WO1990002206A1 - Detection de l'hepatite non a, non b - Google Patents

Detection de l'hepatite non a, non b Download PDF

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Publication number
WO1990002206A1
WO1990002206A1 PCT/US1989/003638 US8903638W WO9002206A1 WO 1990002206 A1 WO1990002206 A1 WO 1990002206A1 US 8903638 W US8903638 W US 8903638W WO 9002206 A1 WO9002206 A1 WO 9002206A1
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WIPO (PCT)
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ala
leu
gln
ser
glu
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Application number
PCT/US1989/003638
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English (en)
Inventor
Belinda Seto
William G. Coleman, Jr.
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The United States Of America, Represented By The Secretary, United States Department Of Commerce
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Publication of WO1990002206A1 publication Critical patent/WO1990002206A1/fr

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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
    • 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
    • G01N33/5767—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis non-A, non-B hepatitis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • 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

  • New clones bearing nucleotide sequences of non-A, non-B hepatitis provides a method for testing blood to detct the presence of non-A, non-B hepatitis materials.
  • the cloned DNA is used as a hybridization probe to screen serum samples or biopsied liver specimens for homologous DNA sequences.
  • Polypeptides expressed by the cloned DNA may be used as antigens in assays to determine whether antibodies to non-A, non-B hepatitis are present.
  • the causative agent of non-A, non-B hepatitis accounts for 90% of the posttransfusion hepatitis in the United States. Of those blood recipients who are infected by transfusion, 50% develop chronic hepatitis. Those chronically infected persons develop chronic hepatitis. Those chronically infected persons continue to be carriers of the infectious disease.
  • the existence of the transmissible agent has been demonstrated by infection of chimpanzees by inoculation with serum from a chronic non-A, non-B hepatitis patient and followed by serial passage of virus into other chimpanzees.
  • the deposited material will be maintained for at least five years after the most recent request for a sample; for a period of 30 years from date of deposit; or for the life of the patent, whichever period is longest. If the deposited sample should become contaminated or non-viable, the sample will be replaced.
  • Plasmid pBR322 and Escherichia coli DH5 are available commercially and were obtained from Bethesda Research Laboratory in Gaithersburg, Maryland. Plasmids pSP65, GEM37, and GEM47 are available from Promega. E. coli strain p678-54 was obtained by request from Dr. Barbara Bachmann, E. coli Genetic Stock Center, Yale University School of Medicine.
  • Figure 1 indicates that hybridization with the DNA insert from clone pSC22 occurred at only the acute phase of the disease in serum and liver (see insert) samples. Note the response at approximately 5 weeks.
  • Figure 2 shows evidence of hybridization of sequences in clone pSC22 with cultures of peripheral blood mononuclear cells isolated from a patient diagnosed as suffering from non-A, non-B hepatitis.
  • Figures 3 and 4 Cultures of mink lung cells superinfected with non-A, non-B hepatitis viral particles evidence sequences hybridizable with sequences from pSC22.
  • Figure 5 shows lack of hybridization of sequences in culture non-A, non-B hepatitis infected mink lung cells (as used in Figures 3 and 4) when vector pBR322 (lacking insert DNA of pSC22) is used.
  • Figure 6 shows the sequence of the DNA insert of clone pSC22 and a sequence of the protein encoded by that DNA.
  • Figure 7 shows the sequence of the DNA insert of clone pSN31 and the protein sequence encoded by the DNA.
  • Figure 8 shows the bands of immunoreactive protein from the 764 DNA-insert clones.
  • Figure 9 is a diagramatic presentation of clones pSN31 and pSN32.
  • the proteins expressed by the clones may be used as immunogens to elicit production of antibodies against the non-A, non-B hepatitis antigens.
  • the clones pSC22 pLC30, pLC50, pSN30, pSN31, and pSN32 disclosed herein can be used to propagate non-A, non-B sequences for use as probes which will hybridize with a complementary strand DNA in infected test samples.
