WO2009020628A1 - Diagnostic d'infection par le virus de l'herpès associé au sarcome de kaposi (kshv) - Google Patents

Diagnostic d'infection par le virus de l'herpès associé au sarcome de kaposi (kshv) Download PDF

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WO2009020628A1
WO2009020628A1 PCT/US2008/009466 US2008009466W WO2009020628A1 WO 2009020628 A1 WO2009020628 A1 WO 2009020628A1 US 2008009466 W US2008009466 W US 2008009466W WO 2009020628 A1 WO2009020628 A1 WO 2009020628A1
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kshv
lanal
protein
antibodies
antigen
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PCT/US2008/009466
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Michael Houghton
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Epiphany Biosciences, Inc.
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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
    • G01N33/56994—Herpetoviridae, e.g. cytomegalovirus, Epstein-Barr virus
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • 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
    • G01N2333/01—DNA viruses
    • G01N2333/03—Herpetoviridae, e.g. pseudorabies virus

Definitions

  • the present invention relates to methods and compositions for diagnosis of
  • KSHV KSHV.
  • this invention relates to production of KSHV viral proteins and peptides in mammalian cells, purification of KSHV viral proteins and polypeptides from KSHV- infected cells in culture and their utility in detecting serum antibodies.
  • Kaposi's sarcoma is the most common cancer associated with the acquired immunodeficiency syndrome (AIDS).
  • Classic Kaposi's sarcoma is a relatively benign and indolent disease usually seen in older, heterosexual, HIV-negative men of Jewish, Mediterranean or Eastern European descent and typically involves only the skin.
  • AIDS-related Kaposi's sarcoma is rapidly progressive and almost always develops into disseminated disease.
  • Immunosuppressed individuals who have received organ transplants are also at an increased risk of Kaposi's sarcoma that can be aggressive and spread beyond the skin to the mucous membranes or other organs.
  • Kaposi's sarcoma is also endemic in Equatorial Africa, where the disease is similar to the classic form although it usually develops at a younger age.
  • Kaposi's sarcoma is caused by infection with Kaposi's sarcoma-associated herpesvirus (KSHV), which is also known as human herpesvirus-8.
  • KSHV infection also causes multicentric Castleman's disease and primary effusion lymphoma. Recent reports have shown that KSHV is present in the blood supply and that it can be transmitted by transfusion. In addition, it can be transmitted from organ-transplant donors to recipients.
  • KSHV The genome of KSHV consists of a 140.5 kilobase long unique coding region flanked by approximately 800 base pairs of tandem repeat noncoding sequence. It is divided into blocks of highly conserved structural and lytic replication genes, similar to those in related herpesviruses, interspersed between blocks showing little or no similarity to other herpesvirus genes (see reference 9). It is predicted to have at least 85 orfs, including those encoding homologues of cellular genes. Like other herpesviruses, KSHV establishes latent infection and can persist as an episomal DNA molecule expressing only a few viral genes, including LANAl encoded by orf 73.
  • orf K8.1 codes for two immunogenic envelope glycoproteins that originate from spliced messages.
  • One of the RNAs from orf K8.1 encodes a unique stretch of 61 amino acids called gp35-37.
  • Kaposi's sarcoma has four major forms: classic, epidemic, immunosuppressive treatment-related and African (10-13).
  • Classic Kaposi's sarcoma is usually seen in older, heterosexual, HIV -negative men.
  • the usual onset of classic Kaposi's sarcoma is at 50 to 70 years of age. It most often has a relatively benign, indolent course, with slow enlargement of the original tumors mostly restricted to skin and gradual development of additional lesions.
  • the epidemic form of Kaposi's sarcoma arose in association with the AIDS epidemic and occurs predominantly in gay or bisexual men.
  • Kaposi's sarcoma in patients with AIDS is much more aggressive than the classic type with multifocal, widespread lesions at the onset of the illness.
  • KSHV infection is also a potential cause of serious illness in organ transplant recipients. Therefore, reduction in the transmission of KSHV would lead to significantly decreased morbidity and mortality, primarily in individuals with AIDS and in organ transplant recipients receiving immunosuppressive treatment.
  • KSHV is also likely transmitted by needle sharing among users of illicit injection drugs (24,29).
  • Kaposi's sarcoma after organ transplantation can occur by two mechanisms: reactivation of latent infection in individuals who were infected prior to receiving immunosuppression and contamination of an allograft with KSHV. While reactivation of latent infection appears to be more common, there are data clearly demonstrating transmissions of KSHV from organ transplant donors to recipients (30-32). Finally, there is strong evidence that KSHV can be transmitted by blood transfusion (33,34).
  • IFAs immunofluorescence assays
  • EIAs enzyme immunoassays
  • individual antibody- capture antigens such as recombinant nucleocapsid (encoded by orf 65), recombinant envelope protein encoded by K8.1 and recombinant latency-associated nuclear antigen (LANAl) encoded by orf 73 (40).
  • LANAl recombinant latency-associated nuclear antigen
  • Semi-purified virions derived from lytically infected cell cultures have also been used as a source of capture antigens for specific antibodies circulating in individuals infected with KSHV (Advanced Biotechnologies, Inc., Columbia, MD). Assays based on these methods have been informative in estimating prevalence and incidence of KSHV infection and its association with Kaposi's sarcoma and related lymphoproliferative disorders in selected cohorts and, importantly, in documenting transmission of virus from American and African blood donors (33,34). However, it is generally agreed that there is still an urgent need to develop a high- throughput assay measuring circulating antibodies to both lytic and latent KSHV antigens with greater sensitivity and specificity, particularly when applied to screening the general population (34-44).
  • orf 65 has been shown to encode B cell epitopes that cross-react with other herpesviruses and to ensure specificity, a synthetic peptide encompassing a major immunodominant epitope has generally been used to detect KSHV-specific antibodies (45).
  • a spliced mRNA from orf K8.1 is known to encode the highly immunodominant envelope glycoprotein gp35-37 but, in past diagnostic use, recombinant antigen has been derived from bacteria and purified under denaturing conditions yielding a non-native antigen (21). Such non-native antigen is likely to be less immunoreactive than appropriately glycosylated protein purified from mammalian cells.
  • recombinant LANAl was originally derived from bacteria but greater efficiency at capturing specific antibodies has been observed when it was derived from insect cells producing protein in a more native conformation (46, 47).
  • KSHV KSHV with appropriate specificity and sensitivity for screening the blood supply, organ donors and organ transplant recipients.
