[go: up one dir, main page]

WO1998029747A1 - Reactifs et procedes permettant de detecter un antigene du virus de l'hepatite gb - Google Patents

Reactifs et procedes permettant de detecter un antigene du virus de l'hepatite gb Download PDF

Info

Publication number
WO1998029747A1
WO1998029747A1 PCT/US1997/023767 US9723767W WO9829747A1 WO 1998029747 A1 WO1998029747 A1 WO 1998029747A1 US 9723767 W US9723767 W US 9723767W WO 9829747 A1 WO9829747 A1 WO 9829747A1
Authority
WO
WIPO (PCT)
Prior art keywords
leu
val
gly
ala
sequence
Prior art date
Application number
PCT/US1997/023767
Other languages
English (en)
Inventor
George J. Dawson
Bruce J. Dille
Robin A. Gutierrez
Isa K. Mushahwar
Original Assignee
Abbott Laboratories
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from PCT/US1995/002118 external-priority patent/WO1995021922A2/fr
Priority claimed from US08/424,550 external-priority patent/US6720166B2/en
Priority claimed from US08/417,629 external-priority patent/US5981172A/en
Priority claimed from US08/473,475 external-priority patent/US5843450A/en
Priority claimed from US08/629,463 external-priority patent/US6156495A/en
Application filed by Abbott Laboratories filed Critical Abbott Laboratories
Publication of WO1998029747A1 publication Critical patent/WO1998029747A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1081—Togaviridae, e.g. flavivirus, rubella virus, hog cholera virus
    • C07K16/109—Hepatitis C virus; Hepatitis G virus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C12Q1/706—Specific hybridization probes for hepatitis
    • C12Q1/707—Specific hybridization probes for hepatitis non-A, non-B Hepatitis, excluding hepatitis D
    • 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
    • G01N33/576—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24211—Hepacivirus, e.g. hepatitis C virus, hepatitis G virus
    • C12N2770/24222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • HGBV HGBV
  • methods such as immunoassays and reagents such antibodies and polypeptides which are useful for detecting the presence, if any, of HGBV antigen in test samples.
  • HGBV-A appears at this time to be of non-human primate source
  • HGBV-C is clearly of human source.
  • the source of HGBV-B is unknown. These viruses are thought to play a role in transmittable hepatitis disease of viral origin.
  • the GB viruses appear to be members of the Flaviviridae family. They possess RNA genomes approximately 9.5 kb in size which contain a single long open reading frame (ORF). Structural and nonst uctural proteins are encoded in the N-terminal one-third and C-terminal two-thirds of the putative viral polyproteins, respectively.
  • GBV-A and GBV-C contain long 5' NTRs that have limited sequence identity al the 5'NTR to each other but no identity to the 5' NTRs GB V- B, HCV or pestiviruses.
  • GBV-A and GBV-C also encode putative envelope proteins that contain relatively lew potential N-linked glycosylation sites. Most strikingly, clearly discernible basic core proteins are not found in the cDNA sequences cloned thus far from these viruses.
  • HGBV antigen detection would comprise not only antibodies, either polyclonal or monclonal, for capture and/or detection of the antigen, but also polypeptides which encode immunogenic epitopes of the HGBV virus.
  • Such methods and reagents could greatly enhance the ability of the medical community to provide a means for determining whether an individual was infected with the GB virus(es). Diagnostic or screening tests developed from these reagents could provide a safer blood and organ supply by helping to eliminate GBV in these blood and organ donations, and could provide a better understanding of the prevalence of HGBV in the population, epidemiology of the disease caused by HGBV and the prognosis of infected individuals. Summary of the Invention
  • the present invention provides a method for determining the presence, if any, of HGBV antigen in a test sample.
  • the method comprises (a) contacting the test sample with an anti-HGBV antibody or fragment thereof, for a time and under conditions sufficient to form a mixture; (b) contacting said mixture with an indicator reagent comprising a antibody or a fragment thereof, which specifically binds to either an HGBV region or which is anti-species to the species of the antibody of step (a) , to which a signal generating compound has been attached, for a time and under conidlions sufficient to form a second mixture; and (c) determining the presence of HGBV antigen present in the test sample by detecting the measurable signal generated by the signal generating compound.
  • the antibodies utilized for step (a) and step (b) can bind specifically to different HGBV epitopes. Further, the antibody of step (a) can be attached to a solid phase prior to performing the method. Further, the amount of HGBV antigen present in the test sample is proportional to the signal generated.
  • the method can comprise step (a) and step (b) being performed simulaneously.
  • the antibodies utilized for step (b) are anti-species to the antibodies of step (a).
  • the antibody of step (a) can be attached to a solid phase prior to performing the method.
  • the amount of HGBV antigen present in the test sample is proportional to the signal generated.
  • the method can comprise step (a) and step (b) being performed simulaneously.
  • the antibody of step (a) is an antibody which specifically binds to Hepatitis GB Virus (HGBV) E2 antigen selected from the group consising of SEQUENCE ID NO:3, SEQUENCE ID NO: 8, SEQUENCE ID NO: 9, SEQUENCE ID NO: 10 and SEQUENCE ID NO: 1 1.
  • the present invention also provides a method for determining the presence, if any, of HGBV antigen in a test sample utilizing an HGBV antigen capture PCR assay.
  • This method comprises (a) contacting the test sample with an anti-HGBV antibody or fragment thereof, for a time and under conditions sufficient to fo ⁇ n a mixture; (b) extracting viral RNA from the mixture of step (a ); (c) amplifying the viral RNA obtained from step (b) by reverse transcriptase PCR using primers specific to the HGBV genome attached to an indicator reagent ; and (d) detecting the presence of viral by detecting a measurable signal generated by the signal generating compound.
  • the antibody of step (a) is attached to a solid phase prior to performing the method.
  • the amount of HGBV antigen present in the test sample is proportional to the signal generated.
  • the antibody of step (a) is an antibody which specifically binds to Hepatitis GB Virus (HGBV) E2 antigen SEQUENCE ID NO:3, SEQUENCE ID NO: 8, SEQUENCE ID NO: 9, SEQUENCE ID NO: 10 and SEQUENCE ID NO: 1 1.
  • the present invention also provides antibodies which are useful in detecting HGBV antigen in test samples. These antibodies can be useful as diagnostic reagents. These antibodies specifically bind to the E2 region of HGBV, and are selected from the group consisting of SEQUENCE ID NO: 3, SEQUENCE ID NO: 8, SEQUENCE ID NO: 9, SEQUENCE ID NO: 10 and SEQUENCE ID NO: 1 1. Test kits useful for detecting hepatitis GB Virus (HGBV) antigen in test samples also are included in the present invention.
  • HGBV hepatitis GB Virus
  • the test kit comprises a container containing an antibody which specifically binds to HGBV E2 antigen, and are selected from the group consisting of SEQUENCE ID NO: 3, SEQUENCE ID NO: 8, SEQUENCE ID NO: 9, SEQUENCE ID NO: 10 and SEQUENCE ID NO: 1 1. These antibodies can be attached to a solid phase.
  • Hepatitis GB Virus or "HGBV”, as used herein, collectively denotes a viral species which causes non-A, non-B, non-C, non-D, non-E hepatitis in man, and attenuated strains or defective interfering particles derived therefrom. This may include acute viral hepatitis transmitted by contaminated foodstuffs, drinking water, and the like; hepatitis due to HGBV transmitted via person to person contact (including sexual transmission, respiratory and parenteral routes) or via intraveneous drug use. The methods as described herein will allow the identification of individuals who have acquired HGBV.
  • the HGBV isolates are specifically referred to as "HGBV-A”, “HGBV-B” and “HGBV-C.”
  • the HGBV genome is comprised of RNA. Analysis of the nucleotide sequence and deduced amino acid sequence of the HGBV reveals that viruses of this group have a genome organization similar to that of the Flaviridae family. Based primarily, but not exclusively, upon similarities in genome organization, the International Committee on the Taxonomy of Viruses has recommended that this family be composed of three genera: Flavivirus, Pestivirus, and the hepatitis C group.
  • HCV-specific ELISAs failed to detect sera containing GB-C sequences in six of eight cases.
  • Second, several human sera that were seronegative for HCV antibodies have been shown to be positive for HCV genomic RNA by a highly sensitive RT- PCR assay (Sugitani, Lancet 339: 1018-1019 (1992). This assay failed to detect HCV RNA in seven of eight sera containing HGB-C sequences (TABLE A).
  • HGBV-C is not a genotype of HCV based on both serologic and molecular assays.
  • HGBV-C is more closely related to I IGBV-A than to any member of the HCV group.
  • sequences of HGBV-C and HGBV-A, while exhibiting an evolutionary distance of 0.42, are not as divergent as HGBV-C is from HGBV-B, which shows an evolutionary distance of 0.92.
  • HGBV-A and HGBV-C may be considered to be members of one subgroup of the GB viruses and GBV-B a member of its own subgroup.
  • the phylogenetic analysis of the helicase sequences from various HCV isolates show that they form a much less diverged group, exhibiting a maximum evolutionary distance of 0.20.
  • a comparison of the HCV group and the HGBV group shows a minimum evolutionary distance between any two sequences from each group of 0.69.
  • the distance values reported hereinabove were used to generate a phylogenic tree.
  • the relatively high degree of divergence among these viruses suggests that the GB viruses are not merely types or subtypes within the hepatitis C group; rather, they constitute their own phyletic group (or groups).
  • amino acid sequence "similarity" and/or 5 “identity” are well-known in the art and include, for example, directly determining the amino acid sequence and comparing it to the sequences provided herein; determining the nucleotide sequence of the genomic material of the putative 11GBV (usually via a cDNA intermediate), and determining the amino acid sequence encoded therein, and comparing the corresponding regions.
  • directly determining the amino acid sequence and comparing it to the sequences provided herein determining the nucleotide sequence of the genomic material of the putative 11GBV (usually via a cDNA intermediate), and determining the amino acid sequence encoded therein, and comparing the corresponding regions.
