WO1998018006A1 - Dispositifs et procedes a base d'un complexe constitue par de l'albumine serique et un antigene cible - Google Patents

Dispositifs et procedes a base d'un complexe constitue par de l'albumine serique et un antigene cible Download PDF

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Publication number
WO1998018006A1
WO1998018006A1 PCT/US1997/018194 US9718194W WO9818006A1 WO 1998018006 A1 WO1998018006 A1 WO 1998018006A1 US 9718194 W US9718194 W US 9718194W WO 9818006 A1 WO9818006 A1 WO 9818006A1
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Prior art keywords
serum albumin
assay
target antigen
sample
complex
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PCT/US1997/018194
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English (en)
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Jaw-Ching Liao
Cheng-Nan Wang
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Genenova Corporation
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Priority to CA002269087A priority Critical patent/CA2269087A1/fr
Priority to JP10519417A priority patent/JP2001502803A/ja
Priority to AU48964/97A priority patent/AU4896497A/en
Priority to EP97911646A priority patent/EP0939901A1/fr
Publication of WO1998018006A1 publication Critical patent/WO1998018006A1/fr

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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
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • 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
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • 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
    • G01N33/54306—Solid-phase reaction mechanisms
    • 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
    • G01N33/54353—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals with ligand attached to the carrier via a chemical coupling agent

Definitions

  • the present invention relates generally to antibodies against a serum albumin, and to methods and compositions for the highly specific, highly sensitive detection of a target antigen that can associate with a serum albumin or antibodies against such a target antigen.
  • the methods and compositions are also suitable for the elicitation of an immune response in an animal, and for the vaccination of an animal, against the target antigen.
  • Serum albumin is a small, relatively symmetrical protein with a calculated molecular weight between about 65,000 and 70,000. Serum albumin functions as a principal protein in plasma, and it has a relatively low intrinsic viscosity and a strong internal structure held together by disulfide bridges. Tullis, J., J.A.M.A. 237(4):355-360, 1977; Tullis, J., J M A. 257(5):460-463, 1977.
  • serum albumin In its native state, serum albumin is a highly soluble molecule carrying a strong net negative charge, and an isoelectric point between about pH 5.4 and 4.4. Despite the high net negative charge of serum albumin, it can bind reversibly to both cations and anions, making it suitable for the transport or inactivation of substances such as trace metals, drugs, dyes, fatty acids, hormones, and enzymes. Id. Principally, albumin has been used in clinical applications for its oncotic action as a plasma volume expander. Id. Serum albumin has been believed to lack either enzymatic or immunological functions. Yeh, P. et al., Proc. Natl. Acad. Sci. USA 89: 1904-1908, 1992.
  • infectious diseases require effective diagnostic assays, adjuvants that enhance the effect of vaccines, and vaccines.
  • infectious diseases include acquired immune deficiency syndrome (AIDS) which is caused by a group of retroviruses known as HIV (Barre-Sinoussi et al., Science 22(9:868-871, 1983; Gallo et al., Science 224:500-503, 1984; Coffin et al., Science 232:691, 1986) and the hepatitis C virus (HCV) (Choo et al., Science 244:359-362, 1989).
  • HIV acquired immune deficiency syndrome
  • HCV hepatitis C virus
  • HIV-1 is typically transmitted by sexual contact, by exposure to blood or certain blood products, or by an infected mother to her fetus or child (Piot et al., Science 25 :573-579, 1988). Examples of transfusion-associated HIV-2 infection have also been disclosed (Courouce et al., AIDS 2:261-265, 1988). Similarly, HCV is often transferred via blood transfusion; post-transfusion hepatitis (PTH) occurs in approximately 10% of transfusion patients, and HCV ( . e. , Non-A, Non-B hepatitis (NANBH)) accounts for up to 90% of these cases.
  • PTH post-transfusion hepatitis
  • NANBH Non-A, Non-B hepatitis
  • EIAs have undesirable, non-specific reaction with specimens from individuals with non-HIV conditions such as autoimmune diseases, a history of multiple pregnancies, anti-HLA, EBV infections or hypergammaglobulinemia.
  • an ELISA has been developed and commercialized by Abbott Laboratories for serological diagnosis of HIV infection using the HIV-1 core and HIV-1 envelope and HIV-2 envelope proteins.
  • these assays have not provided the highly specific, highly sensitive detection needed for superior protection of the blood supply, or for early diagnosis of HIV in a patient.
  • HCV hepatitis C virus
  • ELISA enzyme-linked immunosorbent assay
  • alum aluminum hydroxide and aluminum phosphate
  • Alum has a history of successful use with low toxicity (AIDS Research and Human Retroviruses 11, 1995), but the need for other adjuvants having one or more superior properties has gone unmet.
  • the provision of alternative adjuvants would provides users with a variety of strategies, and the ability to select and choose between superior qualities based upon particular needs. See Vaccines and World Health 227, Oxford, 1994; Mosby, Immunology, 4th ed., p. 19.8-19.9; Mosby, Clinical Immunology, Principles and Practice, Chapter 123, p. 1911-1912.
  • the present invention is directed to the use of serum albumin, including human serum albumin, as an active component of the immune response.
  • the serum albumin aggregates with antigens to provide a complex comprised of invading antigen(s) and serum albumin(s). Pursuant to such complexing, the invading antigen is believed to undergo conformational changes that affect the antigenicity of the antigen.
  • This complexed antigen is then recognized by the immune system, which produces antibodies (such as IgG and IgM antibodies) that have a high specificity and affinity for the complexed antigen, although such antibodies typically do also bind the uncomplexed antigen to a lower specificity and affinity.
  • the immune system produces antibodies against the serum albumin itself.
  • the discovery of this advantage found with a complexed antigen permits the design of assays, methods and compositions that are directed toward the detection of the complexed antigen and the use of the complexed antigen, and the detection and use of antibodies to such a complexed antigen. Such discovery also permits the design of compositions and methods for the induction of an immune response in an animal, including an immunoprotective immune response.
  • the present invention also provides for the provision of anti-serum albumin antibodies, as well as the detection of a diseased state by the detection of anti-serum albumin autoantibodies in a sample.
  • anti-serum albumin antibodies or autoantibodies may interfere with the interactions between the serum albumin and the invading infectious agent, thereby depriving the infectious agent of an interim "host," which might otherwise assist the infectious agent in its invasion of the host animal.
  • the antibodies and autoantibodies discussed throughout this disclosure include all forms of antibodies, and in preferred embodiments comprise IgG and/or IgM antibodies.
  • the present invention provides an assay for the detection of an antibody against a target antigen in a sample, comprising: (a) providing a target antigen complexed with a serum albumin to provide a target antigen- serum albumin complex; (b) contacting the target antigen-serum albumin complex with the sample under conditions suitable and for a time sufficient for the target antigen- serum albumin complex to bind to one or more IgM and/or other forms of antibodies for the target antigen present in the sample, to provide an IgM and/or other forms of antibody-bound complex; and, (c) detecting the IgM antibody-bound complex, and therefrom determining whether the sample contains the antibody for the target antigen.
  • the complex can further comprise a second target antigen complexed with the target antigen-serum albumin complex, to provide a multiple target antigen-serum albumin complex.
  • the assay further comprises the step of binding the target antigen-serum albumin complex to a solid substrate. Such binding to the solid substrate can be performed either before or after the step of contacting the target antigen-serum albumin complex with the sample.
  • the serum albumin is selected from the group consisting of human serum albumin, bovine serum albumin, fetal bovine serum albumin, new born bovine serum albumin and mouse serum albumin.
  • the sample can be either a purified or an unpurified sample, and the methods can further comprise, prior to the step of contacting, isolating the sample from an animal.
  • the sample can be isolated from a human being, and the sample can be a blood sample.
  • the serum albumin is selected from the same species as the sample, although the serum albumin can be from a different species, particularly for assay purposes.
  • the assays of the present invention can be selected from the group consisting of a countercurrent immuno-electrophoresis (CIEP) assay, a radioimmunoassay, a western blot assay, a radioimmunoprecipitation, an enzyme-linked immuno-sorbent assay (ELISA), a dot blot assay, an inhibition or competition assay, a sandwich assay, an immunostick (dip-stick) assay, a simultaneous assay, an immunochromatographic assay, an immunofiltration assay, a latex bead agglutination assay, an immunofluorescent assay, a biosensor assay, and a low-light detection assay.
  • CIEP countercurrent immuno-electrophoresis
  • a radioimmunoassay a western blot assay, a radioimmunoprecipitation
  • ELISA enzyme-linked immuno-sorbent assay
  • a dot blot assay an inhibition or competition assay
  • the present invention provides a method of producing an antibody, such as an IgM or IgG antibody, specific for a target antigen, comprising the following steps: (a) administering to an animal the target antigen complexed with a serum albumin to provide a target antigen-serum albumin complex under conditions suitable and for a time sufficient to induce the production in the animal of antibodies to the target antigen; and, (b) isolating the antibodies to the target antigen.