  • the DNA sequences may be purified by means known in the art. Purification by gel electrophoresis is exemplified. The probe may be radiolabeled by nick translation. Standard procedures such as Southern blot and slot blot tests are appropriate.
  • compositions of the invention may be provided in test kits for use as diagnostic tools.
  • Compositions wherein antigens or antibodies to non-A, non-B viral antigens have been attached to a solid support are particularly useful for such purposes.
  • the clone (pSC22) described in the following examples is constructed by ligating the dephosphorylated EcoRI-restricted pBR322 with the EcoRI digest of liver DNA isolated from a chimpanzee during acute non-A, non-B hepatitis. Competent E. coli strain P678-54 were prepared and transformed by known procedures. Tet, amp transformants were selected on L-broth containing 20 ug/ml of ampicillin and tetracyline. Mini-lysates of tet- and amp-resistant transformants were screened for DNA inserts into pBR322 by the relative migration of plasmid DNA on 0.7% agarose gel. The DNA insert present in clone pSC22 is 764 bp.
  • a single needle biopsied chimpanzee liver speciman (chimpanzee No . CA6 ) was suspended in 1 ml ice- cold TE buffer, pH 8.0 (10 mM Tris-HCl, ImM EDTA) in a Wheaton, Dounce type tissue grinder. The specimen was homogenized with the glass pestle 10 times. One ml of STE, pH 8.0 (100 mM NaCl, 10 mM Tris-HCl, 1 mM EDTA) containing 4 mg/ml protease K (Boehringer-Mannheim) and 0.2% sodium lauryl sulfate was added to the homogenate and incubated for 2 hours at 37°C.
  • the mixture was extracted once with 2 ml phenol.
  • the aqueous phase was extracted with 2 ml phenol-chloroform (1:1 v/v).
  • the chloroform contains a 24:1 v/v ratio of isoamylalcohol.
  • the aqueous phase was treated with NaCl to a final concentration of 0.1 M.
  • Nucleic acids were precipitated twice with absolute alcohol and kept at -20°C for 30 minutes. The precipitate was centrifuged at 8,000 x g for 10 minutes and resuspended in 1 ml TE buffer, pH 8.0.
  • the nucleic acids were digested with 100 g/ml ribonuclease A at 37°C for 1 hour.
  • the phenol and phenol-chloroform extractions as described above were repeated.
  • the DNA is then treated with 0.1 M NaCl ethanol-precipitated, and centrifuged.
  • the restriction process was repeated using pBR322, 15 liters (1 ⁇ g/ul), instead of the liver DNA.
  • the EcoRI restricted CA6 liver DNA was electrophoresed in 0.7% low-melting agarose (Bethesda Research Laboratories) at 100 V in 100 mM Tris base, 100 mM anhydrous boric acid, and 2 mM Na2 EDTA H2O. At the end of the electrophoresis, the gel was divided into 3 seg- ments: top 4.5 cm, middle 3.5 cm, and bottom 2.3 cm. The DNA in each segment was recovered with the Bethesda Research Laboratory Pre-pac columns according to the manufacturer' s procedure.
  • pBR322 (1 mg/ml. New England BioLab) was EcoRI restricted and dephosphorylated with alkaline phosphatase from calf intestine (Boehringer-Mannheim). The treated vector, pBR322 (20 ⁇ l ) was incubated with 8 ul CA6 liver DNA from each of 3 segments separated by electrophoresis, 16 units of T4 DNA ligase (International Biotechnologies, Inc.), and 4 ⁇ l ligase buffer at 16°C for 2-3 minutes. LB broth (1 ml) was added, mixed gently, and incubated for 5-6 hours at 37°C.
  • the mixture was added to 5 ml soft LB agar containing 50 ug/ml amplicillin and 20 ⁇ g/ml tetracycline.
  • the soft agar mixture was immediately poured on top of 2 LB agar plates containing the same antibiotics and incubated overnight at 37°C.
  • pellets were resuspended in 12 ml of cold (sterile) 30 mM CaCl 2 and were allowed to stand on ice for 20 minutes. This incubation step was followed by centrifugation at 4,000 ⁇ g for 5 minutes.
  • the cell pellet was gently resuspended in 2.5 ml of ice-cold 30 mM CaCl 2 plus 15% glycerol. Samples (0.2 ml) of the competent cell suspension were stored in 1.5 ml Eppendorf tubes.
  • Transformants were grown as described and transferred to a nitrocellulose filter.
  • the filter was incubated on LB agar plate at 37°C overnight until colonies were 0.3 mm in diameter. Colony hybridization was performed as described by Grunstein.
  • the hybridization probe was 32p-labeled normal chimpanzee liver DNA. The colonies which did not react with the probe were selected. These were considered possible candidates to contain non-A, non-B hepatitis sequences.
