WO1994014974A1 - Monoclonal antibodies and anti-idiotypic antibodies to hepatitis c virus - Google Patents
Monoclonal antibodies and anti-idiotypic antibodies to hepatitis c virus Download PDFInfo
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- WO1994014974A1 WO1994014974A1 PCT/EP1993/003707 EP9303707W WO9414974A1 WO 1994014974 A1 WO1994014974 A1 WO 1994014974A1 EP 9303707 W EP9303707 W EP 9303707W WO 9414974 A1 WO9414974 A1 WO 9414974A1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/42—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins
- C07K16/4208—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig
- C07K16/4216—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig against anti-viral Ig
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
- C07K16/1081—Togaviridae, e.g. flavivirus, rubella virus, hog cholera virus
- C07K16/109—Hepatitis C virus; Hepatitis G virus
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/10—Cells modified by introduction of foreign genetic material
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
Definitions
- the present invention relates to monoclonal antibodies specifically immunoreactive with hepatitis C viral antigens, cell lines secreting said antibodies, an immunodiagnostic reagent comprising the antibodies, and a method and testkit for the detection of HCV.
- the present invention further relates to anti-idiotypic antibodies, cell lines secreting said anti-idiotypic antibodies, immunodiagnostic reagents comprising said anti-idiotypic antibodies and a test-kit for the detection of anti-HCV antibodies in a sample, using antiidiotypic antibodies.
- Hepatitis C virus is a 9.4-kb, single stranded polyadenylated RNA virus which has been recognized as one of the causative agents of NANB hepatitis (Non-A, Non-B). It causes acute and chronic liver disease and is implicated in hepatocellular carcinoma.
- HCV hepatitis A virus
- HBV hepatitis B virus
- HDV hepatitis delta virus
- HCV hepatitis induced by cytomegalovirus
- EBV Epstein-Barr virus
- Non-A, Non-B Hepatitis was first identified in transfused individuals. Transmission from man to chimpanzee and serial passage in chimpanzees provided evidence that Non-A, Non-B Hepatitis is due to a transmissible infectious agent or agents.
- Non-A, Non-B Hepatitis exist: the water-borne epidemic type; the blood or needle associated type; and the sporadically occurring (community acguired) type.
- the viral genome of HCV encodes a polyprotein of approximately 3010 amino acids that undergoes extensive posttranslational processing.
- the viral structural region is located upstream from the nonstructural region and putatively includes a highly conserved 19-kDa nucleocapsid protein, and two extensively glycosylated envelope polypeptides, gp 33 (E1) and gp72 (E2/NS1).
- HCV envelope may be under strong immune selection.
- a variety of presumed nonstructural proteins are processed from the remainder of the HCV polyprotein, including a membrane-bound 23-kDa protein, NS2, and a soluble protein of approximately 60 kDa, NS3, which corresponds to the viral helicase and may contain a N-terminal serine protease domain, currently thought to be involved in the processing of the NS proteins.
- the function of the NS4 protein is presently unknown, but it comprises the 5-1-1 fragment that contains immunodominant antibody binding sites (Kuo G.
- NS5 contains the viral replicase.
- Clinical studies have shown that, following exposure to HCV, antibodies to conserved regions of the viral nucleoprotein and NS3 may appear several weeks before seroconversion to anti-c100-3, a recombinant protein encompassing the C-terminus of NS3 and part of the NS4 protein.
- serological assays incorporating the highly-conserved HCV nucleocapsid protein as well as NS3 are likely to become useful diagnostic markers of acute HCV infection. It is an object of the present invention to provide novel monoclonal antibodies that specifically react with the NS3 protein of the HCV virus and are particularly useful in immunodiagnostic tests for the detection of the presence or absence of HCV in clinical specimen.
- the present invention provides monoclonal antibodies which bind to an epitope of the NS3 protein of hepatitis C virus, which epitope is specifically recognized by monoclonal antibodies secreted by the Epstein-Barr virus-transformed human lymphocyte-B cell line deposited at the European Collection of Animal Cell Cultures, Porton Down (UK) under deposit No. 92121609.
- a preferred monoclonal antibody according to the invention is secreted by the Epstein-Barr virus-transformed human lymphocyte-B cell line deposited at the European Collection of Animal Cell Cultures, Porton Down (UK) under deposit No. 92121609.
- Peptides comprising the NS-3 epitope recognized by the monoclonal antibodies according to this invention are described in the co-pending and co-owned Patent Application No. PCT/EP93/03478, the contents of which are incorporated herein by reference.