  • identity is meant the exact match-up of either the nucleotide sequence of HGBV and that of another strain(s) or the amino acid sequence of HGBV and that of another strain(s) at the appropriate place on each genome. Also, in general, by “similarity” is meant the exact match-up of amino acid sequence of HGBV and that of another strain(s) at the appropriate place, where the amino acids are identical or
  • HGBV-A HGBV-B or HGBV-C. It is expected that the overall nucleotide sequence identity of the genomes between 5 HGBV-A, HGBV-B or HGBV-C and a strain of one of these hepatitis GB viruses will be about 45% or greater, since it is now believed that the HGBV strains may be genetically related, preferably about 60% or greater, and more preferably, about 80% or greater.
  • HGBV-A 3() between HGBV-A and a strain of HGBV-A at the amino acid level will be about 35% or greater since it is now believed that the HGBV strains may be genetically related, preferably about 40% or greater, more preferably, about 60% or greater, and even more preferably, about 80% or greater.
  • HGBV strains may be genetically related, preferably about 40% or greater, more preferably, about 60% or greater, and even more preferably, about 80% or greater.
  • HGBV-B and a strain of HGBV-B at the amino acid level will be about 35% or greater since it is now believed that the HGBV strains may be genetically related, preferably about 40% or greater, more preferably, about 60% or greater, and even more preferably, about 80% or greater.
  • contiguous sequences of at least about 13 nucleotides which may be provided in combination of more than one contiguous sequence.
  • the overall sequence identity of the genomes between HGBV-C and a strain of HGBV-C at the amino acid level will be about 35% or greater since it is now believed that the HGBV strains may be genetically related, preferably about 40% or greater, more preferably, about 60% or greater, and even more preferably, about 80% or greater.
  • the HGBV strains may be genetically related, preferably about 40% or greater, more preferably, about 60% or greater, and even more preferably, about 80% or greater.
  • compositions and methods described herein will enable the propagation, identification, detection and isolation of HGBV and its possible strains. Moreover, they also will allow the preparation of diagnostics and vaccines for the possible different strains of HGBV, and will have utility in screening procedures for anti-viral agents. The information will be sufficient to allow a viral laxonomisl to identify other strains which fall within the species.
  • LCR ligase chain reaction
  • PCR polymerase chain reaction
  • This epitope is unique to HGBV when compared to other known hepatitis-causing viruses.
  • the uniqueness of the epitope may be determined by its immunological reactivity with HGBV and lack of immunological reactivity with Hepatitis A, B, C, D and E viruses.
  • Methods for determining immunological reactivity include, for example, radioimmunoassay (RIA), enzyme-linked immunosorbant assay (ELISA), hemagglutination (HA), fluorescence polarization immunoassay (FPIA) and several examples of suitable techniques are described herein.
  • a polynucleotide "derived from" a designated sequence for example, the HGBV cDNA, or from the HGBV genome refers to a polynucleotide sequence which is comprised of a sequence of approximately at least about 6 nucleotides, is preferably at least about 8 nucleotides, is more preferably at least about 10-12 nucleotides, and even more preferably is at least about 15-20 nucleotides corresponding, i.e., similar to or complementary to, a region of the designated nucleotide sequence.
  • the sequence of the region from which the polynucleotide is derived is similar to or complementary to a sequence which is unique to the HGBV genome.
  • Whether or not a sequence is complementary to or similar to a sequence which is unique to an HGBV genome can be determined by techniques known to those skilled in the art. Comparisons to sequences in databanks, for example, can be used as a method to determine the uniqueness of a designated sequence. Regions from which sequences may be derived include but are not limited to regions encoding specific epitopes, as well as non-translated and/or non-transcribed regions.
  • the derived polynucleotide will not necessarily be derived physically from the nucleotide sequence of HGBV, but may be generated in any manner, including but not limited to chemical synthesis, replication or reverse transcription or transcription, which are based on the information provided by the sequence of bases in the region(s) from which the polynucleotide is derived.
  • combinations of regions corresponding to that of the designated sequence may be modified in ways known in the art to be consistent with an intended use.
  • polypeptide or amino acid sequence derived from a designated nucleic acid sequence or from the HGBV genome refers to a polypeptide having an amino acid sequence identical to that of a polypeptide encoded in the sequence or a portion thereof wherein the portion consists of at least 3 to 5 amino acids, and more preferably at least 8 to 10 amino acids, and even more preferably 15 to 20 amino acids, or which is immunologically identifiable with a polypeptide encoded in the sequence.
  • a "recombinant polypeptide” as used herein means at least a polypeptide of genomic, semisynthelic or synthetic origin which by virtue of its origin or manipulation is not associated with all or a portion of the polypeptide with which it is associated in nature or in the form of a library and/or is linked to a polynucleotide other than that to which it is linked in nature.
  • a recombinant or derived polypeptide is not necessarily translated from a designated nucleic acid sequence of HGBV or from an HGBV genome. It also may be generated in any manner, including chemical synthesis or expression of a recombinant expression system, or isolation from mutated HGBV.
  • synthetic peptide as used herein means a polymeric form of amino acids of any length, which may be chemically synthesized by methods well- known to the routineer. These synthetic peptides are useful in various applications.
  • polynucleotide as used herein means a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. This term refers only to the primary structure of the molecule. Thus, the term includes double- and single-stranded DNA, as well as double- and single-stranded RNA. It also includes modifications, either by methylation and/or by capping, and unmodified forms of the polynucleotide.
  • HGBV containing a sequence corresponding to a cDNA means that the HGBV contains a polynucleotide sequence which is similar to or complementary to a sequence in the designated DNA.
  • the degree of similarity or complementarity to the cDNA will be approximately 50% or greater, will preferably be at least about 70%, and even more preferably will be at least about 90%.
  • the sequence which corresponds will be at least about 70 nucleotides, preferably at least about 80 nucleotides, and even more preferably al least about 90 nucleotides in length.
  • HGBV and the cDNA can be determined by methods known in the art, and include, for example, a direct comparison of the sequenced material with the cDNAs described, or hybridization and digestion with single strand nucleases, followed by size determination of the digested fragments.
  • "Purified viral polynucleotide” refers to an HGBV genome or fragment thereof which is essentially free, i.e., contains less than about 50%, preferably less than about 70%, and even more preferably, less than about 90% of polypeptides with which the viral polynucleotide is naturally associated.
  • purifying viral polynucleotides include, for example, disruption of the particle with a chaotropic agent, and separation of the polynucleotide(s) and polypeptides by ion-exchange chromatography, affinity chromatography, and sedimentation according to density.
  • purified viral polypeptide means an HGBV polypeptide or fragment thereof which is essentially free, that is, contains less than about 50%, preferably less than about 70%, and even more preferably, less than about 90% of cellular components with which the viral polypeptide is naturally associated. Methods for purifying are known to the routineer.
  • Polypeptide indicates a molecular chain of amino acids and does not refer to a specific length of the product. Thus, peptides, oligopeptides, and proteins are included within the definition of polypeptide. This term, however, is not intended to refer to post-expression modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations and the like.
  • Recombinant host cells refer to cells which can be, or have been, used as recipients for recombinant vector or other transfer DNA, and include the original progeny of the original cell which has been transfected.
  • replicon means any genetic element, such as a plasmid, a chromosome or a virus, that behaves as an autonomous unit of polynucleotide replication within a cell. That is, it is capable of replication under its own control.
  • a “vector” is a replicon in which another polynucleotide segment is attached, such as to bring about the replication and/or expression of the attached segment.
  • control sequence refers to polynucleotide sequences which are necessary to effect the expression of coding sequences to which they are ligated. The nature of such control sequences differs depending upon the host organism. In prokaryotes, such control sequences generally include promoter, ribosomal binding site and terminators; in eukaryotes, such control sequences generally include promoters, terminators and, in some instances, enhancers.
  • control sequence thus is intended to include at a minimum all components whose presence is necessary for expression, and also may include additional components whose presence is advantageous, for example, leader sequences.
  • operably linked refers to a situation wherein the components described are in a relationship permitting them to function in their intended manner.
  • a control sequence "operably linked" to a coding sequence is ligated in such a manner that expression of the coding sequence is achieved under conditions compatible with the control sequences.
  • ORF open reading frame
  • ORF refers to a region of a polynucleotide sequence which encodes a polypeptide; this region may represent a portion of a coding sequence or a total coding sequence.