  • Normal chimpanzee liver DNA was radiolabeled by [ 32 P]ATP using the same procedure as described above except that 2 ⁇ g normal chimpanzee liver DNA was used.
  • Example (b) illustrates the specific association of the cloned DNA insert with acute non-A, non-B hepatitis and the suitability for use in chimpanzees.
  • a chimpanzee is considered a surrogate human, permitting controlled evaluation in a protected environment.
  • a chronic non-A, non-B hepatitis patient known to contain 10 infectious viral particles/ml serum based on transmission of infectivity to chimpanzees was found positive by hybridization to the non-A, non-B hepatitis sequence in clone pSC22. The negative control individuals did not show reaction.
  • Chimpanzee No. 51 was screened to assure the absence of Epstein-Barr virus, cytomegalovirus, hepatitis A virus infection, and hepatitis B virus infection.
  • the animal was inoculated intravenously with 1 ml serum from a patient documented to be a chronic carrier of non-A, non-B hepatitis. Weekly bleedings from the animal were tested for serum aminotransferases.
  • These serial serum samples were also tested in a blind study for hybridization with the DNA insert in clone pSC22. The results shown in Fig. 1 clearly indicate that hybridization occurred only in the acute phase serum and liver samples. No evidence of hybridizable DNA is detected at other intervals.
  • the DNA insert from clone pSC22 can be used to detect infectious non-A, non-B hepatitis samples.
  • the non-A, non-B hepatitis sequence in clone pSC22 was found to hybridize to lijaer biopsied specimens of three non-A, non-B hepatitis infected chimpanzees, Nos. 1290, 1291, and 1279. Reaction was observed between 6 to 16 weeks post-inoculation. In contrast, no hybridization was observed in pre-inoculation specimens. Further, none of the liver specimens from two hepatitis B chimpanzees (Nos. 1196 and 59) showed hybridization (Table 1).
  • Chimpanzee liver cell cultures infected with non-A, non-B hepatitis viral particles from two patients contained sequences which hybridized with sequences from clone pSC22. The uninfected cultures showed no reaction.
  • Clones pLC30 and pLC50 were constructed in the same manner as the clone pSC22, using vector pSP65 instead of pBR322.
  • Clone pSC22 contains the DNA insert of Fig. 6, while pLC30 and pLC50 are complementary strands of DNA, one of which is the DNA insert of Fig. 6.
  • Clones pLC30 and pLC50 are effective for expression of polypeptides.
  • Plasmid pSC22 was subjected to EcoRI restriction to excise the 764 bp DNA insert.
  • the 764 bp DNA segment was then ligated into vector pGEM3Z to provide clone pSN30.
  • the pSN30 plasmid was transfected into E. coli DH50.
  • Plasmid pSN30 was restricted with PstI and religated with T4 DNA polymerase to generate a new recombinant plasmid, pSN31, which contains a 464 bp DNA insert.
  • a clone wherein the 464 bp had the opposite orientation was obtained by insertion of the segment into pGEM4Z to provide plasmid pSN32.
  • Clones pSN31 and pSN32 have been tested in the manner exemplified using pSC22 and have been shown to have similar characteristics. However, the smaller DNA insert provides greater specificity of reaction with nonA, non-B hepatitis virus, viral products, and antibodies to non-A, non-B hepatitis. Hence, the clones containing the 464 bp DNA inserts are preferred embodiments of the invention.
  • the 464 bp inserts may be further digested to provide small fragments of DNA which could be used as probes using hybridization techniques. However, clones from the smaller segments would not be competent for use in production of the full range of viral antigens provided by the clones containing the 464 bp insert. A segment of DNA from the 464 bp insert containing as few as 20 bp could be used as a probe using known hybridization techniques.
  • Antigens expressed by the clones are useful for detection of antibodies to non-A, non-B hepatitis antigens. Enzyme tests such as the ELISA test of RIA are preferred for this purpose.
  • the antigenic polypeptides may also be used as vaccines to elicit antibodies to the virus in at-risk populations.
  • Compositions containing the polypeptides may be formulated using adjuvants customary in the art.
  • Antigenic polypeptides may also be used to elicit antibodies for use in tests for detection of viral products by means known in the art.
  • the use of monoclonal antibodies to the peptides in radioimmunological tests is a preferred means of detection.