  • the present invention also provides antibodies, such as IgM or IgG antibodies, produced according to such methods, as well as other antibody production methods disclosed herein.
  • the present invention provides an antibody specific for a target antigen, the antibody made against a target antigen-serum albumin complex comprising the target antigen complexed with a serum albumin.
  • the antibody is bound to a solid substrate.
  • the present invention provides an assay for the detection of a target antigen in a sample, comprising: (a) contacting the sample with an antibody, such as an IgM or IgG antibody, produced according to the methods discussed above under conditions suitable and for a time sufficient for the antibody to bind the target antigen, to provide a bound antibody; and, (b) detecting the bound antibody , and therefrom determining whether the sample contains the target antigen.
  • an antibody such as an IgM or IgG antibody
  • the present invention provides an assay for the detection of an antibody specific for a serum albumin in a sample, comprising: (a) contacting the serum albumin with the sample under conditions suitable and for a time sufficient for the serum albumin to bind to one or more antibodies, such as IgM or IgG antibodies, specific for the serum albumin present in the sample, to provide an antibody-bound serum albumin; and, (b) detecting the antibody-bound serum albumin, and therefrom determining that the sample contains the antibody specific for the serum albumin.
  • antibodies such as IgM or IgG antibodies
  • the present invention provides a method of producing an antibody, such as an IgM or IgG antibody, against a serum albumin, comprising the following steps: (a) administering to an animal a target antigen-serum albumin complex, comprising the target antigen complexed with a serum albumin, under conditions suitable and for a time sufficient to induce the production in the animal of antibodies specific to the serum albumin; and, (b) isolating the antibodies to the serum albumin.
  • the method can also include the step of isolating the antibody from the animal.
  • the present invention provides an isolated antibody that is specific for a serum albumin, preferably human serum albumin. Such antibody can be bound to a solid substrate.
  • the present invention provides a method for the detection of a serum albumin in a sample, comprising: (a) contacting the sample with an antibody, such as an IgM or IgG antibody, against serum albumin produced as described above under conditions suitable and for a time sufficient for the antibodv to bind the serum albumin, to provide a bound antibody; and, (b) detecting the bound antibody, and therefrom determining whether the sample contains the serum albumin.
  • an antibody such as an IgM or IgG antibody
  • the present invention provides a method for the isolation of a serum albumin from a sample, comprising: (a) contacting the sample with an antibody, such as an IgM or IgG antibody, against serum albumin produced as described above under conditions suitable and for a time sufficient for the antibody to bind the serum albumin, to provide bound serum albumin; and, (b) isolating the bound serum albumin from the sample.
  • an antibody such as an IgM or IgG antibody
  • the present invention also provides a method of producing an assay to detect an antibody, such as an IgM or IgG antibody, against a target antigen comprising contacting the target antigen with a serum albumin under conditions suitable and for a time sufficient for the target antigen to complex with the serum albumin to form a complex, and then binding the complex to a solid substrate.
  • the target antigen is contacted with the serum albumin in a TNSCN buffer.
  • the present invention provides a composition capable of binding to an antibody, such as an IgM or IgG antibody, against a target antigen, the composition comprising an isolated target antigen complexed with a serum albumin to provide an isolated target antigen-serum albumin complex.
  • the isolated target antigen-serum albumin complex is bound to a solid substrate.
  • the present invention provides a composition capable of binding to a target antigen, the composition comprising an antibody, such as an IgM or IgG antibody, produced against a target antigen-serum albumin complex comprised of the target antigen complexed with a serum albumin, the antibody bound to a solid substrate.
  • the present invention provides a composition capable of eliciting an immune response in an animal comprising a target antigen complexed with a serum albumin to provide a target antigen-serum albumin complex, in combination with a pharmaceutically acceptable carrier or diluent.
  • a target antigen is complexed with the target antigen-serum albumin complex, to provide a multiple target antigen-serum albumin complex.
  • the present invention provides a method of enhancing the immune response to a target antigen associated with a pathogenic state, comprising the following steps: (a) complexing the target antigen with a serum albumin to provide a target antigen-serum albumin complex, whereby the serum albumin is an adjuvant; (b) combining the complex with a pharmaceutically acceptable carrier or diluent; and, (c) administering the complex with the pharmaceutically acceptable carrier or diluent to an animal, thereby providing an enhanced immune response to the target antigen.
  • the present invention provides a composition suitable for the treatment of a disease in an animal comprising a selected drug complexed with a serum albumin to provide a drug-serum albumin complex, in combination with a pharmaceutically acceptable carrier or diluent.
  • the present invention provides a kit for the detection of a target antigen, the kit comprising: (a) the target antigen complexed with a serum albumin to provide a target antigen-serum albumin complex; and, (b) one or both of a reagent or a device for detecting the complex or an antibody, such as an IgM or IgG antibody, bound to the complex.
  • the kit for the detection of a target antigen : (a) an antibody (such as an IgM or IgG antibody), produced as discussed herein; and, (b) one or both of a reagent or a device for detecting the antibody.
  • the present invention is based on the discovery that serum albumin in the body, including human serum albumin, is an important component of the irnmune response. It appears that the serum albumin in the body interacts with antigens associated with an infectious agent, such as the core protein of Hepatitis C Virus, to provide a complex comprised of invading antigen(s) and serum albumin(s). Pursuant to such interaction, the invading antigen is believed to undergo conformational changes that affect the antigenicity of the antigen. This conformationally changed antigen is then recognized by the immune system, which produces antibodies that have a high specificity and affinity for the changed antigen, although such antibodies typically do also bind the conformationally unchanged antigen to a lower specificity and affinity. Additionally, when the target antigen is introduced in to an animal, the immune system produces antibodies against the serum albumin itself. Such antibodies may include all forms of antibodies, and in particular, IgM and/or IgG antibodies.
  • the present invention provides improved methods and compositions for the detection and diagnosis of disease wherein the assays utilize conformationally changed target antigens associated with a pathogenic state such as an infectious disease.
  • the present invention also provides antibodies specific for serum albumin and antibodies specific for the conformationally changed target antigens, as well as methods for purifying each using immunologic methods such as affinity columns.
  • the present invention features compositions and methods utilizing a serum albumin aggregated with a target antigen to provide a target antigen-serum albumin complex.
  • the complex provides a synergistic effect when compared to the effect of target antigen alone, and will enhance the reaction of the target antigen with its corresponding antibodies at least about 1.1, and preferably at least about 1.25, 1.5 or 2.0 times more strongly when compared to the reaction of the antibodies with the target antigen alone, when the OD value is measured at 492 nm in a spectrophotometer.
  • the present invention also provides a preferred buffer, TNSCN, comprised of Tris-HCl, NaCl, and NaSCN, typically at a pH of about 7.4 ⁇ 0.2.
  • TNSCN a preferred buffer
  • the buffer facilitates the complexing between the serum albumin and a target antigen, and is useful in assays utilizing such complexing.
  • the buffer is also useful other traditional immuno-based assays, such as ELISAs and RIAs, even if no complexing between targets is involved (for example because there is no molecule with which to complex).
  • the present invention also provides antibodies against serum albumin, methods of purifying serum albumin using such antibodies, and serum albumin so purified.
  • Target antigen is used herein to indicate a selected protein that is capable of inducing an immune response in an animal and is capable of being bound (typically at a particular epitope) by an antibody generated by the animal.
  • the target antigen can be either recombinant or purified from a native source. Further, the target antigen is capable of forming a complex with the serum albumin, which complex, as discussed above, enhances the antigenicity of the target antigen.
  • Determination of whether the target antigen can form such a complex with the serum albumin can be determined by mixing the target antigen with a serum albumin in a coating buffer such as TNSCN, TNEU, carbonate buffer, TNSDS or PBS, and then determining whether a dimer, trimer, multimer, or other oligomer has been formed comprising one or more target antigens and one or more serum albumins.
  • the target antigen is a protein associated with a pathogenic state, such as a cancer or an infectious or other disease.
  • the disease is a blood-borne infectious disease, so that antigens associated with the disease are presented while in the blood stream during normal infection, and therefore have the opportunity to interact with serum albumins during such infection.
  • the infectious disease can be bacterial or viral, and in one preferred embodiment is a positive-stranded RNA virus such as Togaviridae, Coronaviridae, Retroviridae, Picornaviridae, Caliciviridae and Flaviviridae.
  • positive-stranded RNA viruses are discussed in U.S. Application Serial No. 08/447,276 and U.S. Application Serial No. 08/454,928.
  • suitable target antigens include a core antigen of HCV, a core-envelope fusion protein from HCV (such as EN-80-2 (ATCC 55451), described in U.S. Application Serial No.
  • HBV Human Immunodeficiency Virus
  • HBV Human Immunodeficiency Virus
  • HTLV Human T-cell Leukemia virus
  • HBcAg antigen from HBV see, e.g., Principles and Practice of Clinical Virology, 3rd Edition, Chapter 2: Hepatitis Viruses, pp.
