purpose), or by performing the methods of the invention in vivo. Archival tissues, having treatment or outcome history, will be particularly useful. [0096] As used herein, the term "seroconversion" refers to the development of detectable specific antibodies to microorganisms, e.g., bacteria, viruses, in the blood serum of a subject as a result of infection or immunization. Prior to seroconversion, blood tests are seronegative for the antibody; after seroconversion, blood tests are seropositive for the antibody.
[0097] As used herein, the term "solid support" refers to any insoluble material including, without limitation, particles (e.g., beads), fibers, monoliths, membranes, filters, plastic strips and the like.
[0098] The phrase "specifically (or selectively) binds" to an antibody or antigen, such as a HIV antigen, or "specifically (or selectively) immunoreactive with," when referring to an antigen or peptide or antibody, refers to a binding reaction that is determinative of the presence of the antigen or peptide or antibody, in a heterogeneous population of antigens, peptides or antibodies and other biologies. Thus, under designated immunoassay conditions, a specified antibody binds to a particular antigen at least two times the background and more typically more than 10 to 100 times background. Specific binding to an antibody under such conditions requires an antibody that is selected for its specificity for a particular antigen. For example, antibodies raised to a particular protein, polymorphic variants, alleles, orthologs, and conservatively modified variants, or splice variants, or portions thereof, can be selected to obtain only those antibodies that are specifically immunoreactive with a HIV protein and not with other proteins. This selection may be achieved by subtracting out antibodies that cross-react with other molecules. A variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular antigen. For example, solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein (see, e.g., Harlow & Lane, Antibodies, A Laboratory Manual (1988) for a description of immunoassay formats and conditions that can be used to determine specific immunoreacti vity) .
[0099] The terms "subject," "individual, " "host," and "patient," used interchangeably herein, refer to a mammal, including, but not limited to, murines, felines, simians, humans, mammalian farm animals, mammalian sport animals, and mammalian pets. The term includes mammals that are susceptible to infection by a virus, such as a lentivirus.
[0100] As used herein, the term "therapeutic agent" refers to any compound, drug or procedure used to improve the health of a subject. A therapeutic agent includes, without limitations, anti-viral agents. [0101] As used herein, a "tissue biopsy" refers to an amount of tissue removed from a subject for diagnostic analysis. In a patient with cancer, tissue may be removed from a tumor, allowing the analysis of cells within the tumor. In a patient afflicted with AIDS, tissue may be removed, e.g., from a lymph node. "Tissue biopsy" can refer to any type of biopsy, such as needle biopsy, fine needle biopsy, surgical biopsy, etc. [0102] As used herein, the terms "treat", "treatment," "treating," and the like, refer to obtaining a desired pharmacologic and/or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disease and/or adverse affect attributable to the disease. "Treatment," as used herein, covers any treatment of a disease in a mammal, particularly in a human, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, e.g., causing regression of the disease, e.g., to completely or partially remove symptoms of the disease. Treatment means any manner in which the symptoms or pathology of a condition, disorder, or disease are ameliorated or otherwise beneficially altered. In the context of a viral infection, such as lentivirus infection, the term "treatment" encompasses prevention of establishment of a systemic infection following initial contact with the virus; and prophylactic treatment of an individual not yet infected with the virus.
IL COMPOSITIONS
[0103] The invention provides compositions, systems, kits, and methods for the highly sensitive, precise and robust detection of an analyte in a biological sample, and for the determination of the amount or concentration of the analyte in the sample. In particular, the sensitivity and precision of the compositions, systems, kits, and methods of the invention make possible the detection and determination of concentration of analytes, e.g., antigens, such as HIV polypeptides, at extremely low concentrations, e.g., concentrations below about 100, 10, 1, 0.1, or 0.01 pg/ml. In further embodiments, the compositions, systems, kits, and methods of the invention are capable of determining an amount or concentration of an analyte in a biological sample over a large dynamic range of concentrations without the need for dilution or other treatment of the sample, which alter the concentration of the analyte in a sample, e.g., over a concentration range of between about 0.2 pg/ml and about 20,000 pg/ml.
[0104] Examples of molecules which can be detected using the compositions, systems, kits, and methods of the present invention include biopolymers, such as proteins, nucleic acids, carbohydrates, and small molecules, both organic and inorganic. In particular, the compositions, systems, kits, and methods described herein are useful for the detection of proteins in biological samples, and the determination of concentration of such molecules in the sample. [0105] The analyte detected by the present compositions, systems, kits, and methods may be free or may be part of a complex, e.g., an antibody-antigen complex, or more generally a protein-protein complex.
[0106] In one aspect, the present invention provides compositions, systems, kits, and methods comprising two binding partners and a biological sample. In some embodiments, the two binding partners are antibodies. One binding partner, referred to herein as a capture binding partner, is usually immobilized on a solid support. The other binding partner, referred to herein as a detection binding partner, typically comprises a detectable label. Both binding partners bind to the same analyte, such as a HIV antigen, albeit to different or non- overlapping epitopes of the analyte. As such, various combinations of binding partners can be used in the kits, systems, and methods described herein.
A. Analytes [0107] In a preferred embodiment of the present invention, the analyte which is detected or quantified in a biological sample is an antigen. Various antigens can be detected and quantified using methods, compositions, and systems of the present invention. A preferred antigen is an antigen from a pathogen, such as a bacterium or a virus. Thus, preferred antigens include a bacterial antigen and a viral antigen. An preferred viral antigen is a lentivirus antigen. A preferred lentivirus is Human Immunodeficiency Virus (HIV), including HIV-I and HIV-2. A preferred HIV is HIV-I . A preferred lentivirus antigen is a HIV antigen, including HIV-I antigens and HIV-2 antigens. In some aspects of methods of the invention, the presence of HIV-I is determined by determining whether a HIV-I antigen is present in a biological sample. [0108] A preferred HIV-I antigen is a GAG polypeptide, a POL polypeptide, or a NEF polypeptide. A HIV protease mediates the cleavage of GAG, POL, and NEF precursor polypeptides (Jacks et al, 1998, Nature 331 :280-283; Krausslich et al, 1989, Proc Natl Acad Sd USA 86:807-811). Specifically, the main structural polypeptides, GAG polypeptides, are produced by proteolytic cleavage of a GAG polypeptide precursor polypeptide, known as Pr55Gag into matrix (MA, pi 7), core antigen capsid (CA, p24), nucleocapsid (NC, p7), p2, pi, and p6 gag polypeptides. A preferred GAG polypeptide is a p24 polypeptide, but any HIV-I protein can be analyzed.
[0109] HIV-I POL polypeptides include the viral enzymes. They are formed by proteolytic cleavage of a second precursor polypeptide, known as PrI 60gag"po1, a fusion protein derived by ribosomal frame shifting. Although Prl60gag~po! also contains pi 7, p24 and p2, its C-terminal cleavage products are NC, a transmembrane protein (TFP), a p6po1 polypeptide, a protease (PR), a reverse transcriptase, (RTp51), a RT-RNase H (RTp66), and an integrase (IN) (see De Oliveira et al, 2003, J Virol 77(17):9422-9430). In preferred embodiments, a first antibody or fragment thereof binds to a POL polypeptide. In some preferred embodiments, a first antibody or fragment thereof binds to a NEF polypeptide. [0110] As will be appreciated by one skilled in the art, the methods of the present invention are not limited to detection of any particular HIV protein, including any form of Gag, Pol, Env, Nef, Vif, Rev, Vpr, Tat or VpuA, but are useful to also detect non-HIV antigens.
[0111] A preferred HIV-I antigen detected and quantified as described herein, is a GAG polypeptide. A preferred GAG polypeptide is Pr55gag. In some embodiments, a GAG polypeptide is a polypeptide having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence shown in SEQ ID NO: 1. In some embodiments, a GAG polypeptide has the amino acid sequence of SEQ ID NO: 1. SEQ ID NO:1 shows the amino acid sequence of HIV-I (isolate LAV-Ia). Amino acids 2-132 of SEQ ID NO:1 correspond to a pi 7 GAG polypeptide and amino acids 149-363 of SEQ ID NO:1 correspond to a p24 GAG polypeptide. SEQ ID NO:1 can be obtained from GenBank accession number FOVWLV. Another HIV-I strain, HXB2 (GenBank accession Number K03455) is known in the art. Many studies have been performed using HXB2 and the crystal structures for HXB2-related proteins are known. The HXB2 p24 polypeptide sequence (SEQ ID NO:3) is shown in Figure 5 along with a p24 antibody epitope map.
[0112] In some embodiments, a GAG polypeptide is a conservatively modified variant of the GAG polypeptide having the amino acid sequence shown in SEQ ID NO:1. In some embodiments, a GAG polypeptide is a conservatively modified variant of the GAG polypeptide having the amino acid sequence shown in SEQ ID NO:2. In some embodiments, a GAG polypeptide is a conservatively modified variant of the GAG polypeptide having the amino acid sequence shown in SEQ ID NO:3.
[0113] In some embodiments, a GAG polypeptide is a polypeptide having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence shown in SEQ ID NO:2. In some embodiments, a GAG polypeptide has the amino aid sequence of SEQ ID NO:2. Amino acids 2-111 of SEQ ID NO:2 correspond to a pl7 GAG polypeptide and amino acids 146-355 of SEQ ID NO:2 correspond to a p24 GAG polypeptide. SEQ ID NO:2 can be obtained from GenBank accession number ACD80633. [0114] In addition, exemplary GAG polypeptide and p24 polypeptide sequences can be found in GenBank accession numbers AAT80850, ABO61536, Q70622, AAX33047, ABC41796, ABQ82091, NP_057850, AAN08313, AAN08311, P03348, AAX33143, AAB38052, ABY78353, AAT80852, ABP37940, and AAX32976. [0115] In some embodiments, a GAG polypeptide is a p24 polypeptide. A preferred p24 GAG polypeptide is a polypeptide having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to amino acid residues 149-363 of SEQ ID NO:1. Another preferred p24 polypeptide has the amino acid sequence of amino acid residues 149- 363 of SEQ ID NO: 1. In some embodiments, a p24 GAG polypeptide is a polypeptide having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to amino acid residues 146-355 of SEQ ID NO:2. Another preferred p24 polypeptide has the amino acid sequence of amino acid residues 146-355 of SEQ ID NO:2.
[0116] In some embodiments, a p24 polypeptide is a polypeptide having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence shown in SEQ ID NO:3. Another preferred p24 polypeptide has the amino aid sequence of SEQ ID NO:3.
[0117] In some embodiments, a p24 polypeptide is a p24 polypeptide comprising an epitope having an amino acid sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO.14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, and SEQ ID NO:50.
B. Binding Partners And Antibodies
[0118] In some embodiments, the analyte of interest is captured through binding to a capture binding partner. In a preferred embodiment, the capture binding partner is an antibody specifically binding to the analyte of interest, i.e., the analyte to be detected in a biological sample. Other useful capture binding partners are molecules to which the analyte of interest has an affinity, i.e., to which it binds. In a preferred embodiment, a detecting binding partner, e.g., a detecting antibody, binds to the captured analyte. It will be appreciated that an antibody identified herein which is useful as a capture antibody is also useful as a detecting antibody, and vice versa. Binding of a capture antibody will not interfere with the binding of a detecting antibody.
[0119] Antibodies useful to practice methods of the present invention can be monoclonal antibodies, polyclonal antibodies, or fragments thereof. A preferred antibody is a monoclonal antibody.
[0120] Antibodies may exist in a variety of forms, all of which find use in the compositions, kits, systems, and methods of the present invention. These forms include, without limitations, rlgG, Fv, Fab, Fab', F(ab)2, as well as in single chains, scFv. Further, chimeric antibodies, partially or fully humanized antibodies are within the scope of the present invention and can be used in the methods and immunosystems described herein.
[0121] In a preferred embodiment of the present invention, a capture binding partner is an antibody, sometimes referred to herein as a capture antibody or first antibody. A preferred first antibody is an antibody binding to a lentivirus antigen. A preferred lentivirus antigen is a HIV antigen, including HIV-I antigens and HIV-2 antigens. In some aspects of methods of the invention, the presence of HIV is determined by determining whether a HIV antigen is present in a biological sample.
1. Anti-HIV-1 Antibodies
[0122] In some embodiments, a first antibody or fragment thereof binds to a GAG polypeptide. [0123] In some embodiments, a first antibody or fragment thereof binds to a POL polypeptide. [0124] In some embodiments, a first antibody or fragment thereof binds to a NEF polypeptide.
2. Anti-HIV-1 p24 Antibodies
[0125] A preferred HIV-I antigen detected or quantified in a biological sample is a p24 polypeptide. In some embodiments of the present invention, a first antibody is an anti HIV-I p24 antibody or a fragment thereof.
[0126] In some embodiments of the present invention, an anti HIV-I p24 antibody is a monoclonal anti-p24 antibody or a fragment thereof. A preferred monoclonal anti-p24 antibody is 4F6 or a fragment thereof. [0127] Other anti-p24 antibodies are also contemplated for use in the compositions, kits, systems, and methods of the present invention. Preferred anti-HIV-1 p24 antibodies bind to a polypeptide comprising an amino acid sequence selected from the amino acid sequences of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3. In a preferred embodiment of the present invention, a first antibody is an antibody binding to an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3.
[0128] In some embodiments, a first antibody is an anti-HIV-1 p24 antibody selected from a HIV-I antibody binding to a GAG polypeptide having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence shown in SEQ ID NO: 1.
[0129] In some embodiments, a first antibody is an anti-HIV-1 p24 antibody selected from a HIV-I antibody binding to a GAG polypeptide having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence shown in SEQ ID NO:2.
[0130] In some embodiments, a first antibody is an anti-HIV-1 p24 antibody selected from a HIV-I antibody binding to a p24 polypeptide having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence shown in SEQ ID NO:3.
[0131] In some embodiments, a first antibody is an antibody binding to an epitope of a p24 polypeptide comprising the amino acid sequence OfNH2-PIVQNIQGQMVHQAISPRTL- COOH (SEQ ID NO:4). Preferred antibodies binding thereto are 111/182, 112/021, and 112/047 or fragments thereof (Niedrig et al, 1991, J Virol 65:4529-4533) (Figure 5).
[0132] In some embodiments, a first antibody is an antibody binding to an epitope of a p24 polypeptide comprising the amino acid sequence OfNH2-VHQAISPRTLNAWVK-COOH (SEQ ID NO:5). A preferred antibody binding thereto is ID8F6 or a fragment thereof (Ferns et al, 1987, J Gen Virol 68:1543-1551; Ferns et al, 1989, Λ/ZλS 3:829-834) (Figure 5).
[0133] In some embodiments, a first antibody is an antibody binding to an epitope of a p24 polypeptide comprising the amino acid sequence OfNH2-AISPRTLNAW-COOH (SEQ ID NO:6). A preferred antibody binding thereto is F5-2 or a fragment thereof (Kusk et al., 1988, J Acquir Immune Defic Syndr 1 :326-332; Kusk et al. , 1992, AIDS Res Hum Retroviruses 8:1789-1794) (Figure 5).
[0134] In some embodiments, a first antibody is an antibody binding to an epitope of a p24 polypeptide comprising the amino acid sequence OfNH2-NAWVK-COOH (SEQ ID NO:7). A preferred antibody binding thereto is CB- 13/5 or a fragment thereof (Grunow et al., 1990, ZKHn Med 45:367-369; Franke et al, 1992, JMeJ Virol 37:137-142; Kuttner et al, 1992, MoI Immunol 29:561-564; Glaser et al, 1996, J Immunological Methods 189:1-14) (Figure 5).
[0135] In some embodiments, a first antibody is an antibody binding to an epitope of a p24 polypeptide comprising the amino acid sequence OfNH2-SEGATPQDLNTMLNTVG-COOH (SEQ ID NO: 8). A preferred antibody binding thereto is a polyclonal antibody or a fragment thereof (Truong et al, 1997, J Med Virol 51(3):145-51) (Figure 5).
[0136] In some embodiments, a first antibody is an antibody binding to an epitope of a p24 polypeptide comprising the amino acid sequence OfNH2-EGATPQ-COOH (SEQ ID NO:9). A preferred antibody binding thereto is 3D3 or a fragment thereof (Ferns et al, 1987, J Gen Virol 68:1543-1551; Ferns et al, 1989, AIDS 3:829-834) (Figure 5). [0137] In some embodiments, a first antibody is an antibody binding to an epitope of a p24 polypeptide comprising the amino acid sequence OfNH2-GATPQDLNTML-COOH (SEQ ID NO: 10). A preferred antibody binding thereto is CD-4/1 or a fragment thereof (Grunow et al, 1990, ZKHn Med 45:367-369; Franke et al, 1992, J Med Virol 37:137-142; Hohne et al, 1993, MoI Immunol 30:1213-1221 ; Glaser et al, 1996, J Immunological Methods 189:1-14; Ehrhard et al., 1996, Biochemistry 35:9097-9105) (Figure 5). [0138] In some embodiments, a first antibody is an antibody binding to an epitope of a p24 polypeptide comprising the amino acid sequence OfNH2-ATPQDLNTML-COOH (SEQ ID NO:11). A preferred antibody binding thereto is 15F8C7 or a fragment thereof (Janvier et al, 1990, J Virol 64:4258-4263; Janvier et al, 1992, J Virol 66:613) (Figure 5).
[0139] In some embodiments, a first antibody is an antibody binding to an epitope of a p24 polypeptide comprising the amino acid sequence OfNH2-DLNTMLNTVG-COOH (SEQ ID NO:12). A preferred antibody binding thereto is 1 11/052 or a fragment thereof (Niedrig et al, 1991, J Virol 65:4529-4533) (Figure 5).
[0140] In some embodiments, a first antibody is an antibody binding to an epitope of a p24 polypeptide comprising the amino acid sequence of NH2- DLNTMLNTVGGHQAAMQMLKETINEEAAEWDR-COOH (SEQ ID NO: 13). A preferred antibody binding thereto is a polyclonal antibody or a fragment thereof (Pialoux et al, 2001, AIDS 15(10): 1239-49) (Figure 5).
[0141] In some embodiments, a first antibody is an antibody binding to an epitope of a p24 polypeptide comprising the amino acid sequence OfNH2-AAMQMLKETINE-COOH (SEQ ID NO: 14). A preferred antibody binding thereto is 91 -5 or a fragment thereof (Gorny et al , 1989, Proc Natl Acad Sci USA 86:1624-1628; Tyler et al, 1990, J Immunol 145:3276-3282; Robinson et al, 1990, Proc Natl Acad Sci US A 87:3185-3189; Gorny et al, 1998, AIDS Res Hum Retroviruses 14:213-21) (Figure 5).
[0142] In some embodiments, a first antibody is an antibody binding to an epitope of a p24 polypeptide comprising the amino acid sequence of NH2-LKETINEE AAEWDRVHP V-
COOH (SEQ ID NO: 15). Preferred antibodies binding thereto are 47-2 (Robert-Hebmann et al, 1992, MoI Immunol 29:1175-1183; Robert-Hebmann et al, 1992, MoI Immunol 29:729- 738), 714/01 (Robert-Hebmann et al, 1992, MoI Immunol 29:1175-1183; Robert-Hebmann et al, 1992, MoI Immunol 29:729-738), 1109/01 (Robert-Hebmann et al, 1992, MoI Immunol 29:1175-1183; Robert-Hebmann et al, 1992, MoI Immunol 29:729-738), 1G5C8 (Robert- Hebmann et al, 1992, MoI Immunol 29:1175-1183; Robert-Hebmann et al, 1992, MoI Immunol 29:729-738; Janvier et al, J Virol 64:4258-4263; Janvier et al, 1992, J Virol 66:613),14D4E11 (Robert-Hebmann et α/., 1992, MoI Immunol 29:1175-1183; Robert- Hebmann et al, 1992, MoI Immunol 29:729-738; Janvier et al, J Virol 64:4258-4263; Janvier et al, 1992, J Virol 66:613), a polyclonal antibody (Truong et al, J Med Virol 51(3):145-51), or a fragment thereof (Figure 5). [0143] In some embodiments, a first antibody is an antibody binding to an epitope of a p24 polypeptide comprising the amino acid sequence OfNH2-ETINEEAAEWD-COOH (SEQ ID NO: 16). Preferred antibodies binding thereto are 113/038 and 111/073 or a fragment thereof (Niedrig et al, 1991, J Virol 65:4529-4533) (Figure 5).
