WO1994025874A1 - Necessaire et procede de diagnostic simultane des hepatites b et c - Google Patents

Necessaire et procede de diagnostic simultane des hepatites b et c Download PDF

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Publication number
WO1994025874A1
WO1994025874A1 PCT/KR1994/000039 KR9400039W WO9425874A1 WO 1994025874 A1 WO1994025874 A1 WO 1994025874A1 KR 9400039 W KR9400039 W KR 9400039W WO 9425874 A1 WO9425874 A1 WO 9425874A1
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protein
khcv
hepatitis
dna
added
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PCT/KR1994/000039
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English (en)
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WO1994025874A9 (fr
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Joong Myung Cho
Deog Young Choi
Chun Hyung Kim
Hong Seob So
Jae Young Yang
In Soo Kim
Dong Seob Choi
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Lucky Limited
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Priority to JP06524113A priority Critical patent/JP3136327B2/ja
Publication of WO1994025874A1 publication Critical patent/WO1994025874A1/fr
Publication of WO1994025874A9 publication Critical patent/WO1994025874A9/fr

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    • 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
    • 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
    • C12N2730/00—Reverse transcribing DNA viruses
    • C12N2730/00011—Details
    • C12N2730/10011—Hepadnaviridae
    • C12N2730/10111—Orthohepadnavirus, e.g. hepatitis B virus
    • C12N2730/10122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • 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

  • the present invention relates to a diagnostic kit for the simultaneous diagnosis of hepatitis B and hepatitis C and a diagnostic method therefor by employing said diagnostic kit; and, more specifically, to a diagnostic kit for the simultaneous diagnosis of hepatitis B and C comprising a hepatitis B and a hepatitis C viral antigens together, and a diagnostic method utilizing said kit.
  • virus-induced hepatitis is known to be caused by various hepatitis viruses including hepatitis A, B, delta, E, cytomegalo, and Epstein-Barr viruses.
  • HBV hepatitis B virus
  • C gene encoding core protein C gene encoding core protein
  • P gene encoding DNA polymerase X gene encoding unidentified X protein
  • HBV antigens including HBV surface antigen, core antigen and e antigen have been used for diagnosing hepatitis B by detecting antibodies formed after HBV infection in the serum: specifically, the core antigen is useful for detecting hepatitis B at its incipient stage since anti-core antibodies are formed soon after HBV infection and are detectable in the serum of almost every patient infected by HBV; and, the surface antigen is used for determining whether hepatitis B has been cured since anti-surface antigen antibodies are found at the recovery stage of hepatitis B.
  • hepatitis C virus accounts for about 70 to 80% of hepatitis caused by blood transfusion (Alter, H.J., et al., Lancet, 2 , 838-841(1975); and Dienstag, J.L., et al., Seminar Liver Pis., 6, 67-81(1986)); and, such post-transfusion hepatitis frequently progresses into cirrhosis or hepatocellular carcinoma.
  • Said virus is one of RNA viruses consisting of one positive RNA strand and produces a polyprotein precursor from an open reading frame (ORF) of the strand(Choo, Q. L., et al., Science, 244, 359-362(1989); and, Choo, Q. L., et al., Proc. Natl. Acad. Sci. USA, 88, 2451-2455(1991)).
  • hepatitis C virus The gene structure of hepatitis C virus is similar to that of flavivirus or pestivirus (Miller, R. H., et al., Proc. Natl. Acad. Sci. USA, 87, 2057-2061(1990); and, Muraiso, K., et al., Biochem. Biophys. Res.
  • the polyprotein of hepatitis C virus consists of, from N-terminal to C-terminal, core-envelope 1(E1)-envelope 2/non-structural 1 protein(E2/NS1)-non-structural 2 protein(NS2)-non-structural 3 protein(NS3)-non-structural 4 protein(NS4)-non-structural 5 protein(NS5) (Choo, Q, L., et al., Proc. Natl. Acad. Sci. USA, 88 , 2451-2455(1991); Takamizawa. A., et al., J. Virol., 65, 1105-1113(1991); and Kato, N., et al., Proc. Natl. Acad. Sci. USA, 87, 6524-6528(1990)).
  • the infection of hepatitis C virus can be diagnosed by detecting hepatitis C viral RNA directly from a blood sample by using polymerase chain reaction(PCR) (Hosoda, K., et. al., Hepatology, 15, 777-781(1992); Abe, K., et al., Hepatology, 15, 690-695(1992); and Alter, H. J., Annals of Internal Medicine 115, 644-649(1991)), by which the viral RNA can be detected readily, i.e., within 1 to 2 weeks from the infection; however, it entails a high cost and long time due to the need to analyze numerous samples.
  • PCR polymerase chain reaction
  • Another diagnostic method is to detect the antibodies against hepatitis C virus present in the serum sample, e. g., by an enzyme-linked immunoassay using C100-3 protein, (Choo, Q. L., et al., Proc. Natl. Acad. Sci. USA, 88, 2451-2455 (1991); and Kuo, G., et al., Science, 244, 362-384(1989)).
  • Contreras et al. discloses that said diagnostic method using ELISA occasionally exhibits false-positive results on the serum of patients suffering from hepatic diseases other than hepatitis C, for example, biliary cirrhosis, autoimmune disease, cryptogenic hepatocirrhosis, etc. (Contreras M., et. al., Lancet, 2 , 505(1989); Cash J. D., et, al., Lancet, 2 , 505(1989)).
  • Theilmann et al. reports that more than 60% of the patients having rheumatoid arthritis show HCV-positive signal when diagnosed with the first generation diagnostic agent and method of Ortho Diagnostic Systems Inc. (Lancet, 335, 1346(1990)).
  • the above results suggest that the diagnostic method by using ELISA may exhibit false-positive results and, therefore, confirmation tests are also required.
  • Chiron Co. and Ortho Diagnostic Inc. of the U.S. developed an improved diagnostic method by employing a recombinant immunoblot assay(RIBA) to confirm the diagnosis results obtained by using ELISA, in which said improved diagnostic method comprises blotting two HCV-specific antigens of SOD-5-1-1 and SOD-C100-3 to a nitrocellulose filter, reacting said antigens with a serum taken from a hepatitis C patient, reacting the antigen-antibody complex with anti-human IgG antibody labeled with peroxidase, and then determining the existence of anti-HCV antibodies in the serum on the basis of color intensity of each antigen band.
  • Ortho Diagnostic Systems Inc. of the U.S. also reported a second generation RIBA diagnostic agent (RIBA II) having an improved sensitivity to anti-HCV antibodies, which was prepared by adding core antigen C22-3 and non-structural 3 antigen C33C to the pre-existing first generation diagnostic agent.
  • RIBA II second generation RIBA diagnostic agent
  • Vallari et al. reports that anti-HCV antibodies can be detected more earlier and in a higher sensitivity by using dot immunoblot assay with a diagnostic agent comprising said C22-3, C33C and C100-3 proteins than the method using only the C100-3 protein(J. Clin. Microbiol., 30(3), 552(1992)).
  • hepatitis B and hepatitis C have been diagnosed separately with separate diagnostic kits and samples, entailing high costs and requiring a larger amount of samples, but the specificity and accuracy of the existing methods are not satisfactory.
  • the present inventors have endeavored to develop a diagnostic kit and/or method for the simultaneous diagnosis of hepatitis B and C with higher sensitivity and specificity than those of the existing ELISA method or RIBA method.
  • Another object of the present invention is to provide a diagnostic method for the simultaneous diagnosis of hepatitis B and hepatitis C by employing said diagnostic kit.
  • a diagnostic kit which comprises one or more HCV antigenic proteins comprising one or more epitopes of core protein, non-structural 3 protein, non-structural 4 protein, non-structural 5 protein, and envelope protein of hepatitis C virus; and, one or more HBV antigenic proteins comprising one or more epitopes of core protein and surface antigen protein of hepatitis B virus.
  • a diagnostic method for the simultaneous diagnosis of hepatitis B and hepatitis C by employing the diagnostic kit of the present invention.