  • HPV 16 E7 antigen from the Human Papilloma Virus (HPV) (see, e.g., Principles and Practice of Clinical Virology, 3rd Edition, Chapter 21: Papillomaviruses, pp. 621-623).
  • HPV Human Papilloma Virus
  • the determination of whether a given molecule is an antigen, and therefore a target antigen upon selection, is well within the skill of the art in view of the present specification. For example, whether a given molecule is antigenic can be determined by administering the molecule to an animal such as a rabbit or mouse, then observing whether the molecule induces an immune response.
  • Serum albumin is used herein in its traditional sense, to indicate a well known protein found in the blood. Serum albumins are secreted into the blood by liver cells, and bind to and solubilize many small molecules that are only slightly soluble in the blood serum absent such binding by the serum albumin. The folding of the polypeptide chain of serum albumin allows disulfide linkages to form between cysteine residues.
  • the protein contains 3 similar protein domains, and is encoded by a gene having 14 introns and 15 exons. Thus, expression of the gene in eukaryotes and prokaryotes generally includes processing mechanisms or is performed using non-intron containing genes, such as cDNA. Darnell et al., Molecular Cellular Biology, Sci. Am.
  • the serum albumin is human serum albumin, ⁇ -fetoprotein, bovine serum albumin, fetal bovine serum albumin, new born bovine serum albumin or mouse serum albumin.
  • the serum albumin is selected from the same species as the sample source or recipient of the target antigen- albumin complex. However, particularly for in vitro uses such as most diagnostic and detection assays, the albumin can be from another species.
  • the serum albumin can be recombinant, purified from an animal source, or produced synthetically.
  • “Complex,” or aggregate, is also used herein in its traditional sense, to indicate a dimer or multimer formed between one or more target antigens and one or more serum albumins.
  • the complex is believed to be formed by protein-protein interactions between the target antigen and the serum albumin, as opposed, for example, to fusion of ends of the proteins to one another (as through genetic engineering) or covalent cross-linking by intermediate agents such as glutaraldehyde.
  • Complexes of the present invention can include larger multimers, and can have molecular weights in excess of 800kD. Although not required to make or use the present invention, it is believed that the complex may be presented in a micelle form. Upon formation of the complex, it is believed that the conformation of the target antigen is altered. Thus, the presentation of the target antigen in the body, in the presence of serum albumin, is different from the presentation that is found in vitro in the absence of serum albumin.
  • the present invention provides a complex as described above wherein the complex comprises at least three components: a target antigen, a serum albumin, and a second target antigen that is different from the first target antigen.
  • the second target antigen can provide a different antigenic site specific to a second pathogenic state, so that the immuno-inducing composition or assay is effective for more than the pathogenic state associated with the first target antigen.
  • the second target antigen can also provide an even further enhanced immunological response against the first target antigen (and vice-versa), and can similarly provide for an even further enhanced ability to detect the first target antigen in a sample.
  • one or more of the target antigens can be a core-like antigen-adjacent protein, such as those described in U.S. Application Serial No. 08/454,928.
  • Such antigens provide not only enhanced effects in combination with the serum albumin, but also provide enhanced effects in combination with the other target antigen(s).
  • the multiple antigens are selected from two or more of HCV, HIV and HBV, particularly the HBcAg antigen.
  • the present invention also provides antibodies, including monoclonal antibodies, to the conformationally changed target antigens and serum albumins of the present invention, as well as other proteins of the present invention.
  • the antibodies can be used, for example, to provide particularly sensitive and specific detection of the target antigen in a sample.
  • the present invention provides the isolation of serum albumin from a sample by the use of an antibody against the serum albumin.
  • Serum albumin is deemed to be "isolated" from a sample within the context of the present invention if no other (undesired) protein is detected pursuant to SDS-PAGE analysis followed by coomassie blue staining.
  • the serum albumin can be isolated such that no other (undesired) protein is detected pursuant to SDS- PAGE analysis followed by silver staining.
  • the serum albumin is isolated if no other protein having significant antigenic activity that significantly interferes with detection assays or immunological events is included with the serum albumin.
  • the isolation of a protein, including serum albumin from an animal means that the protein has been removed from the animal.
  • the present invention provides compositions and methods for the elicitation of an enhanced immune response in an animal (either humoral, cellular, or both). Even further, the compositions and methods can vaccinate an animal against the pathogenic state, such as an infectious agent associated with the target antigen.
  • the present invention provides the use of a serum albumin complexed with a drug to provide a drug-serum albumin complex.
  • drug is used herein in its traditional meaning and need not be a protein or antigen.
  • the complexing of the serum albumin with the drug enhances the effectiveness of the drug by at least about 1.1 times, and preferably at least about 1.25, 1.5 or 2.0 times when compared to the effectiveness of the drug alone.
  • the drug-serum albumin complex is made and used in accordance with the uses of a target antigen-serum albumin complex discussed herein.
  • the methods and compositions of the present invention are applied to a human being or to samples obtained from a human being.
  • the sample can be obtained from blood, urine, sputum, feces, lymph or other traditional sources.
  • the target antigens and serum albumins of the present invention can be either recombinant or purified from an animal source. Purification can be effected using methods well known in the art, including affinity chromatography, ion exchange ultrafiltration and gel filtration (The National Medical Series For Independent Study, "Chapter 3: Amino Acid and Protein Structure. IV. PURIFICATION OF PROTEINS," Biochemistry, 3d ed., pp. 37-42).
  • Vector constructs suitable for use with a nucleic acid molecule encoding a desired protein can be prepared as a matter of convenience.
  • a vector construct is understood to typically refer to a DNA molecule, or a clone of such a molecule (either single-stranded or double- stranded), that has been modified through human intervention to contain segments of DNA combined and juxtaposed in a manner that as a whole would not otherwise exist in nature.
  • Vector constructs of the present invention comprise a first DNA segment encoding a desired protein operably linked to additional DNA segments required for the expression of the first DNA segment.
  • additional DNA segments will include a promoter and will generally include transcription terminators, and may further include operators, enhancers, mRNA ribosomal binding sites, appropriate sequences that control transcription and translation initiation and termination, and or other elements. See WO 94/25597; WO 94/25598; Pouwels et al., Cloning Vectors: A Laboratory Manual, Elsevier, New York, 1985.
  • the ability to replicate in the desired host cells usually conferred by an origin of replication, and a selection gene by which transformants are identified, may additionally be incorporated into the vector construct.
  • Methods for producing recombinant target antigen or serum ' albumin in a variety of prokaryotic and eukaryotic host cells are generally known in the art (Methods in Enzymology 185, Goeddel (ed.), Academic Press, San Diego, Calif, 1990; see also Methods in Enzymology, Guthrie and Fink (eds.), Academic Press. San Diego, Calif, 1991), and are discussed in more detail below.
  • vectors used for such expression will generally contain a selectable marker, such as a gene for antibiotic resistance, and a promoter that functions in the host cell.
  • the expression units may also include a transcriptional terminator.
  • Appropriate promoters include the following promoter systems: trp (Nichols and Yanofsky, Meth. Enzymol. 101:155-164, 1983), lac (Casadaban et al., J Bacteriol. 143:971-980, 1980; Chang et al.. Nature 275:615, 1978; Goeddel et al., Nature 281:544, 1979), phage ⁇ (Queen, J Mol. Appl. Genet.
  • plasmids useful for transforming prokaryotic hosts include the pUC plasmids (Messing, Meth. Enzymol.
  • Plasmids may contain viral, bacterial and/or eukaryotic elements.
  • prokaryotic host cells may be utilized within the context of the present invention.
  • preferred prokaryotic host cells should have a well- characterized genetic system, including known cloning vectors and methods of genetic manipulation.
  • Representative examples of such host cells include gram negative or gram positive organisms, for example, E. coli, Bacillus subtilis, Salmonella typhimurium, and various other species within the genera Pseudomonas, Enter obacteriaceae, Streptomyces, Staphylococcus, Bacillaceae, Salmonella, Shigella, and Corynebacteria.
  • the host cell is an E.
  • a desired protein may include an N-terminal Methionine residue to facilitate expression of the recombinant polypeptide in the prokaryotic host cell.
  • the N-terminal Met may be cleaved from the expressed recombinant desired protein.
  • Desirable prokaryotes may be readily obtained from a variety of commercial sources including, for example, the American Type Culture Collection (ATCC) (Rockville, Maryland). Alternatively, many of the above-described bacteria may be isolated from sources that are known by those of skill in the art to contain such prokaryotes, based upon techniques that are known in the art. (See Bergy's Shorter Manual of Determinative Bacteriology. 8th ed., Holt, John G. (ed.), Williams & Wilkins, 1977.)