  • a “coding sequence” is a polynucleotide sequence which is transcribed into mRNA and/or translated into a polypeptide when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a translation start codon at the 5' -terminus and a translation stop codon at the 3' -terminus.
  • a coding sequence can include, but is not limited to, mRNA, cDNA, and recombinant polynucleotide sequences.
  • the term "immunologically identifiable with/as” refers to the presence of epitope(s) and polypeptide(s) which also are present in and are unique to the designated polypeptide(s), usually HGBV proteins.
  • Immunological identity may be determined by antibody binding and/or competition in binding. These techniques are known to the routineer and also are described herein.
  • the uniqueness of an epitope also can be determined by computer searches of known data banks, such as GenBank, for the polynucleotide sequences which encode the epitope, and by amino acid sequence comparisons with other known proteins.
  • epitope means an antigenic determinant of a polypeptide.
  • an epitope can comprise three amino acids in a spatial conformation which is unique to the epitope.
  • an epitope consists of at least five such amino acids, and more usually, it consists of at least eight to ten amino acids.
  • Methods of examining spatial conformation include, for example, x-ray crystallography and two-dimensional nuclear magnetic resonance.
  • a polypeptide is "immunologically reactive" with an antibody when it binds to an antibody due to antibody recognition of a specific epitope contained within the polypeptide.
  • Immunological reactivity may be determined by antibody binding, more particularly by the kinetics of antibody binding, and/or by competition in binding using as competitor(s) a known polypeptide(s) containing an epitope against which the antibody is directed.
  • the methods for determining whether a polypeptide is immunologically reactive with an antibody are known in the art.
  • immunogenic polypeptide containing an HGBV epitope means naturally occurring HGBV polypeptides or fragments thereof, as well as polypeptides prepared by other means, for example, chemical synthesis or the expression of the polypeptide in a recombinant organism.
  • transformation refers to the insertion of an exogenous polynucleotide into a host cell, irrespective of the method used for the insertion. For example, direct uptake, transduction, or f-mating are included.
  • the exogenous polynucleotide may be maintained as a non-integrated vector, for example, a plasmid, or alternatively, may be integrated into the host genome.
  • Treatment refers to prophylaxis and/or therapy.
  • the term "individual” as used herein refers to vertebrates, particularly members of the mammalian species and includes but is not limited to domestic animals, sports animals, primates and humans; more particularly the term refers to tamarins and humans.
  • plus strand denotes a nucleic acid that contains the sequence that encodes the polypeptide.
  • minus strand denotes a nucleic acid that contains a sequence that is complementary to that of the "plus” strand.
  • Perfect stranded genome of a virus denotes that the genome, whether
  • test sample refers to a component of an individual's body which is the source of the analyte (such as, antibodies of interest or antigens of interest). These components are well known in the art.
  • test samples include biological samples which can be tested by the methods of the present invention described herein and include human and animal body fluids such as whole blood, serum, plasma, cerebrospinal fluid, urine, lymph fluids, and various external secretions of the respiratory, intestinal and genitorurinary tracts, tears, saliva, milk, white blood cells, myelomas and the like; biological fluids such as cell culture supernatants; fixed tissue specimens; and fixed cell specimens.
  • the recombinant antigen can be used to develop unique assays as described herein to detect either the presence of antigen or antibody to HGBV.
  • These compositions also can be used to develop monoclonal and/or polyclonal antibodies with a specific recombinant antigen which specifically bind to the immunological epitope of
  • HGBV which is desired by the routineer.
  • at least one recombinant antigen of the invention can be used to develop vaccines by following methods known in the art.
  • the reagent employed for the assay can be provided in the form of a test kit with one or more containers such as vials or bottles, with each container containing a separate reagent such as a monoclonal antibody, or a cocktail of monoclonal antibodies, or a recombinant antigen employed in the assay.
  • a separate reagent such as a monoclonal antibody, or a cocktail of monoclonal antibodies, or a recombinant antigen employed in the assay.
  • Other components such as buffers, controls, and the like, known to those of ordinary skill in art, may be included in such test kits.
  • “Analyte,” as used herein, is the substance to be detected which may be present in the test sample.
  • the analyte can be any substance for which there exists a naturally occurring specific binding member (such as, an antibody), or for which a specific binding member can be prepared.
  • an analyte is a substance that can bind to one or more specific binding members in an assay.
  • "Analyte” also includes any antigenic substances, haptens, antibodies, and combinations thereof.
  • the analyte can be detected by means of naturally occurring specific binding partners (pairs) such as the use of intrinsic factor protein as a member of a specific binding pair for the determination of Vitamin B 12, the use of folate-binding protein to determine folic acid, or the use of a lectin as a member of a specific binding pair for the determination of a carbohydrate.
  • the analyte can include a protein, a peptide, an amino acid, a nucleotide target, and the like.
  • a "specific binding member,” as used herein, is a member of a specific binding pair. That is, two different molecules where one of the molecules through chemical or physical means specifically binds to the second molecule. Therefore, in addition to antigen and antibody specific binding pairs of common immunoassays, other specific binding pairs can include biotin and avidin, carbohydrates and lectins, complementary nucleotide sequences, effector and receptor molecules, cofactors and enzymes, enzyme inhibitors and enzymes, and the like. Furthermore, specific binding pairs can include members that are analogs of the original specific binding members, for example, an analyte-analog.
  • lmmunoreactive specific binding members include antigens, antigen fragments, antibodies and antibody fragments, both monoclonal and polyclonal, and complexes thereof, including those formed by recombinant DNA molecules.
  • hapten refers to a partial antigen or non-protein binding member which is capable of binding to an antibody, but which is not capable of eliciting antibody formation unless coupled to a carrier protein.
  • a “capture reagent”, as used herein, refers to an unlabeled specific binding member which is specific either for the analyte as in a sandwich assay, for the indicator reagent or analyte as in a competitive assay, or for an ancillary specific binding member, which itself is specific for the analyte, as in an indirect assay.
  • the capture reagent can be directly or indirectly bound to a solid phase material before the performance of the assay or during the performance of the assay, thereby enabling the separation of immobilized complexes from the test sample.
  • the “indicator reagent” “comprises a “signal generating compound” (label) which is capable of generating and generates a measurable signal detectable by external means conjugated (attached) to a specific binding member for HGBV.
  • Specific binding member as used herein means a member of a specific binding pair. That is, two different molecules where one of the molecules through chemical or physical means specifically binds to the second molecule.
  • the indicator reagent also can be a member of any specific binding pair, including either hapten- anti-hapten systems such as biotin or anti-biotin, avidin or biotin, a carbohydrate or a lectin, a complementary nucleotide sequence, an effector or a receptor molecule, an enzyme cofactor and an enzyme, an enzyme inhibitor or an enzyme, and the like.
  • An immunoreactive specific binding member can be an antibody, an antigen, or an antibody/antigen complex that is capable of binding either to HGBV as in a sandwich assay, to the capture reagent as in a competitive assay, or to the ancillary specific binding member as in an indirect assay.
  • the indicator agent can comprise an antibody which is anti-species to the capture antibody used to complex the antigen in the test sample with the capture reagent (in this example, the antibody which specifically binds to HGBV antigen).
  • anti-species antibodies comprising the indicator reagent will be anti-rabbit when the capture reagent is rabbit and of a species other than rabbit, such as, goat anti-rabbit antisera.
  • Such design choicces are within the skill of the routineer.
  • labels include chromogens, catalysts such as enzymes, luminescent compounds such as fluorescein and rhodamine, chemiluminescent compounds such as dioxetanes, acridiniums, phenanthridiniums and luminol, radioactive elements, and direct visual labels.
  • luminescent compounds such as fluorescein and rhodamine
  • chemiluminescent compounds such as dioxetanes, acridiniums, phenanthridiniums and luminol
  • radioactive elements and direct visual labels.
  • enzymes include alkaline phosphatase, horseradish peroxidase, beta-galactosidase, and the like.
  • the selection of a particular label is not critical, but it will be capable of producing a signal either by itself or in conjunction with one or more additional substances.
  • Solid phases are known to those in the art and include the walls of wells of a reaction tray, test tubes, polystyrene beads, magnetic beads, nitrocellulose strips, membranes, microparticles such as latex particles, sheep (or other animal) red blood cells, duracytes and others.
  • the "solid phase” is not critical and can be selected by one skilled in the art.
  • latex particles, microparticles, magnetic or non-magnetic beads, membranes, plastic tubes, walls of microtiter wells, glass or silicon chips, sheep (or other suitable animal's) red blood cells and duracytes are all suitable examples.
  • Suitable methods for immobilizing peptides on solid phases include ionic, hydrophobic, covalent interactions and the like.
  • a “solid phase”, as used herein, refers to any material which is insoluble, or can be made insoluble by a subsequent reaction.