- the above cell line has been deposited at the ECACC on 16 December 1992, under the terms and conditions of the Budapest treaty, 1977. Cell lines capable of excreting these monoclonal antibodies are also part of the present invention.
- the preparation of cell lines producing monoclonal antibodies may occur by, for example, transformation with Epstein-Barr Virus, the
- a preferred cell line according to the invention is an Epstein-Barr virus-transformed human lymphocyte-B clone cell line capable of excreting monoclonal antibodies which bind to an epitope on the NS3 protein of hepatitis C virus, which cell line is deposited at the European Collection of Animal Cell Cultures, Porton Down (UK), under deposit No. 92121609.
- Epstein Barr virus is capable of transforming and immortalizing human Blymphocytes. With the aid of the Epstein Barvirus immortalized human B-lymphocytes can be obtained without the need of a myeloma partner cell.
- Epstein Barr virus can be obtained from a variety of sources. The most common used source of EBV is the B95-8 marmoset cell line. The B95-8 cell line spontaneously releases Epstein Barr virus into the medium.
- EBV transformed cells The most commonly used are peripheral blood mononuclear cells (PBMC) and fibroblasts.
- PBMC peripheral blood mononuclear cells
- fibroblasts The most commonly used are peripheral blood mononuclear cells (PBMC) and fibroblasts.
- PBMCs consist of monocytes, T lymphocytes and B-lymphocytes (5-10%). Because not all Blymphocytes will provide antibodies of the right specificity, it is advantageous to enrich PBMCs for appropriate cells.
- T-lymphocytes can be removed, prior to EBV infection (with, for example, the supernatant from the EBV-productive B95-8 cell line), by treating the cells with washed sheep red blood cells (In: "Antibodies Vol. I, a practicle approach", Editor: Catty D, IRL Press, Oxford,
- Immortalized B cell lines according to the invention were considered monoclonal if they satisfied the following requirements:
- the monoclonal antibodies according to the invention are extremely suitable to be used in so-called immuno-assays in order to detect HCV or HCV-fragments in a test sample.
- An immunodiagnostic reagent comprising the antibodies according to the invention and a test kit for the detection of HCV in a sample are also part of the present invention.
- the immunochemical reaction that takes place can be a so-called sandwich reaction, an agglutination reaction, a competition reaction or an inhibition reaction.
- the test kit to be used comprises a monoclonal antibody according to the invention coated on a solid support, for example the inner wall of a microtest well, and either a labelled monoclonal antibody or fragment thereof as conjugate.
- Supports which can be used are, for example, the inner wall of a microtest well or a cuvette, a tube or capillary, a membrane, filter, test strip or the surface of a particle such as, for example, a latex particle, an erythrocyte, a dye sol, a metal sol or metal compound as sol particle, a carrier protein such as BSA or KLH.
- Labelling substances which can be used are, inter alia, a radioactive isotope, a fluorescent compound , an enzyme , a dye sol , metal sol or metal compound or other sol as sol particle.
- a further example of an immuno assay that can be used for the detection of HCV is a inhibition assay using human monoclonal antibodies as labelled reagent. The binding of this reagent to antigen on a solid phase can be competed by antibodies in the test sample.
- monoclonal antibodies according to the invention are very suitable in diagnosis, while those antibodies which are neutralizing are very useful in passive immunotherapy.
- the monoclonal antibodies according to the invention can also be used to raise antiidiotypic antibodies.
- Anti-idiotypic antibodies are antibodies directed to the variable part of immunoglobulins. A sub-population of anti-idiotypic antibodies is known as "anti-idiotypic ⁇ " or "internal images". These anti-idiotypic ⁇ antibodies have either a structural or a three dimensional resemblance with the antigen (Uytdehaag, F.G.C.M. et al.
- the antiidiotypic antibodies can be raised in large amounts.
- anti-idiotypic antibodies according to the invention can be obtained by immunizing BALB/c mice with monoclonal antibodies, coupled to KLH with glutaraldehyde according to standard literature procedures, mixed with Freund's complete adjuvant.
- the spleen cells of these mice can be immortalized and the thus obtained hybridomas can be screened for anti-idiotypic antibody production.
- Screening of the hybridomas can be performed, for example but not limited to, by binding a HCV peptide to a solid phase (wells of microtiter plates) and incubating the solid phase with culture supernatant of growing hybridomas and the monoclonal antibodies coupled to Horse Radish Peroxidase (HRP) according to our invention.