  • Recombinant protein produced in prokaryotic culture is usually isolated by initial disruption of the host cells, centrifugation, extraction from cell pellets if an insoluble polypeptide, or from the supernatant fluid if a soluble polypeptide, followed by one or more concentration, salting-out, ion exchange, affinity purification or size exclusion chromatography steps. Finally, RP-HPLC can be employed for final purification steps. Microbial cells can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents. Turning to production of desired protein in eukaryotic cells, eukaryotic host cells suitable for use in practicing the present invention include mammalian, avian, plant, insect and fungal cells such as yeast.
  • Yeast vectors will often contain an origin of replication sequence from a 2 ⁇ yeast plasmid, an autonomously replicating sequence (ARS), a promoter region, sequences for polyadenylation, sequences for transcription termination, and a selectable marker gene.
  • sequences encoding appropriate signal pep tides that are not naturally associated with the desired protein can be incorporated into expression vectors.
  • a DNA sequence for a signal peptide secretory leader
  • a signal peptide that is functional in the intended host cells enhances extracellular secretion of the desired protein.
  • the signal peptide may be cleaved from the desired protein upon secretion of the desired protein from the cell.
  • suitable promoter sequences for yeast vectors include, among others, promoters for metallothionein, 3-phosphoglycerate kinase (Hitzeman et al., J. Biol. Chem. 255:2013, 1980) or other glycolytic enzymes (Hess et al., J. Adv. Enzyme Reg. 7:149, 1968; and Holland et al., Biochem. 17:4900, 1978), such as enolase.
  • glyceraldehyde-3 -phosphate dehydrogenase hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase.
  • Other suitable vectors and promoters for use in yeast expression are further described in Hitzeman, EPA-73,657 or in Fleer et al., Gene 707:285-295, 1991 ; and van den Berg et al., Bio/Technology 5:135-139, 1990.
  • Shuttle vectors replicable in both yeast and E. coli may be constructed by inserting DNA sequences from pBR322 for selection and replication in E. coli (Amp r gene and origin of replication) into the above-described yeast vectors.
  • the yeast ⁇ -factor leader sequence may be employed to direct secretion of an target antigen.
  • the ⁇ -factor leader sequence is often inserted between the promoter sequence and the structural gene sequence.
  • leader sequences suitable for facilitating secretion of recombinant polypeptides from yeast hosts are known to those of skill in the art.
  • a leader sequence may be modified near its 3' end to contain one or more restriction sites. This will facilitate fusion of the leader sequence to the structural gene.
  • Yeast transformation protocols are known to those of skill in the art.
  • One such protocol is described by Hinnen et al., Proc. Natl. Acad. Sci. USA 75:1929, 1978.
  • the Hinnen et al. protocol selects for Trp + transformants in a selective medium, wherein the selective medium consists of 0.67% yeast nitrogen base, 0.5% casamino acids, 2% glucose, 10 ⁇ g/ml adenine and 20 ⁇ g/ml uracil.
  • Yeast host cells transformed by vectors containing ADH2 promoter sequence may be grown for inducing expression in a "rich" medium.
  • a rich medium is one consisting of 1% yeast extract, 2% peptone, and 1% glucose supplemented with 80 ⁇ g/ml adenine and 80 ⁇ g/ml uracil. Derepression of the ADH2 promoter occurs when glucose is exhausted from the medium.
  • Mammalian or insect host cell culture systems can also be employed to express desired recombinant proteins.
  • Baculovirus systems for production of heterologous proteins in insect cells are reviewed by Luckow and Summers, Bio/Technology 6:41, 1988.
  • Established cell lines of mammalian origin also may be employed. Examples of suitable mammalian host cell lines include the COS-7 line of monkey kidney cells (ATCC CRL 1651) (Gluzman et al., Cell 23:175, 1981), L cells. C127 cells, 3T3 cells (ATCC CCL 163), Chinese hamster ovary (CHO) cells.
  • Transcriptional and translational control sequences for mammalian host cell expression vectors may be excised from viral genomes.
  • Commonly used promoter sequences and enhancer sequences are derived from Polyoma virus, Adenovirus 2, Simian Virus 40 (SV40), and human cytomegalo virus.
  • DNA sequences derived from the SV40 viral genome for example, SV40 origin, early and late promoter, enhancer, splice, and polyadenylation sites may be used to provide other genetic elements for expression of a structural gene sequence in a mammalian host cell.
  • Viral early and late promoters are particularly useful because both are easily obtained from a viral genome as a fragment which may also contain a viral origin of replication (Fiers et al., Nature 273:113, 1978). Smaller or larger SV40 fragments may also be used, provided the approximately 250 bp sequence extending from the Hind III site toward the Bgl I site located in the SV40 viral origin of replication site is included.
  • Exemplary expression vectors for use in mammalian host cells can be constructed as disclosed by Okayama and Berg (Mol. Cell. Biol. 5:280, 1983).
  • a useful system for stable high level expression of mammalian cDNAs in C127 murine mammary epithelial cells can be constructed substantially as described by Cosman et al. (Mol. Immunol. 23:935, 1986).
  • a useful high expression vector, PMLSV N1/N4. described by Cosman et al., Nature 312:16%, 1984 has been deposited as ATCC 39890. Additional useful mammalian expression vectors are described in EP-A-0367566, and in U.S. Patent Application Serial No. 07/701,415, filed May 16, 1991.
  • the vectors may be derived from retroviruses.
  • a heterologous signal sequence may be added, such as the signal sequence for IL-7 described in United States Patent 4,965,195; the signal sequence for IL-2 receptor described in Cosman et al., Nature 312:16%, 1984; the IL-4 signal peptide described in EP 367,566; the type I IL-1 receptor signal peptide described in U.S. Patent 4,968,607; and the type II IL-1 receptor signal peptide described in EP 460,846.
  • Preferred eukaryotic cells include cultured mammalian cell lines (e.g., rodent or human cell lines), insect cell lines (e.g., Sf-9) and fungal cells, including species of yeast (e.g., Saccharomyces spp., particularly S. cerevisiae, Schizosaccharomyces spp., Kluyveromyces spp., Pichia, and K. lactis) or filamentous fungi (e.g., Aspergillus spp., Neurospora spp.).
  • yeast e.g., Saccharomyces spp., particularly S. cerevisiae, Schizosaccharomyces spp., Kluyveromyces spp., Pichia, and K. lactis
  • filamentous fungi e.g., Aspergillus spp., Neurospora spp.
  • Transformed yeast host cells are often preferable to mammalian or other eukaryotic systems to express desired protein as a secreted polypeptide in order to simplify purification.
  • Secreted recombinant polypeptide from a yeast host cell fermentation can be purified by methods analogous to those disclosed by Urdal et al. (J. Chromatog. 296:111, 1984).
  • Urdal et al. describe two sequential, reversed-phase HPLC steps for purification of recombinant human IL-2 on a preparative HPLC column.
  • mutations in nucleotide sequences constructed for expression of the inventive proteins preferably preserve the reading frame of the encoding sequences. Furthermore, the mutations preferably do not create complementary regions that could hybridize to produce secondary mRNA structures, such as loops or hairpins, that would adversely affect translation of the mRNA.
  • a mutation site may be predetermined, it is not necessary that the nature of the mutation per se be predetermined. For example, in order to select for optimum characteristics of mutants at a given site, random mutagenesis may be conducted at the target codon and the expressed mutants screened for indicative biological activity.
  • Mutations may be introduced at particular loci by synthesizing oligonucleotides containing a mutant sequence, flanked by restriction sites enabling ligation to fragments of the native sequence. Following ligation, the resulting reconstructed sequence encodes a derivative having the desired amino acid insertion, substitution or deletion.
  • oligonucleotide-directed, site-specific mutagenesis procedures may be employed to provide an altered gene having particular codons altered according to the substitution, deletion, or insertion required.
  • Exemplary methods of making the alterations set forth above are disclosed by Walder et al. (Gene 42:133, 1986); Bauer et al. (Gene 37:13, 1985); Craik (BioTechniques, January 1985, 12-19); Smith et al. (Genetic Engineering: Principles and Methods, Plenum Press, 1981); and Sambrook et al. (supra).
  • the primary amino acid structure of the above-described proteins can also be modified by forming conjugates with other chemical moieties, such as glycosyl groups, lipids, phosphate, acetyl groups, or with other proteins or polypeptides, provided that such modifications do not disrupt the antigenicity and/or cooperative properties of the proteins.
  • modifications See U.S. Patent No. 4,851,341; see also Hopp et al., Bio/Technology (5: 1204, 1988.)
  • modifications should not interfere with the epitopic configuration (including access to the epitope and other antigenic considerations) that is specific to the target antigen when it is complexed with the serum albumin.
  • Cell-free translation systems can also be employed to produce desired proteins using RNAs derived from DNA constructs disclosed herein.
  • the present invention also provides antibodies, i.e., binding partners, such as monoclonal and polyclonal antibodies, directed against the target antigens and serum albumins of the present invention.
  • the antibodies comprise IgM and/or IgG antibodies, further preferably IgM antibodies.
  • the antibodies are produced using a target antigen that has been complexed with a serum albumin to provide a complex as one or both of the immunizing agent or the selective agent.