Abstract

L'invention de clones portant des séquences de nucléotides de l'hépatite non A, non B fournit une méthode de tests sanguins permettant de détecter la présence de matériaux de l'hépatite non A, non B. Le clone ADN est utilisé en tant que sonde d'hybridisation pour analyser des échantillons de sérum ou des échantillons de foie prélevés lors d'une biopsie en vue de détecter des séquences homologues. Des polypeptides exprimés par l'ADN cloné peuvent être utilisés en tant qu'antigènes dans des analyses de détection dans le but de déterminer si des anticorps contre l'hépatite non A, non B sont présents.
PCT/US1989/003638 1988-08-24 1989-08-24 Detection de l'hepatite non a, non b WO1990002206A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US23464188A 1988-08-24 1988-08-24
US234,641 1988-08-24
US34649289A 1989-05-02 1989-05-02
US346,492 1989-05-02

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Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5077193A (en) * 1988-12-20 1991-12-31 Immuno Japan Inc. Non-a, non-b hepatitis virus genome rna, cdna and virus antigen protein
US5106726A (en) * 1990-02-16 1992-04-21 United Biomedical, Inc. Synthetic peptides specific for the detection of antibodies to HCV
EP0531974A1 (fr) * 1991-09-12 1993-03-17 Cedars-Sinai Medical Center Détection directe de l'ARN du virus de l'hépatite-C
EP0544838A1 (fr) * 1990-08-25 1993-06-09 New York Blood Center, Inc. Antigene du virusd e l'hepatite non a, non b, procedes de diagnostics et vaccins
US5350671A (en) * 1987-11-18 1994-09-27 Chiron Corporation HCV immunoassays employing C domain antigens
US5436126A (en) * 1990-02-16 1995-07-25 United Biomedical, Inc. Synthetic peptides specific for the detection of antibodies to HCV, diagnosis of HCV infection and prevention thereof as vaccines
US5635346A (en) * 1991-03-26 1997-06-03 Dade International Inc. Assay for Non-A Non-B hepatitis
US5714314A (en) * 1990-06-12 1998-02-03 National Institute Of Health Hepatitis C virus antigen polypeptide, production method therefor, and antibody detection method
US5747239A (en) * 1990-02-16 1998-05-05 United Biomedical, Inc. Synthetic peptides specific for the detection of antibodies to HCV, diagnosis of HCV infection and preventions thereof as vaccines
US5998130A (en) * 1990-06-25 1999-12-07 The Research Foundation For Microbial Diseases Of Osaka University Non-A, non-B hepatitis virus genomic cDNA and antigen polypeptide
US6210675B1 (en) 1989-12-18 2001-04-03 Glaxo Wellcome Inc. PT-NANB hepatitis polypeptides
US7166287B1 (en) 1989-12-18 2007-01-23 Glaxo Wellcome Inc. Viral agent

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4356164A (en) * 1979-05-21 1982-10-26 Govt. of the U.S., as represented by the Secretary, Dept. of Health & Human Services Detection of non-A, non-B hepatitis associated antigen
US4464474A (en) * 1980-07-09 1984-08-07 Connaught Laboratories Limited Non-A, non-B hepatitis assay and vaccine
US4673634A (en) * 1985-03-08 1987-06-16 The United States Of America As Represented By The Department Of Health And Human Services Purified antigen from non-A, non-B hepatitis causing factor
US4702909A (en) * 1982-05-05 1987-10-27 Louisiana State University A & M Non-A, non-B hepatitis antigen, antigen compositions, vaccine and diagnostic reagent
US4707439A (en) * 1984-10-26 1987-11-17 The United States Of America As Represented By The Department Of Health And Human Services Screening test for reverse-transcriptase containing virus such as non-A, non-B hepatitis, NANBH
EP0263761A2 (fr) * 1986-10-07 1988-04-13 Mitsubishi Kasei Corporation Antigène d'hépatite non-A non-B