[0144] In some embodiments, a first antibody is an antibody binding to an epitope of a p24 polypeptide comprising the amino acid sequence OfNH2-ETINEEAAEWDRVHP-COOH (SEQ ID NO: 17). Preferred antibodies binding thereto are l-E-4 (Niedrig et al, 1989, J Virol 63:3525-3528), l-E-9 (Niedrig et al, 1989, J Virol 53:3525-3528), 2-E-4 (Niedrig et al, 19S8, J Gen Virol 69:2109-2114; Niedrig et al, 1989, J Virol 63:3525-3528), 2-H-4 (Niedrig et al, 1988, J Gen Virol 69:2109-2114; Niedrig et al, 1989, J Virol 63:3525-3528), 8-D-2 (Niedrig et al, 1988, J Gen Virol 69:2109-2114; Niedrig et al, 1989, J Virol 63:3525- 3528; Robert-Hebmann et al, 1992, MoI Immunol 29:1175-1183; Robert-Hebmann et al, 1992, MoI Immunol 29:729-738), 8-H-7 (Niedrig et al , 1988, J Gen Virol 69:2109-2114; Niedrig et al, 1989, J Virol 63:3525-3528; Robert-Hebmann et al, 1992, MoI Immunol 29:1175-1183; Robert-Hebmann et al, 1992, MoI Immunol 29:729-738), 8-G-9 (Niedrig et al, 1989, J Virol 63:3525-3528), 10-E-7 (Niedrig et al, 1988, J Ge« Virol 69:2109-2114; Niedrig et α/., 1989, J Fz>o/ 63:3525-3528), 10-G-9 (Niedrig et al, 1988, J Ge« FzVo/ 69:2109-2114; Niedrig et al, 1989, J Virol 63:3525-3528), 1 l-C-5 (Niedrig et al, 1988, J Gerc Virol 69:2109-2114; Niedrig et α/., 1989, J Fzro/ 63:3525-3528), C5123 (Hinkula et al, 1990, MoI Immunol 27:395-403) or a fragment thereof (Figure 5). [0145] In some embodiments, a first antibody is an antibody binding to an epitope of a p24 polypeptide comprising the amino acid sequence OfNH2-EAAEWDRVHP-COOH (SEQ ID NO:18). Preferred antibodies binding thereto are l-B-7, 3-B-7, 6-D-12, 6-E-7, 8-D-5, or a fragment thereof (Niedrig et al, 1988, J Gen Virol 69:2109-2114; Niedrig et al, 1989, J Virol 63:3525-3528) (Figure 5). [0146] In some embodiments, a first antibody is an antibody binding to an epitope of a p24 polypeptide comprising the amino acid sequence of NH2-EAAEWDRVHPVHAGP-COOH (SEQ ID NO: 19). A preferred antibody binding thereto is FFl or a fragment thereof (Hinkula et al, 1990, MoI Immunol 27:395-403) (Figure 5).
[0147] In some embodiments, a first antibody is an antibody binding to an epitope of a p24 polypeptide comprising the amino acid sequence OfNH2-DRVHPVHAGP-COOH (SEQ ID NO:20). A preferred antibody binding thereto is 113/072 or a fragment thereof (Niedrig et al, 1991, J Virol 65:4529-4533) (Figure 5).
[0148] In some embodiments, a first antibody is an antibody binding to an epitope of a p24 polypeptide comprising the amino acid sequence of NH2-RVHPVHAGPIAPGQMREPRGS- COOH (SEQ ID NO:21). A preferred antibody binding thereto is 25.3 or a fragment thereof (Momany et al. , 1996, Nat Struct Biol 3 :763-770) (Figure 5).
[0149] In some embodiments, a first antibody is an antibody binding to an epitope of a p24 polypeptide comprising the amino acid sequence OfNH2-HPVHAGPIAPG-COOH (SEQ ID NO:22). A preferred antibody binding thereto is 13-102-100 or a fragment thereof (Parker et al, 1996, J Immunol 157:198-206; Qian et al, 1998, Electrophoresis 19:515-9) (Figure 5). [0150] In some embodiments, a first antibody is an antibody binding to an epitope of a p24 polypeptide comprising the amino acid sequence OfNH2-HAGPIAPGQMREPRG-COOH (SEQ ID NO:23). A preferred antibody binding thereto is RL4.72.1 or a fragment thereof (Tatsumi et al, 1990, MoI Cell Biochem 96:127-136; Robert-Hebmann et al, 1992, MoI Immunol 29:1175-1183; Robert-Hebmann et al, 1992, MoI Immunol 29:729-738) (Figure 5). [0151] In some embodiments, a first antibody is an antibody binding to an epitope of a p24 polypeptide comprising the amino acid sequence OfNH2-GSDIAGTTSTLQEQIGWMTNN- COOH (SEQ ID NO:24). Preferred antibodies binding thereto are 406/01 (Robert-Hebmann et al, 1992, MoI Immunol 29:1175-1183; Robert-Hebmann et al, 1992, MoI Immunol 29:729-738) and a polyclonal antibody (Truong et al, J Med Virol 51(3):145-51), or a fragment thereof (Figure 5).
[0152] In some embodiments, a first antibody is an antibody binding to an epitope of a p24 polypeptide comprising the amino acid sequence of NH2-NPPIPVGEIY-COOH (SEQ ID NO:25). A preferred antibody binding thereto is 38:9.6K or a fragment thereof (Hinkula et al, 1990, MoI Immunol 27:395-403) (Figure 5). [0153] In some embodiments, a first antibody is an antibody binding to an epitope of a p24 polypeptide comprising the amino acid sequence of NH2-NPPIPVGEIYKRWII-COOH (SEQ ID NO:26). A preferred antibody binding thereto is EBl A9 or a fragment thereof (Ferns et al, 1987, J Gen Virol 68:1543-1551; Ferns et al, 1989, Λ/DS 3:829-834) (Figure 5).
[0154] In some embodiments, a first antibody is an antibody binding to an epitope of a p24 polypeptide comprising the amino acid sequence of NH2- NPPIPVGEIYKRWIIGLNKIVRMYSPTSILD-COOH (SEQ ID NO.-27). A preferred antibody binding thereto is a polyclonal antibody or a fragment thereof (Pialoux et al, 2001, AIDS 15(10): 1239-49) (Figure 5).
[0155] In some embodiments, a first antibody is an antibody binding to an epitope of a p24 polypeptide comprising the amino acid sequence of NH2- IVRMYSPTSILDIRQGPKEPFRDYVDRFYK-COOH (SEQ ID NO^S). Preferred antibodies binding thereto are EF7 (Hinkula et al, 1990, MoI Immunol 27:395-403; Lundin et al, 1996, Immunology 89:579-586), and 30:3E5 (Hinkula et al, 1990, MoI Immunol 27:395- 403), or a fragment thereof (Figure 5).
[0156] In some embodiments, a first antibody is an antibody binding to an epitope of a p24 polypeptide comprising the amino acid sequence OfNH2-RMYSPT-COOH (SEQ ID NO:29). A preferred antibody binding thereto is LH-104-E or a fragment thereof (Haaheim et al, 1991, Scand J Immunol 34:341-350) (Figure 5).
[0157] In some embodiments, a first antibody is an antibody binding to an epitope of a p24 polypeptide comprising the amino acid sequence OfNH2-SILDIR-COOH (SEQ ID NO:30). Preferred antibodies binding thereto are 1B2C12 (Janvier et al, 1990, J Virol 64:4258-4263; Janvier et al, 1992, J Virol 66:613), and LH-104-K Haaheim et al, 1991, Scand J Immunol 34:341-350), or a fragment thereof (Figure 5).
[0158] In some embodiments, a first antibody is an antibody binding to a discontinuous epitope of a p24 polypeptide comprising the amino acid sequences OfNH2-DIRQGP-COOH (SEQ ID NO:31) and NH2-QGVGGP-COOH (SEQ ID NO:32). A preferred antibody binding thereto is LH- 104- A or a fragment thereof (Haaheim et al, 1991, Scand J Immunol 34:341-350) (Figure 5).
[0159] In some embodiments, a first antibody is an antibody binding to an epitope of a p24 polypeptide comprising the amino acid sequence of NH2-DIRQGPKEPFRD YVDRF YKTL- COOH (SEQ ID NO:33). Preferred antibodies binding thereto are 1A7, 1.17.3, and 1F6, or a fragment thereof (Otteken 1992, J Gen Virol 73:2721-2724) (Figure 5). [0160] In some embodiments, a first antibody is an antibody binding to an epitope of a p24 polypeptide comprising the amino acid sequence of NH2-IRQGPKEPFRD YVDRF YKTL- COOH (SEQ ID NO:34). Preferred antibodies binding thereto are 23A5G5 (Robert- Hebmann et al, 1992, MoI Immunol 29:1175-1183; Robert-Hebmann et al, 1992, MoI Immunol 29:729-738), 23A5G4 (Janvier et al, 1990, J Virol 64:4258-4263; Janvier et al. 1992, J Virol 66:613; Jannvier et al, 1996, AIDS Res Hum Retroviruses 12:519-525), 3D10G6 (Janvier et al, 1990, J Virol 64:4258-4263; Janvier et al, 1992, J Virol 66:613), and a polyclonal antibody (Truong et al, 1997, J Med Virol 51(3):145-51), or a fragment thereof (Figure 5). [0161] In some embodiments, a first antibody is an antibody binding to an epitope of a p24 polypeptide comprising the amino acid sequence OfNH2-
IRQGPKEPFRDYVDRFYKTLRAE-COOH (SEQ ID NO:35). A preferred antibody binding thereto is F5-4 or a fragment thereof (Kusk et al , 1988, J Acquir Immune Defic Syndr 1 :326- 332; Kusk et al, 1992, AIDS Res Hum Retroviruses 8:1789-1794) (Figure 5). [0162] In some embodiments, a first antibody is an antibody binding to an epitope of a p24 polypeptide comprising the amino acid sequence of NH2-
IRQGPKEPFRDYVDRFYKTLRAEQAS-COOH (SEQ ID NO:36). Preferred antibodies binding thereto are MO9.42.2 and MO9.50.2, or a fragment thereof (Robert-Hebmann et al, 1992, MoI Immunol 29:1175-1183; Robert-Hebmann et al, MoI Immuniol 29:729-738) (Figure 5).
[0163] In some embodiments, a first antibody is an antibody binding to an epitope of a p24 polypeptide comprising the amino acid sequence OfNH2-QGPKEPFRDYVDRFY-COOH (SEQ ID NO:37). A preferred antibody binding thereto is VlO or a fragment thereof (Matsuo et al, 1992, J Gen Virol 73:2445-2450) (Figure 5). [0164] In some embodiments, a first antibody is an antibody binding to an epitope of a p24 polypeptide comprising the amino acid sequence of NH2-
QGPKEPFRDYVDRFYKTLRAEQA-COOH (SEQ ID NO:38). A preferred antibody binding thereto is Vl 07 or a fragment thereof (Matsuo et al, 1992, J Gen Virol 73:2445- 2450) (Figure 5). [0165] In some embodiments, a first antibody is an antibody binding to a discontinuous epitope of a p24 polypeptide comprising the amino acid sequences OfNH2-GPKEPF-COOH (SEQ ID NO:39) and NH2-QGVGGP-COOH (SEQ ID NO:32). A preferred antibody binding thereto is LH-104-C or a fragment thereof (Haaheim et al, 1991, Scand J Immunol 34:341-350) (Figure 5).
[0166] In some embodiments, a first antibody is an antibody binding to an epitope of a p24 polypeptide comprising the amino acid sequence OfNH2-FRDYVDRFYK-COOH (SEQ ID NO:40). Preferred antibodies binding thereto are 12-B-4 (Niedrig et al , 1988, J Gen Virol 69:2109-2114; Niedrig et al, 1989, J Virol 63:3525-3528) and C5122 (Hinkula et al, 1990, MoI Immunol 27:395-403), or a fragment thereof (Figure 5).
[0167] In some embodiments, a first antibody is an antibody binding to an epitope of a p24 polypeptide comprising the amino acid sequence of NH2-KTLRAEQASQEVKNWMTET- COOH (SEQ ID NO:41 ) A preferred antibody binding thereto is 9A4C4 or a fragment thereof (Janvier et al, 1990, J Virol 64:4258-4263; Janvier et al, 1992, J Virol 66:613; Robert-Hebmann et al, 1992, MoI Immunol 29: 1175-1183; Robert-Hebmann et al, 1992, MoI Immunol 29:729-738) (Figure 5).
[0168] In some embodiments, a first antibody is an antibody binding to an epitope of a p24 polypeptide comprising the amino acid sequence of NH2-TLRAEQASQEVKNWM-COOH (SEQ ID NO:42). Preferred antibodies binding thereto are 1 IDl 1F2 (Janvier et al, 1990, J Virol 64:4258-4263; Janvier et al, 1992, J Virol 66:613), 1 IClOBlO (Janvier et al, 1990, J Virol 64:4258-4263; Janvier et al, 1992, J Virol 66:613) and CD12B4 (Ferns et al, 1987, J Gen Virol 68:1543-1551; Ferns et al, 1989, AIDS 3:829-834), or a fragment thereof (Figure 5).
[0169] In some embodiments, a first antibody is an antibody binding to an epitope of a p24 polypeptide comprising the amino acid sequence OfNH2-QASQEVKNWMTETLL-COOH (SEQ ID NO:43). Preferred antibodies binding thereto are BE3 and Ll 4, or a fragment thereof (Hinkula et al, 1990, MoI Immunol 27:395-403) (Figure 5). [0170] In some embodiments, a first antibody is an antibody binding to an epitope of a p24 polypeptide comprising the amino acid sequence OfNH2-VKNWMTETLL-COOH (SEQ ID NO:44). Preferred antibodies binding thereto are 110/015 and 108/03, or a fragment thereof (Niedrig et al, 1991, J Virol 65:4529-4533) (Figure 5).
[0171] In some embodiments, a first antibody is an antibody binding to an epitope of a p24 polypeptide comprising the amino acid sequence of NH2-
KTILKALGPAATLEEMMTACQGVG-COOH (SEQ ID NO:45). Preferred antibodies binding thereto are 32:32K and C5200, or a fragment thereof (Hinkula et al, 1990, MoI Immunol 27:395-403) (Figure 5).
[0172] In some embodiments, a first antibody is an antibody binding to an epitope of a p24 polypeptide comprising the amino acid sequence OfNH2-ILKALGPAATLEEMM-COOH (SEQ ID NO:46). A preferred antibody binding thereto is FH2 or a fragment thereof (Hinkula et al, 1990, MoI Immunol 27:395-403) (Figure 5).
[0173] In some embodiments, a first antibody is an antibody binding to an epitope of a p24 polypeptide comprising the amino acid sequence OfNH2-LGPAATLEEMM-COOH (SEQ ID NO:47). A preferred antibody binding thereto is 13B5 or a fragment thereof (Berthet- Colominas et al, 1999, EMBO J 18:1124-36) (Figure 5).
[0174] In some embodiments, a first antibody is an antibody binding to an epitope of a p24 polypeptide comprising the amino acid sequence of NH2-LEEMMTACQGVGGPGHKARV- COOH (SEQ ID NO:48). A preferred antibody binding thereto is 106/01 or a fragment thereof (Niedrig et al, 1991, J Virol 65-4529-4533) (Figure 5). [0175] In some embodiments, a first antibody is an antibody binding to an epitope of a p24 polypeptide comprising the amino acid sequence OfNH2-GHKARV-COOH (SEQ ID NO:49). A preferred antibody binding thereto is LH-104-B or a fragment thereof (Haaheim et al, 1991, Scand J Immunol 34:341-350) (Figure 5).
[0176] In some embodiments, a first antibody is an antibody binding to an epitope of a p24 polypeptide comprising the amino acid sequence OfNH2-HKARVL-COOH (SEQ ID
NO:50). A preferred antibody binding thereto is LH-104-1 or a fragment thereof (Haaheim et al, 1991, Scand J Immunol 34:341-350) (Figure 5).
[0177] In some embodiments of the present invention, a first antibody is an anti-HIV-1 p24 antibody binding to a p24 polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID
NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31 , SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, and SEQ ID NO:50.
[0178] In some embodiments of the present invention, a first antibody is an anti-HIV-1 p24 antibody selected from the group consisting of 4F6, KC57, 111/182, 112/021, 112/047, ID8F6, F5-2, CB-13/5, 3D3, CD-4/1, 15F8C7, 111/052, 91-5, 47-2, 714/01, 1109/01, IG5C8, 14D4E11, 113/038, 111/073, l-E-4, l-E-9, 2-E-4, 2-H-4, 8-D-2, 8-H-7, 8-G-9, 10-E-7, 10-G- 9, l l-C-5, C5123, l-B-7, 3-B-7, 6-D-12, 6-E-7, 8-D-5, FFl, 113/072, 25.3, 13-102-100, RL4.72.1, 406/01, 38:9.6k, EB1A9, EF7, 30:3E5, LH-104-E, 1B2C12, LH-104-K, LH-104- A, 1A7, 1.17.3, 1F6, 23A5G5, 23A5G4, 3D10G6, F5-4, MO9.42.2, MO9.50.2, V-10, Vl 07, LH-104-C, 12-B-4, 9A4C4, 11D11F2, 1 IClOBlO, CD12B4, BE3, L14, 110/015, 108/03, 32:32K, C5200, FH2, 13B5, 106/01, LH-104-B, LH-104-B, and fragments thereof.
[0179] Some monoclonal anti-HIV-1 p24 antibodies described herein and others have been reported to also bind to HIV-2 core protein p26. These monoclonal antibodies bind to shared epitopes on HIV-I protein p24 and HIV-2 protein p26 and include, for example, 120A-270, 115B-151, 103-350, 115B-303, 117-289, and 108-394 (US 2002/0106636). Thus, in some embodiments of the present invention, a first antibody is an antibody that binds to both HIV-I p24 and HIV-2 p26. As such, using the monoclonal antibodies of above and the methods of the present invention, in addition to HIV-I p24 polypeptide, a HIV-2 p26 polypeptide can be detected in a biological sample. [0180] In some embodiments of the present invention, a capture binding partner is labeled. For example, when using the Luminex technology, as further described herein, a capture binding partner, such as a first antibody is labeled by being bound to a fluorescently-labeled solid support, such as fluorescently-labeled microspheres.
3. Detecting Binding Partner [0181] The present invention provides for a detecting binding partner which binds to the same antigen as the capture binding partner, albeit to a non-overlapping epitope on the antigen, thereby allowing both capture binding partner and detecting binding partner to bind to the same antigen. A preferred detecting binding partner is an antibody, referred to herein as a second antibody to distinguish it from the capture binding partner, which is referred herein also as a first antibody. [0182] In some embodiments, a second antibody is an antibody binding to a HIV-I antigen, such as a GAG polypeptide, preferably, a p24 polypeptide. Thus, in a preferred embodiment, a second antibody is an anti -HIV-I antigen antibody. In some embodiments, the second antibody is an anti-HIV-1 GAG polypeptide antibody, preferably, an anti-HIV-1 p24 antibody.
[0183] In some embodiments, the second antibody is KC57 or a fragment thereof. KC57 binds to p24 and its GAG precursor polypeptides. In some preferred embodiments, a first antibody is 4F6 and the second antibody is KC57.
[0184] Other second anti-p24 antibodies are also contemplated for use in the compositions, kits, systems, and methods of the present invention. In some embodiments, a second antibody is an anti-HIV-1 p24 antibody that binds to a polypeptide comprising an amino acid sequence selected from the amino acid sequences of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3. In a preferred embodiment of the present invention, a second antibody is an anti- HIV-1 antibody binding to an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3.
[0185] In some embodiments, a second antibody is an anti-HIV-1 p24 antibody selected from a HIV-I antibody binding to a GAG polypeptide having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence shown in SEQ ID NO: 1.
[0186] In some embodiments, a second antibody is an anti-HIV-1 p24 antibody selected from a HIV-I antibody binding to a GAG polypeptide having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence shown in SEQ ID NO:2.
[0187] In some embodiments, a second antibody is an anti-HIV-1 p24 antibody selected from a HIV-I antibody binding to a p24 polypeptide having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence shown in SEQ ID NO:3.