  • this invention provides a sensitive and specific serological assay for the detection of KSHV in a biological sample comprising (1) expressing K8.1 and LANAl in mammalian cells; (2) purifying K8.1 and LANAl proteins under non-denaturing conditions; (3) using the purified proteins herein in EIAs to detect KSHV-specific antibodies present in the biological sample.
  • This assay can be used in a high-throughput manner for purposes such as screening the blood supply.
  • antigens are derived from orf K8.1 , a spliced mRNA encoding the virion envelope glycoprotein gp35-37, and orf 73, a major latency antigen (LANAl) found in the nucleus of latently infected cells.
  • LANAl major latency antigen
  • K8.1 and LANAl produced in mammalian cells and purified under non-denaturing conditions are vastly superior to previously used recombinant antigens and are key ingredients of high sensitivity and specificity assays to detect KSHV infection.
  • this invention provides a method for producing native K8.1 antigen, said method comprising expressing K8.1 in mammalian cells and purifying K8.1 protein under non-denaturing conditions.
  • this invention provides a method for producing native
  • LANAl antigen said method comprising expressing LANAl in mammalian cells and purifying LANAl protein under non-denaturing conditions.
  • the K8.1 and LANAl antigens produced using the methods of this invention can be used in combination, optionally with other polypeptides derived from KSHV, for example, a nucleocapisd protein encoded by orf 65, to provide a high-throughput assay of high sensitivity and specificity that can be used to screen the general population, including the blood supply, for KSHV infection.
  • other polypeptides derived from KSHV for example, a nucleocapisd protein encoded by orf 65
  • KSHV Sarcoma-associated Herpes Virus
  • provided herein is a method for producing native K8.1 antigen, said method comprising expressing K8.1 in mammalian cells and purifying K8.1 protein under non-denaturing conditions.
  • a method for producing native LANAl antigen said methods comprising expressing LANAl in mammalian cells and purifying LANAl protein under non-denaturing conditions.
  • KSHV antibodies said composition comprising 1) a K8.1 antigen produced using a method comprising expressing K8.1 in mammalian cells and purifying K8.1 protein under non- denaturing conditions; 2) a LANAl antigen produced using a method comprising expressing LANAl in mammalian cells and purifying LANAl protein under non-denaturing conditions; and optionally 3) one or more polypeptide(s) derived from KSHV.
  • the optional polypeptide(s) derived from KSHV is a nucleocapsid protein encoded by orf65.
  • a method detecting KSHV-specific antibodies comprising: 1) incubating a biological sample suspected of containing KSHV- specific antibodies with isolated K8.1 and isolated LANA 1; and 2) detecting antibody- antigen complexes formed thereby; wherein the K8.1 and LANAl have been isolated from KSHV-infected cells under non-denaturing conditions.
  • KSHV antibodies said composition comprising: 1) native K8.1 protein isolated from KSHV- infected cells under non-denaturing conditions; 2) native LANAl protein isolated from KSHV-infected cells under non-denaturing conditions; and optionally 3) one or more polypeptide(s) derived from KSHV.
  • the optional polypeptide(s) derived from KSHV is a nucleocapsid protein encoded by orf65.
  • SEQ ID NO: 1 is the DNA sequence of the open reading frame encoding the full-length K8.1 protein of KSHV, with a poly-Histidine tag added to the carboxyl-terminus.
  • SEQ ID NO:2 is the amino acid sequence of the full-length K8.1 protein of
  • SEQ ID NO:3 is the DNA sequence of an open reading frame encoding a truncated version of the K8.1 protein of KSHV, lacking 34 amino acids at the carboxyl- terminus, with a poly-Histidine tag added to the carboxyl-terminus.
  • SEQ ID NO:4 is the amino acid sequence of a truncated version of the K8.1 protein of KSHV, lacking 34 amino acids at the carboxyl-terminus, with a poly-Histidine tag added to the carboxyl-terminus.
  • SEQ ID NO:5 is the DNA sequence of an open reading frame encoding the full-length LANAl protein of KSHV, with a poly-Histidine tag added to the carboxyl- terminus.
  • SEQ ID NO:6 is the amino acid sequence of the full-length LANAl protein of
  • SEQ ID NO:7 is the 17-base pair core LANAl binding sequence (LBS). DETAILED DESCRIPTION OF THE INVENTION
  • This invention is directed toward a highly sensitive and specific EIA to detect antibodies against KSHV in blood from individuals infected with the virus.
  • an assay should contain antigens expressed in both lytically and latently infected cells.
  • KSHV polypeptides K8.1 (gp35-37) and nucleocapsid, which are expressed in lytically infected cells, have been shown to be useful in capturing antibodies from serum samples of individuals infected with the virus (45). As other herpesviruses do not have K8.1 homologues, this antigen also provides specificity in detecting KSHV.
  • LANAl which is expressed in nuclei and is the most prominently detected latent antigen, is frequently detected by immunofluorescence microscopy and immunoblot by antibodies from individuals infected with KSHV.
  • LANAl produced in insect cells has greater sensitivity in detecting antibodies in sera of subjects infected with KSHV than protein produced in bacteria; however, even insect cell derived LANAl has suboptimal sensitivity in discriminating sera from subjects infected with KSHV compared to controls (45-47). Therefore, we expect that K8.1 and LANAl produced in mammalian cells and purified under non-denaturing conditions would be vastly superior to previously used recombinant antigens to detect serum antibodies against KSHV.
  • a method for producing native K8.1 antigen comprising expressing K8.1 in mammalian cells and purifying K8.1 protein under non-denaturing conditions.
  • Full-length K8.1 or truncated protein can be used.
  • Full-length K8.1 typically has the sequence of the naturally occurring protein (residues 1-228). In some embodiments a variant is used that differs from the naturally occurring protein by virture of a small number of amino acid substitutions (in some embodiments fewer than 5 substitutions).
  • truncated K8.1 lacks some or all of the carboxyl -terminal transmembrane domain of the full-length protein. In one embodiment the truncated K8.1 comprises amino acids 1-196 of the full-length protein.
  • DNA fragments encoding full-length K8.1 and the protein lacking its carboxyl-terminal transmembrane segment are cloned into pBlueScript vector and then sequenced. DNA fragments contain coding sequences at their 3 ' ends so that hexahistidine tags are at the carboxyl-termini to facilitate characterization and purification.