  • Fig. 1 shows the nucleotide sequence encoding KHCV CORE 14 protein, and the amino acid sequence of the polypeptide encoded therein;
  • Fig. 2 depicts the nucleotide sequence encoding KHCV 897 protein, and the amino acid sequence of the polypeptide encoded therein;
  • Fig. 3 describes the nucleotide sequence encoding KHCV NS4 protein, and the amino acid sequence of the polypeptide encoded therein;
  • Fig. 4 represents the nucleotide sequences encoding KHCV E1G, KHCV E2A and KHCV E2E, respectively, and the amino acid sequences of the polypeptide encoded therein;
  • Fig. 5 shows the nucleotide sequence encoding KHCV NS5-1.2 protein, and the amino acid sequence encoded therein;
  • Fig. 6 depicts the nucleotide sequence of HBV core gene, and the amino acid sequence of the polypeptide encoded therein;
  • Fig. 7 represents the nucleotide sequence encoding an HBV surface antigen, and the amino acid sequence of the polypeptide encoded therein;
  • Fig. 8 portrays, an expression vector for the expression of a nucleotide sequence encoding KHCV CORE 14 protein and KHCV 897 protein fused with ubiquitin gene;
  • Fig. 9 A shows the result of SDS-polyacrylamide gel electrophoresis (SDS-PAGE) after the expression of a nucleotide sequences encoding KHCV UBCORE 14 protein and KHCV UB897 protein respectively, in E. coli cells;
  • Fig. 9 B shows the result of western blotting analysis with the gel of Fig. 9 A by employing a serum of hepatitis C patient;
  • Fig. 10 delineates the preparation strategy for preparing an expression vector for the expression of a nucleotide sequence encoding KHCV NS4E protein fused with ubiquitin gene;
  • Fig. 11 A shows the result of SDS-PAGE after the expression of a nucleotide sequence encoding KHCV UBNS4E protein in E. coli cells;
  • Fig. 11 B shows the result of western blotting analysis with the gel of Fig. 11 A by using a serum of hepatitis C patient;
  • Fig. 12 depicts the strategy for ligating the DNA fragments encoding KHCV E1G, KHCV E2A and KHCV E2E and a ubiquitin gene, and preparing an expression vector for the expression of the ligated DNA(UB E1E2 DNA);
  • Fig. 13 A represents the result of SDS-PAGE after the expression of UB E1E2 DNA in E. coli cells
  • Fig. 13 B represents the result of western blotting analysis with the gel of Fig. 13 A by a using a serum of hepatitis C patient;
  • Fig. 14 shows a schematic diagram for preparing an expression vector for expressing a recombinant DNA encoding UBNS4E1E2 protein comprising ubiquitin, KHCV NS4E protein and KHCV E1E2 protein;
  • Fig. 15 A represents the result of SDS polyacrylamide gel electrophoresis (SDS-PAGE) after the expression of the recombinant DNA encoding UBNS4E1E2 protein in E. coli cells;
  • Fig. 15 B represents the result of western blotting analysis of the gel of Fig. 15 A by using serum of hepatitis
  • Fig. 16 shows a schematic diagram for preparing an expression vector for the expression of a nucleotide sequence encoding KHCV NS5-1.2 protein fused with ubiquitin gene;
  • Fig. 17 A shows the result of SDS polyacrylamide gel electrophoresis (SDS-PAGE) after the expression of the recombinant DNA encoding KHCV UBNS5-1.2 protein in E. coli cells;
  • Fig. 17 B shows the result of western blotting analysis with the gel of Fig. 17 A by using a serum of hepatitis C patient;
  • Fig. 18 depicts a schematic diagram for preparing an expression vector for the expression of a fused gene(UB HBV CORE DNA) of HBV CORE gene and a ubiquitin gene;
  • Fig. 19 A shows the result of SDS-PAGE after the expression of UB HBV CORE DNA in E. coli cells
  • Fig. 19 B shows the result of western blotting analysis with the gel of Fig. 19 A by using a serum of hepatitis B patient;
  • Fig. 20 depicts a schematic diagram for preparing an expression vector for the expression of a DNA fragment encoding HBV surface antigen in yeast cells
  • Fig. 21 shows the result of SDS-PAGE after the expression of the DNA fragment encoding HBV surface antigen in yeast cells.
  • Fig. 22 discloses the results of diagnosing serum samples by employing immunoblot assay with the diagnostic kit of the present invention.
  • hepatitis C virus refers to a virus causative of non-A non-B hepatitis or hepatitis C.
  • HCV and hepatitis C are used interchangeably herein.
  • KHCV Korean-type hepatitis C virus
  • Korean hepatitis C patients whose cDNA has an open reading frame of a nucleotide sequence encoding the amino acid sequence, wherein the amino acids having the numbers of 842, 849 and 853 are phenylalanine, leucine and threonine; or leucine, phenylalanine and alanine, respectively.
  • epitope refers to an antigenic determinant of a polypeptide which is capable of eliciting an immune response in an immunologically competent host organism and/or is capable of specifically binding itself to a complementary antibody.
  • An epitope of the present invention generally consists of at least 6 amino acids, preferably 7 or 8 amino acids.
  • the number of a nucleotide of HCV cDNA or of an amino acid of HCV protein is based on the fulll KHCV nucleotide sequence or amino acid sequence disclosed in Korean Patent Laid-open Publication No. 93-683.
  • KHCV KHCV-LBC1
  • ATCC American Type Culture Collection
  • ATCC 75008 accession number of ATCC 75008 under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganism for the Purpose of Patent Procedure; see Korean Patent Laid-open Publication No. 93-683
  • primers for polymerase chain reaction which correspond to the 5'- and 3'-ends of cDNA fragments encoding KHCV897 protein, envelope 1 and envelope 2 proteins, or non-structural 5 protein.
  • a polymerase chain reaction is carried out by using said primers and KHCV897 gene (which was deposited at ATCC on June 27, 1991 with an accession number of ATCC 68640 under the Budapest Treaty), KHCV envelope gene(which was deposited at ATCC on December 11, 1991 with an accession numbers of ATCC 68878, 69866 and 74117) and KHCV NS5 gene (see Korean Patent Laid-open Publication No. 93-683) as templates to obtain cDNA fragments of KHCV897 gene, KHCV envelope 1 and 2 gene, and KHCV NS5 gene. Each cDNA fragment is inserted into a vector, and the expression vector is used to transform a suitable host organism such as E. coli.
  • polypeptides produced by the transformed host cells are subjected to an electrophoresis on polyacrylamide gel and then to a western blotting analysis by using sera of hepatitis C patients to confirm which polypeptides react specifically with anti-KHCV antibodies as epitopes of KHCV antigens. The location of confirmed epitopes in full sequence of KHCV cDNA is also examined.
  • epitopes are found to exist in the carboxyl terminal of KHCV envelope 1 protein which is expressed from the 309 base pairs corresponding to from the 1201st to the 1509th nucleotides of KHCV cDNA(ElG protein); in the amino terminal of KHCV envelope 2 protein which is expressed from the 240 base pairs corresponding to from the 1510th to the 1749th nucleotides of KHCV cDNA(E2A protein); and in the carboxyl terminal of KHCV envelope 2 protein which is expressed from the 249 base pairs corresponding to from the 2281st to the 2529th nucleotides of KHCV cDNA(E2E protein) (see Fig. 4 for amino acid of KHCV E1G, KHCV E2A and KHCV E2E protein and nucleotide sequences encoding them).
  • epitopes of NS5 protein exist in the amino terminal of NS5 protein encoded by 1,200 base pairs corresponding to from the 6649th to the 7284th nucleotides including a KHCV403 cDNA fragment and reacts specifically with a serum of KHCV patient in a higher sensitivity than KHCV403.
  • Epitopes of HCV or HBV antigens are very important for the development of efficient and economical diagnostic agents.
  • the fusion proteins comprising one or more epitopes are more preferable in terms of economy, efficiency and accuracy; and the fusion proteins comprising more than one epitopes are most preferable.
  • HCV recombinant protein comprising more than one HCV epitopes
  • KHCV CORE 518 fusion protein comprising the epitopes of KHCV core protein and NS3 protein
  • KHCV NS4E1E2 fusion protein comprising the epitopes of KHCV El, E2 and NS4 proteins.
  • the recombinant proteins may be prepared by employing various expression vector systems containing a nucleotide sequence encoding said fusion proteins; and, the vector may be capable of directing production of a recombinant fusion protein comprising said fusion proteins and other specific proteins, preferably, ubiquitin which can increase the expression rate of the protein.
  • the recombinant fusion protein comprising ubiquitin as well as the KHCV fusion protein can be used for the purpose of the invention as long as it retains the necessary characteristic of KHCV protein, e.g., antigenicity of HCV.
  • the above expression system may be effectively employed where the desired protein is unstable and can be digested easily by proteinases in a host cell since the ubiquitin can protect the desired protein from the protease attack or stabilize it. Moreover, the expression of a desired recombinant protein fused with ubiquitin can be confirmed by using anti-ubiquitin antibodies and easily purified by using the properties of ubiquitin.
  • HBV core antigen and surface antigen comprising one or more epitopes thereof, respectively.
  • the amino acid sequences of HBV core antigen and surface antigen(Pre S2 S Ag) are exemplified in Figs. 6 and 7, respectively. Since many types of HBV are known to exist, the amino sequences of HBV core antigen and surface antigen may be slightly different in each type of HBV, which may also be employed for the present invention.
  • the HBV antigenic proteins may be prepared by expressing a DNA fragment which encodes one of the HBV antigenic proteins by employing various expression vector systems.
  • the DNA fragment encoding the HBV antigenic protein may be prepared, for example, by employing PCR with the synthetic primers which correspond to the 5'- and 3'-ends of cDNA fragments encoding HBV core antigen and surface antigen, respectively.
  • the HBV antigenic proteins may be prepared as a form of recombinant protein comprising the HBV antigenic protein and other specific proteins which can increase the protein stability or facilitate the purification procedure, preferably, ubiquitin, as described in section 2 above.
  • a compatible host cell is transformed with an expression vector containing an HCV or HBV cDNA fragment encoding HCV or HBV epitopes; and the transformed cell is cultured under a condition that allows the expression.
  • Selection of an appropriate host organism is affected by a number of factors as known in the art. These factors include, for example, compatibility with the chosen vector, toxicity of the proteins encoded by the recombinant plasmid, ease of recovery of the desired protein, protein characteristics, biosafety and costs. A balance of these factors must be considered, and it must be understood that not all hosts will be equally effective for the expression of a particular recombinant DNA molecule.
  • Suitable host organisms which can be used in this invention include, but are not limited to, bacteria such as
  • Escherichia coli and yeasts such as Saccharomyces cerevisiae.
  • polypeptides produced in a host cell may be isolated and purified by a combined use of conventional methods, e.g., cell disruption, centrifugation, dialysis, salting-out, chromatography, gel filtration, electrophoresis and electroelution.
  • polypeptides of this invention can also be chemically synthesized by suitable methods such as exclusive solid phase synthesis, partial solid phase method, fragment condensation or classical solution synthesis. Solid phase synthesis as disclosed by Merrifield(J. Am. Chem. Soc., 85, 2149(1963)) is preferred.