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4356164A (en) * 1979-05-21 1982-10-26 Govt. of the U.S., as represented by the Secretary, Dept. of Health & Human Services Detection of non-A, non-B hepatitis associated antigen
US4464474A (en) * 1980-07-09 1984-08-07 Connaught Laboratories Limited Non-A, non-B hepatitis assay and vaccine
US4702909A (en) * 1982-05-05 1987-10-27 Louisiana State University A & M Non-A, non-B hepatitis antigen, antigen compositions, vaccine and diagnostic reagent
US4707439A (en) * 1984-10-26 1987-11-17 The United States Of America As Represented By The Department Of Health And Human Services Screening test for reverse-transcriptase containing virus such as non-A, non-B hepatitis, NANBH
US4673634A (en) * 1985-03-08 1987-06-16 The United States Of America As Represented By The Department Of Health And Human Services Purified antigen from non-A, non-B hepatitis causing factor
EP0263761A2 (fr) * 1986-10-07 1988-04-13 Mitsubishi Kasei Corporation Antigène d'hépatite non-A non-B

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
INTERNATIONAL SYMPHOSIUM ON VIRAL HEPATITIS AND LIVER DISEASE, 26 May 1988 to 28 May 1988, published in 1988, (Alan R. Liss, Inc., London, England); Z. SCHAFF et al.: "Morphology, Immunohistochemistry, and In-Situ Hybridization of Experimental and Human Non-A, Non-B Hepatitis", pages 580-587, see especially the MATERIALS AND METHODS section on pages 580-581. *
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES (USA), Volume 82, published in 1985, (The National Academy of Sciences, Washington DC.),; B. SETO et al.: "A Glycoprotein associated with the non-A, non-B hepatitis agents", pages 4934-4938, see the Abstract. *
SCIENCE, Volume 244, published in 1989 (American Association for the Advancement of Science, Washington DC.); Q. CHOO et al.: "Isolation of a cDNA Clone Derived from a Blood Borne Non-A, Non-B Viral Hepatitis Genome", pages 359-362, see especially the Abstract. *

Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5350671A (en) * 1987-11-18 1994-09-27 Chiron Corporation HCV immunoassays employing C domain antigens
US5077193A (en) * 1988-12-20 1991-12-31 Immuno Japan Inc. Non-a, non-b hepatitis virus genome rna, cdna and virus antigen protein
US7166287B1 (en) 1989-12-18 2007-01-23 Glaxo Wellcome Inc. Viral agent
US6210675B1 (en) 1989-12-18 2001-04-03 Glaxo Wellcome Inc. PT-NANB hepatitis polypeptides
US5106726A (en) * 1990-02-16 1992-04-21 United Biomedical, Inc. Synthetic peptides specific for the detection of antibodies to HCV
US5436126A (en) * 1990-02-16 1995-07-25 United Biomedical, Inc. Synthetic peptides specific for the detection of antibodies to HCV, diagnosis of HCV infection and prevention thereof as vaccines
US5747239A (en) * 1990-02-16 1998-05-05 United Biomedical, Inc. Synthetic peptides specific for the detection of antibodies to HCV, diagnosis of HCV infection and preventions thereof as vaccines
US5714314A (en) * 1990-06-12 1998-02-03 National Institute Of Health Hepatitis C virus antigen polypeptide, production method therefor, and antibody detection method
US5734019A (en) * 1990-06-12 1998-03-31 National Institute Of Health Hepatitis C virus antigen polypeptide, production method therefor, and antibody detection method
US5998130A (en) * 1990-06-25 1999-12-07 The Research Foundation For Microbial Diseases Of Osaka University Non-A, non-B hepatitis virus genomic cDNA and antigen polypeptide
US6217872B1 (en) 1990-06-25 2001-04-17 The Research Foundation For Microbial Diseases Of Osaka University Non-A, non-B hepatitis virus genomic cDNA and antigen polypeptide
EP0544838A1 (fr) * 1990-08-25 1993-06-09 New York Blood Center, Inc. Antigene du virusd e l'hepatite non a, non b, procedes de diagnostics et vaccins
EP0544838A4 (en) * 1990-08-25 1997-09-10 New York Blood Center Inc Non-a, non-b hepatitis virus antigen, diagnostic methods and vaccines
US5635346A (en) * 1991-03-26 1997-06-03 Dade International Inc. Assay for Non-A Non-B hepatitis
EP0531974A1 (fr) * 1991-09-12 1993-03-17 Cedars-Sinai Medical Center Détection directe de l'ARN du virus de l'hépatite-C

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IL91371A0 (en) 1990-04-29
AU4193789A (en) 1990-03-23

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