  • Antibodies including all forms of antibodies such as IgG and IgM antibodies, can be produced by using a protein/aggregate of the invention as an immunogen through standard procedures for preparing a hybridoma, and/or via other methods. The resulting antibodies are particularly useful for detecting the target antigen in a sample, preferably a sample from a human being. See WO 94/25597 and WO 94/25598. Polyclonal antibodies can be readily generated by one of ordinary skill in the art from a variety of warm-blooded animals such as horses, cows, goats, sheep, dogs, chickens, turkeys, rabbits, mice, or rats.
  • the desired protein or peptide is utilized to immunize the animal, typically through intraperitoneal, intramuscular, intraocular, or subcutaneous injections.
  • the immunogenicity of the protein or peptide of interest may be increased through the use of an adjuvant such as Freund's complete or incomplete adjuvant.
  • an adjuvant such as Freund's complete or incomplete adjuvant.
  • small samples of serum are collected and tested for reactivity to the desired protein or peptide.
  • polyclonal antisera may be readily obtained either by weekly bleedings, or by exsanguinating the animal.
  • Monoclonal antibodies can also be readily generated using well-known techniques (see U.S. Patent Nos. RE 32,011, 4,902,614, 4,543,439, and 4,411,993; see also Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, Kennett et al. (eds.), Plenum Press, 1980, and Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, 1988). Briefly, in one embodiment, a subject animal such as a rat or mouse is injected with a desired protein or peptide. If desired, various techniques may be utilized in order to increase the resultant immune response generated by the protein, in order to develop greater antibody reactivity.
  • the desired protein or peptide may be coupled to another protein such as ovalbumin or keyhole limpet hemocyanin (KLH), or through the use of adjuvants such as Freund's complete or incomplete adjuvants.
  • KLH keyhole limpet hemocyanin
  • the initial elicitation of an immune response may be through intraperitoneal, intramuscular, intraocular, or subcutaneous routes.
  • the animal may be reirnmunized with booster immunization.
  • the animal may then be test bled and the serum tested for binding to the unprocessed polypeptide using assays as described above. Additional immunizations may also be accomplished until the animal has reached a plateau in its reactivity to the desired protein or peptide.
  • the animal may then be given a final boost of the desired protein or peptide, and three to four days later sacrificed.
  • the spleen and lymph nodes may be harvested and disrupted into a single cell suspension by passing the organs through a mesh screen or by rupturing the spleen or lymph node membranes which encapsulate the cells.
  • red cells are subsequently lysed by the addition of a hypotonic solution, followed by immediate return to isotonicity.
  • suitable cells for preparing monoclonal antibodies are obtained through the use of in vitro immunization techniques. Briefly, an animal is sacrificed, and the spleen and lymph node cells are removed as described above. A single cell suspension is prepared, and the cells are placed into a culture containing a form of the protein or peptide of interest that is suitable for generating an immune response as described above. Subsequently, the lymphocytes are harvested and fused as described below.
  • Cells that are obtained through the use of in vitro immunization or from an immunized animal as described above may be immortalized by transfection with a virus such as the Epstein-Barr Virus (EBV). (See Glasky and Reading, Hybridoma 5(4):377-389, 1989.)
  • EBV Epstein-Barr Virus
  • the harvested spleen and/or lymph node cell suspensions are fused with a suitable myeloma cell in order to create a "hybridoma" which secretes monoclonal antibodies.
  • Suitable myeloma lines are preferably defective in the construction or expression of antibodies, and are additionally syngeneic with the cells from the immunized animal.
  • myeloma cell lines are well known in the art and may be obtained from sources such as the American Type Culture Collection (ATCC), Rockville, Maryland (see Catalogue of Cell Lines & Hybridomas, 6th ed., ATCC, 1988).
  • Representative myeloma lines include: for humans, UC 729-6 (ATCC No. CRL 8061), MC/CAR-Z2 (ATCC No. CRL 8147), and SKO-007 (ATCC No. CRL 8033); for mice, SP2/0-Agl4 (ATCC No. CRL 1581), and P3X63Ag8 (ATCC No. TIB 9); and for rats, Y3-Agl.2.3 (ATCC No.
  • Preferred fusion lines include NS-1 (ATCC No. TIB 18) and P3X63-Ag 8.653 (ATCC No. CRL 1580), which may be utilized for fusions with either mouse, rat, or human cell lines. Fusion between the myeloma cell line and the cells from the immunized animal can be accomplished by a variety of methods, including the use of polyethylene glycol (PEG) (see Antibodies: A Laboratory Manual, supra) or electrofusion (see Zimmerman and Vienken, J. Membrane Biol. (57:165-182, 1982).
  • PEG polyethylene glycol
  • electrofusion see Zimmerman and Vienken, J. Membrane Biol. (57:165-182, 1982).
  • the cells are placed into culture plates containing a suitable medium, such as RPMI 1640 or DMEM (Dulbecco's Modified Eagles Medium, JRH Biosciences, Lenexa, Kan.).
  • a suitable medium such as RPMI 1640 or DMEM (Dulbecco's Modified Eagles Medium, JRH Biosciences, Lenexa, Kan.).
  • the medium may also contain additional ingredients, such as fetal bovine serum (FBS, e.g., from Hyclone, Logan, Utah, or JRH Biosciences), thymocytes that were harvested from a baby animal of the same species as was used for immunization, or agar to solidify the medium.
  • FBS fetal bovine serum
  • thymocytes that were harvested from a baby animal of the same species as was used for immunization
  • agar to solidify the medium e.g., from Hyclone, Logan, Utah, or JRH Biosciences
  • HAT medium hyperxanthine, aminopterin, and thymidine
  • HAT medium hyperxanthine, aminopterin, and thymidine
  • the resulting fused cells or hybridomas may be screened in order to determine the presence of antibodies which recognizes the proteins of the present invention.
  • a hybridoma producing antibodies that bind to the protein of interest can be isolated.
  • mRNA is isolated from a B cell population and utilized to create heavy and light chain immunoglobulin cDNA expression libraries in the ⁇ MMUNOZAP(H) and ⁇ MMUNOZAP(L) vectors.
  • vectors may be screened individually or co-expressed to form Fab fragments or antibodies (see Huse et al., supra; Sastry et al., supra). Positive plaques can subsequently be converted to a non-lytic plasmid that allows high level expression of monoclonal antibody fragments from E. coli.
  • antibodies can also be constructed utilizing recombinant DNA techniques to incorporate the variable regions of a gene that encodes a specifically binding antibody.
  • the construction of these antibodies can be readily accomplished by one of ordinary skill in the art given the disclosure provided herein. (See Larrick et al..
  • DNA segments encoding the desired protein or peptide interest-specific antigen binding domains are amplified from hybridomas that produce a specifically binding monoclonal antibody, and are inserted directly into the genome of a cell that produces human antibodies. (See Verhoeyen et al., supra; see also Reichmann et al., supra.) This technique allows the antigen-binding site of a specifically binding mouse or rat monoclonal antibody to be transferred into a human antibody. Such antibodies are preferable for therapeutic use in humans because they are not as antigenic as rat or mouse antibodies.
  • Monoclonal antibodies and other antibodies can be produced in a number of host systems, including tissue cultures, bacteria, eukaryotic cells, plants and other host systems known in the art.
  • suitable antibodies may be isolated or purified by many techniques well known to those of ordinary skill in the art (see Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, 1988; U.S. Patent No. 4,736,110; and U.S. Patent No. 4,486,530).
  • Suitable isolation techniques include peptide or protein affinity columns, HPLC or RPHPLC, purification on protein A or protein G columns, or any combination of these techniques.
  • the term "isolated" as used to define antibodies means substantially free of other blood components.
  • the antibodies of the present invention have many uses. As discussed further below, the antibodies of the present invention are particularly useful for the detection and diagnosis of target antigens and the pathogenic agents associated therewith. Other uses include, for example, flow cytometry to sort cells displaying one more of the target antigens of the present invention. Briefly, in order to detect the target antigens of interest on cells, the cells are incubated with a labeled monoclonal antibody which specifically binds to the protein of interest, followed by detection of the presence of bound antibody. These steps may also be accomplished with additional steps such as washings to remove unbound antibody.
  • Labels suitable for use within the present invention are well known in the art including, among others, flourescein isothiocyanate (FITC), phycoerythrin (PE), horse radish peroxidase (HRP), and colloidal gold.
  • FITC flourescein isothiocyanate
  • PE phycoerythrin
  • HRP horse radish peroxidase
  • colloidal gold particularly preferred for use in flow cytometry is FITC, which may be conjugated to a purified antibody according to the method of Keltkamp in "Conjugation of Fluorescein Isothiocyanate to Antibodies. I. Experiments on the Conditions of Conjugation," Immunology 75:865-873, 1970. (See also Keltkamp, "Conjugation of Fluorescein Isothiocyanate to Antibodies. II. A Reproducible Method," Immunology 75:875-881, 1970; Goding, “Conjugation of Antibodies with Fluorochromes: Modification
  • the present invention provides methods for detecting a target antigen in a sample.