  • In vitro transcription-translation in reticulocyte lysates with and without microsomal membranes can be performed to confirm the molecular masses of the encoded proteins.
  • DNA coding regions are inserted into a mammalian expression vector such as pCMVIII for expression in transfected COS7 or 293T cells.
  • a mammalian expression vector such as pCMVIII for expression in transfected COS7 or 293T cells.
  • Cells are maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum at 37°C and 5% carbon dioxide in air.
  • Cells are transfected with expression plasmids by use of a transfection kit (51,52).
  • Expression yields of full-length K8.1 and the truncated form lacking the transmembrane segment can be compared.
  • the full-length protein remains cell-associated in the endoplasmic reticulum, Golgi or plasma membrane while the truncated protein is secreted into the culture medium.
  • Glycosylation of full-length and truncated K8.1 glycoprotein can be confirmed by digestion with glycosylases that selectively remove carbohydrate residues with different linkages. Digestion can be monitored by SDS-polyacrylamide electrophoresis followed by immunoblotting with anti-histidine monoclonal antibody. Purification of K8.1 or Truncated K8.1 under Non-denaturing Conditions
  • cells are washed with ice-cold phosphate-buffered saline and lysed by homogenization or sonication in hypotonic buffer, such as 20 mM 3- morpholinopropanesulfonic acid [pH 7.4], 10 mM NaCl, 1.5 mM MgCl 2 , and 1% Triton X- 100, containing protease inhibitors. DNAse and RNAse are optionally added. After incubation on ice, the debris is cleared by centrifugation. The ionic strength and detergent type/concentration of the solution are adjusted then for subsequent purification steps (see below). For secreted truncated polypeptide, media is collected after low-speed centrifugation of cell cultures and concentrated by ultrafiltration. This solution is diluted into buffers of appropriate ionic strength and with appropriate detergents for subsequent purification steps (see below).
  • hypotonic buffer such as 20 mM 3- morpholinopropanesulfonic acid [pH 7.4], 10
  • NTA Ni- nitrilotriacetic acid
  • Ni-NTA Magnetic Agarose Beads Ni-NTA Magnetic Agarose Beads; Qiagen. They are pre-charged with nickel for capturing hexahistidine-tagged proteins under native or denaturing conditions for small-scale purification.
  • These beads can provide a one- step purification process for native KSHV K8.1 and native truncated K8.1 as illustrated in Qiagen' s Ni-NTA Magnetic Agarose Beads Handbook (Second Edition) .
  • the purification involves capture of the polypeptide with a hexahistadine tag from a cell lysate or a protein solution by binding to nickel followed by washing. Protein-bound Ni-NTA magnetic beads are collected from the lysate or solution by attracting them to the side of a vessel, including 96 well plates, after placing near a magnet for 30-60 seconds. Hexahistidine-tagged protein can be eluted from the beads using imidazole or alternatively retained on beads.
  • Biochemical properties of proteins are dependent on the individual protein, and an optimal protocol for purification of hexahistidine-tagged K8.1 and truncated K8.1 proteins under native conditions will be determined empirically.
  • Some general guidelines to optimize the purification procedure include addition of low concentrations of imidazole to lysis and binding buffers to inhibit binding of non-tagged contaminating proteins. According to the manufacturer, for most hexahistidine-tagged proteins, up to 20 mM imidazole can be used without affecting the binding properties.
  • To inhibit protein degradation cells and buffers are kept at 0— 4 0 C at all times and a cocktail of protease inhibitors added to lysis and binding buffers as may be necessary.
  • All buffers are of sufficient ionic strength to prevent nonspecific interactions between proteins and the Ni-NTA matrix.
  • the minimum salt concentration during binding and washing steps is about 300 mM NaCl and increased as may be necessary.
  • Low concentrations of a non-ionic detergent, such as Tween 20, can also be included in binding and washing buffers.
  • protein retained on beads can be directly collected using a magnet on 96-well plates.
  • protein can be eluted from the beads using higher imidazole concentrations and subjected to further purification steps. Protein captured by the Ni-NTA beads can be analyzed for purity and lack of degradation by Coomassie blue staining of SDS-polyacrylamide slab gels.
  • Ni-NTA Magnetic Agarose Beads have a binding capacity of 300 micrograms of protein per milliliter of suspension for hexahistidine-tagged dihydrofolate reductase (24 kDa). Adjusting the amount of Ni-NTA Magnetic Agarose Beads and therefore the binding capacity allows flexible choice of the amount of tagged protein captured. The protocols involving these magnetic beads are easily fully automated using Qiagen BioRobo Systems. In addition, the QIAexpress range of Ni-NTA matrices offers purification products for protein quantities up to large-scale assay production.
  • a method for producing native K8.1 protein comprising isolating intact KSHV virions from lytic cultures in vitro and purifying native K8.1 protein under non-denaturing conditions.
  • Infectious KSHV virions can isolated from lytic cultures in vitro, for example using the BCBL-I cell line, a body cavity lymphoma cell line derived from a primary effusion lymphoma latently infected with KSHV.
  • BCBL-I cells can be cultured according to standard procedures known to those skilled in the art. For example, BCBL-I cells can be grown in 2 liter liquid cultures using RPMI- 1640 medium supplemented with 10% (v/v) fetal bovine serum, 2 mM L-glutamine, and penicillin-streptomycin (5U/ml and 5 ⁇ g/ml, respectively).
  • the cultures Once the cultures reach a density between 2 x 10 5 and 3 x 10 5 cells per milliliter (ml), they lytic growth is induced by treatment with both 20 ng/ml of 12-0- tetradecanoyl phorbol 13 -acetate (TPA) and 0.3 mM sodium butyrate for 12 to 18 hours.
  • TPA 12-0- tetradecanoyl phorbol 13 -acetate
  • the cells are then gently pelleted, washed, and transferred to fresh RPMI- 1640 medium supplemented as described herein, and cultured for another 6 to 7 days.
  • the medium is then centrifuged at 600 ⁇ g for five minutes, then at 2,000 ⁇ g for thirty minutes to pellet cells, nuclei, and other debris from the culture medium.
  • Intact KSHV particles are then pelleted from the cleared medium by filtration or ultracentrifugation, for example, at 50,000xg for two hours.
  • the isolated virions are resuspended in an appropriate buffer supplemented with protease inhibitors as necessary.
  • Isolated KSHV particles can then be subject to further purification to obtain full-length native K8.1 protein.