  • the solid phase can be chosen for its intrinsic ability to attract and immobilize the capture reagent.
  • the solid phase can retain an additional receptor which has the ability to attract and immobilize the capture reagent.
  • the additional receptor can include a charged substance that is oppositely charged with respect to the capture reagent itself or to a charged substance conjugated to the capture reagent.
  • the receptor molecule can be any specific binding member which is immobilized upon (attached to) the solid phase and which has the ability to immobilize the capture reagent through a specific binding reaction.
  • the receptor molecule enables the indirect binding of the capture reagent to a solid phase material before the performance of the assay or during the performance of the assay.
  • the solid phase thus can be a plastic, derivatized plastic, magnetic or non- magnetic metal, glass or silicon surface of a test tube, microtiter well, sheet, bead, microparticle, chip, sheep (or other suitable animal's) red blood cells, duracytes and other configurations known to those of ordinary skill in the art.
  • the solid phase also can comprise any suitable porous material with sufficient porosity to allow access by detection antibodies and a suitable surface affinity to bind antigens.
  • Microporous structures are generally preferred, but materials with gel structure in the hydrated state may be used as well. All of these materials may be used in suitable shapes, such as films, sheets, or plates, or they may be coated onto or bonded or laminated to appropriate inert carriers, such as paper, glass, plastic films, or fabrics.
  • An immobilizable immune complex is separated from the rest of the reaction mixture by ionic interactions between the negatively charged poly-anion/immune complex and the previously treated, positively charged porous matrix and detected by using various signal generating systems previously described, including those described in chemiluminescent signal measurements as described in co-pending U.S. Patent Application Serial No. 921 ,979 corresponding to EPO Publication No. 0 273,1 15.
  • the methods of the present invention can be adapted for use in systems which utilize microparticle technology including in automated and semi- automated systems wherein the solid phase comprises a microparticle (magnetic or non-magnetic).
  • Such systems include those described in pending U. S. Patent Applications 425,651 and 425,643, which correspond to published EPO applications Nos. EP 0 425 633 and EP 0 424 634, respectively.
  • SPM scanning probe microscopy
  • the capture phase for example, at least one of the monoclonal antibodies of the invention
  • a scanning probe microscope is utilized to detect antigen/antibody complexes which may be present on the surface of the solid phase.
  • the use of scanning tunneling microscopy eliminates the need for labels which normally must be utilized in many immunoassay systems to detect antigen/antibody complexes.
  • SPM to monitor specific binding reactions can occur in many ways.
  • one member of a specific binding partner is attached to a surface suitable for scanning.
  • the attachment of the analyte specific substance may be by adsorption to a test piece which comprises a solid phase of a plastic or metal surface, following methods known to those of ordinary skill in the art.
  • covalent attachment of a specific binding partner (analyte specific substance) to a test piece which test piece comprises a solid phase of derivatized plastic, metal, silicon, or glass may be utilized.
  • Covalent attachment methods are known to those skilled in the art and include a variety of means to irreversibly link specific binding partners to the test piece.
  • test piece is silicon or glass
  • the surface must be activated prior to attaching Ihe specific binding partner.
  • polyelectrolyte interactions may be used to immobilize a specific binding partner on a surface of a test piece by using techniques and chemistries described by pending U. S. Patent applications Serial No. 150,278, filed January 29, 1988, and Serial No. 375,029, filed July 7, 1989.
  • the preferred method of attachment is by covalent means.
  • the surface may be further treated with materials such as serum, proteins, or other blocking agents to minimize nonspecific binding.
  • the surface also may be scanned either at the site of manufacture or point of use to verify its suitability for assay purposes. The scanning process is not anticipated to alter the specific binding properties of the test piece.
  • the antibodies of the present invention can be employed in various assay systems to determine the presence, if any, of HGBV proteins in a test sample. Fragments of these antibodies provided also may be used.
  • a polyclonal or monoclonal anti-HGBV antibody or fragment thereof, or a combination of these antibodies, which has been coated on a solid phase is contacted with a test sample which may contain HGBV proteins, to form a mixture. This mixture is incubated for a time and under conditions sufficient to form antigen/antibody complexes.
  • an indicator reagent comprising a monoclonal or a polyclonal antibody or a fragment thereof, which specifically binds to an HGBV region, or a combination of these antibodies, to which a signal generating compound has been attached, is contacted with the antigen/antibody complexes to form a second mixture.
  • This second mixture then is incubated for a time and under conditions sufficient to form antibody/antigen/antibody complexes.
  • the presence of HGBV antigen present in the test sample and captured on the solid phase, if any, is determined by detecting the measurable signal generated by the signal generating compound.
  • the amount of HGBV antigen present in the lest sample is proportional to the signal generated.
  • a polyclonal or monoclonal anti-HGBV antibody or fragment thereof, or a combination of these antibodies which is bound to a solid support the test sample and an indicator reagent comprising a monoclonal or polyclonal antibody or fragments thereof, which specifically binds to HGBV antigen, or a combination of these antibodies to which a signal generating compound is attached, are contacted to form a mixture.
  • This mixture is incubated for a time and under conditions sufficient to form antibody/antigen/antibody complexes.
  • the presence, if any, of HGBV proteins present in the test sample and captured on the solid phase is determined by detecting the measurable signal generated by the signal generating compound.
  • the amount of HGBV proteins present in the test sample is proportional to the signal generated.
  • one or a combination of at least two monoclonal antibodies which specifically bind to HGBV protein can be employed as a competitive probe for the detection of antibodies to HGBV protein.
  • HGBV proteins such as the recombinant antigens disclosed herein, either alone or in combination, can be coated on a solid phase.
  • a test sample suspected of containing antibody to HGBV antigen then is incubated with an indicator reagent comprising a signal generating compound and at least one antibody of the invention for a time and under conditions sufficient to form antigen/antibody complexes of either the test sample and indicator reagent to the solid phase or the indicator reagent to the solid phase.
  • the reduction in binding of the antibody to the solid phase can be quantitatively measured.
  • a measurable reduction in the signal compared to the signal generated from a confirmed negative NANB, non-C, non- D, non-E hepatitis test sample indicates the presence of anti-HGBV antibody in the test sample.
  • the antibodies of the present invention can be employed in the detection of HGBV antigens in fixed tissue sections, as well as fixed cells by immunohistochemical analysis. Cytochemical analysis wherein these antibodies are labeled directly (fluorescein, colloidal gold, horseradish peroxidase, alkaline phosphatase, etc.) or are labeled by using secondary labeled anti-species antibodies (with various labels as exemplified herein) to track the histopathology of disease also are within the scope of the present invention.
  • these antibodies can be bound to matrices similar to CNBr-activated Sepharose and used for the affinity purification of specific HGBV proteins from cell cultures, or biological tissues such as blood and liver such as to purify recombinant and native viral HGBV antigens and proteins.
  • the antibodies of the invention can also be used for the generation of chimeric antibodies for therapeutic use, or other similar applications.
  • the antibodies or fragments thereof as described herein can be provided individually to detect HGBV antigens.
  • Combinations of the antibodies (and fragments thereof) provided herein also may be used together as components in a mixture or "cocktail" of at least one anti-HGBV antibody of the invention with antibodies to other HGBV regions, each having different binding specificities.
  • this cocktail can include the antibodies of the invention which are directed to HGBV proteins and other antibodies to other antigenic determinants of the HGBV genome. It is preferred that monoclonal antibodies, which specifically bind to one epitope of HGBV, be utilized in this embodiment.
  • the polyclonal antibody or fragment thereof which can be used in the assay formats should specifically bind to a specific HGBV region or other HGBV proteins used in the assay.
  • the polyclonal antibody used preferably is of mammalian origin; human, goat, rabbit or sheep anti-HGBV polyclonal antibody can be used. Most preferably, the polyclonal antibody is rabbit polyclonal anti- HGBV antibody.
  • the polyclonal antibodies used in the assays can be used either alone or as a cocktail of polyclonal antibodies.
  • the cocktails used in the assay formats are comprised of either monoclonal antibodies or polyclonal antibodies having different HGBV specificity, they would be useful for diagnosis, evaluation and prognosis of HGBV infection, as well as for studying HGBV protein differentiation and specificity.
  • the presence of antibody and/or antigen to HGBV can be detected in a simultaneous assay, as follows.
  • a test sample is simultaneously contacted with a capture reagent of a first analyte, wherein the capture reagent comprises a first binding member specific for a first analyte attached to a solid phase and contacted with a capture reagent for a second analyte, wherein the capture reagent comprises a first binding member for a second analyte attached to a second solid phase, to thereby form a mixture.