  • the methods, or assays are typically based on the detection of antigens associated with a pathogenic agent, such as the antigens displayed by a positive-stranded RNA virus.
  • the present invention also provides methods of detecting antibodies produced against a target antigen.
  • the methods are characterized by the ability of the target antigen-serum albumin complex of the present invention to be bound by antibodies against the target antigen, and the ability of antibodies produced against the target antigen-serum albumin complex of the present invention to bind to target antigens in a sample.
  • these aspects of the invention preferably include the use of IgM and/or IgG antibodies.
  • the target antigen-serum albumin complex, or antibodies produced against the complex, of the present invention provide significantly enhanced detection of target antigen.
  • the use of both an core-env target antigen complexed with a serum albumin in the assay provides a synergistic effect that permits significantly more sensitive detection of the target antigen than when either the core-env antigen or the serum albumin is utilized alone (indeed, the serum albumin alone provides essentially no detection of the target antigen or antibodies thereto).
  • a preferred assay for the detection of the target antigen is a sandwich assay such as an enzyme-linked immunosorbent assay (ELISA).
  • the ELISA comprises the following steps: (1) coating target antigen complexed with at least one serum albumin onto a solid phase, (2) incubating a sample suspected of containing antibodies against the target antigen with the complex coated onto the solid phase under conditions that allow the formation of an antigen-antibody complex, (3) adding an anti-antibody (such as anti-IgG) conjugated with a label to be captured by the resulting antigen-antibody complex bound to the solid phase, and (4) measuring the captured label and determining therefrom whether the sample has antibodies against the target antigen.
  • an anti-antibody such as anti-IgG
  • assays include: countercurrent immuno-electrophoresis (CIEP), radioimmunoassays, radioimmunoprecipitations, enzyme-linked immunosorbent assays (ELISA), dot blot assays, inhibition or competition assays, sandwich assays, immunostick (dip-stick) assays, simultaneous assays, immunochromatographic assays, immunofiltration assays, latex bead agglutination assays, immunofluorescent assays, biosensor assays, and low-light detection assays (see U.S. Patent Nos. 4,376,110 and 4,486,530; WO 94/25597; WO/25598; see also Antibodies: A Laboratory Manual, supra).
  • CIEP countercurrent immuno-electrophoresis
  • ELISA enzyme-linked immunosorbent assays
  • dot blot assays inhibition or competition assays
  • sandwich assays sandwich assays
  • immunostick (dip-stick) assays simultaneous assays
  • a fluorescent antibody test uses a fluorescently labeled antibody able to bind to one of the target antigens of the invention. For detection, visual determinations are made by a technician using fluorescence microscopy, yielding a qualitative result. In one embodiment, this assay is used for the examination of tissue samples or histological sections.
  • antibodies made using the target antigen-serum albumin complex of the present invention are conjugated to latex beads.
  • the antibodies conjugated to the latex beads are then contacted with a sample under conditions permitting the antibodies to bind to target antigens in the sample, if any.
  • the results are then read visually, yielding a qualitative result.
  • this format can be used in the field for on-site testing.
  • Enzyme immunoassays include a number of different assays able to utilize the antibodies provided by the present invention.
  • a heterogeneous indirect EIA uses a solid phase coupled with an antibody of the invention and an affinity purified, anti-IgG immunoglobulin preparation.
  • the solid phase is a polystyrene microtiter plate.
  • the antibodies and immunoglobulin preparation are then contacted with the sample under conditions permitting antibody binding, which conditions are well known in the art.
  • the results of such an assay can be read visually, but are preferably read using a spectrophotometer, such as an ELISA plate reader, to yield a quantitative result.
  • An alternative solid phase EIA format includes plastic- coated ferrous metal beads able to be moved during the procedures of the assay by means of a magnet.
  • Yet another alternative is a low-light detection immunoassay format. In this highly sensitive format, the light emission produced by appropriately labeled bound antibodies are quantitated automatically. Preferably, the reaction is performed using microtiter plates.
  • a radioactive tracer is substituted for the enzyme mediated detection in an EIA to produce a radioimmunoassay (RIA).
  • the target antigen is bound between an antibody attached to a solid phase, preferably a polystyrene microtiter plate, and a labeled antibody.
  • a solid phase preferably a polystyrene microtiter plate
  • the results are measured using a spectrophotometer, such as an ELISA plate reader.
  • This assay is one preferred embodiment for the present invention.
  • reagents are allowed to incubate with the capture antibody in a step-wise fashion.
  • the test sample is first incubated with the capture antibody.
  • an incubation with the labeled antibody occurs.
  • the two incubation periods described in the sequential assay are combined. This eliminates one incubation period plus a wash step.
  • a dipstick/immunostick format is essentially an immunoassay except that the solid phase, instead of being a polystyrene microtiter plate, is a polystyrene paddle or dipstick. Reagents are the same and the format can either be simultaneous or sequential.
  • a capture antibody and a labeled antibody are dried onto a chromatographic strip, which is typically nitrocellulose or nylon of high porosity bonded to cellulose acetate.
  • the capture antibody is usually spray dried as a line at one end of the strip. At this end there is an absorbent material that is in contact with the strip.
  • the labeled antibody is deposited in a manner that prevents it from being absorbed into the membrane.
  • the label attached to the antibody is a latex bead or colloidal gold.
  • the assay may be initiated by applying the sample immediately in front of the labeled antibody.
  • Immunofiltration/immunoconcentration formats combine a large solid phase surface with directional flow of sample/reagents, which concentrates and accelerates the binding of antigen to antibody.
  • the test sample is preincubated with a labeled antibody then applied to a solid phase such as fiber filters or nitrocellulose membranes or the like.
  • the solid phase can also be precoated with latex or glass beads coated with capture antibody. Detection of analyte is the same as standard immunoassay.
  • the flow of sample/reagents can be modulated by either vacuum or the wicking action of an underlying absorbent material.
  • a threshold biosensor assay is a sensitive, instrumented assay amenable to screening large numbers of samples at low cost.
  • such an assay comprises the use of light addressable potentiometric sensors wherein the reaction involves the detection of a pH change due to binding of the desired protein by capture antibodies, bridging antibodies and urease-conjugated antibodies. Upon binding, a pH change is effected that is measurable by translation into electrical potential ( ⁇ volts).
  • the assay typically occurs in a very small reaction volume, and is very sensitive. Moreover, the reported detection limit of the assay is 1,000 molecules of urease per minute.
  • compositions and methods for the elicitation of an immune response to the target antigen-serum albumin complex which response may be either humoral, cellular, or both.
  • the immune response is induced by a vaccine against the pathogenic agent associated with a target antigen, such as a positive-stranded RNA virus associated with its core/gag protein, and is therefore an immunoprotective immune response.
  • a vaccine against the pathogenic agent associated with a target antigen such as a positive-stranded RNA virus associated with its core/gag protein
  • compositions and methods typically involve an immunogen comprising a target antigen-serum albumin complex of the present invention in combination with a pharmaceutically acceptable carrier or diluent.
  • compositions can also include a cocktail of differing target antigens so that multiple disease states can be affected, or so that multiple aspects of a single disease state can be affected.
  • a cocktail can include a variety of target antigens, with each complexed with its own serum albumin, or the cocktail can include a plurality of target antigens aggregated with the serum albumin in a single complex.
  • the target antigen may comprise an inactivated preparation or an attenuated preparation.
  • target antigen-serum albumin complexes capable of eliciting an immune response, preferably capable of immunizing an animal.
  • target antigens of the present invention slight deviations of the amino acid sequences can be made without affecting the immunogenicity of the immunogen.
  • Substantial equivalents of the target antigens include conservative substitutions of amino acids that maintain substantially the same charge and hydrophobicity as the original amino acid. Conservative substitutions include replacement of valine for isoleucine or leucine, and aspartic acid for glutamic acid, as well as other substitutions of a similar nature (see Dayhof et al. (eds.), "Atlas of Protein Sequence and Structure," Natl. Biomed. Res. Fdn., 1978).
  • the immunogens listed above, including their substantial equivalents may stimulate different levels of response in different animals.
  • the immunogens listed above, including their substantial equivalents can be tested for effectiveness as a vaccine. These tests include T-cell proliferation assays, determination of lymphokine production after stimulation, and immunoprotection trials. Briefly, T-cell proliferation assays can be utilized as an indicator of potential for cell-mediated immunity. Additionally, evidence of lymphokine production after stimulation by an immunogen can be utilized to determine the potential for protection provided by an immunogen.
  • PBLs peripheral blood lymphocytes
  • pathogenic agents in the following manner.
  • PBLs can be isolated from diluted whole blood using Ficoll density gradient centrifugation and utilized in cell proliferation studies with [ 3 H]-thymidine as described below. Positive peptides are then selected and utilized in primate trials.
  • the immunogens, or polypeptides, of the present invention can be readily produced utilizing many other techniques well known in the art (see Sambrook et al., supra, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989).