  • amino acid substitutions in proteins which do not substantially alter biological and immunological activities are known to occur and have been described, e.g., by Neurath et al., in The Proteins, Academic Press, New York(1979).
  • Such functionally equivalent amino acid substitutions are within the scope of the invention as long as the resulting proteins retain the same antigenic properties.
  • the diagnostic kit in accordance with the present invention comprises at least one HCV antigenic protein comprising one or more HCV epitopes, preferably, including KHCV NS4E protein, KHCV E1G protein, KHCV E2A protein, KHCV E2E protein, KHCV COREEPI protein, KHCV 518 protein and KHCV NS5-1,2 protein, and at least one HBV antigenic protein comprising one or more epitopes of HBV core protein or surface antigen protein.
  • HCV antigenic protein comprising one or more HCV epitopes, preferably, including KHCV NS4E protein, KHCV E1G protein, KHCV E2A protein, KHCV E2E protein, KHCV COREEPI protein, KHCV 518 protein and KHCV NS5-1,2 protein, and at least one HBV antigenic protein comprising one or more epitopes of HBV core protein or surface antigen protein.
  • HCV or HBV antigenic proteins which are in the form of a recombinant protein wherein the epitope(s) is fused with another protein, preferably, ubiquitin.
  • the diagnostic kit in accordance with the present invention may comprises KHCV CORE14 protein, KHCV UB897 protein, KHCV UBNS4E protein, KHCV UBNS4E1E2 protein, KHCV UBNS5-1,2 protein, HBV CORE protein and HBV S2 S Ag protein. Therefore, the present kit can give more accurate diagnostic results in detecting HCV owing to their ability to detect antibodies specific to HCV envelope protein and NS5 protein, which has not been detectable by using other preexisting diagnostic kits. Moreover, HBV CORE protein and HBV S2 surface antigen which are also comprised in the present kit allow the diagnosis of hepatitis B simultaneously with hepatitis C.
  • the amount of each of the antigenic proteins included in the diagnostic kit of the present invention may be optionally adjusted, and it is preferable to use each protein in an equal molar amount.
  • ubiquitin is also included in the kit as a control protein.
  • the diagnostic kit of the present invention may comprise buffers, human immunoglobulin as a positive control, supporting materials or other agents which may be necessary, depending on the diagnostic method employed with the kit.
  • the diagnosis for hepatitis B and C can be carried out by using any known method in the art with the diagnostic kit of the present invention; and, preferably, by using immunoblot assay.
  • the present invention also relates to a diagnostic method employing a diagnostic kit in accordance with the present invention by using immunoblot assay.
  • the diagnostic method of the present invention is more specific and accurate for detecting anti-HBV and anti-HCV antibodies in the serum of hepatitis patients than any of the existing methods, and therefore, it can be used as a diagnostic method and confirm test for hepatitis C and as a diagnostic method for diagnosing HBV and HCV at the same time.
  • the diagnostic method by employing the antigenic proteins may comprise the following steps:
  • each of one or more HBV antigenic proteins and one or more HCV antigenic proteins are added to a solid support, e.g., a nitrocellulose filter or well of microliter to make said antigenic protein adsorb onto the surface of the material;
  • a solid support e.g., a nitrocellulose filter or well of microliter to make said antigenic protein adsorb onto the surface of the material;
  • a putative sample diluted with a diluent is added to the antigen-coated solid support where the antigen- antibody complex would be formed if there were anti-HBV antibodies or anti-HCV antibodies in the serum;
  • an enzyme e.g., HRP(horseradish peroxidase), or alkaline phosphatase-conjugated anti-human IgG is added to the supporting material to allow the enzyme to bind the antibodies of the complex formed in the second step; and
  • substrates for the enzyme e.g., O-phenylene diamine dihydrochloric acid(OPD) and hydrogen peroxide for peroxidase are added to the material to develop a color reaction.
  • O-phenylene diamine dihydrochloric acid(OPD) and hydrogen peroxide for peroxidase are added to the material to develop a color reaction.
  • the degree of color intensity can be measured with a densitometer or a microwell reader; and the existence of anti-HCV antibodies or anti-HBV antibodies can be determined on the basis of the result.
  • the solid support used in the first step may include nitrocellulose filter membrane, vinyl membrane and immobilon P R , preferably nitrocellulose filter membrane.
  • HBV antigenic proteins may be adsorbed onto the solid support in admixture, or separately; and so may HCV antigenic proteins.
  • the diagnostic method by employing the diagnostic kit of the present invention comprises the following steps:
  • (F) determining the color density of each band after developing a color reaction on the strip obtained in (E).
  • the recombinant protein(s) of the present invention which comprises more than one epitope is used for preparing a diagnostic agent, it would allow more sensitive and accurate diagnosis than the case using existing antigens with only one epitope.
  • the diagnostic kit of the present invention which comprises more than one recombinant proteins comprising more than one HBV or HCV epitopes show an excellent diagnostic result.
  • restriction endonuclease and reaction buffers were added to be a reaction volume ranging from 50 to 100 ⁇ l, and the reaction was carried out at a temperature of 37 °C for 1 to 2 hours.
  • the reaction mixture was heat-treated at 65°C for 15 minutes (or extracted with phenol and precipitated with ethanol in the case of a heat-resistant endonuclease) to inactivate the restriction endonuclease.
  • Restriction enzymes and reaction buffers used in this example were purchased from NEB (New England Biolabs, Jolla, MA, U.S.A.).
  • 10 ⁇ reaction buffer for the reaction of a restriction endonuclease has the following composition: 10 ⁇ NEB reaction buffer 1: 100mM bis Tris propane-HCl, 100mM MgCl 2 , 10mM dithiothreitol(DTT), pH 7.0
  • 10 ⁇ NEB reaction buffer 2 100mM Tris-HCl, 100mM MgCl 2 , 500mM NaCl, 10mM DTT, pH 7.0
  • 10 x NEB reaction buffer 3 100mM Tris-HCl, 100mM MgCl 2 , 1000mM NaCl, 10mM DTT, pH 7.0
  • 10 ⁇ NEB reaction buffer 4 200mM Tris-acetate, 100mM magnesium acetate, 500mM potassium acetate, 10mM DTT, pH 7.0
  • reaction mixture was extracted with phenol for the purpose of inactivating the enzyme or recovering the DNA in the reaction mixture, wherein phenol preequilibrated with a buffer containing 10mM Tris-HCl (pH 8.0) and ImM EDTA was used.
  • Phenol extraction was carried out by mixing equal volumes of the sample and the phenol with vigorous shaking; centrifuging the mixture at 15,000rpm for 5 minutes; and transferring the aqueous layer into a new tube. The above procedure was repeated three times.
  • Reference Example 3 Ligation Reaction
  • Ligation reaction of DNA was carried out by employing T4 DNA ligase and 10 ⁇ ligation reaction buffer(0.5M Tris-HCl, pH 7.0, 0.1M MgCl 2 , 0.2M DTT, 10mM ATP, 0.5mg/ml bovine serum albumin(BSA) ) purchased from NEB.
  • the reaction volume was generally 20 ⁇ l, and 10 units of T4 ligase was used for the ligation of cohesive ends of DNA, while 100 units was used for the ligation of blunt ended DNAs.
  • the reaction was carried out at 16°C for 5 hours or at 4°C for over 14 hours; and, after the reaction was completed, the reaction mixture was heated at 65°C for 15 minutes to inactivate T4 DNA ligase.
  • E. coli strains e. g., E. coli HB101(ATCC 33694), E. coli W3110(ATCC 27325) or E. coli JM105(ATCC 47016)
  • E. coli HB101(ATCC 33694) E. coli W3110(ATCC 27325) or E. coli JM105(ATCC 47016)
  • a method known in the art e.g., as described by Maniatis et al., in Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, N.Y.(1982), or by Cohen in Proc. Natl. Acad. Sci. U.S.A. 69, 2110(1972).
  • Oligonucleotides were synthesized by employing a DNA synthesizer (Applied Biosystems Inc., model No. 380B, U.S.A.) using automatic solid phase phosphoamidite chemistry.
  • the synthesized oligonucleotides were purified by using denaturing polyacrylamide gel(2M urea, 12% acrylamide and bis(29:1), 50mM Tris, 50mM boric acid, ImM EDTA-Na 2 ) electrophoresis and C 18 SEP-PAK(Waters Inc., U.S.A) column chromatography by using acetonitrile:water (50 :50) as an eluent; and the amount was determined by measuring O.D. at 260nm.
  • 10 ⁇ l of 10 ⁇ Taq polymerase reaction buffer (10mM Tris-HCl, pH 8.3, 500mM KCl, 15mM MgCl 2 , 0.1% (w/v) gelatin), 10 ⁇ l of a mixture of dNTP's(each of dGTP, dATP, TTP and dCTP is 10mM), 2 ⁇ g of each primer (generally, 2 primers were used for a reaction, and in the case that 3 primers were used, the primer located in the middle was used in an amount of 0.02 ⁇ g), and 0.5 ⁇ l of AmpliTaq DNA polymerase (Perkin Elmer Cetus, U.S.A.) was added distilled water in an amount to make a total volume of 100 ⁇ l; and 50 ⁇ l of mineral oil was added thereto to protect the reaction mixture from evaporation.
  • 10 ⁇ Taq polymerase reaction buffer 10mM Tris-HCl, pH 8.3, 500mM KCl, 15mM MgCl 2 , 0.1% (w
  • the PCR was carried out by using a thermal cycler (Perkin Elmer Cetus, U.S.A.); and the thermal cycle was programmed to repeat 25 times or more, the cycle of: 95°C for 1 minute ⁇
  • Antibodies against the Fc region of human IgG were purified by chromatography using human IgG-attached sepharose CL-4B affinity column and protein-G column(Pharmacia LKB, Sweden) to obtain said antibodies with a purity over 90%.