  • This mixture is incubated for a time and under conditions sufficient to form capture reagent/first analyte and capture reagent/second analyte complexes.
  • Such so-formed complexes then are contacted with an indicator reagent comprising a member of a binding pair specific for the first analyte labeled with a signal generating compound and an indicator reagent comprising a member of a binding pair specific for the second analyte labeled with a signal generating compound, to form a second mixture.
  • This second mixture is incubated for a time and under conditions sufficient to form capture reagent/first analyte/indicator reagent complexes and capture reagent/second analyte/indicator reagent complexes.
  • the presence of one or more analytes is determined by delecting a signal generated in connection with the complexes formed on either or both solid phases as an indication of the presence of one or more analytes in the test sample.
  • recombinant antigens derived from human expression systems may be utilized as well as antibodies produced from the proteins derived from the mammalian expression systems or produced by synthetic means known in the art, as disclosed herein.
  • Such assay systems are described in greater detail in pending U.S. Patent Application Serial No. 07/574,821 entitled Simultaneous Assay for Detecting One Or More Analytes, which corresponds to EP Publication No. 0473065.
  • RT-PCR polymerase chain reaction
  • RT- AGLCR asymmetric gap ligase chain reaction
  • a one step RT-PCR assay method can be utilized.
  • target-specific primers are designed to detect the above target sequence by reverse transcription PCR by methods known in the art.
  • One step RT-PCR is a sequential procedure that performs both RT and PCR in a single reaction mixture.
  • the procedure usually is performed in a 200 ⁇ l reaction mixture containing 50 mM (N,N,-bis[2-Hydroxyethyl]glycine), pH 8.15, 81.7 mM KOAc, 33.33 mM KOH, 0.01 mg/ml bovine serum albumin, 0.1 mM elhylene diaminetetraacetic acid, 0.02 mg/ml NaN 3 , 8% w/v glycerol, 150 ⁇ M each of dNTP, 0.25 ⁇ M each primer, 5U rTth polymerase, 3.25 mM Mn(OAc) 2 , and 5 ⁇ l blood equivalents of target.
  • 50 mM (N,N,-bis[2-Hydroxyethyl]glycine) pH 8.15, 81.7 mM KOAc, 33.33 mM KOH, 0.01 mg/ml bovine serum albumin, 0.1 mM elhylene diaminetetraace
  • RNA and the rTth polymerase enzyme are unstable in the presence of Mn(OAc) 2 , the Mn(OAc) 2 should be added just before target addition.
  • Optimal conditions for cDNA synthesis and thermal cycling readily can be determined by those skilled in the art. The reaction is incubated in a Perkin-Elmer Thermal Cycler 480. Optimal conditions for cDNA synthesis and thermal cycling can readily be determined by those skilled in the art. Conditions which may be found useful include cDNA sysnthesis at 60°-70° for 15-45 min, and 30-45 amplification cycles at 94°C, 1 min; 55°C-70°C, 1 min; 72°C, 2 min.
  • One step RT-PCR also may be performed by using a dual enzyme procedure with Taq polymerase and a reverse transcriptase enzyme, such as MMLV or AMV RT enzymes.
  • a traditional two step RT-PCR reaction may be performed; for example, as described by K.-Q.
  • the extracted mRNA is transcribed in a 25 ⁇ l reaction mixture containing 10 mM Tris-HCl, pH 8.3, 5 mM MgCl 2 , 500 ⁇ M dNTP, 20 U RNasin, 1 ⁇ M antisense primer, and 25 U AMV (avian myeloblastosis virus) or MMLV (Moloney murine leukemia virus) reverse transcriptase. Reverse transcription is performed at 37-45°C for 30-60 min, followed by further incubation at 95°C for 5 min to inactivate the RT.
  • PCR is performed using 10 ⁇ l of the cDNA reaction in a final PCR reaction volume of 50 ⁇ l containing 10 mM Tris-HCl, pH 8.3, 50 mM KC1, 2 mM MgCl 2 , 200 ⁇ M dNTP, 0.5 ⁇ M of each primer and 2.5 U of Taq polymerase.
  • Optimal conditions for cDNA synthesis and thermal cycling can be readily determined by those skilled in the art.
  • the reaction is incubated in a
  • Perkin-Elmer Thermal Cycler 480 Conditions which may be found useful include 30-45 cycles of amplification (94°C, 1 min; 55°-70°C, 1 min; 72°C, 2 min), final extension (72°C, 10 min) and soak at 4°C.
  • the present invention discloses the preference for the use of solid phases, it is contemplated that the reagents such as antibodies, proteins and peptides of the present invention can be utilized in non-solid phase assay systems. These assay systems are known to those skilled in the art, and are considered to be within the scope of the present invention.
  • Example 1 Preparation of Immunogens An E. coli derived recombinant antigen encoded by GBV-C sequence from the putative E2 region of the GBV-C genome and expressed as a fusion protein with CMP-KDO synthetase (CKS), designated as pHGBV 180 (SEQUENCE ID NO: 3), was employed as an immunogen for the generation of polyclonal rabbit antisera.
  • the protein was prepared as follows.
  • the cloning vector pJO200 allowed the fusion of recombinant proteins to the CKS protein.
  • the plasmid consisted of the plasmid pBR322 with a modified lac promoter fused to a kdsB gene fragment (encoding the first 239 of the entire 248 amino acids of the E.
  • the synthetic linkers included multiple restriction sites for insertion of genes, translational stop signals, and the trpA rho-independent transcriptional terminator.
  • the unique restriction sites in this linker region include, from 5' to 3', EcoRI, Sad, Kpnl, Smal, BamHI, Xbal, Pstl, Sphl, and Hindlll.
  • the CKS method of protein synthesis as well as CKS vectors are disclosed in U.S. Patent No. 5, 124,255, which enjoys common ownership and which is incorporated herein by reference, and the use of CKS fusion proteins in assay formats and test kits is described in United States Serial No.
  • a PCR product encoding a 212 amino acid region of the HGBV-C E2 protein (amino acids 226-437 of SEQUENCE I.D. NO: 1 , designated as SEQUENCE ID NO: 3) which corresponds to nucleotides 1 158-1793 of SEQUENCE I.D. NO: 2 (and designated as SEQUENCE ID NO: 4) was cloned and expressed in E. coli as a CKS fusion by the following method.
  • the PCR product was generated using as template a plasmid containing a 1008 nucleotide fragment of HGBV-C (nucleotides 1 143-2150 of SEQUENCE I.D. NO: 2; desginated as SEQUENCE ID NO: 5).
  • the sense primer (SEQUENCE I.D. NO: 6) had a BamHI restriction site on the 5' end and the antisense primer (SEQUENCE I.D. NO: 7) had a Hindlll site on the 5' end to facilitate cloning.
  • a 25 ⁇ l PCR was set up using lx PCR buffer II (Perkin Elmer), 2 mM MgC12, 200 ⁇ M dNTPs, 1 ⁇ M each of primers, 1.5 ng plasmid DNA, and 1.25 units of AmpliTaq ® polymerase (Perkin Elmer). The reaction was incubated at 94°C for 20 sec, 50°C for 30 sec, and 72°C for 60 sec. for a total of 35 cycles of amplification followed by a final extension at 72°C for 10 minutes. The resulting product was digested with the restriction enzymes BamHI and Hindlll (10 units, NEB) and then cloned into the CKS expression vectors pJO200 as described in Example 1 of U.S.
  • This protein was prepared for immunization as follows. E. coli cells were lysed by homogenization (BIOSPEC Products Inc. Bartlesville, OK) for 45 seconds in 50 mM CAPS(3-[cyclohexylamino]- 1 -propanesulfonic acid) buffer, pH 10 containing 200 ug/ml Lysozyme ® , 10 ug/ml DNase in the presence of the following protease inhibitors: 1.5 mM MgCl 2 , 0.5 ug/ml leupeptin, 0.7 ug/ml pepstatin and 2 mM phenylmethanesulphonyl fluoride (PMSF).
  • CAPS(3-[cyclohexylamino]- 1 -propanesulfonic acid) buffer pH 10 containing 200 ug/ml Lysozyme ®
  • 10 ug/ml DNase 10 ug/ml DNase in the presence of the following protea
  • lysis buffer Approximately 8 is of lysis buffer was used per gram of wet pellet weight. Following a 30 minute incubation at 37°C with occasional swirling, the mixture was placed on ice until the temperature was between 10-15°C. The mixture was then sonicated (Vibracell - Sonics and Materials Inc., Danbury, Conn.) on ice for five minutes on 50% duty cycle, pulsed setting. Lysis was assessed by visualization under a phase contrast microscope. Lysis was considered to be complete when less than 15% intact cells were remaining. The lysed cell extract was centrifuged (Beckman Instruments) at 4°C for 30 minutes at 10,000 rpm. Five successive washes were performed on the insoluble protein extract.
  • Pellet washes 1 - 3 were performed with dH 2 O ("Quickwash", available from Abbott Laboratories, Abbott Park, IL), followed by 5% Triton X- 100*/50mM Tris Buffer, pH8.5 and finally 50 mM Tris Buffer, pH8.0. Briefly, each wash step consisted of a 45 second homogenization 5 followed by centrifugation (as described hereinabove) and the decanting of the extract supernatant. The resulting pellet was then solubilized in 20 mM Tris, pH8.5 with 0.5% SDS on a rotating platform for 1 -2 hours/room temperature until no visible pellet remained. The protein was purified using gel filtration chromatography, following procedures as known in the art. Protein purity was
  • synthetic peptides were produced based on sequence from the putative E2 region of the GBV-C genome. These peptides constituted a second type of immunogen used for generation of polyclonal antibodies and were designated as spGBVC-9 (GBV-C E2 aa 17-46, SEQUENCE ID NO: 8); spGE2- 0 36 (GBV-C E2 aa 133- 162, SEQUENCE ID NO: 9); and spGE2-37 (GBV-C E2 aa 147-176, SEQUENCE ID NO: 10). These peptides were purified by High Performance Liquid Chromatography (HPLC) and assessed for purity by mass spectroscopy. All peptides used as immunogens were coupled to Keyhole Limpet Hemocyanin (KLH), as follows. Briefly, KLH at approximately 1-2 nM was
  • Example 2 Generation of high-titered rabbit GBV-C E2 antibody
  • the immunization schedule for the pHGBV180 immunogen (SEQUENCE ID NO: 3) (prepared as descibed in Example 1), was as follows: One mg for primary inoculation, 0.5 mg boost at month 1 , 0.2 mg boost at month 2, and 0.5 mg boosts at months 4 and 6.
  • the immunization schedule for the synthetic peptide immunizations was as follows: 1 mg for primary inoculation, 0.5 mg boost al month one, 0.5 mg boost at month 2, and 0.2 mg boost al month 4.
  • the rabbits were bled approximately two weeks after every boost and once in the months in which there was no boost.
  • Sera from pre-immunization and post-immunization bleeds were serially diluted in specimen dilution buffer (Tris Buffered Saline containing 1 % BSA/21 % normal goat serum/ 10% Calf Serum with EGTA, EDTA and 0.2% Triton X-100 w .
  • specimen dilution buffer Tris Buffered Saline containing 1 % BSA/21 % normal goat serum/ 10% Calf Serum with EGTA, EDTA and 0.2% Triton X-100 w .