  • Immunogens comprising the target antigen-serum albumin complexes of the present invention in combination with a pharmaceutically acceptable carrier or diluent can be administered to a patient in accordance with a number procedures known in the art. See WO 94/25597 and WO 94/25598.
  • warm-blooded animals include, among others, humans, primates, rabbits and mice, chickens and other fowl, cats and dogs, cattle and horses.
  • Suitable carriers or diluents can be utilized in the present invention, including among others saline, buffered saline, and saline mixed with nonspecific serum albumin.
  • the pharmaceutical composition may also contain other excipient ingredients, including adjuvants, buffers, antioxidants, carbohydrates such as glucose, sucrose, or dextrins, and chelating agents such as EDTA.
  • the serum albumin itself acts as an adjuvant, thereby enhancing the immune response to the target antigen, other adjuvants may also be utilized along with the immunogen, if desired. Examples of such adjuvants include alum or aluminum hydroxide for humans.
  • immunizations will involve oral administration.
  • the vaccine can be parenterally administrated via the subcutaneous route, or via other routes.
  • quantities of injected immunogen will vary from 50 ⁇ g to several milligrams in combination with a pharmaceutically acceptable carrier or diluent, and will typically vary from about 100 ⁇ g to 1 mg.
  • Booster immunizations can be given from 4-6 weeks later.
  • kits for analyzing samples for the presence of target antigens or antibodies comprise a target antigen-serum albumin complex or antibody against the same and an appropriate solid phase.
  • the target antigen-serum albumin complex or antibody is bound to the solid phase.
  • the kits can also provide one or more reagents and/or devices for the detection of the target antigen-serum albumin complex or antibodies.
  • a variety of formats, reagents and devices for inclusion within the kits, including means for detecting the antigens or antibodies, are discussed herein.
  • kits for the induction of an immune response comprise compositions comprising a target antigen-serum albumin complex of the invention in combination with an pharmaceutically acceptable carrier or diluent, and can also provide devices for administering or assisting in the administration of the composition.
  • kits suitable for use with the features of the present invention are also provided herewith.
  • HCV Hepatitis C Virus
  • HSA human serum albumin
  • Human serum albumin (No. A-8763, Sigma, St. Louis, Missouri, USA) in a stock solution at 1 mg/ml in distilled water was added to coating buffer to provide a final concentration of 1.5 ⁇ g/ml, and mixed at room temperature for 60 minutes. 100 ⁇ l/well of this solution was coated onto a microtiter plate (Nunc-Immuno Strip, MaxiSorp, Denmark) and blocked with a buffer containing bovine serum albumin. The plate was incubated for 24 hours in a 40°C incubator.
  • postcoating buffer (0.01M NaH 2 P0 4 H 2 O Na,HPO 4 7H 2 O/0.15N NaCl/1% BSA, pH 7.2-7.4) was added into each of the wells. The plate was then let stand either overnight at 4°C or at room temperature for 3 hours.
  • Anti-hlgG Purified anti-human Immunoglobulin G
  • HRPO horse radish peroxidase
  • the conjugate was purified by S-200 gel filtration and was diluted in sample diluent (0.1M Tris-HCl pH: 7.4 ⁇ 0.2, 40% NBBS, 1% BSA, 2% mouse serum).
  • TNSCN coating buffer 50 mM Tris-HCl/0.15 N NaCl 3 M NaSCN, pH: 7.4 ⁇ 0.2.
  • TNEU coating buffer 50 mM Tris-HCl/0.15 N NaCl/1 mM EDTA/6 M urea, pH 7.2-7.4.
  • Carbonate coating buffer 0.1 M pH 9.6 ⁇ 0.2.
  • PBS (phosphate buffered saline) coating buffer 0.01 M Na phosphate buffer (NaH 2 PO 4 -H 2 O/Na 2 HPO 4 -7H 2 O/0.15 N NaCl, pH 7.2-7.4).
  • TNSDS coating buffer 50 mM Tris-HCl/0.15 N NaCl/0.02% SDS, pH 7.4 ⁇ 0.2.
  • wash Solution Phosphate buffer, 0.01 M (KT PO./Na HPOJ/O.lS N NaCl/0.05% Tween 20/0.001% thimerosol.
  • O-phenylene diamine dissolved in citrate-phosphate buffer containing H 2 O 2 (if the solution becomes orange, it means that the solution has been contaminated and cannot be used any more).
  • Substrate diluent Citrate-phosphate buffer (4.666 g Citric acid and 7.299 g Na 2 HPO 4 dissolved in 1 liter distilled water) containing H 2 O 2 (final concentration: 0.0125%).
  • Substrate solution 1 OPD tablet dissolved in 5 ml substrate diluent.
  • Stopping Solution 2N H 2 SO 4 .
  • the OD value per well was measured at 492 nm in a spectrophotometer.
  • PCx-NCx mean of the readings of the positive controls (PCx) and that of the negative controls (NCx) is equal to or more than 0.5.
  • the cut-off value (CO) is calculated by the following formula:
  • the samples were expected to be positive; however, it is preferred to repeat the assay for the samples in duplicate. If the readings of either of the duplicate samples were less than the CO value, the samples were considered to be negative. If the duplicate samples were both more than or equal to the Cut-off value, the samples were considered to be positive. When the readings of test samples are more than NCx but less than the
  • HCV Hepatitis C Virus
  • Anti-HCV positive samples were N8, N12, N9, N81, N302, N141, N93, N210, N207, Nl 17, N10, N71, N49, N451, N505, N103 and N26.
  • Anti-HCV negative samples were N626, N639, N634, N632, N209, N202, N203, N997, N986 and N208.
  • Sample diluent 0.1 M Tris-HCl, pH: 7.4 ⁇ 0.2 with 40% new born bovine serum, 1% BSA and 2% mouse serum.
  • ELISA was performed as set forth in the first assay of this Example 2, except that recalcified human serum (HS; non-reactive for HBV, HCV, HIV and HTLV) was mixed with the core-envelope fusion protein of Hepatitis C Virus (HCV) to provide a complex of the serum albumin in the HS and the HCV antigen.
  • HS human serum
  • HCV Hepatitis C Virus
  • An ELISA was performed as set forth in the first and second assays of this Example 2, except that bovine serum albumin (BSA; Sigma, St. Louis, Missouri, USA) was mixed with the core-env fusion protein of Hepatitis C Virus (HCV) to provide a complex of the BSA and the HCV antigen.
  • BSA bovine serum albumin
  • HCV Hepatitis C Virus
  • ELISA was performed as set forth in the first and second assays of this Example 2, except that fetal bovine serum (FBS; CSL, Victoria, Australia) was mixed with the core-env fusion protein of Hepatitis C Virus (HCV) to provide a complex of the serum albumin in the FBS and the HCV antigen.
  • FBS fetal bovine serum
  • HCV Hepatitis C Virus
  • HSA in carbonate buffer to provide a complex of the HSA and the HCV antigen.
  • HIV-1 Human Immunodeficiency Virus- 1
  • EN-I-6 a recombinant fusion protein comprising the amino-terminal fragment of ⁇ -galactosidase (31 1 a.a.) fused to amino acids 474-863 of env, i.e., gpl60, having 705 a.a. (including spacer amino acids) and a Mw of 80.7 kDa; see U.S. Application Serial No. 08/454,928) was mixed with the HSA in TNEU buffer to provide a complex of the HSA and the HIV-1 env antigen.
  • the samples for the assay were as follows. Anti-HIV-1 positive samples were Tl, T2, T3, T4, T5, T10 and T14. Anti-HIV-1 negative samples were N977, N966, N968, N764, N766 and N952.
  • HIV-1 Human Immunodeficiency Virus- 1
  • EN-I-5 a recombinant fusion protein comprising the amino-terminal fragment of ⁇ -galactosidase (377 a.a.) fused to gag pi 7 (a.a. 15-132) followed by gag p24 (a.a. 133-363) followed by gag pi 5 (a.a. 364-437), having 831 a.a. (including spacer amino acids) and a Mw of 92.8 kDa; see U.S. Application Serial No. 08/454,928) was mixed with the HSA in TNSCN buffer to provide a complex of the HSA and the HTV-l gag antigen.
  • Table 12 a gag antigen of the Human Immunodeficiency Virus- 1 (HIV-1) (EN-I-5, a recombinant fusion protein comprising the amino-terminal fragment of ⁇ -galactosidase (377 a.a.) fused to gag pi 7 (a.a
  • Example 3 using TNEU as the coating buffer, and, as antigens, HSA, EN-I-5, EN-I-6, and EN-80-2 as set forth in Table 13.
  • An ELISA was performed as set forth in the first assay of this Example 3, except that the EN-I-5 gag antigen of the Human Immunodeficiency Virus 1 (HIV-1) (EN-I-5) was mixed with the HSA in PBS to provide a complex of the HSA and the HIV-1 gag antigen.