  • ⁇ obtained antibodies were labelled with horseradish peroxidase according to sodium periodate method described in Nakane, et al., J. Histochemcytochem. 22, 1084(1974) as follows:
  • KHCV CORE14 and KHCV 897 DNAs which consist of the region from the 343rd to 726th nucleotides and the region from the 3916th to the 4713th nucleotides of KHCV-LBC1, respectively
  • primers were synthesized:
  • TATAC 3'-end two stop codons
  • nucleotides on the 5'-end region comprising 25 nucleotides on the 5'-end region overlapping with those on the 3'-end region of the ubiquitin gene, and other nucleotides designed to initiate translation from the 3916th nucleotide of KHCV-LBC1.
  • Test tube A was provided with the primer PCOREUB1 2 ⁇ g and primer PSALC0RE14 2 ⁇ g.
  • Test tube B was provided with the primer PK897SAL 2 ⁇ g and primer PK897UB1 2 ⁇ g.
  • To each of the tubes were added 50ng of KHCV-LBC1 DNA(ATCC 75008), 10 ⁇ l of 10 ⁇ polymerase buffer solution, 10 ⁇ l of 2mM dNTP(2mM dGTP, 2mM dATP, 2mM TTP, 2mM dCTP), 2.5 unit of Taq polymerase; and distilled water was added thereto to adjust the total volume to be 100 ⁇ l.
  • the PCR products obtained in the above (1-2) were subjected to 5% polyacrylamide gel electrophoresis. As a result, it was confirmed that about 384 bp of DNA in Tube A, about 897 bp of DNA in Tube B were amplified.
  • the DNA fragments were purified by the same polyacrylamide gel electrophoresis as (1-2) above and named fragment K384 and K897, respectively.
  • TGTAGTCAGACAAGGTTCTACCATCTTCTAGTTGCTTACCAGCAAAAA-3' UBI1 was designed to have a recognition site of Nde
  • UBI3 was designed to have .a recognition site of Sac II (5'-CCGCGG-3') without any change in the amino acid sequence encoded therein.
  • Tube A 2 ⁇ g of primer UBI1 and 2ug of primer PSALCORE14; and Tube B: 2 ⁇ g of primer UBI1 and 2 ⁇ g of primer PK897SAL.
  • the fragments obtained in tubes A and B were named fragments KHCV UBCORE14 and KHCV UB897, respectively.
  • Ligation Tube A was provided with 100ng of KHCV UBCORE14; and ligation Tube B was provided with 100 ng of KHCV UB897.
  • To each of the tubes were added 100ng of PL, 100ng of PT, 2 ⁇ l of 10 ⁇ ligation buffer solution and 10 units of T4 DNA ligase; and distilled water was added thereto to adjust the total volume to be 20 ⁇ l. The reaction was carried out at 16°C for 12 hours.
  • Each of the ligated vectors was isolated; and E. coli HB101(ATCC 33694) was transformed with each of the vectors.
  • the vector containing fragment UBCORE14 was isolated and named ptrpH-UB-CORE14; the vector containing fragment KHCV UB897 was isolated and named ptrpH-UB-KHCV897; ⁇ Step 4> Expression of KHCV UB CORE14 and KHCV UB897 DNAs
  • E. coli W3110(ATCC 37339) cells were transformed with each of the plasmids prepared in the above ⁇ Step 3>.
  • E. coli W3110 transformed with ptrpH-UB-KHCV897 was deposited at ATCC on June 27, 1991 with the accession number of ATCC 69640; and E. coli W3110 transformed with ptrpH-UB-CORE14 was deposited at ATCC on July 1, 1991 with the accession number of ATCC 68642 under the terms of Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure.
  • E. coli W3110(ATCC 37339) cells transformed with the plasmids ptrpH-UB-CORE14 and ptrpH-UB-KHCV897 was cultured with shaking in liquid LB medium(6% Bacto-tryptone, 0.5% yeast extract, 1% NaCl) containing 50 ⁇ g/ml ampicillin at 37°C for 12 hours. 5ml of the culture was transferred into 19 of M9 medium( 40mM K 2 HPO 4 , 22mM KH 2 PO 4 , 8.5mM NaCl, 18.7mM NH 4 C1, 1% glucose, 0. ImM MgSO 4 , 0.1mM CaCl 2 , 0.4% casamino acid, 10 ⁇ l/ml Vit.
  • M9 medium 40mM K 2 HPO 4 , 22mM KH 2 PO 4 , 8.5mM NaCl, 18.7mM NH 4 C1, 1% glucose, 0. ImM MgSO 4 , 0.1mM CaCl 2 , 0.4%
  • lane M represents the standard molecular size marker, i.e., 72, 43, 29 and 18 kilodaltons from the top;
  • lane 1 shows the products of E. coli having plasmid without KHCV gene;
  • lane 2 shows the products of E. coli transformed with ptrpH-UB-CORE14 wherein about 23kd of protein was produced;
  • lane 5 shows the products of E. coli transformed with ptrpH-UB-KHCV897 wherein about 40kd protein was produced; and lanes 3, 4 and 6 show another KHCV proteins.
  • the proteins separated on the gel were blotted onto a nitrocellulose filter (Bio-Rad Lab., pore size 0.22 ⁇ m, CA, U.S.A) according to the methods described in Towbin, Proc. Natl. Acad. Sci. U.S.A. 76, 4750(1979).
  • the filter was put in PBS(10mM phosphate, pH 7.0, 0.15M NaCl) containing 0.2% Tween 20; and shaken at room temperature for 2 hours to block the nonspecific binding of IgG to the proteins.
  • the filter was put in IgG solution prepared by diluting IgG(8.2mg/ml) purified from Korean HCV patients with 200-fold volume of PBS containing 0.5% gelatin and 0.05% Tween 20; and mildly shaken for 1 hour at room temperature to react the protein and IgG. The filter was then washed four times with PBS containing 0.2% Tween 20, each for 5 minutes. The filter was put in an anti-human IgG antibody solution prepared by diluting goat anti-human IgG labeled with horseradish peroxidase (Bio-Rad Lab., CA, U.S.A.) with 200-fold volume of PBS containing 0.5% gelatin and 0.05% Tween 20, and shaken at room temperature for 1 hour. The filter was washed four times with PBS containing 0.2% Tween 20, each for 5 minutes and then, twice with 50mM Tris buffer solution(pH 7.0).
  • IgG solution prepared by diluting IgG(8.2mg/ml
  • E. coli cell precipitate obtained in ⁇ Step 4> was suspended in 20ml of buffer 1(50mM Tris, pH 7.5, 5mM EDTA, 10mM ⁇ -mercaptoethanol, 1mM phenyl methyl sulfonyl fluoride, 1 ⁇ g/ml pepstatin A) at 4°C.
  • buffer 1 50mM Tris, pH 7.5, 5mM EDTA, 10mM ⁇ -mercaptoethanol, 1mM phenyl methyl sulfonyl fluoride, 1 ⁇ g/ml pepstatin A
  • 9M urea was added to the supernatant to the final concentration of 8M and the resultant was stirred for 12 hours at room temperature.
  • the solution containing KHCV UB-CORE 14 protein obtained in (6-A-3) was passed over a column (2.5cm ⁇ 10cm) having 25ml of CM-Sepharose resin (Pharmacia, Sweden) equilibrated with buffer 2(8M urea, ImM EDTA, 10mM ⁇ -mercaptoethanol and 10mM acetate, pH 5.0) at a flow rate of 1ml/min. Materials remaining in the column in free form were thoroughly washed with said buffer solution. Then, 500ml of the above buffer 2 having a concentration gradient of 0 to 0.5M sodium chloride was added at a flow rate of 3ml/min. to elute the bound proteins.
  • the eluate was subjected to SDS-polyacryl amide gel electrophoresis, which indicated that KHCV UBCORE 14 protein was eluted at about 0.3M.
  • the fractions containing KHCV UBCORE 14 were collected for use in the next step.
  • the fractions collected in (6-A-4) were concentrated to a volume of 10ml with YM5 ultrafiltration membrane (Amicon, U.S.A.). The concentrate was passed over S-200 Sephacryl column(2.5cm ⁇ 100cm, Pharmacia, Sweden) equilibrated with PBS solution containing 6M urea, ImM EDTA and 1 mM ⁇ -mercapto-ethanol at a flow rate of 0.5ml/min to separate proteins according to their molecular weight. The protein fractions were subjected to SDS-polyacrylamide gel electrophoresis. Fractions comprising KHCV UBCORE 14 protein were collected.
  • the fractions comprising KHCV UBCORE 14 protein obtained in (6-A-5) were diluted with the same volumes of buffer 3 ( 6M urea, 1mM EDTA, 1mM ⁇ -mercaptoethanol and 10 mM phosphate, pH 7.0), and then passed over FPLC Mono-S column (HR 5/5, Pharmacia, Sweden) equilibrated with buffer A.
  • the column was washed with said buffer A, and thereafter, buffer B containing 6M urea, ImM EDTA, 1mM ⁇ -mercaptoethanol, 10mM phosphate and 0.4M sodium chloride was added gradually to an amount of 35% for first 5 minutes, 70% for next 55 minutes and 100% for final 10 minutes at a flow rate of 0.8ml/min to elute the bound proteins.
  • buffer B containing 6M urea, ImM EDTA, 1mM ⁇ -mercaptoethanol, 10mM phosphate and 0.4M sodium chloride was added gradually to an amount of 35% for first 5 minutes, 70% for next 55 minutes and 100% for final 10 minutes at a flow rate of 0.8ml/min to elute the bound proteins.