  • the samples were incubated with the antigen coated beads for lhr at RT in the Dynamic Incubator (available from Abbott Laboratories, Abbott Park, IL) in dynamic mode (i.e., with agitation).
  • the beads After performing a wash step with dH 2 O (Qwikwash, Abbott Laboratories, Abbott Park, IL ) the beads were reacted with horseradish peroxidase (HRPO)-labeled goat anti-rabbit (H+L) conjugate (avalilable from Kirkegaard & Perry Laboratories Gaithersburg Md.) at a concentration of 0.2 ug/ml for one hr at RT in the Dynamic Incubator in dynamic mode. After a final wash with Qwikwash, the beads were transferred to tubes and 300 ul of o-phenylenediamine-2HCl in citrate buffer conataining 0.02% hydrogen peroxide (OPD) was added per tube and allowed to develop for 30 minutes at RT.
  • HRPO horseradish peroxidase
  • H+L horseradish peroxidase conjugate
  • OPD hydrogen peroxide
  • the reaction was terminated by the addition of 1ml of IN H 2 SO 4 and the resulting absorbance at 492/600 nm was recorded.
  • the signal was determined to be specific when the post-immunization response was titratable against the respective immunogen.
  • the endpoint liter was determined as the dilution where the signal of the post-immunization bleed was greater than or equal to 5 times the absorbance value of the pre-immunization sera at a 1: 10,000 dilution.
  • TABLE 1 shows the endpoint titers of the rabbit bleeds that were monitored starting one month after the 3rd boost.
  • Example 3 Preparation of reagents for antigen test IgG was purified using a HiTrap Protein A column (available from Pharmacia, Uppsala, Sweden) as follows. Briefly, 1 ml of sera was used for purification and diluted 1 :5 in 20 mM Phosphate Buffer, pH 7.0. This material was filtered thruogh a 0.45 ⁇ m syringe filter and the applied to the column at a flow rate of 1 ml/minute. The diluted antisera was cycled through the column three times; the column was then washed with 20 mM Phosphate Buffer and the absorbance at A 280 of the resultant eluate was monitored until the absorbance was below 0.05.
  • HiTrap Protein A column available from Pharmacia, Uppsala, Sweden
  • the IgG then was eluted using 0.1 M citric acid, pH 3.5. Fractions with absorbance values greater than 1.0 were pooled, dialyzed into 20mM Phosphate Buffer and stored at -70°C. The so-purified IgG preparation from rabbits identified as 10363 and
  • the purified IgG preparation was biotinylated as follows.
  • Biotinylated rabbit IgG probes were prepared by adding Sulfo-NHS-LC-Biotin (available from Pierce Chemical Co., Rockford, IL) to 600 ugs IgG in order to give molar ratios ranging from 5: 1 to 2158: 1. Each mixture was rotated end-over- end for 2 hours at 4°C. The reaction was quenched using 100 mM Tris Buffer, pH 9.0 at a 1 : 10 ratio. The material was dialyzed ON/4°C vs. 20 mM Tris, pH8.5 with 200 mM NaCl. A 1 : 10 dilution of 10% BSA was then added for stability. The preparation was aliquoted and stored at -70°C.
  • the biotinylated rabbit IgG probes then were qualified in two complementary ELISA's, as follows.
  • the first assay was used to determine whether the probes had been biotinylated. This was performed by reacting the biotinylated probes, which had been diluted in 10% fetal calf serum (FCS)/0.1 % Twecn-20"7Tris Buffered Saline (TBS), with beads coated with the immunogen prepared as described in Example 2 as well as beads coated with unrelated protein (following the general coating procedure exemplified in Example 2). The probes were diluted appropriately and reacted with the solid phase. This first incubation was either done overnight at RT or for 2 hours at 37°C using a Dynamic Incubator in dynamic mode. After a wash step using Qwikwash, HRPO-Streptavidin
  • the probes were considered to be biotinylated if they were reactive with HRPO-Streptavidin. They were considered to be specific toward their respective immunogen if they were reactive with it on the solid phase, but unreactive against the unrelated protein.
  • the second assay compared die reactivity of the biotinylated probes toward their respective immunogen and an unrelated protein with the non-biotinylated IgG at the same concentrations-. This was performed as described above except that the conjugate in this case was HRPO-labeled goat anti-rabbit (H+L) conjugate (avalilable from Kirkegaard & Perry Laboratories Gaithersburg Md.).
  • This assay allowed the determination of whether or not the immunoreactivity of the biotinylated IgG had been compromised relative to that of the non-biotinylated IgG. Reactivity was considered compromised if the absorbance (A 49260 o) of the biotinylated reagents was less than half the absorbance (A 49260 o) of the non-biotinylated IgG.
  • Example 4 Sandwich Antigen Assay Polystyrene beads (1/4") were coated with rabbit 10363 purified IgG which had been prepared as described in Example 3 following the coating procedure described previously in Example 2, and at concentrations ranging from 83 pM to 330 nM. pHGBVl 80 was used as a model antigen source and a standard curve was run at concentrations ranging from 15 ng/ml to 500 ug/ml. The antigen standard curve was prepared in 0.1% Tween-20 ® /10% fetal calf serum (FCS)/Tris Buffered Saline (TBS).
  • FCS fetal calf serum
  • TBS Tris Buffered Saline
  • the pHGBVl 80 antigen (SEQUENCE ID NO:3) was incubated with the antibody coated solid phase (prepared as described above in Example 1 , 2 and 3) overnight at room temperature or for two hours at 37°C in the Dynamic Incubator in the dynamic mode.
  • the solid phase was then washed with dH 2 O (Qwikwash, Abbott Laboratories) and Biotinylated-Rabbit 10369 IgG (at a molar ratio of 1075: 1) diluted to 1 ⁇ g/ml in 10% FCS/0.1% Tween-20 ® /TBS was added.
  • Example 5 Immunization of Rabbits with GBV-C CHO-315 Purified Antigen
  • GBV-C CHO-315 antigen SEQUENCE ID NO:l 1
  • Both rabbits were inoculated both intramuscularly (IM) and subcutaneously for all inoculations.
  • the immunization schedule for both CHO- 315 rabbits was as follows: 0.5 mg for primary inoculation, 0.25 mg boost at month one, and 0.25 mg boost at month two. Rabbits were bled approximately two weeks after every boost.
  • the immune response of each rabbit was assessed by a CHO-315 ELISA where CHO-315 antigen was coated onto polystyrene beads at 5 ug/ml in 100 mM Phosphate Buffer, pH 7.5. Briefly, 233 ul coating solution per bead was added and incubated on a rotating wheel at 40°C for 2 hours. After washing with 0.1 % Triton X- 100 ® /PBS for 1 hour/rotating at 40°C, the beads were washed with PBS three times. The free sites on the bead were blocked by incubation with a 5% Bovine Serum Albumin (BSA)/PBS solution, again rotating for 1 hour at 40°C.
  • BSA Bovine Serum Albumin
  • Specimen Diluent Tris Buffered Saline containing 1 % BSA/21 % Normal Goat Serum 10% Calf Serum with EGTA, EDTA and
  • the beads were transferred to tubes and 300 ⁇ l of OPD (o- phenylenediamine-2HC! in citrate-phosphate buffer conataining 0.02% hydrogen peroxide) was added per tube and allowed to develop for 30 minutes at RT. The reaction was terminated by the addition of 1 N H 2 SO 4 and the resulting absorbance at 492/600 nm was read in the Abbott Quantum . The signal was determined to be specific if the post-immunization bleeds were tilratable against their respective immunogen.
  • OPD o- phenylenediamine-2HC! in citrate-phosphate buffer conataining 0.02% hydrogen peroxide
  • the endpoint titer was determined as the dilution where the signal of the post-immunization bleed was greater than or equal to 5 times the absorbance value of the pre-immunization sera at a 1 : 10,000 dilution.
  • TABLE 3 presents the absorbance values and S/N ratios of the rabbit bleeds.
  • Example 6 GBV-C Antigen Assay on GBV-C PCR Positive Human Donors
  • Specimens were evaluated in the presence of 0.5% Tween-20 ® throughout the entire assay. Specimens were diluted 1 :2 in specimen diluent (SpD) containing 20% Normal Goat Serum(NGS)/10% Fetal Calf Serum (FCS) in Tris Buffered Saline, pH 7.5 containing 1% Tween-20 ® . A total of 200 ⁇ l was added per well and allowed to incubate overnight at room temperature. An antigen standard curve consisting of CHO-315 antigen diluted from 5 ng/ml to 78 pg/ml in specimen diluent was run as a positive control and a measure of assay sensitivity. A PCR negative human commercial donor served as the negative control.
  • the beads were washed with dH 2 O (Qwikwash) and 200 ul of Biotinylated-Rabbit IgG (20: 1 molar ratio as described in Example 3) at 5 ⁇ g/ml diluted in SpD with 0.5% Tween-20 ® was added per well. This was allowed to incubate at 37°C in the dynamic mode (Dynamic Incubator, Abbott Laboratories) for two hours. The beads were then washed with Qwikwash and 200 ⁇ l HRPO-Streptavidin (available from Kirkegaard & Perrry Laboratories, Gaithersburg, Md) at a concentration of 0.1 ⁇ g/ml was added per well.
  • the diluent for the conjugate was SpD containing either 0.5%J Tween-20 ® . This was allowed to incubate for 30 minutes at 37°C in the dynamic mode (Dynamic Incubator, Abbott Laboratories). The solid phase was again washed as described above and color development using OPD as the chromagen was allowed to proceed for 30 minutes at room temperature. The reaction was stopped using 1 ml of IN H 2 SO 4 . The absorbance values at A 492/6 ⁇ 0 were read in the Abbott Quantum'". The data are presented in TABLE 4. A slightly elevated absorbance value was seen in the PCR-positive material over that of the PCR-negative sample.
  • Example 7 GBV-C Antigen-Capture Polymerase Chain Reaction
  • HGBV-C E2 the viral structural proteins
  • a less complex one e.g. one containing antibody and the virus alone
  • an antigen-capture PCR procedure was developed. Briefly, beads were coated with purified IgG derived from rabbits as described in Example 3 or from high titered human sera as described in Example 5. Human samples, previously screened by PCR, were diluted 1: 10 in 50% FCS in Tris Buffered Saline and 200 ⁇ l added to a reaction well.
  • NAME POREMBSKI , PRISCILLA E.
  • Trp Met Gly Pro Gin Arg Leu Met Phe Leu Val Leu Trp Lys Leu Ala 645 650 655
  • MOLECULE TYPE DNA (genomic)
  • GGACAACTCC CTTCACCATA AGGGGGCCCC TGGGCAACCA GGGGCGAGGC GACCCGGTGC 1800 GGTCGCCCTT GGGTTTTGGG TCCTACACCA TGACCAAGAT CCGAGACTCC TTACACTTGG 1860
  • GRCCCATCCT CATGGTGGGC TTGGCCATAG CGGGCGGCAT GATCTACGCC TCTTACACTG 5160 GGTCGCTAGT GGTGGTAACA GACTGGAATG TGAAGGGAGG TGGCAATCCC CTTTATAGGA 5220
  • CTAGGGGCCT GTTGTGGCAT CCAGGACTCC GGCTTCCTCC CCCTGAGATT GCTGGTATCC 8940
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • CTTTCTTGTC CCCAATTTGT CTACGGCGCC GTTTCAGTGA CCTGCGTGTG GGGTTCTGTG 300
  • GAGCTCTCCG AATGGGGAAT CCCCTGCGCC ACTTGTATCC TGGACAGGCG GCCTGCCTCG 480
  • MOLECULE TYPE DNA (genomic)