  • biochemical properties of proteins are dependent on the individual protein, and an optimal protocol for purification of native K8.1 protein from intact KSHV virions will be determined empirically. Because the size of native K8.1 is known, a preparation of intact KSHV virions is first solubilized under non-denaturing conditions and fractionated using a size exclusion column spanning the appropriate size range. Fractions containing proteins in the desired size range are combined and concentrated by standard methods, such as ultrafiltration or precipitation, and further purified. Because K8.1 is a glycoprotein, purification on lectin columns can also be attempted. Hydroxyapatite and ion exchange resins can also be used.
  • buffers, columns, and other reagents used in the purification scheme are kept at 0-4 0 C at all times and a cocktail of protease inhibitors added to buffers as may be necessary. All buffers are of sufficient ionic strength to prevent nonspecific interactions between proteins and particular column matrices.
  • the minimum salt concentration during binding and washing steps is about 300 mM NaCl and increased as may be necessary.
  • Low concentrations of a non-ionic detergent, such as Tween 20, can also be included in binding and washing buffers.
  • Standard cloning methods and bacterial expression vectors are used to synthesize KSHV K8.1 and truncated K8.1 in prokaryotes for comparison to proteins synthesized in mammalian cells. Coding DNA fragments are the same as those generated for use in the mammalian expression system. Inoue et al. (49) have published methods for purification of K8.1 from Escherichia coli.
  • the immunoreactivities of native KSHV K8.1 and/or truncated K8.1 polypeptides can be compared with the corresponding forms derived from bacterial expression in EIAs. Briefly, 96 well plates are coated with antigens by standard methods. Blocking solutions contain 5% non-fat milk. Additional wells are coated with irrelevant proteins such as bovine serum albumin as negative control. Serum samples are added at various dilutions, plates washed and secondary enzyme-linked antibodies added against human immunoglobulin and washing repeated. Absorbance can be measured using a microplate reader. Results of EIAs can be compared with standard IFAs using cell cultures lytically infected with KSHV to detect antibodies (39-43).
  • Immunoreactivity can be assessed using well-characterized serum and plasma samples derived from patients with Kaposi's sarcoma infected with KSHV alone or co- infected with HIV-I. Importantly, serum samples from asymptomatic individuals infected with KSHV are assayed also since such individuals are known to have lower titers of anti- KSHV antibodies than individuals with Kaposi's sarcoma. As controls, sample from uninfected blood donors are screened to assess the specificity of diagnosis using these antigens.
  • this invention provides a method for producing native K8.1 or native truncated K8.1 antigen produced in mammalian cells.
  • said method comprises expressing LANAl in mammalian cells and purifying LANAl protein under non-denaturing conditions.
  • LANAl encoded by orf 73 has been shown to play a key role in the latency of
  • KSHV infection It is expressed in all KSHV-associated tumor cells, binds to tumor suppressors p53 and retinoblastoma protein (56) and is responsible for the maintenance of the KSHV episome (57). It also inhibits antigen processing when in cis and appears to have a similar function to that of EBNA-I encoded by Epstein Barr virus (58).
  • LANAl is of key diagnostic significance since antibodies against it are present in blood of approximately 85% of subjects with Kaposi's sarcoma (59,60). Furthermore, antibodies against LANAl can be present when antibodies to lytic antigens are absent (45). LANAl has also been shown to be the main immunoreactive component of latent- IFA cell-based assays (60).
  • the sensitivity of immunoblots of bacterial-derived LANAl in detecting patient antibodies is low (47).
  • an improvement in sensitivity has been obtained by expression in insect cells, possibly reflecting a more native product (46).
  • the recombinant LANAl produced in mammalian cells and purified under non-denaturing conditions according the methods of invention is a more immunoreactive antigen than that derived from insect cells.
  • LANAl as a hexahistidine-tagged protein in transfected cells are the same as those described above for K8.1.
  • Constructs to express LANAl are generated by standard methods using polymerase chain reaction and KSHV template DNA (50). Inoue et al. (49) have provided sequences of polymerase chain reaction primers for amplification of DNA encoding LANAl. DNA fragments encoding LANAl are cloned into pBlueScript vector and then sequenced. In vitro transcription- translation in reticulocyte lysates can be performed to confirm the molecular mass of the encoded proteins.
  • the DNA coding region is inserted then into a mammalian expression vector such as pCMVIII for expression in transfected COS7 or 293T cells.
  • a mammalian expression vector such as pCMVIII for expression in transfected COS7 or 293T cells.
  • Cells are maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum at 37 0 C and 5% carbon dioxide in air.
  • Cells are transfected with expression plasmids by use of a transfection kit (51,52).
  • LANAl is normally a nuclear protein
  • immunofluorescence microscopy can be performed using antibodies against hexahistidine on cultured cells to confirm a nuclear localization of the protein encoded by the construct.
  • Cells are lysed as described above. Similarly, initial purification of histidine- tagged protein is carried out using Ni-NTA Magnetic Agarose Beads. Optimal purification conditions can be established empirically following the general guidelines described above. When purification on Ni-NTA agarose alone does not generate sufficiently pure protein for EIA, additional purification steps, such as hydroxyapatite and ion exchange chromatography are necessary.
  • a method for producing native LANAl protein comprising preparing nuclear extracts from KSHV-infected cells cultured in vitro and purifying LANAl protein under non-denaturing conditions.
  • LANAl is a DNA-binding protein
  • native LANAl can be isolated from nuclear extracts prepared from latently infected cells grown in culture, using, for example KSHV-positive primary effusion lymphoma cell lines such as BC-3 or BCBL-I.
  • BC-3 and BCBL-I cells can be cultured according to standard procedures known to those skilled in the art. For example, BC-3 and BCBL-I cells are grown in 2 liter liquid cultures using RPMI- 1640 medium supplemented with 10% (v/v) fetal bovine serum, 2 mM L-glutamine, and penicillin-streptomycin (5U/ml and 5 ⁇ g/ml, respectively).
  • the cells are collected by centrifugation and washed with cold phosphate- buffered saline (PBS).
  • PBS cold phosphate- buffered saline
  • the cells are then resuspended in Buffer A (10 mM HEPES, 10 mM KCl, 1.5 mM MgCl 2 , 5 mM dithiothreitol (DTT), 0.5 mM phenylmethylsulfonyl fluoride (PMSF), and 10 ⁇ g/ml aprotinin), incubated on ice for an hour, and gently homogenized in a Dounce homogenizer.