- HRP Horse Radish Peroxidase
- the presence of anti-idiotypic antibodies in culture supernatant will then be indicated by inhibition of the binding of the monoclonal antibodies according to our invention and the HCV peptide coated on the solid phase.
- Anti-idiotypic antibodies reactive with the monoclonal antibodies according to the invention, as described above, are part of the present invention.
- Anti-idiotypic antibodies can be used in mimicking antigen in a diagnostic test. Beside the use in diagnostic tests anti-idiotypic antibodies are also useful for prevention and/or treatment of Non-A, Non-B Hepatitis, as well as for the elucidation of important epitopic regions of HCV-antigens.
- Preferred anti-idiotypic antibodies according to the present invention are the antiidiotypic antibodies secreted by the cell lines which are deposited at the European Collection of
- Anti-idiotypic antibodies according to the invention preferably are anti-idiotypic antibodies capable of competing with an epitope for the binding to the monoclonal antibody according to the invention, where the epitope is the epitope recognized by the monoclonal antibodies secreted by the Epstein-Barr virus- transformed human lymphocyte-B cell line deposited with the European Collection of Animal Cell Cultures (ECACC), Porton Down (UK), under deposit No. 92121609.
- ECACC European Collection of Animal Cell Cultures
- UK Porton Down
- Immunodiagnostic reagents comprising the anti-idiotypic antibodies according to the invention, and a test kit for the detection of anti-HCV antibodies in a sample using an immunodiagnostic reagent comprising antiidiotypic antibodies are also part of the present invention.
- the preparation of cell lines producing anti-idiotypic antibodies may occur by, for example, transformation with Epstein-Barr Virus, the K ⁇ hler and Milstein technique (K ⁇ hler and Milstein devised the techniques that resulted in the formation monoclonal antibody-producing hybridomas (G. Köhler and C. Milstein, 1975, Nature 256:495-497; 1976, Eur. J. Immunol.
- HCVID.OT2A under deposit No. 93122307
- HCVID.OT2D under deposit No. 93122310
- HCVID.OT2E under deposit No. 93122311, HCVID.OT2I under deposit No. 9312231
- HCVID.OT2J under deposit No. 93122313
- HCVID.OT2M under deposit No. 93122314
- HCVID.OT2P under deposit No. 93122315
- HCVID.OT2Q under deposit No.
- HCVID.OT3A under deposit No. 93122317
- HCVID.OT3D under deposit No. 93122318
- HCVID.OT3E under deposit No. 93122319
- HCVID.OT3H under deposit No. 93122320
- HCVID.OT3L under deposit No. 93122321
- HCVID.OT30 under deposit No. 93122322.
- An immunodiagnostic reagent comprising the anti-idiotypic antibodies according to the invention and a test kit for the detection of HCV in a sample are also part of the present invention.
- Example 1 illustrates the way in which a cell line, producing monoclonal antibodies, according to the invention may be derived.
- Example 2 further exemplifies the specific immune reactivity of monoclonal antibodies according to the invention.
- Example 3 exemplifies the production of anti-idiotypic antibodies and the specific ability of these anti-idiotypic antibodies to mimic a immunodominant NS-3 epitope.
- EXAMPLE 1 Preparation of B-cell line clone.
- Peripheral blood mononuclear cells (PBMC) were obtained from one patient with chronic HCV infection and circulating antibodies to the NS3 and nucleocapsid proteins of HCV but not to c100- 3/NS4(HCV 1st generation antibody test, Ortho Diagnostic Systems, Raritan, NJ) as shown by reactivity of the patient's serum with a commercial assay that incorporates a recombinant protein designated C22-3, the viral nucleoprotein, and the recombinant nonstructural protein c200 encoded by the NS3 and NS4 regions of the HCV genome (Ortho HCV ELISA Test System, 2nd generation).
- PBMC peripheral blood mononuclear cells
- RPMI-1640 medium containing 10% heat-inactivated pooled human serum, supplemented with 10% human endothelial culture supernatant (HECS) + 20 U/ml rIL6 and cultured in 25 cm2 flasks at the concentration of 4 x 10E6/ml in the presence of recombinant NS3 protein (Organon Teknika) at the concentration of 25, 125, and 625 ng/ml for 6 days at 37 °C in 5% CO2.