  • the KHCV UB-CORE 14 protein was eluted when the amount of buffer B reached 60%, i.e., when the concentration of sodium chloride became 0.25M.
  • the fraction was dialyzed against PBS solution at 4 °C to obtain 4mg of KHCV UB-CORE 14 protein having a purity of at least 90% .
  • the precipitate obtained in (6-B-1) was suspended in 50ml of buffer 4(50mM Tris, pH 8.5, 5mM EDTA, 2mM ⁇ -mercaptoethanol) containing 1% Triton X-100.
  • the suspension was stirred at a room temperature for 30 minutes and centrifuged at 11,000rpm for 25 minutes with a centrifuge (Beckman J2-21, Rotor JA 14) to remove the supernatant and obtain insoluble precipitate.
  • the precipitate was resuspended in 50ml of buffer 4. The suspension was stirred and centrifuged once again to remove the supernatant and obtain insoluble precipitate.
  • KHCV UB 897 protein having a purity of at least 60% was obtained through the above simple washing procedure only. (6-B-3): Dissolution of insoluble precipitate with 8M urea
  • the insoluble precipitate containing KHCV UB 897 protein obtained in (6-B-2) was suspended in 50ml of a buffer 5(20mM phosphate, pH 6.0, 2mM EDTA, 2mM ⁇ -mercaptoethanol) containing 8M urea. The suspension was stirred at a room temperature for 1 hour and centrifuged to remove insoluble precipitate and obtain the supernatant.
  • the supernatant obtained in (6-B-4) was passed over S-Sepharose column(Pharmacia, FF, 2.5cm ⁇ 7cm, U.S.A.) equilibrated with buffer 5 containing 4M urea and was eluted with 600ml of the buffer having a concentration gradient of
  • Protein fractions were subjected to SDS-PAGE to collect the fractions comprising highly purified KHCV UB 897 protein.
  • the protein fractions comprising KHCV UB 897 protein collected in (6-B-4) were dialyzed against buffer 6(10mM Tris, pH 8.5, 2mM EDTA, 2mM ⁇ -mercaptoethanol) to remove urea, loaded over FPLC-Mono Q ion exchange resin column (Pharmacia, HR 5/5) equilibrated with said buffer and eluted with 40ml of the buffer having a concentration gradient of 0 to 0.4M sodium chloride.
  • the fractions comprising highly purified KHCV UB 897 protein were collected to obtain KHCV UB 897 protein having a purity of at least 90%.
  • KHCV NS4E DNA which consists of the region from the 5422nd to the 5547th nucleotides of KHCV-LBC1
  • trp promotor the following primers were synthesized:
  • Primer PNS4ET2 5' -TGAGACTCCGCGGTGGTATCATCCCCGATAGGGAAGTT-3' comprising a recognition site of SacII and the 5422nd to the 5442nd nucleotides of KHCV-LBC1;
  • Primer PNS4ESAL 5' -AAAAAAGTCGACTATTACAACCCGAGCGCCTTCTGTTT-3' comprising a stop codon to terminate translation after the 5547th nucleotide of KHCV-LBC1, and a recognition site of SalI.
  • a test tube was provided with the primer PNS4ET2 2 ⁇ g and primer PNS4ESAL 2 ⁇ g.
  • To the tube were added 50ng of KHCV-LBC1 DNA(ATCC 75008), 10 ⁇ l of 10 ⁇ polymerase buffer solution, 10 ⁇ l of 2mM dNTP(2mM dGTP, 2mM dATP, 2mM TTP, 2mM dCTP), 2.5 unit of Taq polymerase; and distilled water was added thereto to adjust the total volume to be 100 ⁇ l.
  • the PCR product obtained in the above (1-2) was subjected to 5% polyacrylamide gel electrophoresis. As a result, it was confirmed that about 130bp of DNA was amplified.
  • the DNA was purified by the same polyacrylamide gel electrophoresis as above and named fragment NS4E.
  • a ligation tube was provided with 100ng of DNA fragment obtained above. To the tube were added 50ng of fragment ptrpH-UB-T2/L obtained in the above, 2 ⁇ l of 10x ligation buffer solution, 10 units of T4 DNA ligase; and distilled water was added to adjust the total volume to be 20 ⁇ l. The ligation was carried out at 16°C for 12 hours.
  • E. coli W3110 was transformed with the ligation mixture to obtain recombinant E. coli cell containing plasmid ptrpH-UB-NS4E comprising fragment NS4E(Fig. 10).
  • ⁇ Step 3> Preparation of KHCV NS4E protein
  • E. coli W3110(ATCC 37339) was transformed with the plasmid ptrpH-UB-NS4 prepared in the above ⁇ Step 2>.
  • Transformed E. coli cells were cultured with shaking in liquid Luria medium(6% Bacto-tryptone, 0.5% yeast extract, 1% NaCl) containing 50 ⁇ g/ml of ampicillin at 37°C for 12 hours. 3ml of the culture was transferred into 300 ml of M9 medium (40mM K 2 HPO 4 , 22mM KH 2 PO 4 , 8.5mM NaCl, 18.7mM NH 4 Cl, 1% glucose, 0.1mMMgSO 4 , 0.1mM CaCl 2 , 0.4% casamino acid, 10 ⁇ g/ml Vit. B 1 , 40 ⁇ g/ml ampicillin); and cultured with shaking at 37°C for 4 hours. When its O.D.
  • indole acrylic acid(IAA) was added to the culture to adjust the final concentration to be 50 ⁇ g/ml. After 5 hours, the resulting culture was centrifuged at 11,000rpm for 25min. to collect the E. coli cell precipitates.
  • lane 1 represents the standard molecular size marker
  • lanes 2 to 13 show the products of E. coli transformed with plasmid ptrpH-UB-NS4E
  • lane 14 shows the products of E. coli having plasmid without any KHCV DNA fragment.
  • the proteins separated on the gel were blotted onto a nitrocellulose filter (Bio-Rad Lab., pore size 0.22 ⁇ m, CA, U.S.A.) by employing Towbin's method (Towbin, et al., Proc. Natl. Acad. Sci. U.S.A., 76, 4750(1979)).
  • the filter was put in PBS(10mM phosphate, pH 7.0, 0.15M NaCl) containing 0.5% Tween 20; and shaken gently at room temperature for 2 hours to block the nonspecific binding of IgG to the proteins.
  • the filter was put in IgG solution prepared by diluting IgG purified from Korean HCV patients with PBS containing 0.5% gelatin and 0.05% Tween 20 to adjust the final concentration to be 16 ⁇ g/ml; and mildly shaken for 1 hour at room temperature to react the protein and IgG.
  • the filter was then washed 4 times with PBS containing 0.2% Tween 20, each for 5 minutes.
  • the filter was put in an anti-human IgG antibody solution prepared by diluting goat anti-human IgG labeled with horseradish peroxidase (goat anti-human IgG-HRP, Bio-Rad Lab., CA., U.S.A.
  • E. coli cell precipitate obtained in the above ⁇ Step 3> was suspended in 40ml of buffer 7(20mM Tris, pH 8.0, ImM EDTA, 10mM ⁇ -mercaptoethanol, ImM phenyl methyl sulfonyl fluoride, I ⁇ g/ml pepstatin A). To the suspension was added lysozyme to the final concentration of 0.2mg/ml, and the resulting solution was left on ice for 30 minutes.
  • the resultant was subjected to ultrasonication in an ice bath for 15 minutes with an ultrasonicater(HEAT SYSTEMS ULTRASONICS INC., W225, U.S.A.) at an output of 80% and 50% duty-cycle to disrupt the cell and obtain a homogenate of E. coli cell.
  • HEAT SYSTEMS ULTRASONICS INC. W225, U.S.A.
  • the cell homogenate obtained in the above was centrifuged at 15,000 rpm for 25 minutes with a centrifuge(Beckman J2-21, Rotor JA 20) to remove insoluble precipitate and obtain dissolved proteins.
  • the protein fractions collected in (5-2) were concentrated to a volume of 5ml with YM10 ultrafiltration membrane (Amicon, U.S.A.), and then, passed over S-200 resin column (Pharmacia, 2.5cm ⁇ 90cm) equilibrated with PBS at a flow rate of lml/min.
  • the eluted protein was collected by 3ml fractions and subjected to 15% SDS-PAGE to collect the fractions comprising highly pure UBNS4E protein.
  • the protein fractions were subjected to 15% SDS-PAGE to collect the fractions comprising UBNS4E protein having a purity of at least 95%; and the antigenic specificity of the purified protein was confirmed by employing a western blotting analysis.
  • Primer PE2ET2 5'-TGAGACTCCGCGGTGGTACTCGGGGAGAGCGTTGTGAC-3' comprising a recognition site of SacII and the 2281st to the 2298th nucleotides of KHCV-LBC1;
  • a test tube was provided with the primer PE2EGE1G 2 ⁇ g and primer PE2AXHO 2 ⁇ g.
  • To the tube were added 50ng of KHCV-LBC1 DNA(ATCC 75008) as a template, 10 ⁇ l of 10x polymerase buffer solution, 10 ⁇ l of 2mM dNTP(2mM dGTP, 2mM dATP, 2mM TTP, 2mM dCTP), 2.5 unit of Taq polymerase; and distilled water was added thereto to adjust the total volume to be 100 ⁇ l.
  • the PCR product obtained in the above (1-2) was subjected to 5% polyacrylamide gel electrophoresis. As a result, it was confirmed that about 550bp of DNA was amplified.