  • HAV-1 Human Immunodeficiency Virus 1
  • An assay was performed as set forth in the Example 1 , above, except that an HI antigen from the HTLV-l envelope region was mixed with the HSA in TNEU buffer to provide a complex of the HSA and the HTLV-l env antigen.
  • the samples for the assay were as follows. Anti-HTLV-1 positive samples were SP, BBI 23 and BBI 25. Anti-HTLV-1 negative samples were N966 and N968.
  • HBcAg antigen identified as A5
  • HSA Hepatitis B Virus
  • Example 1 An assay was performed as set forth in the Example 1, above, except that a human papillomavirus (HPV) type 16 E7 antigen, identified as EN-P-1 was mixed with the HSA in TNEU buffer to provide a complex of the HSA and the E7 antigen of HPV.
  • HPV human papillomavirus
  • the samples for the assay were as follows. Anti-HPV positive samples were B7, B9 and B12. Anti-HPV negative samples were N966, N968, N977 and B14.
  • ELISA was performed as set forth in Example 2, except that an ⁇ - fetoprotein (AFP), purified from human cord blood using a monoclonal anti-AFP affinity column, was mixed with the core-env fusion protein of Hepatitis C Virus
  • HCV HCV
  • the samples for the assay were as follows. Anti-HCV positive samples were N49, N451 , and N 103. Anti-HCV negative samples were N949 and N950.
  • the 02 immunogen was prepared by dialyzing a core-envelope fusion antigen from HCV (EN-80-2) against PBS. After dialysis, the antigen was used to immunize ICR mice.
  • the M+02 immunogen was prepared by mixing the core-envelope fusion antigen from HCV (EN-80-2) with mouse serum albumin (MSA) in TNEU buffer for one hour. ' After mixing, the complex was dialyzed against PBS. Then, the complex was used to immunize ICR mice.
  • the 02 and M+02 immunogens were administered to ICR mice at 6-8 weeks of age.
  • the first administration, boost and sampling schedule was as follows:
  • Test group 1 (ID nos. 1-1 and 1-2):
  • Test group 2 (ID nos. 2-1 and 2-2): Day 0: 100 ⁇ g of M + 02 immunogen (50 ⁇ g of EN-80-2 antigen complexed with
  • the resulting samples were assayed for the presence of antibodies against HCV using methods described above.
  • the assay antigens were MSA and EN-80-2, which had been allowed to form an aggregate in TNEU coating buffer. Rat anti-mouse:HRPO conjugate was used for detection in this assay.
  • the conjugate was a monclonal anti-hIgM:HRPO conjugate, which had been purified by conjugating anti-human immunoglobulin M (anti-hlgM) with horse radish peroxidase (HRPO) using NaIO 4 to obtain the anti-IgM:HRPO conjugate.
  • the conjugate was purified by S-200 gel filtration and was diluted in a sample diluent (0. IM Tris-HCl pH: 7.4 ⁇ 0.2, 40% NBBS, 1% BSA, 2% mouse serum).
  • the procedure was modified in that the samples were diluted by putting 20 ⁇ l of sample into a test tube, and then adding 1.0 ml of sample diluent (0.1M Tris-HCl ph: 7.4 ⁇ 0.2, 40% NBBS, 1% BSA and 2% mouse serum). Next, 200 ⁇ l of the sample diluent and 10 ⁇ l of the diluted sample were added into each well of a treated microtiter plate, with some wells retained as blanks. In part (D) procedure of Example 1, lOO ⁇ l of anti-human IgM:HRPO conjugate solution was added to each well instead of the anti-human IgG:HRPO conjugate solution.
  • Table 21 indicates that IgG anti-HSA antibody was found in HBV positive, HCV negative samples.
  • Example 1 Four assays in accordance with the assay set forth in Example 1 were performed on random human serum samples.
  • the samples comprised the following groups: Group 1 : Anti-HCV positive, HBsAg positive 2 samples
  • Example 2 An assay in accordance with the assays set forth in Example 1 was performed on sample N1029, which is HBV positive and HCV negative.
  • the IgG for the assay was human and purified from sample N1029 by using 33% saturated ammonium sulfate and a PROTEIN A affinity column (Pharmacia Biotech). The results are set forth below in Table 24.
  • An assay was performed as set forth in Example 4, except that an antigen from the HTLV-l envelope region was mixed with a synthetic peptide in TNEU buffer to provide a complex of the synthetic peptide and the HTLV-l env antigen.
  • the sequences of synthetic peptides were as follows.
  • SP1 NH2-VEVSRSLGKVGTR-COOH (SEQ ID No._ , derived from Bovine serum albumin.
  • SP2 NH2-VEVSRNLGKVGSK-COOH (SEQ ID No._ j, derived from Human serum albumin.
  • SP3 NH2-VEAARNLGRVGTK-COOH (SEQ ID No, j, derived from Rat serum albumin.
  • SP4 NH2-RRRSRNLGKVIDT-COOH (SEQ ID No, _), derived from HCV serum albumin. Human serum albumin was also used in this assay as a control.
  • the samples for the assay were as follows: Anti-HTLV-1 positive samples were SP and POOL. Anti-HTLV-1 negative samples were N522 and N530.

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Abstract

Procédés et compositions améliorés servant à effectuer la détection et le diagnostic de maladies au moyen d'un complexe constitué par de l'albumine sérique et un antigène ciblé, ce qui permet d'obtenir un antigène ciblé dont la conformation a été modifiée. Anticorps comprenant à la fois des anticorps d'IgM et de IgG, ainsi que toutes les autres formes d'anticorps, spécifiques pour l'albumine sérique ou pour les antigènes ciblés à conformation modifiée, ainsi que procédés servant à purifier chacun au moyen de méthodes immunologiques, telles que des colonnes d'affinité. Procédés et compositions améliorés contenant un antigène ciblé mis en complexe avec une albumine sérique afin de provoquer une réaction immune chez un animal, ainsi que vaccination améliorée de cet animal. De plus, tampon préféré, TNSCN, composé de Tris-HCl, NaCl et NaSCN, normalement à un pH d'environ 7,4+/-0,2.
PCT/US1997/018194 1996-10-18 1997-10-17 Dispositifs et procedes a base d'un complexe constitue par de l'albumine serique et un antigene cible WO1998018006A1 (fr)

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JP10519417A JP2001502803A (ja) 1996-10-18 1997-10-17 血清アルブミン―標的抗原複合体を含むデバイスおよび方法
AU48964/97A AU4896497A (en) 1996-10-18 1997-10-17 Devices and methods comprising a serum albumin - target antigen complex
EP97911646A EP0939901A1 (fr) 1996-10-18 1997-10-17 Dispositifs et procedes a base d'un complexe constitue par de l'albumine serique et un antigene cible

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WO2004063371A1 (fr) * 2003-01-10 2004-07-29 Protein Crystal Co., Ltd. Complexe de proteines, processus de fabrication et d'utilisation dudit complexe

Citations (1)

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Publication number Priority date Publication date Assignee Title
JPH03156369A (ja) * 1989-11-14 1991-07-04 Tosoh Corp ヒトアルブミンの測定方法

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Publication number Priority date Publication date Assignee Title
JPH03156369A (ja) * 1989-11-14 1991-07-04 Tosoh Corp ヒトアルブミンの測定方法

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Title
CHEMICAL ABSTRACTS, vol. 120, no. 25, 20 June 1994, Columbus, Ohio, US; abstract no. 321040, XP002055507 *
D.C. PHIPPS ET AL.: "An ELISA employing a Haemophilus influenzae type b oligosaccharide-human serum albumin conjugate correlates with the radioantigen binding assy.", JOURNAL OF IMMUNOLOGICAL METHODS, vol. 135, no. 1-2, 1990, AMSTERDAM NL, pages 121 - 128, XP002055506 *
N. KOBAYASHI ET AL.: "Specificity of the polyclonal antibodies raised against a novel 25-hydroxyvitamin d3-bovine serum albimin conjugate linked through the c-11 alpha position.", JOURNAL OF STEROID BIOCHEMISTRY AND MOLECULAR BIOLOGY, vol. 48, no. 5-6, 1994, NEW YORK NY USA, pages 567 - 572 *
PATENT ABSTRACTS OF JAPAN vol. 15, no. 391 (P - 1259) 3 October 1991 (1991-10-03) *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004063371A1 (fr) * 2003-01-10 2004-07-29 Protein Crystal Co., Ltd. Complexe de proteines, processus de fabrication et d'utilisation dudit complexe
JPWO2004063371A1 (ja) * 2003-01-10 2006-06-01 株式会社プロテインクリスタル タンパク質複合体及びその製造方法並びにその用途
CN1323164C (zh) * 2003-01-10 2007-06-27 株式会社蛋白质晶体 蛋白质复合物及其制备方法以及其用途
JP5253715B2 (ja) * 2003-01-10 2013-07-31 株式会社プロテインクリスタル タンパク質複合体及びその製造方法並びにその用途

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