  • Buffer A 10 mM HEPES, 10 mM KCl, 1.5 mM MgCl 2 , 5 mM dithiothreitol (DTT), 0.5 mM phenylmethylsulfonyl fluoride (PMSF), and 10 ⁇ g/ml aprotinin
  • the cell homogenate is then centrifuged to collect intact nuclei, washed once with Buffer A, and resuspended in Buffer B (20 mM HEPES, 10% (v/v) glycerol, 420 mM NaCl, 1.5 mM MgCl 2 , 5 mM DTT, 0.5 mM PMSF, and 10 ⁇ g/ml aprotinin). After thirty minutes of incubation on ice, nuclear debris is removed by centrifugation at high speed.
  • the resulting supernatant contains a mixture of nuclear protein, and is resuspended in an equal volume of Buffer C (20 mM HEPES, 0.2 mM EDTA, 5 mM DTT, 0.5 mM PMSF, and 10 ⁇ g/ml aprotinin). Protein concentration in the extract is determined by Bradford assay or other appropriate method known to those skilled in the art.
  • Native LANAl protein is then purified from the nuclear extract, for example, by DNA affinity chromatography.
  • DNA oligonucleotides containing the 17- base pair core LANAl binding sequence (LBS) (SEQ ID NO:7) are ligated to cyanogen bromide (CNBr)-activated Sepharose beads according to the manufacturer's instructions, and the LBS-Sepharose beads loaded into a column.
  • LBS 17- base pair core LANAl binding sequence
  • CNBr cyanogen bromide
  • the nuclear extracts are diluted in an equal volume of binding buffer plus 0.1% (w/v) BSA, 0.4 mg/ml single-stranded sonicated calf thymus DNA, and 10 ⁇ g/ml poly (dl-dC) and loaded on the column.
  • the column is allowed to stand for thirty minutes at 4°C and then passed through the affinity resin by gravity flow.
  • the resin is washed three times with binding buffer to eliminate non-specific protein binding to the LBS-Sepharose.
  • Bound LANAl is then eluted with binding buffer supplemented with a high concentration of NaCl (e.g., between 500 and 1000 mM NaCl).
  • a high concentration of NaCl e.g., between 500 and 1000 mM NaCl.
  • Other suitable methods for purifying LANAl from nuclear extracts can be used as necessary.
  • buffers, columns, and other reagents used in the purification scheme are kept at 0 ⁇ °C at all times and a cocktail of protease inhibitors added to buffers as may be necessary. All buffers are of sufficient ionic strength to prevent nonspecific interactions between proteins and particular column matrices. Low concentrations of a non-ionic detergent, such as Tween 20, can also be included in binding and washing buffers.
  • a non-ionic detergent such as Tween 20
  • Standard cloning methods and baculovirus expression vectors are used to express LANAl in insect cells for comparison to protein synthesized in mammalian cells.
  • the coding DNA fragment is the same as that used in the mammalian expression system.
  • Inoue et al. (49) have published methods for expression of LANAl in insect cells. Immunoreactivity of Native Antigen Compared to Antigen Purified from Insect Cells and Other Assays
  • the immunoreactivity of native KSHV LANAl can be compared with the corresponding form derived from insect cells in EIAs. Briefly, 96 well plates are coated with antigens and blocked by standard methods. Serum samples are added at various dilutions, plates washed and secondary enzyme-linked antibodies added against human immunoglobulin and washing repeated. Absorbance can be measured using a microplate reader. Results of EIAs are compared also with standard IFA using latently infected cell cultures treated with phorbol-12-myristate- 13 -acetate to detect anti-LAN Al antibodies (39- 43).
  • Immunoreactivity can be assessed using well-characterized serum and plasma samples as well as control samples from blood donors as described above.
  • EIAs to analyze defined serum samples from individuals with Kaposi's sarcoma infected with KSHV (many are infected also with HIV), asymptomatic carriers infected with KSHV but without a diagnosis of Kaposi's sarcoma and uninfected controls.
  • the sensitivities and specificities of these antigens can be compared to those for antigens expressed in bacterial and insect cells to show superiority.
  • this invention provides a sensitive and specific serological assay for the detection of KSHV in a biological sample comprising (1) expressing K8.1 and LANAl in mammalian cells; (2) purifying K8.1 and LANAl proteins under non- denaturing conditions; (3) using the purified proteins herein in EIAs to detect KSHV-specif ⁇ c antibodies present in the biological sample.
  • This assay can be used in a high-throughput manner for purposes such as screening the blood supply.
  • K8.1 and LANAl antigens produced using the methods of this invention can be used in combination, optionally with other polypeptides derived from KSHV, for example, a nucleocapisd protein encoded by orf 65, to provide a high-throughput assay of high sensitivity and specificity that can be used to screen the general population, including the blood supply, for KSHV infection.
  • the antigens produced according to methods described herein may be used in any of a variety of immunoassay methods to analyze biological samples (e.g., blood, serum, and the like) from individuals with Kaposi's sarcoma infected with KSHV (many are infected also with HIV), asymptomatic carriers infected with KSHV but not diagnosed with Kaposi's sarcoma, and uninfected controls.
  • biological samples e.g., blood, serum, and the like
  • Immunoassay methods that can be used to analyze biological samples include Western blots, enzyme-labeled and enzyme-mediated immunoassays, such as enzyme-linked immunosorbent assays (ELISAs), biotin/avidin type assays, antigen sandwich assays, antibody sandwich assays, antigen/antibody combination assays, radioimmunoassay, immunoelectrophoresis, immunoprecipitation, and the like.
  • the assay mixtures generally include detectable labels such as fluorescent, chemiluminescent, radioactive, enzymatic labels or dye molecules, but may also use other methods for detecting the formation of a complex between the antigen and the antibody or antibodies reacted therewith.
  • the aforementioned assays may involve separation of unbound antibody or antigen in a liquid phase from a solid phase support to which antigen-antibody complexes are bound.
  • Solid supports that can be used in such immunoassays include substrates such as nitrocellulose (e.g., in membrane or microtiter well form), polyvinylchloride (e.g., sheets or microtiter wells), polystyrene latex (e.g., beads or microtiter plates), polyvinylidine fluoride, diazotized paper, nylon membranes, activated beads, magnetically responsive beads, and the like.
  • the native immunogenic KSHV polypeptides and compositions described herein can be used for capture or detection or both of anti-KSHV antibodies in a biological sample.