[0188] In some embodiments of the present invention, a second antibody is an anti-HIV-1 p24 antibody binding to a p24 polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO: 16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, and SEQ ID NO:50. Preferred antibodies binding to a p24 having any of the sequences SEQ ID NOS:4-50 are set forth herein. Any of combinations of the p24 antibodies described herein can be used as a first antibody and as a second antibody for use in the kits, systems, and methods of the present invention, as long as the epitopes to which the antibodies bind are non-overlapping.
[0189] In some embodiments of the present invention, a second antibody is an anti-HIV-1 ρ24 antibody selected from the group consisting of 4F6, KC57, 111/182, 112/021, 112/047, ID8F6, F5-2, CB-13/5, 3D3, CD-4/1, 15F8C7, 111/052, 91-5, 47-2, 714/01, 1109/01, IG5C8, 14D4E11, 113/038, 111/073, l-E-4, l-E-9, 2-E-4, 2-H-4, 8-D-2, 8-H-7, 8-G-9, 10-E-7, 10-G- 9, l l-C-5, C5123, l-B-7, 3-B-7, 6-D-12, 6-E-7, 8-D-5, FFl, 113/072, 25.3, 13-102-100, RL4.72.1, 406/01, 38:9.6k, EB1A9, EF7, 30:3E5, LH-104-E, 1B2C12, LH-104-K, LH-104- A, 1A7, 1.17.3, 1F6, 23A5G5, 23A5G4, 3D10G6, F5-4, MO9.42.2, MO9.50.2, V-10, V107, LH-104-C, 12-B-4, 9A4C4, 11D11F2, 1 IClOBlO, CD12B4, BE3, L14, 1 10/015, 108/03, 32:32K, C5200, FH2, 13B5, 106/01, LH-104-B, LH-104-B, and fragments thereof.
[0190] In some embodiments, a second antibody or fragment thereof binds to a GAG polypeptide. In some embodiments, a second antibody or fragment thereof binds to a POL polypeptide. In some embodiments, a second antibody or fragment thereof binds to a NEF polypeptide. a) Labeling the Detecting Binding Partner
[0191] In some embodiments, the invention provides methods and compositions that include labels for the highly sensitive detection and quantification of an analyte, e.g., of an antigen in a sample. In some embodiments of the present invention, the detecting binding partner comprises a label. In some embodiments of the present invention, the detecting binding partner generates a detectable signal. In some embodiments of the present invention, the detecting binding partner is an antibody which comprises a label. In some embodiments of the present invention, the detecting binding partner is an antibody which generates a detectable signal. [0192] This signal-generating detecting binding partner, such as a second antibody, comprises a compound or "label" which is in itself detectable or may be reacted with one or more additional compounds to generate a detectable product or detectable signal. Examples of signal-generating compounds include chromogens, radioisotopes (e.g., 1251, 1311, 32P, 3H, 35S and 14C), chemiluminescent compounds (e.g., acridinium), particles (visible or fluorescent), nucleic acids, complexing agents, or catalysts such as enzymes (e.g., luciferase, alkaline phosphatase, acid phosphatase, horseradish peroxidase, beta-galactosidase and ribonuclease). In the case of enzyme use (e.g., alkaline phosphatase or horseradish peroxidase), addition of a chromo-, fluro-, or lumino-genic substrate results in generation of a detectable signal. Other detection systems such as time-resolved fluorescence, internal- reflection fluorescence, amplification (e.g., polymerase chain reaction) and Raman spectroscopy are also useful.
(1) Fluorescent Moieties
[0193] A preferred label is a fluorescent moiety, also referred to herein as a fluorochrome. In some embodiments, a fluorescent moiety is attached to a detecting binding partner. The compositions and methods of the invention may utilize highly fluorescent moieties wherein the fluorescent moiety is capable of emitting photons when simulated by a laser emitting light at the excitation wavelength of the moiety.
[0194] In some embodiments, the fluorescent moiety comprises an average of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 fluorescent entities, e.g., fluorescent molecules. In some embodiments, the fluorescent moiety comprises an average of no more than about 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 fluorescent entities, e.g., fluorescent molecules. In some embodiments, the fluorescent moiety comprises an average of about 1 to 11, or about 2 to 10, or about 2 to 8, or about 2 to 6, or about 2 to 5, or about 2 to 4, or about 3 to 10, or about 3 to 8, or about 3 to 6, or about 3 to 5, or about 4 to 10, or about 4 to 8, or about 4 to 6, or about 2, 3, 4, 5, 6, or more than about 6 fluorescent entities. By "average" is meant that, in a given sample that is a representative sample of a group of labels of the invention, where the sample contains a plurality of the binding partner- fluorescent moiety units, the molar ratio of the particular fluorescent entity of which the fluorescent moiety is comprise, to the binding partner, as determined by standard analytical methods, corresponds to the number or range of numbers specified. For example, in embodiments in which the label comprises a detecting binding partner that is an antibody and a fluorescent moiety that comprises a plurality of fluorescent dye molecules of a specific absorbance, a spectrophotometric assay may be used in which a solution of the label is diluted to an appropriate level and the absorbance at 280 nm is taken to determine the molarity of the protein (antibody) and an absorbance at, e.g., 650 nm (for AlexaFluor 647) is taken to determine the molarity of the fluorescent dye molecule. The ratio of the latter molarity to the former represents the average number of fluorescent entities (dye molecules) in the fluorescent moiety attached to each antibody.
[0195] In some embodiments, the moiety comprises a plurality of fluorescent entities, e.g., about 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, or 3-5, 3-6, 3-7, 3-8, 3-9, or 3-10 fluorescent entities. In some embodiments, the moiety comprises about 2 to 4 fluorescent entities. In some embodiments, the moiety comprises one fluorescent entity. [0196] In some embodiments, the fluorescence of the fluorescent moiety is such that it allows detection and/or quantification of an analyte, e.g., an antigen, within a dynamic range of between about 20,000 pg/ml and about 0.2 pg/ml and with a coefficient of variation of less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1%.
[0197] Fluorescent moieties, e.g., a single fluorescent dye molecule or a plurality of fluorescent dye molecules, that are useful in some embodiments of the invention may be defined in terms of their photon emission characteristics when stimulated by EM radiation. For example, in some embodiments, the invention utilizes a fluorescent moiety, e.g., a moiety comprising a single fluorescent dye molecule or a plurality of fluorescent dye molecules, that is capable of emitting an average of at least about 10, 20, 30, 40, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 350, 400, 500, 600, 700, 800, 900, or 1000 photons when simulated by a laser emitting light at the excitation wavelength of the moiety.
[0198] The fluorescence generated by the fluorescent-generating moiety is dependent upon the presence, absence or concentration of the analyte. Examples of suitable fiuorescence- generating moieties include rhodamine 110; rhodol; coumarin or a fluorescein compound. Derivatives of rhodamine 110, rhodol, or fluorescein compounds that have a 4' or 5' protected carbon may likewise be employed. Preferred examples of such compounds include 4'(5')thiofluorescein, 4'(5')-amino fluorescein, 4'(5')-carboxyfluorescein , 4'(5')- chlorofluorescein, 4'(5')-methylfluorescein, 4'(5')-sulfofluorescein, 4'(5')-aminorhodol, 4'(5')- carboxyrhodol, 4'(5')-chlororhodol, 4'(5')-methylrhodol, 4'(5')-sulforhodol; 4'(5')- aminorhodamine 110, 4'(5')-carboxyrhodamine 110, 4'(5')-chlororhodamine 110, 4'(5')- methylrhodamine 110, 4'(5')-sulforhodamine 110 and 4'(5')thiorhodamine 110. "4'(5')" means that at the 4' or 5' position the hydrogen atom on the carbon atom is substituted with a specific organic group or groups. (a) Fluorescent Dyes
[0199] In some embodiments of the present invention, a fluorescent entity is a fluorescent dye molecule. The following provides a non-inclusive list of useful fluorescent dyes for use as fluorescent moieties: Bimane, Dapoxyl, Dimethylamino coumarin-4-acetic acid, Marina blue, 8-Anilino naphthalene- 1 -sulfonic acid, Cascade blue, Alexa Fluor 405, Cascade blue, Cascade yellow, Pacific blue, PyMPO, Alexa 430, Atto-425, NBD, Alexa 488, Fluorescein, Oregon Green 488, Atto 495, Cy2, DY-480-XL, DY-485-XL, DY-490-XL, DY-500-XL, DY-520-XL, Alexa Fluor 532, BODIPY 530/550, 6-HEX, 6-JOE, Rhodamine 6G, Atto-520, Cy3B, Alexa Fluor 610, Alexa Fluor 633, Alexa Fluor 647, BODIPY 630/650, Cy5, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, Alexa Fluor, B-phycoerythrin, R- phycoerythrin, Allophycocyanin, PBXL-I, PBXL-3, Atto 425, Atto 495, Atto 520, Atto 560, Atto 590, Atto 610, Atto 655, Atto 680, DY-495/5, DY-495/6, DY-495X/5, DY-495X/6, DY- 505/5, DY-505/6, DY-505X/5, DY-505X/6, DY-550, DY-555, DY-610, DY-615, DY-630, DY-631, DY-633, DY-635, DY-636, DY-650, DY-651, DYQ-660, DYQ-661, DY-675, DY- 676, DY-680, DY-681, DY-700, DY-701, DY-730, DY-731, DY-750, DY-751, DY-776, DY-780-OH, DY-780-P, DY-781 , DY-782 , EVOblue-10, and EVOblue-30. Either one of these individual dyes or combinations thereof may be used as a fluorescent moiety. A detailed characterization of these dyes can be found in US2006/0078998 and US2008/0064113, which are herein incorporated by reference in their entirety. [0200] In some embodiments, a fluorescent dye molecule comprises at least one substituted indolium ring system in which the substituent on the 3 -carbon of the indolium ring contains a chemically reactive group or a conjugated substance.
[0201] Suitable dyes for use in the invention include modified carbocyanine dyes. The modification of carbocyanine dyes includes the modification of an indolium ring of the carbocyanine dye to permit a reactive group or conjugated substance at the number 3 position. The modification of the indolium ring provides dye conjugates that are uniformly and substantially more fluorescent on proteins, nucleic acids and other biopolymers, than conjugates labeled with structurally similar carbocyanine dyes bound through the nitrogen atom at the number one position. In addition to having more intense fluorescence emission than structurally similar dyes at virtually identical wavelengths, and decreased artifacts in their absorption spectra upon conjugation to biopolymers, the modified carbocyanine dyes have greater photostability and higher absorbance (extinction coefficients) at the wavelengths of peak absorbance than the structurally similar dyes. Thus, the modified carbocyanine dyes result in greater sensitivity in assays that use the modified dyes and their conjugates.
[0202] Preferred modified dyes include compounds that have at least one substituted indolium ring system in which the substituent on the 3 -carbon of the indolium ring contains a chemically reactive group or a conjugated substance. Other dye compounds include compounds that incorporate an azabenzazolium ring moiety and at least one sulfonate moiety. Modified carbocyanine dyes that can be used in the present invention are described in U.S. Pat. No. 6,977,305, which is herein incorporated by reference in its entirety. Thus, in some embodiments the labels of the invention utilize a fluorescent dye that includes a substituted indolium ring system in which the substituent on the 3 -carbon of the indolium ring contains a chemically reactive group or a conjugated substance group. [0203] In some embodiments, the label comprises a fluorescent moiety that includes one or more Alexa dyes (Molecular Probes, Eugene, Oreg.). The Alexa dyes are disclosed in U.S. Pat. Nos. 6,977,305, 6,974,874, 6,130,101, and 6,974,305, which are herein incorporated by reference in their entirety. Some embodiments of the invention utilize a dye selected from the group consisting of AlexaFluor 647, AlexaFluor 488, AlexaFluor 532, AlexaFluor 555, AlexaFluor 610, AlexaFluor 680, AlexaFluor 700, and AlexaFluor 750. Some embodiments of the invention utilize a dye selected from the group consisting of AlexaFluor 488, AlexaFluor 532, AlexaFluor 647, AlexaFluor 700 and AlexaFluor 750. Some embodiments of the invention utilize a dye selected from the group consisting of AlexaFluor 488, AlexaFluor 532, AlexaFluor 555, AlexaFluor 610, AlexaFluor 680, AlexaFluor 700, and AlexaFluor 750. In some embodiments, a dye molecule is a AlexFluor molecule selected from the group consisting of AlexaFluor 488, AlexaFluor 532, AlexaFluor 647, AlexaFluor 680, and AlexaFluor 700. In some embodiments, the dye molecule is a AlexaFluor molecule selected from the group consisting of AlexaFluor 488, AlexaFluor 532, AlexaFluor 680, and AlexaFluor 700. In some embodiments of the invention the fluorescent dye is a AlexaFluor 647 molecule, which has an absorption maximum between about 650 and 660 nm and an emission maximum between about 660 and 670 nm. The AlexaFluor 647 dye may be used alone or in combination with other AlexaFluor dye.
[0204] In some embodiments, a fluorescent entity comprises a first type and a second type of dye molecule, e.g., two different AlexaFluor molecules, e.g., where the first type and second type of dye molecules have different emission spectra. The ratio of the number of first type to second type of dye molecule may be, e.g., 4:1, 3:1, 2:1, 1 :1, 1:2, 1 :3 or 1 :4. The binding partner may be, e.g., a polypeptide.
[0205] A preferred fluorescent dye is R-phycoerythrin.
(b) Quantum Dots
[0206] In some embodiments of the present invention, a fluorescent entity is a quantum dot.
Thus, in some embodiments, the fluorescent label moiety that is used to detect an analyte in a sample is a quantum dot. Quantum dots (QDs), also known as semiconductor nanocrystals or artificial atoms, are semiconductor crystals that contain anywhere between 100 to 1,000 electrons and range from 2-10 nm. Some QDs can be between 10-20 nm in diameter. QDs have high quantum yields, which makes them particularly useful for optical applications. QDs are fluorophores that fluoresce by forming excitons, which can be thought of the excited state of traditional fluorophores, but have much longer lifetimes of up to 200 nanoseconds. This property provides QDs with low photobleaching. The energy level of QDs can be controlled by changing the size and shape of the QD, and the depth of the QDs' potential. One of the optical features of small exci tonic QDs is coloration, which is determined by the size of the dot. The larger the dot, the redder, or more towards the red end of the spectrum the fluorescence. The smaller the dot, the bluer or more towards the blue end it is. The bandgap energy that determines the energy and hence the color of the fluoresced light is inversely proportional to the square of the size of the QD. Larger QDs have more energy levels which are more closely spaced, thus allowing the QD to absorb photons containing less energy, i.e. those closer to the red end of the spectrum. Because the emission frequency of a dots dependent on the bandgap, it is therefore possible to control the output wavelength of a dot with extreme precision.
[0207] Colloidally prepared QDs are free floating and can be attached to a variety of molecules via metal coordinating functional groups. These groups include but are not limited to thiol, amine, nitrile, phosphine, phosphine oxide, phosphonic acid, carboxylic acids or other ligands. By bonding appropriate molecules to the surface, the quantum dots can be dispersed or dissolved in nearly any solvent or incorporated into a variety of inorganic and organic films. Quantum dots (QDs) can be coupled to streptavidin directly through a maleimide ester coupling reaction or to antibodies through a maleimide-thiol coupling reaction. This yields a material with a biomolecule covalently attached on the surface, which produces conjugates with high specific activity. In some embodiments, the protein that is detected is labeled with one quantum dot. In some embodiments the quantum dot is between 10 and 20 nm in diameter. In other embodiments, the quantum dot is between 2 and 10 nm in diameter. Useful Quantum Dots include QD 605, QD 610, QD 655, and QD 705. A particularly preferred Quantum Dot is QD 605.
[0208] Detecting binding partners labeled with a fluorescent dye or a quantum dot can be detected as described herein and as further described in US2006/0078998 and US 2008/0064113. b) Attaching a Fluorescent Moiety
[0209] One skilled in the art will recognize that many strategies and a variety of suitable means can be used for the attachment of a fluorescent moiety, or fluorescent entities that make up the fluorescent moiety, to a detecting binding partner, e.g., an antibody as described herein. The label may be attached by any known means, including methods that utilize non- specific or specific interactions of label and target. Labels may provide a detectable signal. In addition, labeling can be accomplished directly or through binding partners.
[0210] In some embodiments, after attachment of the fluorescent moiety to the binding partner to form a label for use in the methods of the invention, and prior to the use of the labeled detecting binding partner to bind to an analyte of interest, it is useful to perform a filtration step. E.g., an antibody-dye label may be filtered prior to use, e.g., through a 0.2 micron filter, or any suitable filter for removing aggregates. Other reagents for use in the assays of the invention may also be filtered, e.g., e.g., through a 0.2 micron filter, or any suitable filter. Without being bound by theory, it is thought that such filtration removes a portion of the aggregates of the, e.g., antibody-dye labels. As such aggregates will bind as a unit to the analyte of interest, but upon release in elution buffer are likely to disaggregate, false positives may result; i.e., several labels will be detected from an aggregate that has bound to only a single analyte molecule of interest. Regardless of theory, filtration can reduce false positives in the subsequent assay and improve accuracy and precision.
C. Solid Supports
[0211] In some embodiments of the present invention, a capture binding partner, such as an antibody is attached to a solid support, preferably to a plurality of solid support, hi some embodiments, e.g., when using microspheres, beads, etc., the plurality of solid support comprises at least about 10 members, at least about 50 members, at least about 100 members, at least about 300 members, at least about 500 members, at least about 1,000 members, at least about 3,000 members, at least about 5,000 members, at least about 10,000 members, at least about 30,000 members, at east about 50,000 members, at east about 100,000 members, at least about 500,000 members, at least about 1 ,000,000 members, at least about 5,000,000 members, at least about 10,000,000 members, or at least about 50,000,000 members. The plurality of solid support can be the same solid support or each member can be different. Individual members of solid support may be different with respect to size or form.
[0212] The choice of solid support for use in the compositions, kits, systems, and methods described herein is determined based upon desired assay format performance characteristics. Acceptable supports for use in the present invention can vary widely. A support can be porous or nonporous. It can be continuous or non-continuous, flexible or nonflexible. A support can be made of a variety of materials including ceramic, glassy, metallic, organic polymeric materials, or combinations thereof. [0213] Preferred supports include organic polymeric supports, such as particulate or beaded supports, woven and nonwoven webs (such as fibrous webs), microporous fibers, microporous membranes, hollow fibers or tubes. Polyacrylamide and mineral supports such as silicates and carbonates (e.g., hydroxyl apatite) can also be used. Woven and nonwoven webs may have either regular or irregular physical configurations of surfaces. Particularly preferred embodiments include solid supports in the form of spherical or irregularly-shaped beads or particles. [0214] Porous materials are useful because they provide large surface areas. The porous support can be synthetic or natural, organic or inorganic. Suitable solids with a porous structure having pores of a diameter of at least about 1.0 nanometer (run) and a pore volume of at least about 0.1 cubic centimeter/gram (cm3/g). Preferably, the pore diameter is at least about 30 nm because larger pores will be less restrictive to diffusion. Preferably, the pore volume is at least about 0.5 cm3/g for greater potential capacity due to greater surface area surrounding the pores. Preferred porous supports include particulate or beaded supports such as agarose, hydrophilic polyacrylates, polystyrene, mineral oxides and Sepharose, including spherical and irregular-shaped beads and particles. [0215] For significant advantage, the supports for binding antibodies are preferably hydrophilic. Preferably, the hydrophilic polymers are water swellable to allow for greater infiltration of analytes. Examples of such supports include natural polysaccharides such as cellulose, modified celluloses, agarose, cross-linked dextrans, amino-modified cross-linked dextrans, guar gums, modified guar gums, xanthan gums, locust bean gums and hydrogels. Other examples include cross-linked synthetic hydrophilic polymers such as polyacrylamide, polyacrylates, polyvinyl alcohol (PVA) and modified polyethylene glycols.
[0216] A preferred solid support is a plurality of microspheres. Another preferred solid support is paramagnetic beads. Magnetic beads, typically, comprise a ferromagnetic oxide particle, such as ferromagnetic iron oxide, maghemite, magnetite, or manganese zinc ferrite (see, e.g., U.S. Pat. No. 6,844,426). The paramagnetic material may be constituted of very fine particles of mineral oxides with paramagnetic properties such as magnetite (a mixed iron oxide), hematite (an iron oxide), chromite (a salt of iron and chrome) and all other material attracted by a permanent magnet of electromagnet. Also ferrites such as iron tritetraoxide (Fe3O4), γ-sesquioxide (γ-Fe2O3), MnZn-ferrite, NiZn-ferrite, YFe-garnet, GaFe-garnet, Ba- ferrite, and Sr-ferrite; metals such as iron, manganese, cobalt, nickel, and chromium; alloys of iron, manganese, cobalt, nickel, and the like, but not limited thereto, can be used. The preferred material is magnetite because of its availability and low cost. It is supplied as particles of different size, dry or as an aqueous stabilized suspension.