- HECS human endothelial culture supernatant
- lymphocytes-B After washing, cells were enriched in lymphocytes-B by removing lymphocytes-T with S- (2-amino-ethyl)-isothiouronium-bromide- hydrobromide treated sheep red blood cells and infected with supernatant from the EBV-productive B95-8 cell line as described in Cerino A et al. J. Immunol., 147, p2692, 1991.
- IgG heavy and light (L) chains were determined as described in Cerino A et al. J.Tmiminol., 147, p2692, 1991. Briefly, B cell line supernatants were incubated on NS3 coated wells for 1 hour at 37 °C and, after washing, human IgG subclass-specific murine mAb were added for 1 hour followed by a peroxidase-conjugated rabbit anti-mouse Ig for another hour. To determine IgG L chains, peroxidase-conjugated rabbit anti-human Ig kappa- or lambda-chain were used in a direct assay. In both cases the reaction was developed with orthophenylen- diamine/2 HCl as substrate.
- Recombinant immunoblot assay (RIBA II generation, Ortho Diagnostics). This is a nitrocellulose-based assay that includes 4 recombinant HCV antigens:
- Sueernatant from clone HCVHU.OT3 was analyzed for specificity in the above described way.
- Figure 1 illustrates the specific binding capacity of antibodies according to the invention.
- the binding of the antibodies according to the invention (HCVHU.OT3) to different HCV derived proteins is compared with the binding of antibodies specific for HCV core and NS4 proteins respectively.
- the monoclonal antibodies according to the invention (HCVHU.OT3) recognized a recombinant NS3 prctein preparation and gave a clear positive reaction in Ortho II generation assay, whereas no binding to recombinant HCV core and NS-5 proteins could be documented.
- HCVHU.OT3 supernatant by recombinant immunoblot revealed clear binding to the c33c polypeptide only, further attesting to the specificity of the mAb as can be seen from figure 2, where lane 2 represents an antibody according to the invention (HCVHU.OT3).
- Monoclonal human IgG (HCVHU.OT3) was purified from the culture supernatant of the cell-line deposited at the European Collection of Animal Cell Cultures, Porton Down (UK) under deposit No. 92121609. This IgG was conjugated to KLH (Oosterlaken T.A.M. et al., (1990) Scandinavian Journal of Immunology, 31, 159) mixed with Complete Freunds adjuvant or Quil A and injected into 4 mice and 4 rats. Immunizations were performed according to the scheme in table 1. After 2 immunizations the animal with the highest serum titer, one mouse and one rat, was used for making monoclonal antibodies, essentially as described in
- Clones secreting putative anti-idiotypic antibodies were also detected with the assay described in figure 3. In this way 16 clones were obtained that reacted well in this assay.
- the clones secreting putative anti-idiotypic antibodies were cultured and subcloned to 100% clonality according to standard procedures (Köhler, G. and Milstein, C.
- the cell lines were grown in higher cell densities and cultured for 5 days in serumfree medium. Antibodies were purified from the culture supernatant using a Protein A-Sepharose R column. Purification was performed according to the manufacturers instructions (Pharmacia-LKB).
- the anti-idiotypic antibodies were further analysed for their ability to mimic the NS-3 epitope recognized by the human monoclonal antibody HCVHU.OT3.
- the purified monoclonal antibodies were conjugated to HRP with the Actizyme-peroxidase kit (Zymed laboratories). The procedure was performed according to the manufacturers instructions.
- Serum dilutions or dilutions of HCVHU.OT3 were added to the wells followed by 1 hour incubation at 37 °C.
- reaction with HCVHU.OT3 is shown as black bars
- reaction with an irrelevant human monoclonal antibody with the same subclass as HCVHU.OT3 is shown as shaded bars.
- Other monoclonal anti-idiotypic antibodies gave comparable results.
- Table 3 shows the reactivity of human sera from HCV infected patients and normal controls with anti-idiotypic antibodies HCVID.OT2A and HCVID.OT2I.
- N.R. normalised response, calculated as OD450 of serum sample divided by cut off value.
- Figure 1 is a graph illustrating the binding specificity of monoclonal antibody HCVHU.OT3 for the NS3 protein.
- Figure 2 is a photograph of a recombinant immunoblot assay.
- the characters A-G represent the following proteins;
- Lane 1 represents negative control serum
- lane 2 represents an anti NS-3 monoclonal antibody (HCVHU.OT3)
- lane 3 represents an anti-core monoclonal (HCVHU.OT2)
- lane 4 represents polyclonal serum from a HCV- infected patient.