  • capture of an analyte (i.e., anti- KSHV antibodies in a sample) refers to the fact that the analyte can be separated from other components of the biological sample by virtue of its specific binding to the capture molecule.
  • the capture molecule is directly or indirectly associated with a solid support.
  • the detection molecule is directly or indirectly associated with a detectable label.
  • a solid support is first reacted with a solid phase component (e.g., one or more native immunogenic KSHV polypeptides, such as full-length K8.1 polypeptide, C-terminal truncated K8.1 polypeptide, full-length LANAl polypeptide, or a mixture thereof) under suitable binding conditions so that the component is sufficiently immobilized to the solid support.
  • a solid phase component e.g., one or more native immunogenic KSHV polypeptides, such as full-length K8.1 polypeptide, C-terminal truncated K8.1 polypeptide, full-length LANAl polypeptide, or a mixture thereof
  • Immobilization to the solid support can be enhanced if necessary by first coupling the solid phase component (e.g., one or more native immunogenic KSHV polypeptides, such as full-length K8.1 polypeptide, C-terminal truncated K8.1 polypeptide, full-length LANAl polypeptide, or a mixture thereof) to a protein with better binding properties for coupling to the desired solid support.
  • suitable coupling proteins include, but are not limited to, serum albumins, including bovine serum albumin (BSA), keyhole limpet hemocyanin (KLH), immunoglobulin molecules, thyroglobulin, ovalbumin, and other proteins known to those skilled in the art.
  • Other molecules that can be used to bind the antigen or antibody to the solid support include polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and the like. Such molecules and methods of coupling proteins to those molecules are known in the art. See, e.g., Brinkley, M. A., BIOCONJUGATE CHEM. (1992) 3:2- 13; Hashida et al., J. APPL. BIOCHEM. (1984) 6:56-63; and Anjaneyulu and Staros, INT'L J. PEPT. PROT. RES. (1987) 30:117-124.
  • any remaining non-immobilized solid-phase component is removed from the support by washing, and the support-bound component is then contacted with a biological sample suspected of containing the analyte component (i.e., anti-KSHV antibodies) under suitable binding conditions.
  • a biological sample suspected of containing the analyte component i.e., anti-KSHV antibodies
  • a secondary binding agent can be added under suitable binding conditions, wherein the secondary binding agent associates selectively with the bound analyte. The presence of the secondary binding agent can then be detected using techniques well known in the art.
  • anti-KSHV antibodies are detected by an ELISA method, according to which the wells of one or more microtiter plates are coated with one or more native immunogenic KSHV polypeptides or compositions (e.g., full-length K8.1 polypeptide, C-terminal truncated K8.1 polypeptide, full-length LANAl polypeptide, or a mixture thereof) prepared by the methods described herein.
  • KSHV polypeptides or compositions e.g., full-length K8.1 polypeptide, C-terminal truncated K8.1 polypeptide, full-length LANAl polypeptide, or a mixture thereof
  • the one or more microtiter plates can be washed to remove unbound antibodies and a detectably-labeled secondary binding agent added.
  • the secondary binding agent is allowed to react with any captured sample, the plate washed and the presence of the secondary binding agent detected using standard methods known to those skilled in the art.
  • anti-KSHV antibodies are detected by an ELISA antigen sandwich format.
  • the solid support e.g., a microtiter plate or an immunoblot strip
  • a native immunogenic KSHV polypeptide such as full- length K8.1 polypeptide, C-terminal truncated K8.1 polypeptide, full-length LANAl polypeptide, or a mixture thereof.
  • the sample is then contacted with the support under conditions that allow anti-KSHV antibodies, if present, to bind the native immunogenic KSHV polypeptide or mixture of polypeptides, thereby forming an antigen/antibody complex.
  • Unbound sample is removed and an enzymatically-labeled secondary binding agent that reacts with the bound antigen/antibody complex, such as an enzymatically-labeled rabbit anti-human IgG antibody, is added.
  • an enzymatically-labeled secondary binding agent that reacts with the bound antigen/antibody complex such as an enzymatically-labeled rabbit anti-human IgG antibody.
  • a number of anti-human immunoglobulin (Ig) antibodies are known in the art which can be readily conjugated to a detectable enzyme label, such as horseradish peroxidase, alkaline phosphatase or urease, using methods known to those skilled in the art.
  • An appropriate enzyme substrate is then used to generate a detectable signal.
  • competitive-type ELISA techniques can be practiced using methods known to those skilled in the art.
  • the native immunogenic KSHV polypeptides of the present invention can be used with any known immunoassay format that employs a native immunogenic KSHV polypeptide or mixture thereof, such as the indirect IgG ELISA described in Johnson et al. (J. CLIN MICROBIOL. 2000 38:1827-1831).
  • Other useful formats include a microsphere immunoassay (Wong et al., J. CLIN. MICROBIOL. 2004 42:65-72) and epitope-blocking ELISA (Blitvich et al., J. CLIN. MICROBIOL. 2003 41 :2676-2679).
  • the immunogenic composition comprising a KSHV heterodimer can be used in an indirect IgG ELISA as follows.
  • An antibody specific for a native immunogenic KSHV polypeptide e.g., full-length K8.1 polypeptide, C-terminal truncated K8.1 polypeptide, or full-length LANAl polypeptide, or a mixture thereof
  • the support is then contacted with the native KSHV antigen-containing composition under conditions that would allow binding to the anti-KSHV antibody bound to the support to form an antibody/antigen complex.
  • Unbound antigen is removed and the support is contacted with a sample to be tested for the presence of human IgG to KSHV under conditions that would allow binding of human anti-KSHV IgG, if present, to the heterodimer in the antibody/antigen complex.
  • the presence of bound anti-KSHV IgG can be detected using a detectably labeled anti-human IgG antibody.
  • ELISAs assay formats described herein are referred to as "ELISAs," though a skilled artisan would recognize that detectable labels other than those that are “enzyme-linked” may be used in certain situations. Other suitable detectable labels are known in the art and described herein. Any of the ELISA assays described herein may be automated or otherwise easily adapted for use in a high-throughput screen.
  • Immunoassays can also be conducted in solution, such that the native immunogenic KSHV antigens and antibodies specific for those molecules form antigen- antibody complexes under precipitating conditions.
  • those antigen- antibody complexes can be detected directly in solution using a labeled secondary binding agent (i. e. , a labeled goat anti-human IgG antibody), as described for ELISAs herein.