[0217] Non-paramagnetic material on which a capture binding partner can be attached are made of polymeric materials. Among the most common polymeric materials are cross-linked acrylates, polystyrene, polyurethane, polyvinyl, nylon, and polysaccharides. More specifically, these polymeric materials include organic polymers produced by polymerization of a polymerizable monomer: the monomer including styrenic polymerizable monomers such as styrene, α-methylstyrene, β-methylstyrene, o-methylstyrene, m-methylstyrene, p- methylstyrene, 2,4-dimethylstyrene, p-n-butylstyrene, p-t-butylstyrene, p-n-hexylstyrene, p- n-octyl styrene, p-n-nonylstyrene, p-n-decylstyrene, p-n-dodecylstyrene, p-methoxystyrene, and p-phenylstyrene; acrylic polymerizable monomers such as methyl acrylate, ethyl acrylate, n-propyl acrylate, isopropyl acrylate, n-butyl acrylate, isobutyl acrylate, t-butyl acrylate, n- amyl acrylate, n-hexyl acrylate, 2-ethylhexyl acrylate, n-octyl acrylate, n-nonyl acrylate, cyclohexyl acrylate, benzyl acrylate, dimethylphosphatoethyl acrylate, diethylphosphatoethyl acrylate, dibutylphosphatoethyl acrylate, and 2-benzoyloxyethyl acrylate; methacrylic polymerizable monomer such as methyl methacrylate, ethyl methacrylate, n-propyl methacrylate, isopropyl, methacrylate, n-butyl methacrylate, isobutyl methacrylate, t-butyl methacrylate, n-amyl methacrylate, n-hexyl methacrylate, 2-ethylhexyl methacrylate, n-octyl methacrylate, n-nonyl methacrylate, diethylphosphatoethyl methacrylate, acrylamide, methacrylamide and derivatives; dibutylphosphatoethyl methacrylate; aliphatic monocarboxylic acid esters; vinyl polymerizable monomer such as vinyl esters, vinyl acetate, vinyl propionate, vinyl benzoate, vinyl butyrate, vinyl benzoate, and vinyl formate; vinyl ethers such as vinyl methyl ether, vinyl ethyl ether, and vinyl isobutyl ether; and vinyl ketones such as vinyl methyl ketone, vinyl hexyl ketone, and vinyl isopropyl ketone. Other examples of the polymeric structures are those made of inorganic solids, including clay minerals such as kaolinite, bentonite, talc, and mica; metal oxides such as alumina, titanium dioxide, and zinc oxide; insoluble inorganic salts such as silica gel, hydroxyapatite, and calcium phosphate gel; metals such as gold, silver, platinum, and copper; and semiconductor compounds such as GaAs, GaP, and ZnS. The material is not limited thereto. The polymeric structure may be used in combination of two or more thereof. [0218] In some embodiments, the solid support is a magnetic or ferromagnetic bead or microsphere, preferably a carboxymethylated magnetic or ferromagnetic bead or microsphere, more preferably, a fluorescent carboxymethylated magnetic or ferromagnetic bead or microsphere. Such beads or microspheres are commercially available, e.g., from Spherotech, Inc., Lake Forest, IL. [0219] Magnetic beads for use in the present invention include, but are not limited to, e.g., magnetic particles and ferromagnetic particles, including SPHERO™ Magnetic Polystyrene Particles, SPHERO™ Carboxyl Magnetic Particles, SPHERO™ Jeffamine Magnetic Particles, SPHERO™ Amino Magnetic Particles, SPHERO™ Magnetic Polystyrene Particles, Smooth Surface, SPHERO™ Carboxyl Magnetic Particles, Smooth Surface, SPHERO™ Amino Magnetic Particles, Smooth Surface, SPHERO™ Magnetic Cross-linked Particles, SPHERO™ Diethylamino Magnetic Particles, SPHERO™ Dimethylamino Magnetic Particles, SPHERO™ Epoxy Magnetic Particles, Smooth Surface, SPHERO™ Hydroxyethyl Magnetic Particles, Smooth Surface.
[0220] Also preferred magnetic or ferromagnetic beads are fluorescent magnetic and fluorescent ferromagnetic beads. Fluorescent magnetic particles for use in the present invention include SPHERO™ Fluorescent Magnetic Particles, such as Fluorescent Nile Red Magnetic (Spherotech, Inc., catalog # FPM-4056-2) and Fluorescent UV Magnetic Particles (Spherotech, Inc., catalog # FPM-5041-2). Also for use in the present invention are
SPHERO™ Amino Fluorescent Magnetic Particles, such as Fluorescent Yellow Amino Magnetic (Spherotech, Inc., catalog # FAM-2052-2), Fluorescent Pink Amino Magnetic (Spherotech, Inc., catalog # FPM-2058-2), and Fluorescent Nile Red Amino Magnetic (Spherotech, Inc., catalog # FAM-4056-2). Also for use in the present invention are SPHERO™ Carboxyl Fluorescent Magnetic Particles, such as Fluorescent Nile Red
Carboxyl Magnetic (0.2-0.39 μm; Spherotech, Inc., catalog # FCM-02556-2), Fluorescent Yellow Carboxyl Magnetic (1.0-1.4 μm; Spherotech, Inc., catalog # FCM-1052-2), Fluorescent Pink Carboxyl Magnetic (1.0-1.4 μm; Spherotech, Inc., catalog # FCM-1058-2), Fluorescent Yellow Carboxyl Magnetic (2.0-2.4 μm; Spherotech, Inc., catalog # FCM-2052- 2), Fluorescent Pink Carboxyl Magnetic (2.0-2.4 μm; Spherotech, Inc., catalog # FCM-2058- 2), Fluorescent Yellow Carboxyl Magnetic (4.0-4.9 μm; Spherotech, Inc., catalog # FCM- 4052-2), Fluorescent Nile Red Carboxyl Magnetic (4.0-4.9 μm; Spherotech, Inc., catalog # FCM-4056-2), Fluorescent Pink Carboxyl Magnetic (4.0-4.9 μm; Spherotech, Inc., catalog # FCM-4058-2), Fluorescent UV Carboxyl Magnetic (7.0-7.9 μm; Spherotech, Inc., catalog # FCM-7041-2), Fluorescent Yellow Carboxyl Magnetic (28.0-9.9 μm; Spherotech, Inc., catalog # FCM-8052-2), and Fluorescent Nile Red Carboxyl Magnetic (8.0-9.9 μm; Spherotech, Inc., catalog # FCM-8056-2).
[0221] Ferromagnetic fluorescent particles for use in the present invention include SPHERO™ Fluorescent Carboxyl Ferromagnetic Particles, such as Fluorescent Yellow Carboxyl Ferromagnetic (2.0-2.9 μm; Spherotech, Inc., catalog # FCFM-2052-2), Fluorescent Yellow Carboxyl Ferromagnetic (4.0-4.9 μm; Spherotech, Inc., catalog # FCFM-4052-2), and Fluorescent Nile Red Carboxyl Ferromagnetic (4.0-4.9 μm; Spherotech, Inc., catalog # FCFM4056-2). 1. Modification of Beads
[0222] In some embodiments a solid support is a solid support for use in the Luminex technology, such as Luminex's xMAP technology (multi-analyte profiling beads; Luminex Corporation; Durham, NC). This technology is described herein and further in US Pat. No. 6,268,222, incorporated herewith by reference in its entirety. Luminex's xMAP technology is built on flow cytometry, microspheres. LASERs, digital signal processing and traditional chemistry that have been combined in a unique way. Luminex color-codes tiny beads (e.g., polystyrene beads), called microspheres into distinct sets of beads. Each bead set can be coated with a reagent specific to a particular assay, e.g., with a capture binding partner, preferably, a first antibody, as described herein. The capture binding partner then allows the capture and detection of an analyte of interest from a sample, e.g., a GAG polypeptide. Within the Luminex compact analyzer, lasers excite the internal dyes that identify each microsphere particle, and also any reporter dye captured during the assay. In some embodiments, a reporter dye, e.g., is a fluorescent moiety, as described herein, attached to a second antibody. By changing the relative amounts or density of two or more spectrally distinct fluorochromes (e.g., red and infrared fluorophores) distinct sets of microsphere beads are generated each of which has a unique spectral signature. Based on the numbers of distinct beads sets used in a testing (e.g., 100), the xMAP technology allows multiplexing of several distinct assays (e.g., 100) within a single sample. In these embodiments, a third fluorochrome coupled to the detection binding partner, e.g., a second antibody, as described herein, quantifies the amount of analyte captured by the capture binding partner, e.g., first antibody, bound to the solid support.
[0223] In some embodiments, a solid support is a fluorescent magnetic or ferromagnetic bead or a microsphere as described herein that can be detected using a conventional fluorimeter and subjected to washing using a conventional plate washer equipped with an appropriate magnet (e.g., Magna Sim96B, commercially available from SiMBiOTiX Controls, Inc., Mississauga, Ontario, CA). Such solid supports are commercially available, e.g., through Spherotech, Lake Forest, IL. As described herein the fluorescent magnetic or ferromagnetic beads are coupled to an anti-HIVl antibody. The amount of captured HIVl antigen is detected using a second, fluorescently labeled antibody against a non-overlapping epitope of HIVl . The fluorescent magnetic beads fluoresce at a wavelength that is separately detectable from the wavelength of the fluorophore label on the second antibody. The assay is read at two different wavelengths: at a first wavelength to quantify the number of beads and at a second wavelength to quantify the amount of antigen. For example, in one embodiment, yellow fluorescent magnetic beads are used in combination with a second antibody labeled with phycoerythrine. By using a conventional fluorimeter to read the assay, results can be determined in a matter of minutes (e.g., about 10 minutes). Concentrations of HIVl antigen as low as about 33 pg/ml can be detected.
[0224] Using a conventional fluorimeter and magnetic or ferromagnetic beads, the assay as described herein can be converted into a high throughput assay.
D. Biological Samples
[0225] Methods of the present invention include detecting an analyte of interest in a biological sample. The biological sample may be any suitable biological sample. Typically, the biological sample is a biological fluid. In some embodiments the biological sample is selected from the group consisting of serum, blood, lymph, and plasma. In some embodiments the sample is a serum sample. In some embodiments, the sample is a blood sample. In some embodiments the sample is a plasma sample. In some embodiments the sample is a lymph sample. In some embodiments, the sample is a urine sample. In some embodiments, the sample is saliva. In some embodiments, the sample is a mouth swab.
[0226] In some embodiments, the biological sample is a sample of genital tract cells and/or genital tract secretions from a woman. Such a sample may be obtained by, e.g., pap smear, cervicovaginal lavage, as a cytobrush, as a biopsied cervix tissue, or as an endocervical curettage. Pap smear or genital tract cell and secretion collection methods such as cervicovaginal lavage (CVL), offer clinicians and investigators a simple noninvasive method of sampling cells and mucus secreted by the uterus, cervix and vaginal tissues in a state readily amenable to in vitro studies and methods described herein. CVL sampling is a routine gynecological procedure. [0227] A preferred biological sample is obtained from a human. Another preferred biological sample is obtained from subject having or suspected of having a lentivirus infection. Another preferred biological sample is obtained from subject having or suspected of having AIDS. Another preferred biological sample is obtained from subject treated with an antiviral agent. [0228] Biological samples may be prepared for use in the methods described herein. Sample preparation includes steps necessary to prepare a raw sample for analysis. These steps can involve, by way of example, one or more steps of: separation steps such as centrifugation, filtration, distillation, chromatography; concentration, cell lysis, alteration of pH, addition of buffer, addition of diluents, addition of reagents, heating or cooling, addition of label, binding of label, cross-linking with illumination, separation of unbound label, inactivation and/or removal of interfering compounds and any other steps necessary for the biological sample to be prepared for analysis using the methods described herein. In some embodiments, blood is treated to separate out plasma or serum.
[0229] In general, any method of sample preparation may be used that produces a detectable label corresponding to the analyte of interest, e.g., an antigen. In some embodiments of the present invention, the method of detecting an analyte in a biological sample comprises the step of lysing the biological sample, preferably prior to the contacting the capture binding partner, such as the first antibody. As known in the art, lysing a biological sample can be done using a variety of conditions, including, but not limited to, heat-induced lysis, acidic or basic lysis, lysing cells and virus particles using enzymes, such as proteases, lysis by ultrasound, mechanical lysis, and cytolysis (lysing cells by osmotic shock). In other embodiments of the present invention, a biological sample is filtered and/or centrifuged.
[0230] Microfluidics systems may also be used for biological sample preparation, especially for samples suspected of containing concentrations of analytes high enough that detection requires smaller samples. Principles and techniques of micro fluidic manipulation are known in the art. See, e.g., U.S. Pat. Nos. 4,979,824; 5,770,029; 5,755,942; 5,746,901;
5,681,751; 5,658,413; 5,653,939; 5,653,859; 5,645,702; 5,605,662; 5,571,410; 5,543,838;
5,480,614, 5,716,825; 5,603,351 ; 5,858,195; 5,863,801; 5,955,028; 5,989,402; 6,041,515;
6,071,478; 6,355,420; 6,495,104; 6,386,219; 6,606,609; 6,802,342; 6,749,734; 6,623,613; 6,554,744; 6,361,671; 6,143,152; 6,132,580; 5,274,240; 6,689,323; 6,783,992; 6,537,437;
6,599,436; 6,811,668 and published PCT patent application No. WO9955461(A1).
[0231] In some embodiments, a biological sample comprises a buffer. The buffer may be mixed with the biological sample. While any suitable buffer can be used, the preferable buffer has low fluorescence background, is inert to the detectably labeled particle, and can maintain the working pH. The buffer concentration can be any suitable concentration, such as in the range from about 1 to about 200 mM. Any buffer system may be used as long as it provides for solubility, function, and detection of the analyte of interest. Preferably, the buffer is selected from the group consisting of phosphate, glycine, acetate, citrate, acidulate, carbonate/bicarbonate, imidazole, triethanolamine, glycine amide, borate, MES, Bis-Tris, ADA, aces, PIPES, MOPSO, Bis-Tris Propane, BES, MOPS, TES, HEPES, DIPSO, MOBS, TAPSO, Trizma, HEPPSO, POPSO, TEA, EPPS, Tricine, Gly-Gly, Bicine, HEPBS, TAPS, AMPD, TABS, AMPSO, CHES, CAPSO, AMP, CAPS, and CABS. The buffer can also be selected from the group consisting of Gly-Gly, bicine, tricine, 2-morpholine ethanesulfonic acid (MES), 4-morpholine propanesulfonic acid (MOPS) and 2-amino-2-methyl-l-propanol hydrochloride (AMP). A useful buffer is 2 mM Tris/borate at pH 8.1, but Tris/glycine and Tris/HCl are also acceptable. Other buffers are as described herein (see, e.g., Examples). 1. Biological Samples Comprising an Analyte
[0232] There are numerous analytes and markers of interest, which can be detected using compositions, kits, systems, and methods described herein. The analytical methods and compositions of the present invention provide levels of sensitivity and precision that allow the detection of many markers over a dynamic range at which the markers have been previously undetectable. Using methods described herein, normal and abnormal ranges for such markers can de determined. Virtually any compound within a biological sample can be employed as an analyte in the presence invention. Without limitation, such analytes may be enzymes, co-factors, receptors, receptor ligands, hormones, cytokines, blood factors, viruses, antigens, steroids, drugs, antibodies, etc. For example, an analyte of the present invention may include without limitations, drugs, metabolites, co-factors, hormones, cytokines, cell- surface-receptors, antibodies, antigens, enzymes or other proteins whose expression is characteristic of a disease, disorder, or pathological condition.
2. Biological Samples Comprising a HIV-I Antigen
[0233] In a preferred embodiment of the present invention, a biological sample is suspected of comprising a HIV antigen, preferably a HIV-I antigen. Thus, within the meaning of a biological sample are samples comprising HIV-I, inactivated HIV-I or a partially purified or recombinant HIV-I antigen. Preferred HIV-I antigens are set forth herein.
III. METHODS
A. Detecting An Analyte In A Biological Sample [0234] The compositions, systems, kits, and methods of the present invention make possible the detection and quantification of an analyte, such as an antigen, preferably an antigen from a pathogen, such as a bacterium or virus, more preferably, a virus antigen, more preferably a lentivirus antigen, and even more preferably, a HIV-I antigen, in samples within a dynamic range previously not described.
[0235] In one aspect, the invention provides a method for detecting an analyte of interest, e.g., an antigen, in a biological sample. This method is useful for determining the presence or absence of an analyte of interest in a biological sample. In a preferred embodiment of the present invention, a method for detecting an analyte of interest, such as an antigen, preferably, a HIV-I antigen, in a biological sample, comprises using an assay where the assay has a dynamic range of between about 0.2 pg/ml and about 20,000 pg/ml and a coefficient of variation of less than 10%.
[0236] Methods and assays of the present invention are characterized by having a dynamic range of at least four logs, preferably at least 5 logs, more preferably at least 6 logs. In some embodiments, the method of detecting an analyte of interest is capable of determining a concentration for this analyte in a sample where the analyte may range in concentration over a range of at least about 100-fold, or 1 ,000-fold, or 10,000-fold, or 30,000-fold, or 100,000- fold, or 300,000-fold, or 1 ,000,000-fold, or 3,000,000-fold, or 10,000,000-fold. In a preferred embodiment of the present invention, the method of detecting an analyte in a biological sample has a dynamic range of between about 0.2 pg/ml and about 20,000 pg/ml. This means that e.g., if a microtiter plate contains (i) a first sample having about 0.2 pg/ml of an analyte of interest in one well, (ii) a second sample having about 2 pg/ml of an analyte of interest in a second well, a third sample having about 20 pg/ml of an analyte of interest in third well, a fourth sample having about 200 pg/ml of an analyte of interest in a fourth well, a fifth sample having about 2,000 pg/ml of an analyte of interest in fifth well, and a sixth sample having about 20,000 pg/ml of an analyte of interest in a sixth well, an assay having a dynamic range of five logs and a lower limit of detection of about 0.2 pg/ml will accurately quantify the concentration of the analytes in all the samples without the need for further treatment to adjust the analyte concentration, e.g., dilution and/or concentration.
[0237] Accuracy may be determined by standard methods, e.g., using a series of standards of concentrations that span the dynamic range and constructing a standard curve (see Figure 1 and Examples herein). Standard measures of fit of the resulting standard curve may be used as a measure of accuracy, e.g., an R2 greater than about 0.7, 0.75, 0.8, 0.85, 0.9, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98, 0.99, or 0.995. In a preferred embodiment of the present invention, the accuracy of the assay described herein is greater than 0.99, preferably greater than 0.995, more preferably greater than 0.9975.
[0238] Further, methods and assays of the present invention are characterized by having a low detection limit of less than about 100 pg/ml, less than about 90 pg/ml, less than about 80 pg/ml, less than about 70 pg/ml, less than about 60 pg/ml, less than about 50 pg/ml, less than about 40 pg/ml, less than about 30 pg/ml, less than about 20 pg/ml, less than about 10 pg/ml, less than about 9 pg/ml, less than about 8 pg/ml, less than about 7 pg/ml, less than about 6 pg/ml, less than about 5 pg/ml, less than about 4 pg/ml, less than about 3 pg/ml, less than about 2 pg/ml, less than about 1 pg/ml, less than about 0.9 pg/ml, less than about 0.8 pg/ml, less than about 0.7 pg/ml, less than about 0.6 pg/ml, less than about 0.5 pg/ml, less than about 0.4 pg/ml, less than about 0.3 pg/ml, less than about 0.2 pg/ml, and less than about 0.1 pg/ml analyte less than about 0.05 pg/ml, or less than about 0.01 pg/ml.