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Virology (AREA)
  • Communicable Diseases (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Biophysics (AREA)
  • Medicinal Chemistry (AREA)
  • Hematology (AREA)
  • Biotechnology (AREA)
  • Physics & Mathematics (AREA)
  • Biomedical Technology (AREA)
  • Zoology (AREA)
  • Analytical Chemistry (AREA)
  • Urology & Nephrology (AREA)
  • Microbiology (AREA)
  • Wood Science & Technology (AREA)
  • Pathology (AREA)
  • Cell Biology (AREA)
  • General Physics & Mathematics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Food Science & Technology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La présente invention concerne des procédés de détection d'un antigène du virus de l'hépatite GB (HGVB) dans des échantillons de test. Des anticorps dirigés contre la région E2 de HGVB sont utilisés de manière à capturer un antigène dans des échantillons de test. Les procédés comprennent un dosage immunologique et une amplification en chaîne par polymérase de capture d'antigène. La présente invention concerne également des anticorps utiles comme réactifs de diagnostic, ceux-ci étant fournis dans des nécessaires de test.
PCT/US1997/023767 1994-11-23 1997-12-23 Reactifs et procedes permettant de detecter un antigene du virus de l'hepatite gb WO1998029747A1 (fr)

Applications Claiming Priority (11)

Application Number Priority Date Filing Date Title
US34419094A 1994-11-23 1994-11-23
US34418594A 1994-11-23 1994-11-23
US37755795A 1995-01-30 1995-01-30
PCT/US1995/002118 WO1995021922A2 (fr) 1994-02-14 1995-02-14 Reactifs pour l'hepatite non-a, non-b, non-c, non-d et procede pour leur utilisation
US08/424,550 US6720166B2 (en) 1994-02-14 1995-02-14 Non-a, non-b, non-c, non-c, non-d, non-e hepatitis reagents and methods for their use
US08/417,629 US5981172A (en) 1994-02-14 1995-04-06 Non-A, non-B, non-C, non-D, non-E Hepatitis reagents and methods for their use
US48099595A 1995-06-07 1995-06-07
US08/473,475 US5843450A (en) 1994-02-14 1995-06-07 Hepatitis GB Virus synthetic peptides and uses thereof
US08/629,463 US6156495A (en) 1994-02-14 1996-04-19 Hepatitis GB virus recombinant proteins and uses thereof
US77837796A 1996-12-31 1996-12-31
US08/778,377 1996-12-31