- Figure 3 represents a screening procedure used for detection of anti-idiotypic antibodies against human monoclonal antibody HCVHU.OT3 in serum and in culture supernatant of monoclonal antibody producing cells.
- Figure 4 represents an assay procedure used to validate putative anti-idiotypic antibodies which emerged from the screening procedure described in figure 3.
- Figure 5 represents a sandwich assay (as shown in figure 4) to determine the specificity of 6 different anti-idiotypic antibodies (2A, 2B, 2I, 2P, 2Q and 3A) for HCVHU.OT3.
- Reaction with HCVHU.OT3 is shown as black bars, reaction with an irrelevant human monoclonal antibody with the same subclass as HCVHU.OT3 is shown as shaded bars. Other monoclonal anti-idiotypic antibodies gave comparable results.
Abstract
The present invention relates to monoclonal antibodies specifically immunoreactive with hepatitis C viral antigens, cell lines secreting said antibodies, an immunodiagnostic reagent comprising the antibodies, and a method and test-kit for the detection of HCV. The present invention further relates to anti-idiotypic antibodies, cell lines secreting these anti-idiotypic antibodies, immunodiagnostic reagents comprising said anti-idiotypic antibodies and a test-kit for the detection of anti-HCV antibodies in a sample, using anti-idiotypic antibodies.
Description
Title: Monoclonal antibodies and antiidiotypic antibodies to Hepatitis C virus.
The present invention relates to monoclonal antibodies specifically immunoreactive with hepatitis C viral antigens, cell lines secreting said antibodies, an immunodiagnostic reagent comprising the antibodies, and a method and testkit for the detection of HCV.
The present invention further relates to anti-idiotypic antibodies, cell lines secreting said anti-idiotypic antibodies, immunodiagnostic reagents comprising said anti-idiotypic antibodies and a test-kit for the detection of anti-HCV antibodies in a sample, using antiidiotypic antibodies.
Hepatitis C virus (HCV) is a 9.4-kb, single stranded polyadenylated RNA virus which has been recognized as one of the causative agents of NANB hepatitis (Non-A, Non-B). It causes acute and chronic liver disease and is implicated in hepatocellular carcinoma.
It can be distinguished from other forms of viral-associated liver diseases, including those caused by known hepatitis viruses, i.e., hepatitis A virus (HAV), hepatitis B virus (HBV), and hepatitis delta virus (HDV), as well as the hepatitis induced by cytomegalovirus (CMV) or Epstein-Barr virus (EBV). Evidence based on hydrophobicity plots and seguence homologies suggests that HCV may be distantly related to the family Flaviviridae (Houghton M. et al., Hepatology, 14:381, 1991). Non-A, Non-B Hepatitis was first identified in transfused individuals. Transmission from man to chimpanzee and serial
passage in chimpanzees provided evidence that Non-A, Non-B Hepatitis is due to a transmissible infectious agent or agents.
Epidemiologic evidence is suggestive that three types of Non-A, Non-B Hepatitis exist: the water-borne epidemic type; the blood or needle associated type; and the sporadically occurring (community acguired) type. The viral genome of HCV encodes a polyprotein of approximately 3010 amino acids that undergoes extensive posttranslational processing. The viral structural region is located upstream from the nonstructural region and putatively includes a highly conserved 19-kDa nucleocapsid protein, and two extensively glycosylated envelope polypeptides, gp 33 (E1) and gp72 (E2/NS1). Recent studies indicate that substantial sequence heterogeneity exists among virtually all HCV isolates in the N-terminal region of E2/NS1, suggesting that this region of the HCV envelope may be under strong immune selection. A variety of presumed nonstructural proteins are processed from the remainder of the HCV polyprotein, including a membrane-bound 23-kDa protein, NS2, and a soluble protein of approximately 60 kDa, NS3, which corresponds to the viral helicase and may contain a N-terminal serine protease domain, currently thought to be involved in the processing of the NS proteins. The function of the NS4 protein is presently unknown, but it comprises the 5-1-1 fragment that contains immunodominant antibody binding sites (Kuo G. et al., Science 244:362, 1991: Cerino A. et al.J. Immunol., 147:2692) ; NS5 contains the viral replicase. Clinical studies have shown that, following exposure to HCV, antibodies to conserved regions of the viral nucleoprotein and
NS3 may appear several weeks before seroconversion to anti-c100-3, a recombinant protein encompassing the C-terminus of NS3 and part of the NS4 protein. Thus, serological assays incorporating the highly-conserved HCV nucleocapsid protein as well as NS3 are likely to become useful diagnostic markers of acute HCV infection. It is an object of the present invention to provide novel monoclonal antibodies that specifically react with the NS3 protein of the HCV virus and are particularly useful in immunodiagnostic tests for the detection of the presence or absence of HCV in clinical specimen.