[0239] The "detecting" of an analyte includes detecting the analyte directly or indirectly. In the case of indirect detection, a label that has been attached to the analyte, may be detected. [0240] In a preferred embodiment, the method for detecting an analyte in a biological sample using an assay having a dynamic range of between about 0.2 and about 20,000 pg/ml and a coefficient of variation of less than 10%, comprises the steps of (a) binding a capture binding partner specifically binding an analyte of interest to a solid support, (b) contacting the capture binding partner with a biological sample suspected of containing the analyte of interest for a time sufficient to allow binding of the analyte of interest to the capture binding partner, thereby forming a capture binding partner/analyte complex, (c) contacting the capture binding partner/analyte complex with a detecting binding partner specifically binding the analyte of interest for a time sufficient to allow binding of the detecting binding partner to the analyte of the capture binding partner/analyte complex, thereby forming a capture binding partner/analyte/detecting binding partner complex, wherein the detecting binding partner produces a detectable signal, and (d) detecting the detectable signal. The capture binding partner and the detecting binding partner bind to different binding sites, e.g., epitopes on the analyte of interest. Thereby the analyte of interest in the biological sample is detected.
[0241] A preferred method for detecting an antigen in a biological sample using an assay having a dynamic range of between about 0.2 and about 20,000 pg/ml and a coefficient of variation of less than 10%, comprises the steps of (a) binding a first antibody specifically binding an antigen to a solid support, (b) contacting the first antibody with a biological sample suspected of containing the antigen for a time sufficient to allow binding of the antigen to the first antibody, thereby forming a first antibody/antigen complex, (c) contacting the first antibody/antigen complex with a second antibody specifically binding the antigen for a time sufficient to allow binding of the second antibody to the antigen of the first antibody/antigen complex, thereby forming a first antibody/antigen/second antibody complex, wherein the second antibody produces a detectable signal, and (d) detecting the detectable signal. The first antibody and the second antibody bind to different binding sites, e.g., epitopes on the antigen. Thereby the analyte in the biological sample is detected.
[0242] In a preferred embodiment of the present invention, this method comprises an assay, in which binding partner pairs, e.g. antibodies, to the same analyte, e.g., same antigen, are used. The invention also encompasses binding partner pairs, e.g., antibodies, wherein both antibodies are specific to the same analyte, e.g., the same antigen, and wherein at least one member of the pair comprises a label as described herein. Thus, for any label that includes a binding-partner and a labeling moiety, the invention also encompasses a pair of binding partners wherein the first binding partner, e.g., a first antibody, is, typically, unlabeled and serves as a capture binding partner and the second binding partner, e.g., a second antibody, is typically labeled and serves as detecting binding partner.
[0243] A preferred antigen detected by the methods of the present invention, is a HIV-I antigen. Thus, one particularly useful assay format is a sandwich assay, e.g., a sandwich immunoassay. In this format, an antigen, such as a HIV-I antigen, is captured, e.g., by using a capture binding partner attached to a solid support. A preferred capture binding partner is a first antibody which binds to the HIV-I antigen. This is followed by binding of a detecting binding partner, such as a second antibody, comprising a detectable label, e.g., fluorescent moiety, to the HIV-I antigen. Then the detectable label is detected as described herein. [0244] Thus, in a preferred embodiment of the present invention a method for detecting a HIV-I antigen in a biological sample using an immunoassay having a dynamic range of between about 0.2 and about 20,000 pg/ml and a coefficient of variation of less than 10% is provided. In a preferred embodiment, this method comprises the steps of (a) binding a first antibody specifically binding a HIV-I antigen to a solid support, (b) contacting the first antibody with a biological sample suspected of containing the HIV-I antigen for a time sufficient to allow binding of the HIV-I antigen to the first antibody, thereby forming a first antibody/HIV- 1 antigen complex, (c) contacting the first antibody/HIV- 1 antigen complex with a second antibody specifically binding the HIV-I antigen for a time sufficient to allow binding of the second antibody to the HIV-I antigen of the first antibody/HIV- 1 antigen complex, thereby forming a first antibody/HIV- 1 antigen/second antibody complex, wherein the second antibody produces a detectable signal, and (d) detecting the detectable signal. The first antibody and the second antibody bind to different epitopes of the HIV-I antigen. Thereby the HIV-I antigen in the biological sample is detected.
1. Binding A Capture Binding Partner To A Plurality Of Solid Support
[0245] In one embodiment of the present invention, the method of detecting an analyte in a biological sample comprises the step of binding a capture binding partner specifically binding an analyte of interest to a plurality of a solid support.
[0246] A capture binding partner may be attached to a solid support. A preferred solid support is a microtiter plate or a plurality of microspheres, e.g., paramagnetic beads. In some embodiments, the invention provides a capture binding partner for an analyte of interest, e.g., an antigen, such as a HIV-I antigen, attached to a microtiter plate. In some embodiments, the invention provides a capture binding partner for an analyte of interest, e.g., an antigen, such as a HIV-I antigen, attached to a plurality of microspheres. In some embodiments, the invention provides a binding partner for an analyte of interest, e.g., an antigen, such as a HIV-I antigen, attached to paramagnetic beads. [0247] Any suitable capture binding partner that is specific for the analyte to be captured may be used. A preferred capture binding partner is an antibody, more preferred, a monoclonal antibody. Attachment of an antibody to a solid support may be accomplished through a variety of methods known in the art. Binding moieties, such as an antibody, may be coupled to a solid support using reversible or non-reversible interactions. [0248] A non-reversible or covalent attachment in which the orientation of the capture binding partner is such that capturing of the analyte of interest is optimized, is particularly useful. In some embodiments, a capture binding partner is covalently attached to a solid support in an oriented way. In some embodiments, a capture binding partner is covalently attached to a microtiter plate in an oriented way. In some embodiments, a capture binding partner is covalently attached to a plurality of microspheres in an oriented way. In some embodiments, a capture binding partner is covalently attached to paramagnetic beads in an oriented way. The oriented way is usually achieved by reacting the carboxylic groups of sugars residues found in the Fc region and hydrazide moieties typically present on a solid support. Alternative methods in the art are known, including, but not limited to, using a solid phase coupled to protein A or protein G.
[0249] Non-reversible or covalent interactions may be made using a support that includes at least one reactive functional group, such as a hydroxyl, carboxyl, sulfhydryl, or amino group that chemically binds to the binding moiety, optionally through a spacer group. Suitable functional groups include N-hydroxysuccinimide esters, sulfonyl esters, iodoacetyl groups, aldehydes, epoxy, imidazolyl carbamates, and cyanogen bromide and other halogen- activated supports. Such functional groups can be provided to a support by a variety of known techniques. For example, a glass surface can be derivatized with aminopropyl triethoxysilane in a known manner. Thus, in one preferred embodiment of the present invention, the step of binding a capture binding partner, such as a first antibody to a solid support comprises binding of the binding of the capture binding partner, such as the first antibody to an activated solid support. [0250] In a preferred embodiment of the present invention the first antibody which is bound to the solid support is an anti -HIV-I p24 antibody, preferably monoclonal antibody 4F6 or a fragment thereof. In some embodiments, a first antibody bound to a solid support, is antibody KC57. Other anti-HIV-1 p24 antibodies described herein (e.g., see Figure 5) can also be attached to a solid support. [0251] An exemplary protocol for the oriented attachment of a capture binding partner, exemplified by the capture antibody 4F6, to a solid support, is described in Example 2. A preferred non-reversible or covalent attachment comprises a two-step carbodiimide chemical reaction as described in Example 2.
[0252] Alternatively, the capture binding partner is bound to a solid support by reversible or non-covalent (e.g., ionic, hydrophobic, etc.) interactions between a solid support and a binding moiety. Such attachments may be made using linker moieties associated with the solid support and/or the binding moiety. A variety of linker moieties suitable for use with the present invention are known in the art and include, without limitations, biotin- avidin/streptavidin interactions. Use of linker moieties for coupling diverse agents is well known to one of ordinary skill in the art, who can apply this common knowledge to form solid support/binding moiety couplings suitable for use in the present invention with no more that routine experimentation. [0253] In another preferred embodiment of the present invention, the amount of the solid support having bound a capture binding partner, such as a first antibody, is determined. This can be done by a variety of assays as known in the art, including, but not limited to, titration of fluorescently-labeled goat anti-mouse antibodies where the first antibody is a mouse antibody, or by titration of fluorescently-labeled goat anti -human antibodies where the first antibody is a human antibody.
[0254] In yet another preferred embodiment of the present invention, the solid support having bound a capture binding partner, such as a first antibody, is added to a container. Useful containers include, without limitation, tubes, multi-well container, micro-titer plates, and the like.
2. Contacting The Capture Binding Partner With A Biological Sample Suspected of Containing An Analyte Of Interest
[0255] In one embodiment of the present invention, the method of detecting an analyte in a biological sample comprises the step of contacting the capture binding partner, such as a first antibody, with a biological sample suspected of containing an analyte of interest, such as a HIV-I antigen, for a time and under conditions sufficient to allow binding of the analyte of interest, such as the HIV-I antigen, to the capture binding partner, such as the first antibody, thereby forming a capture binding partner/analyte complex, such as a first antibody/HIV- 1 antigen complex. [0256] Typically, the biological sample suspected of containing the analyte of interest is added to a container (or vessel, plastic tube, etc.), containing a suspension comprising the capture binding partner bound to the solid support. Thereby the biological sample is contacted with the capture binding partner, such as a first antibody. In some embodiments of the present invention the first antibody is an anti-HIV-1 p24 antibody, preferably monoclonal antibody 4F6 or a fragment thereof.
[0257] Methods of the present invention require only minute amount of a biological sample. Thus, in some embodiments of the present invention, the biological sample contacted to the capture binding partner, such as the first antibody, is about 10 μl, about 20 μl, about 30 μl, about 40 μl, about 50 μl, about 100 μl, or about 200 μl. In some embodiments of the present invention, the volume of the biological sample contacted to the capture binding partner, such as the first antibody, is less than 10 μl. In some embodiments of the present invention, the volume of the biological sample contacted to the capture binding partner, such as the first antibody, is less than 20 μl. In some embodiments of the present invention, the volume of the biological sample contacted to the capture binding partner, such as the first antibody, is less than 30 μl. In some embodiments of the present invention, the volume of the biological sample contacted to the capture binding partner, such as the first antibody, is less than 40 μl. In some embodiments of the present invention, the volume of the biological sample contacted to the capture binding partner, such as the first antibody, is less than 50 μl. In some embodiments of the present invention, the volume of the biological sample contacted to the capture binding partner, such as the first antibody, is less than 100 μl. In some embodiments of the present invention, the volume of the biological sample contacted to the capture binding partner, such as the first antibody, is less than 200 μl. In some embodiments of the present invention, the volume of the biological sample contacted to the capture binding partner, such as the first antibody, is between about 10 μl and about 200 μl, preferably between about 20 μl and about 100 μl, more preferably between about 20 μl and about 50 μl. [0258] The time and conditions sufficient to allow for binding of the analyte, such as a
HIV-I antigen, to the capture binding partner, such as a first antibody, will vary depending on the conditions. It will be apparent that shorter binding times are desirable in some settings, especially in a clinical setting. The use of, e.g., paramagnetic beads can reduce the time required for binding. In some embodiments, the time allowed for binding of the analyte of interest, such as a HIV-I antigen, to the capture binding partner, e.g., a first antibody, is less that about 12, 10, 8, 6, 4, 3, 2, or 1 hours, or less than about 60, 50, 40, 30, 25, 20, 15, 10, or 5 minutes. In some embodiments, the time allowed for binding of the analyte of interest, such as a HIV-I antigen, to the capture binding partner, e.g., a first antibody, is less than about 2 hours. In some embodiments, the time allowed for binding of the analyte of interest, such as a HIV-I antigen, to the capture binding partner, e.g., a first antibody, is less than about 60 minutes. In some embodiments, the time allowed for binding of the analyte of interest, such as a HIV-I antigen, to the capture binding partner, e.g., a first antibody, is less than about 40 minutes. In some embodiments, the time allowed for binding of the analyte of interest, such as a HIV-I antigen, to the capture binding partner, e.g., a first antibody, is less than about 30 minutes. In some embodiments, the time allowed for binding of the analyte of interest, such as a HIV-I antigen, to the capture binding partner, e.g., a first antibody, is less than about 20 minutes. In some embodiments, the time allowed for binding of the analyte of interest, such as a HIV-I antigen, to the capture binding partner, e.g., a first antibody, is less than about 10 minutes.
[0259] Unwanted molecules and other substances that may be present in the biological sample analyzed for the presence or absence of an analyte, such as a HIV-I antigen, may then optionally be washed away. Thus, in some embodiments, following the binding of the analyte, such as the HIV-I antigen, to the capture binding partner, e.g., first antibody, particles that may have bound nonspecifically, as well as other unwanted substances in the biological sample, are washed away leaving substantially bound to the solid support only capture binding partner/analyte complexes, such as first antibody/HIV- 1 antigen complexes. [0260] In other embodiments, no washing is performed. Thus, in alternative embodiments, biological sample and detection binding partner comprising a detectable label are added to the capture binding partner without a wash in between, e.g., at the same time. Other variations will be apparent to one of skill in the art.
3. Contacting The Capture Binding Partner/ Analyte Complex With A Detecting Binding Partner
[0261] In one aspect of the present invention, the method of detecting an analyte in a biological sample comprises the step of contacting the capture binding partner/analyte complex, such as the first antibody/HIV- 1 antigen complex with a detecting binding partner, such as a second antibody, specifically binding the analyte of interest, such as the HIV-I antigen, for a time sufficient to allow binding of the detecting binding partner, such as the second antibody, to the analyte of interest, such as the HIV-I antigen, of the capture binding partner/analyte complex, such as the first antibody/HIV- 1 antigen complex, thereby forming a capture binding partner/analyte/detecting binding partner complex, such as a first antibody/HIV- 1 antigen/second antibody complex, wherein the detecting binding partner, such as the second antibody, produces a detectable signal.
[0262] Typically, the detecting binding partner is added to a container (or vessel, or plastic tube, etc.) containing a suspension comprising the capture binding partner/analyte complex.
[0263] The time allowed for binding of the detecting binding partner, such as a second antibody, to the analyte, such as a HIV-I antigen, of the capture binding partner/analyte complex, such as the first antibody/HIV- 1 antigen complex, will vary depending on the conditions. It will be apparent that shorter binding times are desirable in some settings, especially in a clinical setting. In some embodiments, the time allowed for binding of the detecting binding partner, such as a second antibody, to the analyte of interest, such as the HIV-I antigen, of the capturing binding partner/analyte complex, such as a first antibody/HIV- 1 antigen complex is less that about 12, 10, 8, 6, 4, 3, 2, or 1 hours, or less than about 60, 50, 40, 30, 25, 20, 15, 10, or 5 minutes. In some embodiments, the time allowed for binding of the detecting binding partner, such as the second antibody, to the analyte of interest, such as the HIV-I antigen, is less than about 2 hours. In some embodiments, the time allowed for binding of the detecting binding partner, such as the second antibody, to the analyte of interest, such as the HIV-I antigen, is less than about 60 minutes. In some embodiments, the time allowed for binding of the detecting binding partner, such as the second antibody to the analyte of interest, such as the HIV-I antigen, is less than about 40 minutes. In some embodiments, the time allowed for binding of the detecting binding partner, such as the second antibody to the analyte of interest, such as the HIV-I antigen, is less than about 30 minutes. In some embodiments, the time allowed for binding of the detecting binding partner, such as the second antibody to the analyte of interest, such as the HIV-I antigen, is less than about 20 minutes. In some embodiments, the time allowed for binding of the detecting binding partner, such as the second antibody to the analyte of interest, such as the HIV-I antigen, is less than about 10 minutes.
[0264] Unbound label may then optionally be washed away. Thus, in some embodiments, following the binding of the detecting antibody, such as the second antibody to the analyte of interest, such as the HIV-I antigen, unbound label and detecting binding partner that may have bound nonspecifically, are washed away leaving substantially bound to the solid support only capture binding partner/analyte/detecting binding partner complexes, such as first antibody/HIV- 1 antigen/second antibody complexes. In other embodiments, no washing is performed. Other variations will be apparent to one of skill in the art. 4. Detecting The Detectable Signal
[0265] In one aspect of the present invention, the method of detecting an analyte in a biological sample comprises the step of detecting the detectable signal produced by the detecting binding partner, such as a second antibody.
[0266] It will be appreciated that detecting a detectable signal depends on the kind of detectable signal produced by the detecting binding partner. In some embodiments, detecting the detectable signal comprises resuspending the solid support having bound the capture binding partner/analyte/detecting binding partner complex, such as the first antibody/HIV- 1 antigen/second antibody complex in an assay buffer.
[0267] In a preferred embodiment, a fluorescence signal is detected. The step of detecting a fluorescence signal may comprise measuring a fluorescence signal for each individual member of the plurality of solid support having bound the capture binding partner/analyte/detecting binding partner complex, such as the first antibody/HIV- 1 antigen/second antibody complex. In some embodiments, the fluorescence signal is detected in a fluorimeter. In preferred embodiments, the fluorescence signal is detected using the Luminex technology as described herein and further in published U.S. Patent Application Nos. 2008/0151240. 2007/0207513, 2006/0159962, 2005/0277197, and 20050118574, incorporated herewith by reference in their entireties.
[0268] Microtiter or multi-well plates are preferred formats for detecting an analyte, such as HIV-I antigen, using compositions, kits, systems, and methods described herein. High- density format plates, such as 96, 384, 864 and 1536 well plates can be used. Assays conducted in multiwell plates employ some type of light detection from the plate as the reporter for positive or negative assays results. Such assays include fluorescence assays, chemiluminescence assays (e.g., luciferase-based assays), phosphorescence assays, scintillation assays, and the like. In particular, with the advent of solid phase scintillating materials, capsules and beads, homogeneous scintillation proximity assays (SPA) are now being performed with increasing frequency in multiwell plates.
[0269] A preferred light signal detected is fluorescence. A preferred detecting partner produces a fluorescence signal. Detection of a fluorescence signal can be performed as described herein, e.g., in Example 3. Alternative detection methods are described in US2006/0078998 and US 2008/0064113, incorporated herein by reference in their entirety. [0270] Detection of light signals from multiwell plates can be done using plate readers, which generally employ a photodetector, an array of such photodetectors, photomultiplier tubes or a photodiode array to quantify the amount of light emitted from different wells. Plate readers have been disclosed, for example, in U.S. Pat. Nos. 4,810,096 and U.S. Pat. No. 5,198,670. [0271] Detection of light from multiwell plates can also be accomplished by imaging.
Imaging systems typically comprise a standard 50-55 mm fl.4 photographic lens coupled to a camera. Such systems can be used to image an entire multiwell plate. Further, a detectable signal can also be detected using a charge coupled device (CCD) as described in US 2003/0078737. Suitable CCD cameras are available from Alpha- Innotech (San Leandro, CA), Stratagene (La Jolla, CA), BioRad (Richmond, CA), and Beckman-Coulter (Fullerton, CA). [0272] Another manner of detecting the analyte, such as a HIV-I antigen, is to utilize a conjugate comprising a third antibody attached to a signal -generating compound. In particular, once the first antibody/HIV- 1 antigen/second antibody complexes described above have formed (i.e., the latter antibody being the second antibody which is unlabeled), one may then add a conjugate which binds to the second unlabeled antibody. The conjugate may comprise, for example, an antigen or anti-antibody capable of binding to the bound second antibody (e.g., anti-mouse antibody or an anti- human antibody) attached to a signal- generating compound capable of generating a detectable signal. Detection of the signal, thus, indicates presence of the complexes and, thus, presence of the analyte, such as the HIV-I antigen in the sample. The signal generated is actually proportional to the amount of antigen present in the sample. (See, e.g., U.S. Pat. No. 6,015,662.). The design of the assay is dependent upon the affinities and specificities of the third antibodies used, accuracy of results obtained, convenience, the nature of the solid phase, etc. (See U.S. Pat. No. 5,104,790 for a discussion of different antigen assay formats).
5. Determining The Amount Of An Analyte In A Biological Sample [0273] The amount of the analyte, such as a HIV-I antigen, in a biological sample may also be determined, as the signal generated by the detecting binding partner, such as a second antibody, is proportional to the amount of the analyte or HIV-I antigen in the sample. Thus, in another aspect, the invention provides a method for determining the amount of an analyte, e.g., an antigen, in a biological sample. A preferred antigen for which the amount in a biological is determined by the methods of the present invention, is a HIV-I antigen. As used herein, determining the amount of an analyte in a biological sample is synonymous with determining a concentration of an analyte in a biological sample.