Publications (1)

Publication Number Publication Date
WO1998029747A1 true WO1998029747A1 (fr) 1998-07-09

Family

ID=27578817

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1997/023767 WO1998029747A1 (fr) 1994-11-23 1997-12-23 Reactifs et procedes permettant de detecter un antigene du virus de l'hepatite gb

Country Status (1)

Country Link
WO (1) WO1998029747A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2267018A3 (fr) * 2000-08-18 2011-04-20 Human Genome Sciences, Inc. Polypeptides de liaison pour la protéine stimulatrice des lymphocytes B (BLyS)
WO2015200833A3 (fr) * 2014-06-27 2016-02-18 Abbott Laboratories Compositions et procédés pour détecter le pegivirus humain 2 (hp-v-2)

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995021922A2 (fr) * 1994-02-14 1995-08-17 Abbott Laboratories Reactifs pour l'hepatite non-a, non-b, non-c, non-d et procede pour leur utilisation

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995021922A2 (fr) * 1994-02-14 1995-08-17 Abbott Laboratories Reactifs pour l'hepatite non-a, non-b, non-c, non-d et procede pour leur utilisation

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
J. SIMONS ET AL.: "Isolation of novel viris-like sequences associated with human hepatitis.", NATURE MEDICINE, vol. 1, no. 6, June 1995 (1995-06-01), NEW YORK, USA, pages 564 - 569, XP000508526 *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2267018A3 (fr) * 2000-08-18 2011-04-20 Human Genome Sciences, Inc. Polypeptides de liaison pour la protéine stimulatrice des lymphocytes B (BLyS)
WO2015200833A3 (fr) * 2014-06-27 2016-02-18 Abbott Laboratories Compositions et procédés pour détecter le pegivirus humain 2 (hp-v-2)
US9777340B2 (en) 2014-06-27 2017-10-03 Abbott Laboratories Compositions and methods for detecting human Pegivirus 2 (HPgV-2)
EP3161490A4 (fr) * 2014-06-27 2017-11-08 Abbott Laboratories Compositions et procédés pour détecter le pegivirus humain 2 (hp-v-2)
US9938589B2 (en) 2014-06-27 2018-04-10 Abbott Laboratories Compositions and methods for detecting human pegivirus 2 (HPgV-2)
US10501816B2 (en) 2014-06-27 2019-12-10 Abbott Laboratories Compositions and methods for detecting human pegivirus 2 (HPgV-2)
EP3594684A1 (fr) * 2014-06-27 2020-01-15 Abbott Laboratories Compositions et procédés pour détecter le pegivirus humain 2 (hpgv-2)

Similar Documents

Publication Publication Date Title
JP2809956B2 (ja) Ｎａｎｂｖの診断用薬
US5683864A (en) Combinations of hepatitis C virus (HCV) antigens for use in immunoassays for anti-HCV antibodies
EP0627000B1 (fr) Systemes d'expression mammiferes generant des proteines du vhc
JPH06107687A (ja) 抗ウイルス剤
IE20020123A1 (en) New HCV Isolates
AU2585092A (en) Monoclonal antibodies to putative hcv e2/ns1 proteins and methods for using same
EP0741787B1 (fr) Systemes d'expression mammaliens pour des genes d'enveloppe du virus de l'hepatite c
EP0642666B2 (fr) Analyse de detection de l'hepatite c
EP0747482A2 (fr) Protéines récombinantes de virus de l'hépatite GB et leur utilisations
JP3061258B2 (ja) 非ａ非ｂ非ｃ非ｄ非ｅ型肝炎試薬及びそれらの使用方法
US5843450A (en) Hepatitis GB Virus synthetic peptides and uses thereof
JP4641695B2 (ja) 新規なｈｅｖ抗原性ペプチド及び方法
US5620843A (en) Non-A non-B sequences
WO1998029747A1 (fr) Reactifs et procedes permettant de detecter un antigene du virus de l'hepatite gb
EP0599913B1 (fr) Anticorps monoclonaux se fixant sur les proteines ns5 putatives du virus de l'hepatite c, et procedes d'utilisation de ces anticorps
EP0600018B1 (fr) Depistage de l'hepatite c a l'aide d'antigenes recombines diriges contre la region c-100
JPH10337193A (ja) 肝炎ｇｂウイルスエピトープに対する抗体及びその使用

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): CA JP

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FI FR GB GR IE IT LU MC NL PT SE

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
122 Ep: pct application non-entry in european phase