  • the DNA was purified by the same polyacrylamide gel electrophoresis as above and named fragment GE1GE2A.
  • a test tube was provided with the primer PE2ET2 2 ⁇ g and primer PE2AXHO 2 ⁇ g.
  • 50ng of plasmid pYLBC-A/G-UBE2C(ATCC 74117, see Korean Patent Laid-open Publication No. 93-683) and 50ng of fragment GE1GE2A as templates 10 ⁇ l of 10x polymerase buffer solution, 10 ⁇ l of 2mM dNTP(2mM dGTP, 2mM dATP, 2mM TTP, 2mM dCTP), 2.5 unit of Taq polymerase; and distilled water was added thereto to adjust the total volume to be 100 ⁇ l.
  • the PCR product obtained in the above (1-4) was subjected to 5% polyacrylamide gel electrophoresis. As a result, it was confirmed that about 800bp of DNA was amplified.
  • the DNA was purified by the same polyacrylamide gel electrophoresis as above and named fragment E1E2.
  • a tube was provided with 100ng of DNA fragment obtained above. To the tube were added 50ng of fragment ptrpH-UB-T2/L obtained in ⁇ Step 2> of Example 2, 2 ⁇ l of 10x ligation buffer solution, 10 units of T4 DNA ligase; and distilled water was added to adjust the total volume to be 20 ⁇ l. The ligation was carried out at 16°C for 12 hours.
  • E. coli W3110 was transformed with the ligation mixture to obtain recombinant E. coli cell containing plasmid ptrpH-UB-E1E2 comprising the fragment E1E2(Fig. 12).
  • E. coli W3110(ATCC 37339) cells transformed with the plasmid ptrpH-UB-ElE2 prepared in the above ⁇ Step 2> were cultured in the same manner as in ⁇ Step 3> of Example 2; and then centrifuged to collect the E. coli cell precipitates.
  • lanes 1 and 4 show the products of E. coli having plasmid without any KHCV DNA fragment
  • lanes 2 and 5 show the products of E. coli transformed with ptrpH-UB-E1E2
  • lane 3 shows the standard molecular size markers, i.e., 43, 29, 18 and 14 kilodaltons from the top of the gel.
  • the precipitate obtained in (5-1) was suspended in 40ml of buffer 8 containing 1% Triton X-100. The suspension was stirred at room temperature for 30 minutes and centrifuged at 15,000 rpm for 25 minutes with a centrifuge (Beckman J2-21, Rotor JA 20) to remove dissolved proteins and obtain insoluble precipitate.
  • the insoluble precipitate of (5-2) was suspended in 100ml of buffer 9(20mM phosphate, pH 6.0, ImM EDTA and 10mM ⁇ -mercaptoethanol) containing 8M urea and 0.5% Triton X-100. The suspension was stirred at room temperature for 2 hours and centrifuged to remove dissolved proteins and obtain insoluble precipitate.
  • buffer 9 20mM phosphate, pH 6.0, ImM EDTA and 10mM ⁇ -mercaptoethanol
  • the insoluble precipitate of (5-3) was suspended in 10ml of PBS(10mM phosphate, pH 7.0, 150mMNaCl) containing 1% SDS. The suspension was stirred at room temperature for 12 hours and centrifuged to remove insoluble precipitate and obtain supernatant.
  • HCV envelope protein and KHCV NS4E DNA and to clone it into an expression vector comprising ubiquitin gene under the control of trp promotor, the following primers were synthesized.
  • Primer PNS4ET2 5'-TGAGACTCCGCGGTGGTATCATCCCCGATAGGGAAGTT-3' comprising a recognition site of SacII and the 5422nd to the 5442nd nucleotides of KHCV-LBC1;
  • Primer PNS4EGE2C3 5'-CAGAAGGCGCTCGGGTTGCCAGGAGGAGGAGGTGGTA
  • Primer PE2AXHO 5'-AAAAAACTCGAGTTACCACCCCTGCGCGAATGTATC-3' comprising a stop codon to terminate translation after the 1749th nucleotide of KHCV-LBC1, and a recognition site of Xhol.
  • a test tube was provided with 2 ⁇ g of primer PNS4EGE2C3 and 2 ⁇ g of primer PE2AXHO.
  • To the tube were added 50ng of ptrpH-UB-E1E2( ⁇ Step 2> of Example 3) as a template, 10 ⁇ l of 10x polymerase buffer solution, 10 ⁇ l of 2mM dNTP(2mM dGTP, 2mM dATP, 2mM TTP, 2mM dCTP), 2.5 unit of Tag polymerase; and distilled water was added thereto to adjust the total volume to be 100 ⁇ l.
  • a test tube was provided with 2 ⁇ g of primer PNS4ET2 and 2 ⁇ g of primer PE2AXHO.
  • 50ng of plasmid ptrpH-UB-NS4E obtained in ⁇ Step 2> of Example 2 as a template 50ng of fragment GENVEPI-III obtained in the above (1-3)
  • 10 ⁇ l of 10x polymerase buffer solution 10 ⁇ l of 2mM dNTP(2mM dGTP, 2mM dATP, 2mM TTP, 2mM dCTP), 2.5 unit of Tag polymerase; and distilled water was added thereto to adjust the total volume to be 100 ⁇ l.
  • a tube was provided with 100ng of DNA fragment obtained above. To the tube were added 50ng of the fragment ptrpH-UB-T2/L obtained in ⁇ Step 2> of Example 2, 2 ⁇ l of 10x ligation buffer solution, 10 units of T4 DNA ligase; and distilled water was added to adjust the total volume to be 20 ⁇ l. The ligation was carried out at 16°C for 12 hours.
  • E. coli W3110 was transformed with the ligation mixture to obtain recombinant E. coli cell containing plasmid ptrpH-UB-NS4E1E2 comprising the fragment NS4E1E2(Fig. 14).
  • ⁇ Step 3> Expression of the fragment NS4E1E2 DNA E. coli W3110(ATCC 37339) transformant harboring plasmid ptrpH-UB-NS4E1E2 prepared in the above ⁇ Step 2> was cultured in the same manner as in ⁇ Step 3> of Example 2; and then centrifuged to collect the E. coli cell precipitates.
  • ⁇ Step 4> Confirmation of expressed UBNS4E1E2 protein and reactivity thereof with a serum taken from a hepatitis C patient
  • lane 2 and 5 show the products of E. coli transformed with ptrpH- UB-NS4E1E2; and lane 3 shows the standard molecular size markers, i.e., 92, 70, 43, 29 and 18 kilodaltons from the top of the gel.
  • the insoluble precipitate obtained in (5-1) was treated as in ⁇ Step 5>(5-2) of Example 3 to remove dissolved proteins and obtain insoluble precipitate.
  • the insoluble precipitate of (5-2) was suspended in 30ml of buffer 2 containing 4M urea. The suspension was stirred at room temperature for 2 hours and centrifuged at 15,000rpm with a centrifuge (Beckman J2-21, Rotor JA 21) to remove dissolved proteins and obtain insoluble precipitate.
  • the insoluble precipitate of (5-3) was suspended in 30ml of buffer 2 containing 6M guanidine chloride. The suspension was stirred at room temperature for 2 hours and centrifuged at 15,000rpm with a centrifuge (Beckman J2-21, Rotor JA 21) to remove dissolved proteins and obtain insoluble precipitate. (5-5) Dissolution of precipitate with 1% SDS
  • the insoluble precipitate of (5-4) was suspended in 10ml of PBS(10mM phosphate, pH 7.0, 150mM NaCl) containing 1% SDS.
  • the suspension was stirred at room temperature for 12 hours and centrifuged at 15,000rpm with a centrifuge (Beckman J2-21, Rotor JA 21) to remove insoluble precipitate and obtain supernatant.
  • 10ml of the supernatant obtained in (5-5) was concentrated to a volume of 4ml with YM10 ultrafiltration membrane (Amicon, U.S.A.), and then, centrifuged at 15,000rpm for 25 minutes with a centrifuge (Beckman J2-21, Rotor JA 21) to remove insoluble precipitate and obtain supernatant.
  • the supernatant was subjected to gel filtration chromatography with S-300 resin column (Pharmacia LKB, 2.5cm x 90cm) equilibrated with PBS containing 0.1% SDS at a flow rate of 40ml/hour.
  • the eluted protein was collected by 2ml fractions and subjected to 15% SDS-PAGE to collect the fractions comprising UBNS4E1E2 protein having a purity of at least 90%; and the antigenic specificity of the purified protein was confirmed by employing a western blotting analysis.
  • Example 5 Preparation of KHCV NS5-1.2 protein
  • Primer PNS5T2 5'-TGAGACTCCGCGGTGGTACGGGCATGACCACTGACAAC-3' comprising a recognition site of SacII and the 6649th to the 6669th nucleotides of KHCV-LBC1;
  • a test tube was provided with 2 ⁇ g of primer PNS5T2 and 2 ⁇ g of primer PNS51.2SAL.
  • To the tube were added 50ng of KHCV-LBC1 DNA(ATCC 75008) as a template, 10 ⁇ l of 10x polymerase buffer solution, 10 ⁇ l of 2mM dNTP(2mM dGTP, 2mM dATP, 2mM TTP, 2mM dCTP), 2.5 unit of Taq polymerase; and distilled water was added thereto to adjust the total volume to be 100 ⁇ l.
  • the PCR product obtained in the above (1-2) was subjected to 5% polyacrylamide gel electrophoresis. As a result, it was confirmed that about 1.2Kb of DNA was amplified.
  • the DNA was purified by the same polyacrylamide gel electrophoresis as above and named fragment NS5-1.2.