[0274] Determination of the amount of an analyte e of interest, such as a HIV-I antigen, and in particular a HIV-I GAG polypeptide, is performed as described herein, e.g., in Example 3. Alternatively, 4p regression and 5p regression can be performed, e.g., as described by Gottschalk and Dunn (2005, Anal Biochem 343:54-65). B. Modifying Assay Sensitivity And Dynamic Range
[0275] In general, fluorescent, chemiluminecent and other assay formats comprise three distinguishable response ranges. Where the amount of analyte being assayed is within the dynamic range of the assay, the reported signal will be dependent upon the amount of analyte present. Where the amount of analyte exceeds the dynamic range of the assay, saturation will occur and the reported signal will not be indicative of the true analyte concentration. Likewise, where the amount of analyte present in the sample falls below the threshold of the assay's dynamic range, the assay may be insufficiently sensitive to the actual analyte concentration, and the reported signal will also not be indicative of the true analyte concentration.
[0276] Two approaches have conventionally been employed to address this problem. In the first, multiple dilutions or concentrations of a sample are made and then assayed for a defined time period and the results are evaluated against that of a "standard curve" of assay results obtained with analyte of varying but known concentration. In the second approach, an amount of sample is assayed for multiple times, and results falling within the dynamic range of the assay are used to calculate the analyte's concentration (see, for example, U.S. Pat. Nos. 5,306,468 and 6,212,291).
[0277] As described herein, e.g., in Example 4, variation of the plurality of solid supports allows changing the dynamic range of the assays described herein. In some embodiments, the changing of the dynamic range of the assay is performed in the context of the Luminex technology. As described herein, using the Luminex technology one detects the fluorescence signal(s) on a single bead. The more fluorescent detector antibody is bound per bead, the more sensitive the assay will be. In other words, the more analyte bound to each bead, the more sensitive the assay will be. When the analyte is abundant, this typically is not a problem, because every bead will have a high number of analyte bound. When, however, the analyte is scarce, then, the limiting quantity of the analyte is shared by all the beads, each of which can only bind a small amount of the analyte, and therefore decreases the fluorescence signal(s) detected on each bead. If less beads are present, then they each individual bead will bind more of the limiting amount of the analyte, and therefore produce a stronger fluorescent signal(s). In contrast thereto, a bulk assay will not have such limitations, and also will not have such a flexibility. C. Adaptation of Assays
[0278] In some embodiments of the present invention, an antigen of interest is detected and quantified using a detecting binding partner, such as a second antibody, which may be labeled with a fluorochrome. Methods described herein can also be adapted to be read by conventional fluorescence plate readers.
[0279] In some embodiments, a label is incorporated in or to a solid support, such as in the capture beads. This provides a normalizing element. Thus, alternatively or in addition to conventional reading at the excitation and emission wavelength of the label attached to the detection binding partner, e.g., a detection antibody, a reading at the excitation and emission wavelength of a label incorporated in or to a solid support is performed. In a preferred embodiment, the step of detecting a detectable signal comprises the step of reading at the excitation and emission wavelength of the label incorporated in or to a solid support, preferably, capture beads. Analyzing the ratio of the reporter signal to the solid bead signal for each well measured, virtually eliminates the variation of bead number as a confounding factor in the assay and data analysis.
[0280] In other embodiments, methods described herein are adapted to a high throughput method. For example, biological samples may be analyzed in standard 96- well plates, 384- well plates, 864-well plates, or 1536-well plates.
[0281] As described herein, assays of the present invention can also be multiplexed so that multiplex assay formats are used. Using a multiplex assay format, assaying for the presence of a second antigen in a biological sample can be performed. The second antigen is different from the first antigen, e.g., different from the HIV p24 polypeptide if it was chosen as the first antigen to be detected in the biological sample.
[0282] Some serologic assays combine antibody and antigen detection. They exhibit superior seroconversion sensitivity compared to assays that detect only antibody, because detection of antigen, which appears prior to antibody, reduces the seroconversion window. An early version of a HIV combination assay is described in Gallarda, et ah, 1992, WO93/21346. Assays described herein may also be included in a HIV-I combination assay, detecting in a biological sample a HIV-I antigen (e.g., a GAG polypeptide) as described herein and an antibody directed against a HIV-I antigen. Any such combination assay, which utilizes one or more of the antibodies described herein, is considered to be within the scope of the present invention. Thus, in some embodiments, methods described herein comprise the step of detecting an antibody against a HIV antigen, preferably, a HIV-I antigen in the biological sample. Methods for detecting HIV antibodies in biological samples are known in the art.
[0283] In some embodiments, methods described herein comprise the step of detecting in the biological sample the absence or presence of a viral antigen wherein the viral antigen is different from a HIV-I GAG polypeptide, preferably different from a p24 polypeptide. Using appropriate antibodies, the presence of, e.g., a Hepatitis A antigen, a Hepatitis B antigen, or a Hepatitis C antigen, can be detected.
IV. DIAGNOSING AND MONITORING [0284] The invention described herein provides methods and compositions for the sensitive detection of analytes in biological samples, and further methods of establishing values for normal and abnormal levels of such analytes by quantification of these analytes. As will be appreciated, abnormal levels of an analyte may be associated with a biological state of a subject, e.g., a disease, disorder, or pathological condition. In further embodiments, the invention provides methods of diagnosis, prognosis, and/or treatment selection based on values established for these analytes. Specifically, where compositions and methods are used to detect a lentivirus antigen, such as a HIV-I antigen, the detection of an amount or concentration of the lentivirus antigen in a biological sample is used for diagnosis, prognosis, and/or treatment selection of a lentivirus infection, such as AIDS. [0285] For a lentivirus antigen, such as a HIV-I antigen, the presence of any lentivirus antigen at all in a biological sample from a subject is an indication of a disease or pathological state, and the threshold is essentially zero. Appearance of a lentivirus antigen in a biological sample taken from a subject, at any level, indicates the subject having a lentivirus infection, e.g., possibly, AIDS. [0286] HIV (including HIV-I and HIV-2) testing is integral to HIV prevention, treatment, diagnosing, monitoring, and care efforts. Knowledge of one's HIV status is critical for preventing the spread of disease. Hence, early diagnosis of a HIV infection is always preferred. Detection of HIV antibodies in the blood of subjects is a preferred method to detect exposure and infection by HIV. The drawback of this method, however, is that the appearance of antibodies against HIV antigens in the blood of a HIV-exposed individual is entirely dependent on the host's immune system and viral characteristics. Unless the host's immune system is challenged enough by the virus, a humoral immune response is not initiated. Further, a lag phase elapses between HIV exposure and initiation of HIV antibody response, the duration of which varies from individual to individual. If we were to rely on the appearance of HIV antibodies in the blood to determine an individual's HIV status, a valuable window of time that could be utilized to prevent the virus from targeting the immune system not only remains unavailable, but more importantly remains unutilized. The mode of transmission of HIV has changed from the past. In the 1980s, needle sharing, homosexual transmission, blood transfusion, or organ donations (sometimes referred to as "high risk population") were the primary modes of HIV transmission. In recent times, however, heterosexual transmission was found to account for 90% of new HIV infection worldwide; and the semen, vaginal secretions, and breast milk of HIV infected individuals became the primary modes of transmission of HIV today. Therefore, HIV antibody screening of the blood may no longer fulfill the criterion of being the most effective strategy and method in determining the HIV status of an individual. Assays for the early detection of HIV exposure and/or infection are needed, especially in a resource-poor setting.
[0287] As shown herein, e.g., Example 5, using compositions, kits, systems, and methods of the present invention, HIV-I antigens, such as a GAG polypeptide, preferably p24 polypeptide, can be detected in biological samples obtained from subjects at the early onset of an infection by HIV-I and/or shortly thereafter. Methods of the present invention positively identified p24 HIV-I antigens during such early time of HIV-I infection when other U.S. FDA-licensed test kits failed to do so (Example 5).
[0288] Thus, in another aspect of the present invention, a method of detecting exposure to HIV in a subject is provided. In a preferred embodiment, this method comprises the steps of (a) evaluating a biological sample from a subject for the presence of HIV and (b) determining whether the subject is HIV-positive or HIV-negative, wherein the presence of HIV in the biological sample indicates the subject is HIV-positive, and the absence of HIV in the sample indicates the subject is HIV-negative. In some embodiments of the present invention, the step of evaluating the biological sample for the presence of HIV is performed using any of the assay methods described herein. In some embodiments of the present invention, the method of detecting exposure to HIV in a subject comprises the step of providing a biological sample from the subject. Methods of the present invention are useful to detect exposure to HIV-I and/or HIV-2 in a subject. [0289] The subject from whom the biological sample is obtained may be HIV-I seronegative and/or may not show any signs of a HIV-I infection. Compositions, systems, kits, and methods of the present invention are useful to detect a HIV-I antigen in a biological sample prior to seroconversion. [0290] In another aspect of the present invention, a method of diagnosing or monitoring HIV in a subject is provided. In a preferred embodiment, this method comprises the steps of (a) evaluating a biological sample from a subject for the presence of HIV, and (b) determining whether the subject is HIV-positive or HIV-negative, wherein the presence of HIV in the biological ample indicates the subject is HIV-positive, and the absence of HIV in the sample indicates the subject is HIV-negative. In some embodiments of the present invention, the step of evaluating the biological sample for the presence of HIV is performed using any of the assay methods described herein. In some embodiments of the present invention, the method of diagnosing or monitoring HIV in a subject comprises the step of providing a biological sample from the subject. Methods of the present invention are useful to diagnose and monitor HIV-I and/or HIV-2 in a subject.
[0291] When monitoring a response to a therapeutic agent, such as an anti-viral agent, typically biological samples obtained from an individual undergoing therapeutic treatment are obtained and analyzed for the absence or presence of a viral antigen at various times during treatment. [0292] In a preferred embodiment of the present invention, a disease status is determined as part of monitoring the effect of an anti-viral drug or a therapy administered to a subject diagnosed with a viral infection, such as a lentivirus infection. The effect of an anti- viral drug or a therapy administered to a subject with a viral infection may include progression of the viral infection (worsening) and regression of the viral infection (improvement). [0293] Using the methods of the invention, analyte levels, e.g., HIV antigen levels, are determined in a biological sample from a subject at various times of having been given an anti-viral drug or a therapy. An analyte, e.g., a HIV antigen, detected in a biological sample from a subject at a first time (tl ; e.g., before giving an anti -viral drug or a therapy) that is lower than the analyte level detected in a comparable biological sample from the same subject taken at a second time (t2; e.g., after giving the anti- viral drug or the therapy), indicates that the viral infection in the subject is progressing. Likewise, a lower HIV antigen level at a second time compared to a HIV antigen level at a first time, indicates that the viral infection in the subject is regressing.
[0294] Similarly, a HIV antigen level detected in a biological sample from a subject at a first time (tl ; e.g., shortly after beginning viral therapy) that is higher than the HIV antigen level detected in a comparable biological sample from the same subject taken at a second time (t2; e.g., weeks or months after beginning anti-viral therapy), may indicate that the viral infection in the subject is not reoccuπϊng. Likewise, a higher HIV antigen level at the second time compared to the HIV antigen level at the first time, may indicate that the viral infection in the subject is reoccurring or the viral therapy is not effective. [0295] The levels of the HIV antigen determined can be used to determine whether to recommend that the subject should continue or discontinue anti-viral therapy or whether a different antiviral therapy than the one used fro the first treatment of the subject should be recommended. For example, if the quantification of the HIV antigen indicates progression of the viral infection, either continuing the anti-viral therapy, increasing the dose of the anti- viral agent and/or recommend another anti-viral therapy should be considered. Likewise, if quantification of the HIV-I antigen indicates regression of the viral infection or no viral infection, the viral therapy may be discontinued or the dose of the viral agent may be lowered.
[0296] In another aspect of the invention, a method of determining whether to recommend that a subject should undergo anti-HIV therapy is provided. In some embodiments of the present invention, this method comprises the steps of (a) evaluating a biological sample from a subject for the presence of HIV, and (b) determining whether the subject is HIV positive or HIV-I negative, wherein the presence of HIV in the biological ample indicates that anti-HIV therapy should be recommended, and the absence of HIV in the sample indicates that anti HIV therapy should not be recommended. Methods of the present invention are useful to determine whether to recommend that a subject should undergo anti HIV-I therapy and/or anti-HIV-2 therapy.
V. KITS AND SYSTEMS
[0297] For use in diagnostic, research, and therapeutic applications described above, kits and systems are also provided by the present invention. In the diagnostic and research applications such kits and systems may include any or all of the following compositions described herein: assay reagents, buffers, a first antibody, a second antibody, a biological sample, a calibration standard, a HIV antigen, a HIV-I antigen or polypeptide, a HIV-I GAG polypeptide, a HIV-I p24 polypeptide, a solid support, a capture binding protein, a capture binding protein bound to a solid support, a first antibody, a first antibody bound to a solid support, a detecting binding partner, a second antibody, a label, a labeled second antibody, or any other compound or composition described herein.
[0298] One or more of the antibodies described herein, and preferably the pairs suggested above, is particularly suitable for use in the form of a kit or system. Kits and systems may comprise one or more containers such as vials or bottles, with each container containing an antibody, a pair of the antibodies, or as cocktails of antibodies. These kits and systems may also contain vials or containers of other reagents needed for performing the assay, such as washing, processing and indicator reagents.
[0299] Reference to particular buffers, media, reagents, cells, culture conditions and the like, or to some subclass of the same, is not intended to be limiting, but should be read to include all such related materials that one of ordinary skill in the art would recognize as being of interest or value in the particular context in which they are presented. For example, it is often possible to substitute one buffer system or culture medium for another, such that a different but known way is used to achieve the same goals as those to which the use of a suggested method, material or composition is directed.
[0300] Typically, the components of a kit or system are provided in a container. In a preferred embodiment of the present invention, a kit or system for detecting an analyte, preferably, a HIV-I antigen, in a biological sample, comprises a container containing a composition as described herein.
[0301] In addition, a kit or system may include instructional materials containing directions {i.e., protocols) for the practice of the methods of this invention. The instructions may be present in the subject kits or systems in a variety of forms, one or more of which may be present in the kit or system. While the instructional materials typically comprise written or printed materials they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this invention. Such media include, but are not limited to electronic storage media {e.g. , magnetic discs, tapes, cartridges, chips), optical media {e.g., CD ROM), and the like. Such media may include addresses to internet sites that provide such instructional materials. [0302] A wide variety of kits and components can be prepared according to the present invention, depending upon the intended user of the kit and the particular needs of the user.
[0303] In one aspect of the present invention, the system is an immunosystem for detecting an amount of an antigen in a biological sample where the immunosystem has a dynamic range of between about 0.2 pg/ml and about 20 ng/ml and a coefficient of variation of less than 10%. In a preferred embodiment of the present invention, the immunosystem comprises (i) a first antibody reactive to an antigen, wherein the first antibody is bound to a solid support, (ii) a biological sample containing or suspected of containing the antigen, and (iii) a second antibody reactive to the antigen, wherein the second antibody binds to a different epitope of the antigen than the first antibody.
[0304] In a preferred embodiment of the present invention, the system is an immunosystem for detecting an amount of a HIV-I antigen in a biological sample where the immunosystem has a dynamic range of between about 0.2 pg/ml and about 20 ng/ml and a coefficient of variation of less than 10%. In a preferred embodiment of the present invention, the immunosystem comprises (i) a first antibody reactive to a HIV-I antigen, wherein the first antibody is bound to a solid support, (ii) a biological sample containing or suspected of containing the HIV-I antigen, and (iii) a second antibody reactive to the HIV-I antigen, wherein the second antibody binds to a different epitope of the HIV-I antigen than the first antibody. [0305] In some embodiments of the present invention, a kit or system comprises (i) a first antibody reactive to an antigen, wherein the first antibody is bound to a solid support, (ii) a calibration sample containing the antigen, and (iii) a second antibody reactive to the antigen, wherein the second antibody binds to a different epitope of the antigen than the first antibody.
[0306] In some preferred embodiments of the present invention, a kit or system comprises (i) a first antibody reactive to a HIV-I antigen, wherein the first antibody is bound to a solid support, (ii) a calibration sample containing the HIV-I antigen, and (iii) a second antibody reactive to the HIV-I antigen, wherein the second antibody binds to a different epitope of the HIV-I antigen than the first antibody.
[0307] Additional kit embodiments of the present invention include optional functional components that would allow one of ordinary skill in the art to perform any of the methods and variations thereof described herein. For example, kits and systems are also provided comprising a capture binding partner and a detection binding partner, optionally comprising a sample or a calibration standard as described herein.
[0308] Although the forgoing invention has been described in some detail by way of illustration and example for clarity and understanding, it will be readily apparent to one of ordinary skill in the art in light of the teachings of this invention that certain variations, changes, modifications and substitutions of equivalents may be made thereto without necessarily departing from the spirit and scope of this invention. As a result, the embodiments described herein are subject to various modifications, changes and the like, with the scope of this invention being determined solely by reference to the claims appended hereto. Those of skill in the art will readily recognize a variety of non-critical parameters that could be changed, altered or modified to yield essentially similar results. The headings used herein are for organizational purposes only and are not meant to be used to limit the scope of the description or the claims. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.
[0309] While each of the elements of the present invention is described herein as containing multiple embodiments, it should be understood that, unless indicated otherwise, each of the embodiments of a given element of the present invention is capable of being used with each of the embodiments of the other elements of the present invention and each such use is intended to form a distinct embodiment of the present invention.
[0310] The referenced patents, patent applications, and scientific literature, including accession numbers to GenBank database sequences, referred to herein are hereby incorporated by reference in their entirety as if each individual publication, patent or patent application were specifically and individually indicated to be incorporated by reference. Any conflict between any reference cited herein and the specific teachings of this specification shall be resolved in favor of the latter. Likewise, any conflict between an art-understood definition of a word or phrase and a definition of the word or phrase as specifically taught in this specification shall be resolved in favor of the latter.
[0311] As can be appreciated from the disclosure above, the present invention has a wide variety of applications. The invention is further illustrated by the following examples, which are only illustrative and are not intended to limit the definition and scope of the invention in any way. VI. EXAMPLES
Example 1: General Methods
(a) Reagents and Reactions
[0312] FlexMap carboxymethylated microspheres were obtained from Luminex Corporation, Austin, TX. Preferably, MicroPlex carboxymethylated microspheres 42 (5.6 μm in diameter, catalog number Ll 00-Cl 42-01, Luminex Corporation, Austin, TX) were used herein.
[0313] Carboxymethylated magnetic fluorescent beads were obtained from Spherotech, Inc., Lake Forest, IL. (b) Antibodies
[0314] High affinity murine anti-p24 monoclonal antibody, clone 4F6, was obtained from ImmunoDiagnostics Inc. Woburn, MA; catalog # 1103). 4F6 reacts with native and recombinant HIV-I p24 HIV-I IHB in ELISA and Western blots. 4F6 also detects HIV-I p24 precursor polypeptides. [0315] RDl -labeled monoclonal anti-p24 antibody KC57 was obtained from Beckman Coulter, Miami, FL (catalog #6604667). The KC57 antibody binds to the 55 kD, 39 IcD, 33 kD, and 24 kD proteins of the core antigen of the HIV-I . The 55 kD protein is the precursor protein for the core antigen (Robey et al, 1985, Science 228:593-595). The 39 kD and 33 kD proteins are intermediate products and the 24 kD protein is the core protein (Chgassagne et al, 1986, J Immunol 136:1442-1445; Schύpbach et al, 1984, Science 224:503-505). KC57- RDl comprises a phycoerythrin label attached at a molar ratio of RDl/protein 0.5-1.5. RDl (orange) excites at 486-580 nm and emits at 568-590 nm.