  • the secondary binding agents specifically bind analyte molecules (i.e., the anti-KSHV antibodies) and can be detectably labeled with a variety of different chemical groups, such as chromophores, fluorophores, radioisotopes, enzymes, and the like, to permit direct detection of antigen-antibody complexes in solution via spectrophotometry or other appropriate instrumentation. Such methods may also be automated or otherwise easily adapted for use in a high-throughput screen.
  • analyte molecules i.e., the anti-KSHV antibodies
  • chemical groups such as chromophores, fluorophores, radioisotopes, enzymes, and the like
  • the molecules e.g., a native immunogenic KSHV polypeptide (e.g., full-length K8.1 polypeptide, C-terminal truncated K8.1 polypeptide, or full-length LANAl polypeptide, or a mixture thereof) are attached to a solid phase particle (e.g., an agarose bead, a magnetic bead or the like) using coupling techniques known in the art, such as direct or indirect chemical coupling.
  • the coated particle is then contacted with a biological sample containing or thought to contain anti-KSHV antibodies under suitable binding conditions. Cross-linking between bound antibodies causes the formation of complex aggregates which can be precipitated and separated from the sample by washing and/or centrifugation.
  • the reaction mixture can be analyzed to determine the presence or absence of complexes using any number of standard methods, such as those immunodiagnostic methods described above.
  • a strip immunoblot assay is used to detect anti-KSHV antibodies in a biological sample using one or more of the above- described native immunogenic KSHV polypeptides immobilized on the test strip.
  • Preferred native immunogenic KSHV polypeptides include, for example, full-length K8.1 polypeptide, C-terminal truncated K8.1 polypeptide, full-length LANAl polypeptide, or a mixture thereof.
  • SIA techniques combining traditional Western and dot-blotting techniques are known in the art, ej ⁇ , the RIBA ® SIA (Chiron Corp., Emeryville, California). The SIA can be conducted in an antigen sandwich format.
  • one or more native immunogenic KSHV polypeptides such as full-length K8.1 polypeptide, C-terminal truncated K8.1 polypeptide, full-length LANAl polypeptide, or a mixture thereof, and optionally, one or more species- specific antiimmunoglobulin antibodies, such as anti-human IgM antibody, anti-human IgG antibody and/or anti-human IgA antibody, are immobilized in discrete positions, e.g., as bands or dots, on a solid support, particularly a membrane support.
  • the terms “discretely immobilized” or “immobilized in discrete positions” on a solid support refers to various reagents that are immobilized on the support as separate components, in discrete and non-overlapping positions, not mixed, such that reactivity or lack thereof with each of the components present can be assessed individually.
  • a biological sample suspected of containing anti-KSHV antibodies is then reacted with the membrane support. Reactivity in the biological sample is visualized using a labeled secondary binding agent capable of binding anti-KSHV antibodies which themselves are bound to immobilized native immunogenic KSHV polypeptides, in conjunction with a colorimetric enzyme substrate.
  • immunoglobulin molecules from the biological sample which have complexed with the antiimmunoglobulin antibodies immobilized on the test strip can also be bound by the native immunogenic KSHV polypeptide-enzyme conjugate.
  • the assay can be performed manually or automated.
  • Internal controls such as antibodies directed against a native Ig polypeptide, particularly KSHV envelope polypeptides, can also be immobilized on the test strip.
  • One particularly convenient method is to include the same antibody in two separate known positions on the test strip, but in high and low concentrations. These controls will be bound by the labeled antibody used for detection of the sample antibodies.
  • the low concentration control is designed to provide the lower cutoff for a positive versus negative result.
  • the higher concentration control is designed to provide a basis for a highly reactive sample. In this configuration, then, a sample is considered positive only if reactivity is greater than or equal to the low level antibody control band, which can be arbitrarily defined to represent a +1 reactivity.
  • a reactivity equivalent to the high level antibody control band is considered to represent, for example, a reactivity of +3.
  • Reactivity intensity intermediate between the low and high level antibody control bands is considered to be +2, and reactivity stronger than the high level antibody band is considered to be +4.
  • Solid supports which can be used in the practice of the strip immunoblot assays include, but are not limited to, membrane supports derived from a number of primary polymers including cellulose, polyamide (nylon), polyacrylonitrile, polyvinylidene difluoride, polysulfone, polypropylene, polyester, polyethylene and composite resins consisting of combinations or derivatives of the above.
  • supports are derived from cellulose, such as nitrocellulose membranes, as well as nylon membranes.
  • the substrate generally includes the desired membrane supported by an inert plastic or other type of backing.
  • the amount of native immunogenic KSHV polypeptide applied to the membrane varies, depending on the polypeptide in question. Generally, the polypeptide will be applied to the strip in an amount of about 20-500 ng/strip, preferably 50-250 ng/strip, more preferably 75-150 ng/strip. One skilled in the art can readily determine the amount of polypeptide necessary to produce a useful result.
  • the antiimmunoglobulin antibodies such as anti-human IgM antibody, anti-human IgG antibody and/or anti-human IgA antibody, can be present in an amount of about 100 to about 2000 ng/strip, preferably about 200 to about 1000 ng/strip, such as 400-900 ng/strip.
  • the low concentration internal control antibody can be present in an amount of e.g., 25-200 ng, such as 50-150 ng, e.g., 100 ng/strip.
  • the high level control will be present in an amount sufficiently higher to give a highly positive result, such as at 200-500 ng, particularly 250-350 ng, e.g., 300 ng/strip.
  • a detectable enzyme label such as horseradish peroxidase (HRP), glucose oxidase, ⁇ -galactosidase, alkaline phosphatase (AP) and urease, among others, using methods known to those skilled in the art.
  • HRP horseradish peroxidase
  • AP alkaline phosphatase
  • urease urease
  • diagnostic kits comprising one or more KSHV antigens produced according to methods described herein in suitable packaging, for example, full-length K8.1 polypeptide, C-terminal truncated K8.1 polypeptide, full-length LANAl polypeptide, or a mixture thereof, either expressed in mammalian cells and purified under non-denaturing conditions, or isolated from KSHV-infected cells under non-denaturing conditions.
  • suitable packaging for compositions (such as KSHV antigens) described herein is known in the art, and includes, for example, vials (such as sealed vials), vessels, ampoules, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like.
  • the KSHV antigens may be provided in any form suitable for use with the methods described herein, including single- or multiple-use aliquots in an appropriate storage buffer, lyophilized, on wet ice at a temperature of 0-4 0 C, or frozen at -20 0 C or -7O 0 C.