  • ⁇ Step 2> Preparation of expression vector 2 ⁇ g of DNA fragment obtained in (1-3) of ⁇ Step 1> was completely digested with SacII and SalI in NEB buffer solution 3 referred to in Reference Example 1.
  • a tube was provided with 100ng of DNA fragment obtained above. To the tube were added 50ng of fragment ptrpH-UB-T2/L obtained in ⁇ Step 2> of Example 2, 2 ⁇ l of 10x ligation buffer solution, 10 units of T4 DNA ligase; and distilled water was added to adjust the total volume to be 20 ⁇ l. The ligation was carried out at 16°C for 12 hours.
  • E. coli W3110 was transformed with the ligation mixture to obtain recombinant E. coli cell containing plasmid ptrpH-UB-NS5-1.2 comprising fragment NS5-1.2(Fig. 16).
  • E. coli W3110(ATCC 37339) cells transformed with the plasmid ptrpH-UB-NS5-1.2 prepared in the above ⁇ Step 2> were cultured in the same manner as in ⁇ Step 3> of Example 2; and then centrifuged to collect the E. coli cell precipitates.
  • ⁇ Step 4> Confirmation of expressed KHCV UBNS5-1.2 protein and reactivity thereof with a serum taken from a hepatitis C patient Production of UBNS5-1.2 protein in E. coli and their reactivity with a serum taken from a hepatitis C patient were confirmed by employing the cell precipitates of ⁇ Step 3> in the same manner as in ⁇ Step 4> of Example 2; and the result is shown in Fig. 17, wherein A is the result of SDS-PAGE and B is the result of western blotting.
  • lane 1 shows the products of E. coli having plasmid without any KHCV DNA fragment
  • lanes 2 and 3 show the products of E. coli transformed with ptrpH-UB-NS5-1.2.
  • the insoluble precipitate of (5-2) was suspended in 100ml of buffer 3(50mM Tris, pH 9.0, ImM EDTA, 10mM ⁇ -mercaptoethanol) containing 8M urea. The suspension was stirred at room temperature for 1 hour and centrifuged to remove insoluble precipitate and obtain the supernatant.
  • buffer 3 50mM Tris, pH 9.0, ImM EDTA, 10mM ⁇ -mercaptoethanol
  • the protein fractions collected in (5-5) were passed over YM10 ultrafiltration membrane (Amicon, U.S.A.) to concentrate to a volume of 4mi.
  • the concentrate was dialyzed against PBS(10mM phosphate, pH 7.0, 15mM NaCl) using a dialysis membrane( Spectrum Medical Industries, Inc., M. W. cut off 6,000-8,000) to remove urea.
  • To the solution was added sodium chloride to a final concentration of 1.5M.
  • Primer PCORET2 5'-TGAGACTCCGCGGTGGTATGGACATTGACCCGTATAAA-3' comprising a recognition site of SacII and the 1st to the 21th nucleotides of HBVCORE DNA.
  • Primer PCORESAL 5'-AAAAAAGTCGACTATTAACATTGAGATTCCCGAGATTG-3' comprising a stop codon to terminate translation on the 3'-end of HBVCORE DNA and a recognition site of SalI.
  • a test tube was provided with 2 ⁇ g of primer PC0RET2 and 2 ⁇ g of primer PCORESAL.
  • To the tube were added 50ng of pHBVadr as a template, 10 ⁇ l of 10x polymerase buffer solution, 10 ⁇ l of 2mM dNTP(2mM dGTP, 2mM dATP, 2mM TTP, 2mM dCTP), 2.5 unit of Taq polymerase; and distilled water was added thereto to adjust the total volume to be 100 ⁇ l.
  • the PCR product obtained in the above (1-2) was subjected to 5% polyacrylamide gel electrophoresis. As a result, it was confirmed that about 550 bp of DNA was amplified.
  • the DNA was purified by the same polyacrylamide gel electrophoresis as above and named fragment HBVCORE.
  • a ligation tube was provided with 100ng of DNA fragment obtained above. To the tube were added 50ng of fragment ptrpH-UB-T2/L obtained ⁇ Step 2> of Example 2, 2 ⁇ l of 10x ligation buffer solution, 10 units of T4 DNA ligase; and distilled water was added to adjust the total volume to be 20 ⁇ l. The ligation was carried out at 16°C for 12 hours.
  • E. coli W3110(ATCC 37339) was transformed with the ligated vector to obtain recombinant E. coli transformant containing plasmid ptrpH-UB-HBVCORE comprising fragment HBVCORE (see Fig. 18).
  • E. coli W3110(ATCC 37339) cells transformed with the plasmid ptrpH-UB-HBVCORE prepared in the above ⁇ Step 2> were cultured in the same manner as in ⁇ Step 3> of Example 2; and then centrifuged to collect the E. coli cell precipitates.
  • lane 1 shows the products of E. coli having plasmid without any HBV DNA fragment
  • lanes 2 and 3 show the products of E. coli transformed with ptrpH-UB-HBVCORE.
  • the resultant was passed over DEAE-Sepharose column(Pharmacia, 1.25cm ⁇ 5cm) equilibrated with buffer 8 containing 6M urea at a flow rate of 3ml/min.; and the same buffer was added to elute free proteins remains in column. Then, 100ml of buffer 8 having a concentration gradient of 0 to 0.5M NaCl was added at a flow rate of 3ml/min. to elute the bound proteins and collect the eluate by 3ml fractions. The protein fractions were subjected to 15% SDS-PAGE to collect the fractions comprising UB HBVCORE protein.
  • the protein fractions obtained in the above (5-2) were concentrated to a volume of 5mi with YM10 ultrafiltration membrane (Amicon, U.S.A.), and then, passed over S-300 resin column(Pharmacia LKB, 2.5cm ⁇ 90cm) eguilibrated with buffer 8 at a flow rate of lml/min.
  • the eluted protein was collected by 3ml fractions and subjected to 15% SDS-PAGE to collect the fractions comprising highly pure UB HBVCORE protein.
  • ⁇ Step 1> Preparation of expression vector 10 ⁇ g of a vector pLBC-PSAG14 (see Korean Patent Publication No. 90-5959) comprising HBV Pre S2 DNA was treated with 10 unit of restriction enzyme BamHI at 37°C for
  • Saccharomyces cerevisiae X400-5B(Yeast Genetics Stock Center, U.S.A.) was transformed with the vector pYPSAG100 obtained in the above ⁇ Step 1>, and the transformed yeast cells were cultured in 3ml of leucine-deficient medium ( 6 . 7g of yeast nitrogen base without amino acids, 182g of sorbitol, 2% glucose, 0.25g of Leu-supplements per 19 of medium) at 30°C for 24 hours.
  • leucine-deficient medium 6 . 7g of yeast nitrogen base without amino acids, 182g of sorbitol, 2% glucose, 0.25g of Leu-supplements per 19 of medium
  • the culture was centrifuged at 3,000 rpm for 5 minutes using a centrifuge (DYNAC, U.S.A) to remove the supernatant.
  • the precipitates were shaken in 5 mi of the culture medium (2% glucose, 1% yeast extract, 2% Bacto peptone) for 8 hours.
  • the culture was centrifuged at 3,000rpm to remove the supernatant and obtain precipitates, which were culture in 5 ml of YEPE medium(5% ethanol, 1% yeast extract, 2% Bacto peptone) at 30°C for 4 hours to induce the expression of pre-surface and surface antigens of hepatitis B virus.
  • the supernatant was passed over the Sepharose CL-4B affinity column (Pharmacia, U.S.A) combined with monoclonal antibodies against HBV surface antigen(CHII 5-60, Lucky Central Research Institute) comprising 8mg of monoclonal antibody per 1 g of dry Sepharose CL-4B.
  • the column was washed sufficiently by eluting the buffer solution( 10mM Tris, pH 7.5, 1M NaCl) to activate the column.
  • the supernatant containing surface antigen was added at a flow rate of lml/hour and the column was washed continuously with 10mM Tris-IM NaCl buffer solution until A 280 of eluent reached 0.
  • the buffer solution (10mM Tris-HCl, pH 7.5) was added to the column to remove nonspecific proteins and to adjust A 280 of eluate to 1.
  • a 280 reached 1
  • a buffer(3M NH ⁇ SCN, 50mM Tris, pH 7.5) was added to elute the bound surface antigen protein and collect 20ml of fractions eluted at the peak of A 280, which was subjected to dialysis against 4i of a buffer (50mM Tris, pH 7.5) at 4°C for 24 hours.
  • the resultant was passed over YM30 ultrafiltration membrane (Amicon, U.S.A) to concentrate to a volume of 1 ml.
  • the concentrate was passed over Sepharose CL-4B Column (1.5 x 90 cm, Pharmacia Co., U.S.A) by adding a buffer(0.1M sodium phosphate, pH 7.0) at a flow rate of 50ml/hour to elute the bound proteins and collect the eluate by 2ml fractions. Using Oszyme II(Abott Co.), each of tubes were tested and fractions exhibiting reactivity were collected.
  • To the obtained solution was added a volume of CsCl 2 solution to a final concentration of 1.2g/cm 3 .
  • the suspension was centrifuged at 35,000 rpm for 24 hours by using a centrifuge (Beckman rotor 41Ti, U.S.A.) to obtain the precipitates.