Example 2: Coupling Microspheres To High Affinity Anti-HIV-1 p24gag Monoclonal Antibody [0316] The capture FlexMap carboxymethylated microspheres or MicroPlex carboxymethylated microspheres (Luminex Corporation, Austin, TX) were coupled to a high affinity anti-p24 monoclonal antibody 4F6, (ImmunoDiagnostics Inc, Woburn, MA). This region can be substituted with any other FlexMap or Microplex microspheres region. The 4F6 antibody was covalently coupled on the surface of carboxylated Luminex beads via a two-step carbodiimide chemical reaction. Specifically, the coupling reaction was performed in a low protein-binding microcentrifuge tube (USA Scientific, Ocala, FL; cat# 1415-2500). All centrifugations were performed for 2 minutes at 8000 x g for 2 min. Pelleted microspheres (12.5 x 106) were resuspended in 160 μl of 0.1 M monobasic sodium phosphate pH 6.2, and were activated for 20 minutes by addition of 20 μL of a freshly prepared 50 mg/ml solution of sulfo-N-hydroxysuccinimide (Pierce, Rockford, IL) in dH2O, and 20 μL of 50 mg/ml solution of l-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) in water. The activated microspheres were washed twice in PBS, pH 7.4, and resuspended in 250μl of PBS, pH 7.4. 100 μl of 1 mg/ml solution of capture antibody (4F6) in PBS were added to the activated microspheres. The reaction volume was brought to a final volume of 1 ml with PBS, pH 7.4, and the reaction was allowed to proceed for 2 hours at RT, with gentle mixing on a thermomixer at 300 rpm. At the end of the coupling reaction, the microspheres were washed twice with 1 ml of PBS, 0.1% BSA, 0.02% Tween-20, 0.05% azide, pH 7.4 (PBS-TBN), and resuspended in a volume of 1 ml of PBS-TBN.
[0317] The microsphere concentration was determined by counting on an automatic hemacytometer (Cellometer AutoMio, Nexcelom Bioscience, Lawrence, MA). Once prepared, the microspheres are stable for extended periods of time, up to 3 years, if properly stored and can be used in the assay as described herein.
Example 3: Cytometric p24 Detection Assay With A Dynamic Range From Less Than 1 pg/ml To 20,000 pg/ml
[0318] In the assay described herein, a first antibody (also referred to herein as 'capture antibody') mediates binding of soluble p24 to the solid support (e.g., beads), while the second antibody ( also referred to herein as 'detection antibody') is used to detect and quantify the p24 antigen. AU assay components were diluted in an assay buffer made of PBS-TBN supplemented with 1% normal mouse serum and 1% goat serum (serum may be used in a range of concentrations from about 0.1% to about 1%). The assay was performed in a 96- well multiscreen filter plates (catalog # MSBVNl 210, 1.2 μm pore size, Millipore
Corporation, Billerica, MA) pre- wetted with 100 μl of PBS. The PBS was removed by vacuum suction using a vacuum manifold (catalog # MAVMO960R). 50 μl of microsphere suspension at various concentrations (as indicated herein), usually corresponding to 1 ,200 microspheres were added to each well of the pre-wetted plate. Biological samples and p24 standard (Alliance p24 Kit standard, Perkin Elmer, Waltham, MA) were lyzed by the addition of 10% volume of 10% Triton XlOO solution, and were diluted in assay buffer supplemented by 1% Triton XlOO. 50 μl of a serial dilution of samples or p24 standard were added to the microsphere suspension and incubated for one hour at 37°C to allow for binding of the p24 antigen to the 4F6 antibody. Following this incubation step, the plates were washed twice with PBS-TBN by using the vacuum manifold to remove any unbound antigen(s). 100 μl of the RDl -labeled monoclonal anti-p24 antibody KC57 (catalog #6604667, Beckman Coulter, Miami, FL) diluted in assay buffer to a concentration of 0.5 μg/ml were added to the beads and incubated for one hour under agitation at RT to allow binding of the KC57 antibody (second antibody) to the p24 antigen. Following incubation with the KC57 antibody, the plates were washed and the beads were resuspended in 150 μl of assay buffer.
[0319] The beads were analyzed on a Luminex 100 controlled by the Bioplex manager software 4.0 or 4.1.1 (Bio-Rad, Hercules, CA) and at least 100 beads per well were analyzed. Figure 1 illustrates the wide dynamic range and the high reproducibility of a typical assay run at 1,200 beads per well (50 μl of a 2.4 x 104 beads/ml solution). The inter assay variation is very low and gives a coefficient of variation (CVs) below 6.5% at all concentrations tested. In this example, the lower limit of p24 antigen detection, as illustrated in Fig. 1 was 0.43 pg/ml and the highest limit was 20,000 pg/ml. [0320] Without being bound by theory, the large dynamic range of the assay reflects the fact that the assay measures the fluorescence bound directly to each bead, as opposed to the measurement of a color resulting from an indirect and dynamic enzymatic reaction. The ability to measure individually the fluorescence bound to each microsphere renders the assay more accurate than assays that rely on a single measurement as the calculations obtained by the instant method are based, e.g., on the median of the data collected for 100 microspheres (or more), each behaving as individual replicates. Hence, the inter assay variability is also low. Four assays performed over a period of a few days each in triplicate had interassay CVs between 14.77 and 4.14% (Figure 2).
Example 4: Varying The Sensitivity Of The Assay [0321] The sensitivity of the assay described herein can be modified by varying the number of beads used to capture an antigen, e.g., p24. While a higher number of beads increases the upper limit of the assay range, decreasing the number of beads lowers the limit of detection by allowing the binding of more antigen per bead.
[0322] Assays with four different bead concentrations, 4,000, 2,000, 1 ,000 and 500 beads per well, each in triplicate, were performed. Statistical analysis (Prism 4, GraphPad software) of the four standard curves fitted by a logistic 4P regression revealed that all the fitted parameters were different for each standard curve (P<0.001) (Table 1) resulting in mid response (EC50) of the assay varying between 12,307 and 5,183 pg/ml for 4,000 and 500 beads per assay, respectively.
[0323] Table 1. Effect of bead concentration on assay performance
Figure imgf000080_0001
a: Parameters fitted on log transformed data according to the following equation: Fluorescence = Bottom+((Top-Bottom)/(l+10Λ(logEC50-Log[p24])-Hill Slope)) b: The limited of detection is the interpolated value obtained for a value of Fluorescence equals to the average background value plus 3 standard deviations)
[0324] The effect of bead concentration was more obvious at the lower limit of detection and at the mid response (EC50) of the assay, which respectively varied by more than a factor of two and nine between 4,000 and 500 beads per assay. The lowest limit of detection obtained by interpolating the average background value plus 3 SD showed that the least detectable concentrations were 0.101 pg/ml for 1,000 beads per well and 0.224 pg/ml for 4,000 beads per well.
[0325] Thus, by varying the quantity of beads used in the assay described herein, the dynamic range of the dose response can be shifted upwards or downwards. Compared to wide dynamic ranges (6-12,500 pg/ml) that have been reported for amplified ELISA monitored by a dedicated kinetic software (Giacomini et al, 1998, J Virol Methods 73:201- 9), the cytometric assay described herein offers a wider dynamic range while requiring smaller sample volume. [0326] Moreover, the assay described herein can easily be multiplexed with additional cytometric assays measuring other biological parameters. Specifically, the assay described herein can be combined with multiplexed detection of HIV antigens (Faucher et al, 2004, Clin Chem 50:1250-3). Similar assays were reported for 4th generation ELISA assays for the diagnostic of HIV infection, which combine the detection of both anti-HIV antibodies and HIV antigens (Ly et al, 2007, J Virol Methods 143:86-94; Yeom et al, 2006, J Virol Methods 137:292-7). The versatility and high sensitivity of the assay described herein makes it an attractive alternative to the more complex and expensive molecular assays in the monitoring of early HIV-I infection, e.g., in the diagnostic of perinatal HIV-I infection (Ficus et al., 2007, J Clin Microbiol 45:2274-7) and in resource poor settings (Fiscus et al., 2007, J Infect Dis 195:416-24; Respess et al, 2005, J Clin Microbiol 43:506-8; Tehe et al, 2006, J Clin Virol 37:199-205.) as well as in research laboratories.
Example 5: Detection of HIV-I Antigens In Seroconversion Panels
[0327] In addition to its usefulness in the research laboratory, the cytometric p24 assay described herein is able to detect HIV p24 in recently infected patients. The assay described herein has been tested on commercially available seroconversion panels and has been shown to detect the presence of HIV-I in these panels.
[0328] Specifically, seroconversion panels AD (PRB929), AZ (PRB950), and Q (PRB917) were obtained from BBI Diagnostics (West Bridgewater, MA, USA). BBI Diagnostics has assembled sets of undiluted plasma samples from serial bleeds from a single plasma donor collected during a period of HIV-I seroconversion for HIV-I antibody, p24 antigen, and HIV-I RNA. All units were maintained in a frozen state, except for the interval of dispensing into vials. No preservatives were added. These panels are provided for use by manufacturers and laboratories in evaluating their anti-HIV- 1, HIV-I antigen and HIV-I RNA assays with well-characterized specimens, and to provide comprehensive data for comparative analysis. Assay data provided by BBI Diagnostics for seroconversion panels analyzed are shown in Tables 2-
[0329] For the data shown in Tables 2-18, all HIV antigen and antibody numeric results are means of duplicates, expressed as specimen absorbance to cutoff ratios (s/co). Ratios >1.0 are considered reactive. Viral RNA was detected using Chiron Quantiplex® HIV RNA 2.0 Assay (bDNA), the Organon Teknika NucliSens™ HIV-I QT (nucleic acid sequence-based amplification; NASBA) Assay and the Roche Amplicor™ HIV-I Monitor™ Test (PCR). POS = positive; IND = indeterminate; NEG = negative; BLD = Below limit of detection; NA = not available; f = faint band. Western Blots are interpreted using ASTPHLD/CDC criteria (MMWR, vol. 38, S-7, 1989). Faintly staining bands are recorded, but interpreted as indeterminate according to specific manufacturer's instructions. All EIA and confirmatory results were generated using commercially available tests (as indicated), performed at BBI and at internationally recognized referee laboratories (RL) by individuals who routinely use these procedures.
[0330] To evaluate the diagnostic performance of the assays described herein, seroconversion panels AD, AZ, and Q, previously analyzed with commercially available detection kits (see Tables 3-7, 9-13, and 15-18) were analyzed using the Luminex assay as described herein. The results, summarized in Tables 2, 8, and 14 represent the results obtained with the assay described herein (sometimes referred to as 'Luminex assay'), using 4F6 as the first anti p24 antibody and fluorescently labeled KC57 antibody. The results represent the ratio of the average of two duplicates per sample divided by the cutoff. The assay cutoff defined as the average background value +3 standard deviations (giving therefore the reading of the lowest significant value above the background with a p value below 0.01). The background was estimated from 17 samples.
[0331] Table 2 shows the assay described herein performed on seroconversion panel AD. Results are given as S/Co.
Figure imgf000082_0001
a: Data obtained with the Luminex assay (as described herein) expressed as signal/cut-off (S/Co), where cut-off is defined as the mean background value +3 standard deviations; a S/Co above 1 (in bold) is considered positive.
[0332] Table 3 shows assay performance using assays detecting HIV-I antigens on seroconversion panel AD (expressed as S/Co) (same samples as in Table 2)
Figure imgf000083_0001
a: Data provided as S/Co by BBI/SeraCare using the Abbott Anti-HIVl antigen immunoassay; b: Coulter Assay
[0333] Table 4 shows assay performance detecting HIV-I RNA (copies/ml) on seroconversion panel AD (same samples as in Table 2)
Figure imgf000083_0002
Figure imgf000084_0001
a: Data provided as positive or negative values using the Roche HIV-I assay.
[0334] Table 5 shows U.S. FDA-Licensed Anti-HIV EIA Test Results expressed as S/Co seroconversion panel AD (same samples as in Table 2)
Figure imgf000084_0002
a: Data provided as S/Co by BBI/SeraCare using the Abbott anti-HIVl-2 enzymatic immunoassay; a S/Co above 1 (in bold) is considered positive. Other FDA approved assays are indicated.
[0335] Table 6 shows U.S. FDA-Licensed Anti-HIV-1 Confirmatory Tests on seroconversion panel AD (same samples as in Table 2)
Figure imgf000084_0003
Figure imgf000085_0001
a: Confirmatory Western blots from Ortho/Cambridge, data interpreted using ASTPHLD/CDC criteria. Faintly stained bands are indicated by "f" . Other FDA approved assays are indicated.
[0336] Table 7 shows assay performance using European test assays detecting HIV-I antigens on seroconversion panel AD (expressed as S/Co) (same samples as in Table 2)
Figure imgf000085_0002
a: Data provided as S/Co by BBI/SeraCare using the 3rd generation enzymatic immunoassay from Abbott 3rd Generation Plus. b: Data provided as S/Co by BBI/SeraCare using the 3rd Generation enzymatic immunoassay from Biotest HIV 1/2.
[0337] Table 8 shows the assay described herein performed on seroconversion panel AZ. Results are given as s/co.
Figure imgf000086_0001
a: Data obtained with the Luminex assay (as described herein) expressed as signal/cut-off (S/Co), where cut-off is defined as the mean background value +3 standard deviations; a S/Co above 1 (in bold) is considered positive.
[0338] Table 9 shows assay performance using assays detecting HIV-I antigens on seroconversion panel AZ (same samples as in Table 8)
Figure imgf000086_0002
a: Data provided as S/Co by BBI/SeraCare using the Abbott Anti-HIVl antigen immunoassay. Other FDA approved assays are indicated.
[0339] Table 10 shows assay performance detecting HIV-I RNA (copies/ml) on seroconversion panel AZ (same samples as in Table 8)
Figure imgf000087_0001
a: Data provided as number of copies per ml of serum using the Roche amplicor assay. Other FDA approved assays are indicated.
[0340] Table 11 shows U.S. FDA-Licensed Anti-HIV EIA Test Results expressed as S/Co seroconversion panel AZ (same samples as in Table 8)
Figure imgf000087_0002
α: Data provided as S/co by BBI/SeraCare using the Abbott anti-HIVl-2 enzymatic immunoassay; a S/Co above 1 (in bold) is considered positive. Other FDA approved assays are indicated.
[0341] Table 12 shows U.S. FDA-Licensed Anti-HIV-1 Confirmatory Tests on seroconversion panel AZ (same samples as in Table 8)
Figure imgf000087_0003
Figure imgf000088_0001
a: Confirmatory Western blots from Ortho/Cambridge, data interpreted using ASTPHLD/CDC criteria. Other FDA approved assays are indicated.
[0342] Table 13 shows assay performance using European test assays detecting HIV-I antigens on seroconversion panel AZ (expressed as S/Co) (same samples as in Table 8)
Figure imgf000088_0002
a: Data provided as S/Co by BBI/SeraCare using the 3 > id generation enzymatic immunoassay from Abbott 3id Generation Plus. b: Data provided as S/Co by BBI/SeraCare using the 31 Generation enzymatic immunoassay from Biotest HIV1/2.
[0343] Table 14 shows the assay described herein performed on seroconversion panel Q. Results are given as S/Co.
Figure imgf000088_0003
Figure imgf000089_0001
a: Data obtained with the Luminex assay (as described herein) expressed as signal/cut-off (S/Co), where cut-off is defined as the mean background value +3 standard deviations; a S/Co above 1 (in bold) is considered positive. NA, samples no longer available.
[0344] Table 15 shows U.S. FDA-Licensed Anti-HIV EIA Test Results expressed as S/Co seroconversion panel Q (same samples as in Table 14)
Figure imgf000089_0002
a: Data provided as S/Co by BBI/SeraCare using the Abbott anti-HIVl-2 enzymatic immunoassay; a S/Co above 1 (in bold) is considered positive. b: Data provided as S/Co by BBI/SeraCare using the Abbott Anti-HIV 1 antigen immunoassay. c: Data provided as positive or negative values using the Roche HIV-I assay. d: Confirmatory Western blots from Ortho/Cambridge, data interpreted using ASTPHLD/CDC criteria. Faintly stained bands are indicated by "f".
[0345] Table 16 shows data for Anti-HIV-1 Seroconversion panel Q (Modified), expressed as S/Co using additional USDA licensed HIV tests (same samples as in Table 14)
Figure imgf000090_0001
1, Abbott HIV-I BBI; 2, CBC HIV-I BBI; 3, CPI HIV-I BBI; 4, Gen Sys HIV-I BBI; 5, Gen Sys HIV- 1/2 BBI; 6, Org Tek HIV-I BBI; 7, Svva HIV-I BBI.
[0346] Table 17 shows assay performance using European test assays detecting HIV-I antigens on seroconversion panel Q (expressed as S/Co) (same samples as in Table 14)
Figure imgf000090_0002
Figure imgf000091_0001
a: Data provided as S/Co by BBI/SeraCare using Diagnostic Pasteur HIV 1/2 assay. b: Data provided as S/Co by BBI/SeraCare using Murex Wellcozyme HIV 1/2 assay.
[0347] Table 18 shows data for Anti-HIV-1 Seroconversion panel Q (Modified), expressed as S/Co using additional European licensed HIV tests (same samples as in Table 14)
Figure imgf000091_0002
1 , Behring Enzygnost HIV 1/2 BBI; 2, Biochem Detect HIV 1/2 BBI; 3, Innotest HIV 1/2 RLl 2. All data provided as S/Co.
[0348] As described above, S/Co ratios of >1.0 are considered reactive. A comparison of the data obtained using the assay of the present invention (presented in Tables 2, 8, and 14) with those obtained using commercially available detection kit demonstrates the superiority of the assay of the present invention. For example, data obtained on seroconversion panel AD using the assay of the invention demonstrated that while none of the U.S. FDA-licensed anti-HIV EIA tests identified HIV-I in the bleeds of days 14, 18, and 21 (see Table 5), the assay of the present invention identified the HIV-I p24 antigen in these samples (Table 2). Further, for bleeds of days 25 and 28, the assay of the present invention positively identified HIV-I p24 antigen in these samples, while only one out 5 commercially available test kits scored (compare Tables 2 and 5). In addition, while none of the U.S. FDA-licensed anti- HIV-I confirmatory tests scored positively in bleeds of days 14, 18, 21, and 25, the assay of the present invention identified HIV-I p24 antigen in these samples (compare Tables 2 and 6). Comparing the data shown in Tables 2 and 4, the assay of the present invention detects the HIV-I p24 antigen as early as day 14, when also HIV-I RNA is detected using a more laborious and more costly PCR test kit. [0349] With regard to seroconversion panel AZ, the assay of the present invention positively detected HIV-I p24 antigen in the samples of bleeds of days 18, 21, and 28 (Table 8). None of the U.S. FDA-licensed anti HIV EIA tests scored positively on the bleed samples day 18 and day 21 (Table 11). Further, three out of four U.S. FDA-licensed anti-HIV-1 confirmatory tests did not identify HIV-I in bleed samples day 18, day 21, and day 28 (Table 12). Only one of these confirmatory tests positively identified HIV-I in bleed sample day 28, but also failed to identify HIV-I in bleed samples taken earlier (Table 12). Again, the assay of the present invention was at least as sensitive as various PCR test kits (compare Tables 8 and 10).
[0350] With regard to seroconversion panel Q, the assay of the present invention positively detected HIV-I p24 antigen in the samples of bleeds of days 53 and 57, which were not detected in 8 out 9 test kits licensed by the U.S. FDA (compare Tables 14, 15 and 16) and not in 5 out of 5 test kits licensed in Europe (compare Tables 14, 17 and 18).
[0351] Thus, the assays described herein outperformed, in the three seroconversion panels (AD, AZ, and Q), all the assays approved by the United States Food and Drug Administration. These data suggest that, instead of nucleic acid detection, the cytometric assay described herein can be used for the diagnostics of HIV-I infection, especially during early times of infection, where HIV-I antigens are not detected at all by other assays. Further, assays of the present invention are particular useful in the diagnosis of perinatal HIV-I infection and in resource poor settings where its cost of about 15 cents per assay makes it an affordable alternative compared to current assays.
Example 6: Detection of HIV-I Antigens Using Fluorescent Magnetic Beads
[0352] This example describes an exemplary alternative embodiment of an assay of the present invention using fluorescent magnetic beads in conjunction with a traditional plate washer equipped with an appropriate magnet and a fluorescent plate reader. By employing fluorescent magnetic beads, the whole plate can be read in about ten minutes and the minimum detectable concentration can be as low as 33 pg/ml. [0353] Briefly, carboxymethylated magnetic yellow fluorescent beads (Spherotech, Inc., product number FCN-4052-2) were used. The fluorescent magnetic beads were coupled using two-step carbodiimide coupling with EDC and NHS to an anti-HIV-1 p24 antibody, e.g., ImmunoDiagnostics #1103, following the protocol described herein using the Luminex beads. Other fluorescent beads having a diameter of about 5 to 8 μm also find use. As a second antibody binding to HIV-I p24, phycoerythrine (PE)-labeled KC57-RD1 from Beckman Coulter was used. The buffers used were the same as those used in the Luminex assay, described herein.