  • the kits provided herein may further comprise additional buffers, including wash solutions, blocking solutions, coupling solutions and the like, as well as labeled secondary binding agents, enzyme substrates, and any other reagent necessary to perform any of the methods described herein.
  • the kits provided herein may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, microtiter plates, or other materials useful in performing any methods described herein.
  • kits may optionally include instructions, generally written instructions, although electronic storage media (e.g., magnetic diskette or optical disk) containing instructions are also acceptable. Those instructions relate to the use of component(s) of the kit in the methods disclosed herein (e.g., methods of detecting KSHV-specific antibodies).
  • the instructions provided with the kit generally include, for example, information describing the individual components of the kit, any additional materials required to perform the disclosed methods, and instructions for using those components and materials to perform an enzyme immunoassay to detect KSHV-specific antibodies, for instance, in a biological sample suspected of containing KSHV-specific antibodies.
  • EXAMPLE 1 DETECTION OF KSHV-SPECIFIC ANTIBODIES BY ELISA
  • a series of microtiter plates is prepared for screening biological samples from patients infected with KSHV by ELISA, each plate adsorbed with (1) full-length K8.1 polypeptide + a polyHistidine tag (SEQ ID NO: 2); (2) C-terminal truncated K8.1 polypeptide + a polyHistidine tag (SEQ ID NO: 4); (3) full-length LANAl polypeptide + a polyHistidine tag (SEQ ID NO: 6); or (4) an equimolar mixture of (2) and (3).
  • the target antigen is adsorbed or covalently linked by standard methods to commercially available microtiter plates in the desired format (e.g., 96-well, 384-well, 1536-well or other desired format).
  • KSHV polypeptide or mixture thereof the wells are washed with a blocking buffer to block non-specific antibody binding and to minimize false positive results. See, e.g., id. at pp. 13-14 (discussing methods and reagents for blocking microtiter plates).
  • the optimum blocking reagent for a particular ELISA is typically chosen empirically, based in part on the type of plate being used, the method of protein adsorption, and the type of protein being used.
  • casein the main protein component of non-fat dry milk
  • caseinate a more soluble version of casein, produced by partial digestion with sodium hydroxide
  • normal serum typically used at concentrations
  • wash buffer to remove any unbound blocking agent.
  • washing reagents are selected for their ability to disrupt low affinity interactions between various reaction components that can affect the ability to detect specific antigen-antibody interactions. See, e.g., id. at pp. 14-15 (discussing methods and reagents for washing microtiter plates). Wash solutions commonly contain a physiological buffer to prevent denaturation of antigens and their cognate antibodies, and to preserve enzyme activity. Buffers such as PBS, Tris-saline, or imidizole-buffered saline at neutral pH are widely used. Specific buffers are typically selected based on the method of detection to be employed in a particular assay.
  • Wash buffers should also include non-ionic detergents such as Tween-20TM, Triton X- 100TM or the like, at concentrations of between 0.01% to 0.05% (v/v), in order to disrupt low-affinity, non-specific interactions between reaction components.
  • non-ionic detergents such as Tween-20TM, Triton X- 100TM or the like, at concentrations of between 0.01% to 0.05% (v/v), in order to disrupt low-affinity, non-specific interactions between reaction components.
  • the adsorbed antigen e.g., (1) full-length K8.1 polypeptide + a polyHistidine tag (SEQ ID NO: 2); (2) C-terminal truncated K8.1 polypeptide + a polyHistidine tag (SEQ ID NO: 4); (3) full-length LANAl polypeptide + a polyHistidine tag (SEQ ID NO: 6); or (4) an equimolar mixture of (2) and (3)
  • a biological sample /. e. , blood or serum obtained from a subject infected with KSHV.
  • the wells are washed, and the presence of autoantibody-autoantigen complexes detected using a secondary antibody labeled with chromogenic (e.g., with horseradish peroxidase and TMB), fluorescent or chemiluminescent (e.g., alkaline phosphatase) means. See, e.g., id. at pp. 15-21 (discussing antibody preparation and use, as well as commonly used detection molecules). The amount of color or fluorescence is measured using a luminometer, a spectrophotometer, or other similar instruments adapted to read microtiter plates in the desired format.
  • chromogenic e.g., with horseradish peroxidase and TMB
  • fluorescent or chemiluminescent e.g., alkaline phosphatase
  • SEQ ID NO: 1 DNA sequence of full-length open reading frame (“ORF") of
  • K8.1 protein from KSHV with poly-Histidine tag (encoding amino acids 1-228+His-tag)]:
  • SEQ ID NO:2 Amino acid sequence of full-length open reading frame
  • SEQ ID NO:3 DNA sequence of C-terminal deletion of K8.1 ORF from
  • SEQ ID NO:4 Amino acid sequence of C-terminal deletion of K8.1 ORF from KSHV with poly-Histidine tag (amino acids 1 to 194 + His-tag)]:
  • SEQ ID NO:5 DNA sequence of full-length ORF of LANAl protein from
  • SEQ ID NO:6 Amino acid sequence of full-length ORF of LANAl protein from KSHV with poly-Histidine tag (amino acids 1-1,129 + His-tag)]: MAPPGMRLRSGRSTGAPLTRGSCRKRNRSPERCDLGDDLHLQPRRKHV ADSVDGRE CGPHTLPIPGSPTVFTSGLPAFVSSPTLPVAPIPSPAPATPLPPPALLPPVTTSSSPIPPSHP VSPGTTDTHSPSPALPPTQSPESSQRPPLSSPTGRPDSSTPMRPPPSQQTTPPHSPTTPPP
  • SEQ ID NO: 7 [DNA sequence of 17-base pair core LANAl binding sequence

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Abstract

L'invention concerne des procédés et des compositions pour le diagnostic du KSHV. Cette invention concerne particulièrement la production de protéines virales et de peptides de KSHV dans des cellules de mammifères, la purification de protéines virales et de polypeptides de KSHV à partir de cellules de culture infectées par le KSHV et leur utilisation pour détecter des anticorps sériques. Les K8.1 et LANAl produits dans des cellules de mammifères et purifiés dans des conditions non-dénaturantes sont extrêmement supérieurs aux antigènes recombinants précédemment utilisés et sont des ingrédients essentiels des essais à sensibilité et spécificité élevées pour détecter l'infection par le KSHV.
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