  • HCV antigenic proteins KHCV CORE14, KHCV NS4E, KHCV NS4E1E2, KHCV 897 and KHCV NS5-1.2
  • 2 kinds of HBV antigenic proteins HBV CORE and HBV Pre S2 S Ag
  • ubiguitin and human immunoglobulin G(hIgG) was diluted with a blotting buffer (100mM sodium carbonate, pH 9.5) to have the following concentrations:
  • hIgG 5 ⁇ g/ml & 0.1 ⁇ g/ml
  • KHCV NS4E 20 ⁇ g/ml
  • KHCV NS4E1E2 10 ⁇ g/ml
  • KHCV NS5-1.2 20 ⁇ g/ml
  • HBV CORE 10 ⁇ g/ml
  • Each of the dilutions containing antigenic proteins was added to a slot (0.7 ⁇ 0.7mm) of a nitrocellulose filter (Schliescher & Schuell, BA 83, pore size 0.22 ⁇ m, U.S.A), which was previously dipped in the blotting buffer by using a slot blotting apparatus (Bio-Rad Lab., Dot SF blotting apparatus, Cat. No. 170-6542, U.S.A.) and then, washed once with the same buffer, in the following orders and volumes :
  • the antigenic proteins were blotted on nitrocellulose filter by applying vaccum pressure of 8 to 10Hg to nitrocellulose filter over 10 to 20 minutes to filter the protein solution, and the membrane was washed once by adding 250 ⁇ l of blotting buffer to each slot and carrying out filtration once again.
  • the nitrocellulose filter was taken off by using a pincettes and dried at 60°C for about 10 minutes.
  • the dried nitrocellulose filter was placed in a phosphate buffered saline containing 0.1% gelatin and shaken gently at room temperature for 30 minutes to block the unspecific binding of proteins to the free blotting sites.
  • the treated filter was dried in the same condition as above; and then numbered in an appropriate method or bound to a support labelled with numbers.
  • the filter was cut into strips comprising all kinds of slots each containing one antigenic protein.
  • Each of strips prepared in the above Example 8 was placed into a glass tube (13 ⁇ 100 mm) and to the tubes was added serum samples, which were 50-fold diluted with PBS (0.25% gelatin, 1% triton X-100, 1mM EDTA and 0.02% Thimerosal) containing 5 ⁇ g/ml of UB, to an amount sufficiently dip the strip.
  • the tubes were sealed with para-film and shaken gently at room temperature for 2 hours to react the antigens on the strips and antibodies in the serum sample.
  • the serum was removed by using a suction apparatus from the glass tube.
  • the strips were washed once by adding 4 mi of washing solution (PBS comprising 0.05 % Tween 20) to each of glass tube, shaking the tube for about 4 minutes and then, removing washing solution.
  • PBS washing solution
  • reaction buffer 50mM Tris, pH7.5.
  • 20ml of reaction buffer containing 400 ⁇ g/ml of 4-chloro-1-naphtol and 0.03% hydrogen peroxide was added thereto and allowed to react with gentle shaking at room temperature for 20 minutes.
  • the membrane was washed 2 times with distilled water, transferred to the absorption paper and dried at room temperature for 20 minutes, visual color bands were shown in strips.
  • Sample No. 4 which had been diagnosed as intermediate by ELISA method of Lucky Ltd. was diagnosed as negative by using the diagnostic kit of the present invention.
  • the diagnostic method of the present invention can reduce the possibility of false diagnosis substantially and can diagnose 2 kinds of hepatitis i.e., hepatitis C and B simultaneously.
  • the present diagnostic kit contains many epitopes KHCV envelope protein, KHCV E1E2 protein and KHCV NS5-1.2 protein, even more accurate diagnosis for HCV infection can be possible.

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  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • General Health & Medical Sciences (AREA)
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  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
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  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

Nécessaire servant au diagnostic simultané de l'hépatite B et de l'hépatite C, et son procédé d'utilisation et, plus particulièrement, nécessaire servant au diagnostic simultané de l'hépatite B et de l'hépatite C comportant une association des antigènes viraux des hépapties B et C, et son procédé d'utilisation.
PCT/KR1994/000039 1993-04-28 1994-04-27 Necessaire et procede de diagnostic simultane des hepatites b et c WO1994025874A1 (fr)

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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0744467A2 (fr) * 1995-03-30 1996-11-27 Eisai Co., Ltd. Peptide multi-antigénique comprenant au moins deux peptides associés avec le virus de l'hépatite C
WO1999006837A1 (fr) * 1997-07-30 1999-02-11 Bionova Corporation Procede pour detecter un hbcag provenant du virus de l'hepatite b
WO1999015901A1 (fr) * 1997-09-22 1999-04-01 Chiron Corporation Tampons pour antigenes de stabilisation
WO1999024466A2 (fr) * 1997-11-06 1999-05-20 Innogenetics N.V. Peptides multimeres derives de proteines d'enveloppe de virus d'hepatite c (hcv) a des fins de diagnostic et de vaccination
US6391540B1 (en) 1997-09-22 2002-05-21 Chiron Corporation Method for detecting antibodies in a sample
EP1767542A2 (fr) * 1996-03-21 2007-03-28 Epimmune Inc. Peptides de fixation de HLA-A2.1 et leurs utilisation
MD34Z5 (ro) * 2008-05-20 2010-01-31 Национальный Центр Общественного Здоровья Министерства Здравоохранения Республики Молдова Metodă de diagnostic al hepatitei virale B la copii de până la un an
CN106771219A (zh) * 2010-06-17 2017-05-31 皇家飞利浦电子股份有限公司 多表位测定
US11167283B2 (en) 2018-05-04 2021-11-09 University Of South Carolina Dot blot box and use thereof

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* Cited by examiner, † Cited by third party
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CN102043050A (zh) * 2009-10-23 2011-05-04 上海荣盛生物药业有限公司 乙肝病毒c抗体的体外检测方法
CN102062779A (zh) * 2009-11-17 2011-05-18 上海荣盛生物药业有限公司 体外检测乙肝病毒c抗体的组合物

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EP0521318A2 (fr) * 1991-06-10 1993-01-07 Lucky Ltd. Diagnostics et vaccins pour l'hépatite C

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EP0105141A3 (fr) * 1982-09-09 1985-12-04 Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. Molécules D'ADN recombinantes, procédé pour leur préparation, leur utilisation dans la production de l'antigène de surface de l'hépatite B (HBsAg) et compositions pharmaceutiques contenant cet HBsAg
JPS61148127A (ja) * 1984-12-21 1986-07-05 Chemo Sero Therapeut Res Inst ＨＢｃ抗原の精製方法
US5091300A (en) * 1989-08-03 1992-02-25 Merck & Co., Inc. Radio-immuno assay for hepatitis b virus pres2 antibodies
CA2041772A1 (fr) * 1990-05-11 1991-11-12 Larry T. Mimms Anticorps monoclonaux diriges contre la polypeptide pres2 et pres1 de l'enveloppe virale de l'hepatite b
EP0473065A3 (en) * 1990-08-29 1992-08-26 Abbott Laboratories Simultaneous assay for detecting two or more analytes

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Cited By (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0744467A3 (fr) * 1995-03-30 1997-07-09 Eisai Co Ltd Peptide multi-antigénique comprenant au moins deux peptides associés avec le virus de l'hépatite C
EP0744467A2 (fr) * 1995-03-30 1996-11-27 Eisai Co., Ltd. Peptide multi-antigénique comprenant au moins deux peptides associés avec le virus de l'hépatite C
EP1767542A2 (fr) * 1996-03-21 2007-03-28 Epimmune Inc. Peptides de fixation de HLA-A2.1 et leurs utilisation
EP1767542A3 (fr) * 1996-03-21 2012-12-12 Epimmune Inc. Peptides de fixation de HLA-A2.1 et leurs utilisation
WO1999006837A1 (fr) * 1997-07-30 1999-02-11 Bionova Corporation Procede pour detecter un hbcag provenant du virus de l'hepatite b
US6153392A (en) * 1997-07-30 2000-11-28 Bionova Corporation Devices and methods comprising an HBcAg from hepatitis B virus
WO1999015901A1 (fr) * 1997-09-22 1999-04-01 Chiron Corporation Tampons pour antigenes de stabilisation
US6261764B1 (en) 1997-09-22 2001-07-17 Chiron Corporation Buffers for stabilizing antigens
US6391540B1 (en) 1997-09-22 2002-05-21 Chiron Corporation Method for detecting antibodies in a sample
US6537745B2 (en) 1997-09-22 2003-03-25 Chiron Corporation Buffers for stabilizing antigens
WO1999024466A2 (fr) * 1997-11-06 1999-05-20 Innogenetics N.V. Peptides multimeres derives de proteines d'enveloppe de virus d'hepatite c (hcv) a des fins de diagnostic et de vaccination
US6855318B1 (en) 1997-11-06 2005-02-15 N.V. Innogenetics S.A. Multi-mer peptides derived from hepatitis C virus envelope proteins for diagnostic use and vaccination purposes
WO1999024466A3 (fr) * 1997-11-06 1999-07-15 Innogenetics Nv Peptides multimeres derives de proteines d'enveloppe de virus d'hepatite c (hcv) a des fins de diagnostic et de vaccination
MD34Z5 (ro) * 2008-05-20 2010-01-31 Национальный Центр Общественного Здоровья Министерства Здравоохранения Республики Молдова Metodă de diagnostic al hepatitei virale B la copii de până la un an
CN106771219A (zh) * 2010-06-17 2017-05-31 皇家飞利浦电子股份有限公司 多表位测定
CN106771219B (zh) * 2010-06-17 2020-04-07 皇家飞利浦电子股份有限公司 多表位测定
US11167283B2 (en) 2018-05-04 2021-11-09 University Of South Carolina Dot blot box and use thereof

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CN1130566C (zh) 2003-12-10
CN1122162A (zh) 1996-05-08
JPH08504954A (ja) 1996-05-28
KR100312534B1 (ko) 2002-05-13

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