[0354] Using magnetic beads allows the use of a standard microtiter plate instead of a filter plate. The magnetic beads allow for washing the assay in a magnetic washer, e.g. , a conventional plate washer which also controls the movements of a strong magnet which keeps the magnetic beads in the plate wells while the liquid is withdrawn and exchanged during the washing process. In this assay, a Biotek EL405 magna washer was used. Unlike the Luminex assay (where each bead is measured as an individual replicate), the beads are not detected individually. Instead the assay using fluorescent magnetic beads requires a sufficient number of beads in a well (e.g., at least about 60 beads) to obtain a reliable statistic. An assay-independent measurement of the number of beads was employed, i.e., a measurement of their own fluorescence which is proportional to their number. Thus, the beads were detected at two wavelengths: at a wavelength to quantify the number of beads (yellow fluorescence) and at a wavelength to quantify the antigen (via the PE-labeled second antibody). By normalizing the antigen detection and the bead fluorescence, reliable detection of the HIV-I antigen with low CVs was obtained. Fluorescence was read using a Tecan Safire II fluorescent plate reader.
[0355] In an exemplary assay using magnetic beads, serial dilution of goat anti -mouse antibodies coupled magnetic beads were incubated for one hour with the labeled monoclonal mouse antibody KC57-RD1 (Beckman Coulter) labeled with phycoerythrine. The assay was performed in a 96 well plate (100 μl per well, room temperature (RT), under agitation) using the same buffers as described herein for non-magnetic beads. The plate was washed with a magnetic washer (Biotek EL405 magna equipped with a Simbiotix MagnaSim96B magnet) and read with a fluorescence plate reader (Tecan Safire II). Excitation was measured at 532 nm and emission was measured at 585 nm. When using this method, a whole plate is read in about 10 minutes. This assay shows that the number of beads used to capture the analyte has a large influence on the outcome of the assay and establishes the linearity of the method of detection as well as validates the washing procedures. Figure 3 shows the linearity of the detection of exemplary 4.98 μm beads coupled to 4F6 monoclonal antibody revealed by phycoerithrine-labeled goat anti-mouse antibodies.
[0356] In another exemplary assay, 7,500 fluorescent yellow carboxyl magnetic beads (diameter 4.98 μm; (Sherotech, Inc.; catalog #FCM-4052-2) coupled to the capture antibody 4F6 (Cat# 1103 from Immunodiagnostics) were incubated with serial dilution of a p24 standard, washed and read on the fluorescence plate reader. The regression analysis of these data show that they are well modeled by a classic 4P logistic curve. A typical standard curve using HIV-I p24 polypeptide as an antigen is shown in Figure 4. In this experiment, the regression shows that the p24 polypeptide antigen can be detected at a dose of about 33 pg/ml. Magnetic beads having a diameter of 1 μm, 2.3 μm, and 7.6 μm were also used and gave similar results. The regression parameters as well as the goodness of fit are reported in Table 19:
Table 19 shows regression analysis of standard curve to a 4P logistic model
Figure imgf000094_0001
[0357] By not requiring the use of a Luminex reader, which is an expensive instrument, the assay using fluorescent magnetic beads is much less expensive. Furthermore, by using a standard fluorescent plate reader, the detection results can be read within about 10 minutes, which is faster than employing the Luminex assay. Example 7: Assay Platforms
[0358] Because of its multiplexing ability, a single Luminex experimental procedure can simultaneously measure, up to a hundred different analytes in 50 μl of sample. Today more than 55 assays related to the direct measurement of infectious agents or to pathogen-specific antibodies are commercially available (Rules Based Medicine, Inc. Austin, TX), and multiplex assays are used for the diagnosis of infectious agents (Brunstein and Thomas, 2006, Diagn MoI Pathol 15, 169-173; Khan et al., 2008, Clin Vaccine Immunol 15, 433-438).
[0359] If only p24 is to be measured, the cytometric assay described herein can be analyzed on a conventional flow cytometer. In this case, all the quality controls that were performed in the Luminex platform, would have to be implemented manually. When using acquisition and analysis software such as CellQuest® or Flow Jo®, in addition to the calibration of the cytometers, care should be taken to analyze only single events by choosing a gating strategy that eliminates the analysis of aggregated beads as they introduce errors in the data. This can be achieved by drawing a tight gate around bead singlets defined in bivariate plots representing the height versus width of both the forward and side scatter parameters and using the median fluorescence value of the reporter fluorochrome, such as phycoerythrine, to perform the regression analysis. In this instance, the assay can be acquired on a single LASER cytometer capable of exciting efficiently phycoerythrine.
[0360] Although the investment in a Luminex instrument might be perceived as a luxury for the measurement of a single analyte, the multiplexing capability of the technology described herein and the high throughput analysis makes it extremely attractive as it saves time and allows the detection of many parameters at once.
[0361] Irrespective of its potential use in a clinical setting, the assays described herein have a large dynamic range, a low cost of about $22 per plate, 60% of which is for the filter plate and can be saved by using magnetic beads. The assay has been used successfully by the inventors to detect HIV p24 antigen in cell culture supernatants, in patient serum, and in culture of human cervico vaginal explants, which require a highly sensitive assay due to the low level of viral replication in this tissue. As it is the case with other filter plate based Luminex assays that detect, in cell lysates, phosphorylated intracellular signaling proteins (Zhou et al. , 2007, J Virol 81 , 7749-7758), the p24 cytometric assay described herein can be adapted to detect intracellular p24 antigen in infected cells or tissues. Because of these traits and because it can be multiplexed with other measurements, therefore saving precious samples, the p24 cytometric assay offers a unique combination of cost, time and sample savings that were missing in the research community and in diagnostics.
Informal Sequence Listing
SEQ ID NO:1 : GAG polypeptide sequence (Genbank Accession No. FOVWLV)
1 MGARASVLSG GELDRWEKIR LRPGGKKKYK LKHIVWASRE LERFAVNPGL LETSEGCRQI
61 LGQLQPSLQT GSEELRSLYN TVATLYCVHQ RIEIKDTKEA LDKIEEEQNK SKKKAQQAAA 121 DTGHSSQVSQ NYPIVQNIQG QMVHQAISPR TLNAWVKVVE EKAFSPEVIP MFSALSEGAT
181 PQDLNTMLNT VGGHQAAMQM LKETINEEAA EWDRVHPVHA GPIAPGQMRE PRGSDIAGTT
241 STLQEQIGWM TNNPPIPVGE IYKRWIILGL NKIVRMYSPT SILDIRQGPK EPFRDYVDRF
301 YKTLRAEQAS QEVKNWMTET LLVQNANPDC KTILKALGPA ATLEEMMTAC QGVGGPGHKA
361 RVLAEAMSQV TNSATIMMQR GNFRNQRKIV KCFNCGKEGH IARNCRAPRK KGCWKCGKEG 421 HQMKDCTERQ ANFLGKIWPS YKGRPGNFLQ SRPEPTAPPE ESFRSGVETT TPSQKQEPID
481 KELYPLTSLR SLFGNDPSSQ
SEQ ID NO:2: GAG polypeptide sequence (Genbank Accession No. ACD80633)
1 MGARASILRG GKLDKWEKIK LRPGGKKPYM LKHLVWASRE LERFALNPGL LETSEGCKQI
61 MKQLHPAIQT GTEELKSLYN TVATLYCVHE GIEVRDTKEA LDKIEEEQNK SQQKAQQAKA 121 ADGKVSQNYP IVQNIQGQMV HQAISPRTLN AWVKVIEEKA FSPEVIPMFT ALSEGATPQD
181 LNTMLNTVGG HQAAMQMLKD TINEEAAEWD RLHPVHAGPI APGQLREPRG SDIAGTTSTL
241 QEQIAWMTNN PPVPVGDIYK RWIILGLNKI VRMYSPVSIL DIKQGPKEPF RDYVDRFFKC
301 LRAEQATQDV KNWMTDTLLV QNANPDCKTI LRALGPAASL EEMMTACQGV GGPSH
SEQ ID NO:3: p24 polypeptide sequence 1 PIVQNIQGQM VHQAISPRTL NAWVKVVEEK AFSPEVIPMF SALSEGATPQ DLNTMLNTVG
61 GHQAAMQMLK ETINEEAAEW DRVHPVHAGP IAPGQMREPR GSDIAGTTST LQEQIGWMTN
121 NPPIPVGEIY KRWIILGLNK IVRMYSPTSI LDIRQGPKEP FRDYVDRFYK TLRAEQASQE
181 VKNWMTETLL VQNANPDCKT ILKALGPAAT LEEMMTACQG VGGPGHKARV L
SEQ ID NO:4: p24 epitope sequence PIVQNIQGQMVHQAISPRTL
SEQ ID NO:5: p24 epitope sequence
VHQAISPRTLNAWVK
SEQ ID NO:6: p24 epitope sequence
AISPRTLNAW SEQ ID NO:7: p24 epitope sequence
NAWVK
SEQ ID NO:8: p24 epitope sequence
SEGATPQDLNTMLNTVG SEQ ID NO:9: p24 epitope sequence
EGATPQ
SEQ ID NO: 10: p24 epitope sequence
GATPQDLNTML SEQ ID NO: 11 : p24 epitope sequence
ATPQDLNTML
SEQ ID NO: 12: p24 epitope sequence
DLNTMLNTVG
SEQ ID NO: 13: p24 epitope sequence DLNTMLNTVGGHQAAMQMLKET INEEAAEWDR
SEQ ID NO: 14: p24 epitope sequence
AAMQMLKETINE
SEQ ID NO: 15: p24 epitope sequence
LKETINEEAAEWDRVHPV SEQ ID NO: 16: p24 epitope sequence
ETINEEAAEWD
SEQ ID NO: 17: p24 epitope sequence
ETINEEAAEWDRVHP
SEQ ID NO: 18: p24 epitope sequence EAAEWDRVHP
SEQ ID NO: 19: p24 epitope sequence
EAAEWDRVHPVHAGP
SEQ ID NO:20: p24 epitope sequence
DRVHPVHAGP SEQ ID NO:21 : p24 epitope sequence
RVHPVHAGPIAPGQMREPRGS
SEQ ID NO:22: p24 epitope sequence
HPVHAGPIAPG SEQ ID NO:23 : p24 epitope sequence
HAGPIAPGQMREPRG
SEQ ID NO:24: p24 epitope sequence
GSDIAGTTSTLQEQIGWMTNN
SEQ ID NO:25: p24 epitope sequence NPPIPVGEIY
SEQ ID NO:26: p24 epitope sequence
NPPIPVGEIYKRWII
SEQ ID NO:27: p24 epitope sequence
NPPIPVGEIYKRWIIGLNKIVRMYSPTSILD SEQ ID NO:28 : p24 epitope sequence
IVRMYS PTS ILDIRQGPKEPFRDYVDRFYK
SEQ ID NO:29: p24 epitope sequence
RMYSPT
SEQ ID NO:30: p24 epitope sequence SILDIR
SEQ ID NO:31 : p24 epitope sequence
DIRQGP
SEQ ID NO:32: p24 epitope sequence
QGVGGP
91 SEQ ID NO:33: p24 epitope sequence
DIRQGPKEPFRDYVDRFYKTL
SEQ ID NO:34: p24 epitope sequence
IRQGPKEPFRDYVDRFYKTL SEQ ID NO:35: p24 epitope sequence
IRQGPKEPFRDYVDRFYKTLRAE
SEQ ID NO:36: p24 epitope sequence
IRQGPKEPFRDYVDRFYKTLRAEQAS
SEQ ID NO:37: p24 epitope sequence QGPKEPFRDYVDRFY
SEQ ID NO:38: p24 epitope sequence
QGPKEPFRDYVDRFYKTLRAEQA
SEQ ID NO:39: p24 epitope sequence
GPKEPF SEQ ID NO:40: p24 epitope sequence
FRDYVDRFYK
SEQ ID NO:41 : p24 epitope sequence
KTLRAEQASQEVKNWMTET
SEQ ID NO:42: p24 epitope sequence TLRAEQASQEVKNWM
SEQ ID NO:43: p24 epitope sequence
QASQEVKNWMTETLL
SEQ ID NO:44: p24 epitope sequence
VKNWMTETLL SEQ ID NO: 45: p24 epitope sequence
KTILKALGPAATLEEMMTACQGVG
SEQ ID NO:46: p24 epitope sequence
ILKALGPAATLEEMM SEQ ID NO:47: p24 epitope sequence
LGPAATLEEMM
SEQ ID NO:48: p24 epitope sequence
LEEMMTACQGVGGPGHKARV
SEQ ID NO:49: p24 epitope sequence GHKARV
SEQ ID NO:50: ρ24 epitope sequence
HKARVL

Claims

WHAT IS CLAIMED IS:
L A method for detecting an antigen in a biological sample using an immunoassay where the immunoassay has a dynamic range of between about 0.2 and about 20,000 pg/ml and a coefficient of variation of less than 10%, the method comprising the steps of: (a) binding a first antibody specifically binding an antigen to a solid support; (b) contacting the first antibody with a biological sample suspected of containing the antigen for a time and under conditions sufficient to allow binding of the antigen to the first antibody, thereby forming a first antibody/ antigen complex; (c) contacting the first antibody/antigen complex with a second antibody specifically binding the antigen for a time and under conditions sufficient to allow binding of the second antibody to the antigen of the first antibody/antigen complex, thereby forming a first antibody/antigen/second antibody complex, wherein the second antibody produces a detectable signal; and (d) detecting the detectable signal; wherein the first antibody and the second antibody bind to different epitopes of the antigen; and thereby detecting the antigen in the biological sample.
2. The method according to claim 1, wherein the antigen is a HIV-I antigen.
3. The method according to claim 2, wherein the HIV-I antigen is a GAG polypeptide.
4. The method according to claim 3, wherein the GAG polypeptide is a p24 polypeptide.
5. The method according to claim 4, wherein the p24 polypeptide has an amino acid sequence which is at least 90% identical to the amino acid sequence of SEQ ID NO:1.
6. The method according to claim 5, wherein the p24 polypeptide has the amino acid sequence of SEQ ID NO: 1.
7. The method according to claim 1 , wherein the first antibody is a monoclonal antibody or a fragment thereof.
8. The method according to claim 4, wherein the first antibody binds to an epitope of the p24 polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO: 12, SEQ ID NO:13, SEQ ID NO: 14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41 , SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, and SEQ ID NO:50.
9. The method according to claim 4, wherein the first antibody is selected from the group consisting of 4F6, KC57, 111/182, 112/021, 112/047, ID8F6, F5-2, CB- 13/5, 3D3, CD-4/1, 15F8C7, 111/052, 91-5, 47-2, 714/01, 1109/01, IG5C8, 14D4E11, 113/038, 111/073, l-E-4, l-E-9, 2-E-4, 2-H-4, 8-D-2, 8-H-7, 8-G-9, 10-E-7, 10-G-9, 1 l-C-5, C5123, l-B-7, 3-B-7, 6-D-12, 6-E-7, 8-D-5, FFl, 113/072, 25.3, 13-102-100, RL4.72.1, 406/01, 38:9.6k, EBl A9, EF7, 30:3E5, LH-104-E, 1B2C12, LH-104-K, LH-104-A, 1A7, 1.17.3, 1F6, 23A5G5, 23A5G4, 3D10G6, F5-4, MO9.42.2, MO9.50.2, V-10, V107, LH-104-C, 12-B-4, 9A4C4, 11D11F2, 1 IClOBlO, CD12B4, BE3, L14, 110/015, 108/03, 32:32K, C5200, FH2, 13B5, 106/01, LH-104-B, LH-104-B, and fragments thereof.
10. The method according to claim 9, wherein the first antibody is 4F6.
11. The method according to claim 1 , wherein the second antibody is a monoclonal antibody or a fragment thereof.
12. The method according to claim 4, wherein the second antibody binds to an epitope of the p24 polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11 , SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO: 14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, and SEQ ID NO:50 and wherein the second antibody binds to an epitope non-overlapping with the epitope to which the first antibody binds.
13. The method according to claim 4, wherein the second antibody is selected from the group consisting of 4F6, KC57, 111/182, 112/021, 112/047, ID8F6, F5-2, CB-13/5, 3D3. CD-4/1, 15F8C7, 111/052, 91-5, 47-2, 714/01, 1109/01, IG5C8, 14D4E11, 113/038, 111/073, l-E-4, l-E-9, 2-E-4, 2-H-4, 8-D-2, 8-H-7, 8-G-9, 10-E-7, 10-G-9, 1 l-C-5, C5123, l-B-7, 3-B-7, 6-D-12, 6-E-7, 8-D-5, FFl, 113/072, 25.3, 13-102-100, RL4.72.1, 406/01, 38:9.6k, EB1A9, EF7, 30:3E5, LH-104-E, 1B2C12, LH-104-K, LH-104-A, 1A7, 1.17.3, 1F6, 23A5G5, 23A5G4, 3D10G6, F5-4, MO9.42.2, MO9.50.2, V-10, V107, LH-104- C, 12-B-4, 9A4C4, 11D11F2, 1 IClOBlO, CD12B4, BE3, L14, 110/015, 108/03, 32:32K, C5200, FH2, 13B5, 106/01, LH-104-B, LH-104-B, and fragments thereof.
14. The method according to claim 13, wherein the second antibody is KC57.
15. The method according to claim 4, wherein the first antibody is 4F6 and the second antibody is KC57.
16. The method according to claim 1 , wherein the second antibody is labeled with a fluorochrome.
17. The method according to claim 16, wherein the fluorochrome is R- phycoerythrin.
18. The method according to claim 16, wherein step (d) comprises measuring a fluorescence signal produced by the second antibody.
19. The method according to claim 18, wherein the solid support comprises a plurality of solid support and step (d) comprises measuring a fluorescence signal for each individual member of the plurality of solid support having bound the first antibody/antigen/second antibody complex.
20. The method according to claim 1 , wherein the solid support is selected from the group consisting of a microsphere, a bead, a particle, a fiber, a monolith, a membrane, a plastic strip, and a microtiter plate.
21. The method according to claim 20, wherein the solid support is a bead or a microsphere.
22. The method according to claim 21, wherein the solid support is carboxymethylated.
23. The method according to claim 21, wherein the solid support is fluorescent.
24. The method according to claim 20, wherein the solid support is magnetic or ferromagnetic.
25. The method according to claim 24, wherein the solid support is fluorescent.
26. The method according to claim 1 , wherein the biological sample is selected from the group consisting of serum, blood, lymph, and plasma.
27. The method according to claim 1, wherein the biological sample is obtained from a human.
28. The method according to claim 27, wherein the human is a patient having or suspected of having AIDS.
29. The method according to claim 28, wherein the patient is treated with an anti-viral drug.
30. The method according to claim 1 , wherein the biological sample is between about 20 μl and about 50 μl in volume.
31. The method according to claim 1 , comprising using a multiplex assay format and assaying for a second antigen present in said biological sample.
32. The method according to claim 28, further comprising the step of: (e) assaying for the presence of a HIV-I antibody in the biological sample.
33. The method according to claim 4, further comprising the step of: (e) assaying for the presence of a second antigen in the biological sample wherein the second antigen is different from the p24 polypeptide.
34. The method according to claim 1, wherein step (a) comprises binding of the first antibody to an activated solid support.
35. The method according to claim 1 , further comprising a washing step after step (a).
36. The method according to claim 1, further comprising the step of: (e) determining the amount of the antigen.
37. The method according to claim 36, wherein the plurality of solid support comprises at least 10 members and step (e) comprises measuring the signal from each of the at least 10 members.
38. The method according to claim 1, further comprising the step of: (e) determining the amount of the solid support having bound the first antibody.
39. The method according to claim 1, further comprising the step of: (e) adding the solid support having bound the first antibody to a multi-well container.
40. The method according to claim 1 , further comprising the step of: (e) lysing the biological sample.
41. The method according to claim 1, wherein step (d) comprises resuspending the solid support having bound the first antibody/antigen/second antibody complex in an assay buffer.
42. The method according to claim 1, which is adapted to a high throughput method.
43. An immunosystem for detecting an antigen in a biological sample where the immunosystem has a dynamic range of between about 0.2 pg/ml and about 20 ng/ml and a coefficient of variation of less than 10%, the immunosystem comprising: (i) a first antibody reactive to an antigen, wherein the first antibody is bound to a solid support; (ii) a biological sample containing or suspected of containing the antigen; and (iii) a second antibody reactive to the antigen, wherein the second antibody binds to a different epitope of the antigen than the first antibody.
44. The immunosystem according to claim 43, wherein the second antibody comprises a label.
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