The present invention provides monoclonal antibodies which bind to an epitope of the NS3 protein of hepatitis C virus, which epitope is specifically recognized by monoclonal antibodies secreted by the Epstein-Barr virus-transformed human lymphocyte-B cell line deposited at the European Collection of Animal Cell Cultures, Porton Down (UK) under deposit No. 92121609.
A preferred monoclonal antibody according to the invention is secreted by the Epstein-Barr virus-transformed human lymphocyte-B cell line deposited at the European Collection of Animal Cell Cultures, Porton Down (UK) under deposit No. 92121609.
Peptides comprising the NS-3 epitope recognized by the monoclonal antibodies according to this invention are described in the co-pending and co-owned Patent Application No. PCT/EP93/03478, the contents of which are incorporated herein by reference.
The above cell line has been deposited at the ECACC on 16 December 1992, under the terms and conditions of the Budapest treaty, 1977. Cell lines capable of excreting these monoclonal antibodies are also part of the present invention.
The preparation of cell lines producing monoclonal antibodies may occur by, for example, transformation with Epstein-Barr Virus, the
Kόhler and Milstein technique (Köhler and
Milstein devised the techniques that resulted in the formation monoclonal antibody-producing hybridomas (G. Kohler and C. Milstein, 1975,
Nature 256:495-497; 1976, Eur. J. Immunol. 6:511-
519)), or a direct transformation technique of Blymphocytes with oncogenic DNA, or a direct fusion of human B-lymphocytes with a fusion partner being either a human or a mouse-human hybrid myeloma cell line, or a direct fusion of an EBV-transformed B cell line with said myeloma cell lines. A preferred cell line according to the invention is an Epstein-Barr virus-transformed human lymphocyte-B clone cell line capable of excreting monoclonal antibodies which bind to an epitope on the NS3 protein of hepatitis C virus, which cell line is deposited at the European Collection of Animal Cell Cultures, Porton Down (UK), under deposit No. 92121609.
The Epstein Barr virus (EBV) is capable of transforming and immortalizing human Blymphocytes. With the aid of the Epstein Barvirus immortalized human B-lymphocytes can be
obtained without the need of a myeloma partner cell. The Epstein Barr virus can be obtained from a variety of sources. The most common used source of EBV is the B95-8 marmoset cell line. The B95-8 cell line spontaneously releases Epstein Barr virus into the medium.
A variety of cell types has been suggested to provide good feeder layers for the cloning of
EBV transformed cells. The most commonly used are peripheral blood mononuclear cells (PBMC) and fibroblasts.
PBMCs consist of monocytes, T lymphocytes and B-lymphocytes (5-10%). Because not all Blymphocytes will provide antibodies of the right specificity, it is advantageous to enrich PBMCs for appropriate cells. To prevent the generation of cytotoxic T-cells against EBV-transformed cells, T-lymphocytes can be removed, prior to EBV infection (with, for example, the supernatant from the EBV-productive B95-8 cell line), by treating the cells with washed sheep red blood cells (In: "Antibodies Vol. I, a practicle approach", Editor: Catty D, IRL Press, Oxford,
England. 1988. Ch. 4).
Immortalized B cell lines according to the invention were considered monoclonal if they satisfied the following requirements:
1) Stability of antibody secretion over time (>6 months),
2) Secretion of only one IgG (H and L) functional molecule,
3) Specificity and stability of antibody secretion following at least 2 sequential subcloning procedures (100% of growing colonies secreting IgG with specificity and genetic phenotype identical to the parental line).
The monoclonal antibodies according to the invention are extremely suitable to be used in so-called immuno-assays in order to detect HCV or HCV-fragments in a test sample.
An immunodiagnostic reagent comprising the antibodies according to the invention and a test kit for the detection of HCV in a sample are also part of the present invention.
Depending on the assay system employed, the immunochemical reaction that takes place can be a so-called sandwich reaction, an agglutination reaction, a competition reaction or an inhibition reaction.
The following assay systems are illustrative only and are not intended to limit the invention.
Carrying out, for instance, a sandwich reaction for the detection of HCV in a test sample the test kit to be used comprises a monoclonal antibody according to the invention coated on a solid support, for example the inner wall of a microtest well, and either a labelled monoclonal antibody or fragment thereof as conjugate.
Supports which can be used are, for example, the inner wall of a microtest well or a cuvette, a tube or capillary, a membrane, filter, test strip or the surface of a particle such as, for example, a latex particle, an erythrocyte, a dye sol, a metal sol or metal compound as sol particle, a carrier protein such as BSA or KLH.
Labelling substances which can be used are, inter alia, a radioactive isotope, a fluorescent compound , an enzyme , a dye sol , metal sol or metal compound or other sol as sol particle. A
further example of an immuno assay that can be used for the detection of HCV is a inhibition assay using human monoclonal antibodies as labelled reagent. The binding of this reagent to antigen on a solid phase can be competed by antibodies in the test sample.
As already mentioned monoclonal antibodies according to the invention are very suitable in diagnosis, while those antibodies which are neutralizing are very useful in passive immunotherapy.
The monoclonal antibodies according to the invention can also be used to raise antiidiotypic antibodies.
Anti-idiotypic antibodies are antibodies directed to the variable part of immunoglobulins. A sub-population of anti-idiotypic antibodies is known as "anti-idiotypic β" or "internal images". These anti-idiotypic β antibodies have either a structural or a three dimensional resemblance with the antigen (Uytdehaag, F.G.C.M. et al.
Immunol.Rev; 90; 93-113: 1986). This type of anti-idiotypic antibodies is widely used as a vaccine against infectious diseases in animal models (Hiernaux J.R.; Infect. Immun.; 56; 1407-
1413; 1988. Kennedy. R.C. et al.; Science 232;
220-223;1986). For use in assays the antiidiotypic antibodies can be raised in large amounts.
Techniques for raising anti-idiotypic antibodies are known in the art. The following example is illustrative only and is not intended to limit the invention; anti-idiotypic antibodies according to the invention can be obtained by immunizing BALB/c mice with monoclonal antibodies, coupled to KLH with glutaraldehyde
according to standard literature procedures, mixed with Freund's complete adjuvant. The spleen cells of these mice can be immortalized and the thus obtained hybridomas can be screened for anti-idiotypic antibody production. Screening of the hybridomas can be performed, for example but not limited to, by binding a HCV peptide to a solid phase (wells of microtiter plates) and incubating the solid phase with culture supernatant of growing hybridomas and the monoclonal antibodies coupled to Horse Radish Peroxidase (HRP) according to our invention. The presence of anti-idiotypic antibodies in culture supernatant will then be indicated by inhibition of the binding of the monoclonal antibodies according to our invention and the HCV peptide coated on the solid phase.
Anti-idiotypic antibodies reactive with the monoclonal antibodies according to the invention, as described above, are part of the present invention.
Anti-idiotypic antibodies, especially representing the internal image, can be used in mimicking antigen in a diagnostic test. Beside the use in diagnostic tests anti-idiotypic antibodies are also useful for prevention and/or treatment of Non-A, Non-B Hepatitis, as well as for the elucidation of important epitopic regions of HCV-antigens.
Preferred anti-idiotypic antibodies according to the present invention are the antiidiotypic antibodies secreted by the cell lines which are deposited at the European Collection of
Animal Cell Cultures (ECACC), Porton Down (UK)
under deposit No. 93122307 (HCVID.OT2A), under deposit No. 93122308 (HCVID.OT2B), under deposit No. 93122309 (HCVID.OT2C), under deposit No. 93122310 (HCVID.OT2D), under deposit No. 93122311
(HCVID.OT2E), under deposit No. 93122312
(HCVID.OT2I), under deposit No. 93122313
(HCVID.OT2J), under deposit No. 93122314
(HCVID.OT2M), under deposit No. 93122315
(HCVID.OT2P), under deposit No. 93122316
(HCVID.OT2Q), under deposit No. 93122317
(HCVID.OT3A), under deposit No. 93122318
(HCVID.OT3D), under deposit No. 93122319
(HCVID.OT3E), under deposit No. 93122320
(HCVID.OT3H), under deposit No. 93122321
(HCVID.OT3L), and under deposit No. 93122322
(HCVID.OT30).
Anti-idiotypic antibodies according to the invention preferably are anti-idiotypic antibodies capable of competing with an epitope for the binding to the monoclonal antibody according to the invention, where the epitope is the epitope recognized by the monoclonal antibodies secreted by the Epstein-Barr virus- transformed human lymphocyte-B cell line deposited with the European Collection of Animal Cell Cultures (ECACC), Porton Down (UK), under deposit No. 92121609.
Immunodiagnostic reagents comprising the anti-idiotypic antibodies according to the invention, and a test kit for the detection of anti-HCV antibodies in a sample using an immunodiagnostic reagent comprising antiidiotypic antibodies are also part of the present invention.
Immortalized cell lines capable of secreting anti-idiotypic antibodies reactive with the monoclonal antibodies according to the invention, as described above, are part of the present invention.
The preparation of cell lines producing anti-idiotypic antibodies may occur by, for example, transformation with Epstein-Barr Virus, the Kόhler and Milstein technique (Kδhler and Milstein devised the techniques that resulted in the formation monoclonal antibody-producing hybridomas (G. Köhler and C. Milstein, 1975, Nature 256:495-497; 1976, Eur. J. Immunol. 6:511- 519)), or a direct transformation technique of B- lymphocytes with oncogenic DNA, or a direct fusion of human B-lymphocytes with a fusion partner being either a human or a mouse-human hybrid myeloma cell line, or a direct fusion of an EBV-transformed B cell line with said myeloma cell lines.
Preferred cell lines according to the invention are deposited at the European Collection of Animal Cell Cultures, Porton Down (UK). HCVID.OT2A under deposit No. 93122307, HCVID.OT2B under deposit No. 93122308, HCVID.OT2C under deposit No. 93122309, HCVID.OT2D under deposit No. 93122310, HCVID.OT2E under deposit No. 93122311, HCVID.OT2I under deposit No. 93122312, HCVID.OT2J under deposit No. 93122313, HCVID.OT2M under deposit No. 93122314, HCVID.OT2P under deposit No. 93122315, HCVID.OT2Q under deposit No. 93122316, HCVID.OT3A under deposit No. 93122317, HCVID.OT3D under deposit No. 93122318, HCVID.OT3E under deposit No. 93122319, HCVID.OT3H under deposit No. 93122320, HCVID.OT3L
under deposit No. 93122321, and HCVID.OT30 under deposit No. 93122322.
An immunodiagnostic reagent comprising the anti-idiotypic antibodies according to the invention and a test kit for the detection of HCV in a sample are also part of the present invention.
The present invention is further exemplified by the following examples:
Example 1 illustrates the way in which a cell line, producing monoclonal antibodies, according to the invention may be derived.
Example 2 further exemplifies the specific immune reactivity of monoclonal antibodies according to the invention.
Example 3 exemplifies the production of anti-idiotypic antibodies and the specific ability of these anti-idiotypic antibodies to mimic a immunodominant NS-3 epitope.
EXAMPLES
EXAMPLE 1: Preparation of B-cell line clone. Peripheral blood mononuclear cells (PBMC) were obtained from one patient with chronic HCV infection and circulating antibodies to the NS3 and nucleocapsid proteins of HCV but not to c100- 3/NS4(HCV 1st generation antibody test, Ortho Diagnostic Systems, Raritan, NJ) as shown by reactivity of the patient's serum with a commercial assay that incorporates a recombinant protein designated C22-3, the viral nucleoprotein, and the recombinant nonstructural protein c200 encoded by the NS3 and NS4 regions of the HCV genome (Ortho HCV ELISA Test System, 2nd generation).
PBMC were resuspended in RPMI-1640 medium containing 10% heat-inactivated pooled human serum, supplemented with 10% human endothelial culture supernatant (HECS) + 20 U/ml rIL6 and cultured in 25 cm2 flasks at the concentration of 4 x 10E6/ml in the presence of recombinant NS3 protein (Organon Teknika) at the concentration of 25, 125, and 625 ng/ml for 6 days at 37 °C in 5% CO2.
After washing, cells were enriched in lymphocytes-B by removing lymphocytes-T with S- (2-amino-ethyl)-isothiouronium-bromide- hydrobromide treated sheep red blood cells and infected with supernatant from the EBV-productive B95-8 cell line as described in Cerino A et al. J. Immunol., 147, p2692, 1991.
After overnight incubation, cells were seeded at a density of 3* 104/well in U-bottom 96-well microtiter plates. After 20-30 days in culture, cultures secreting antibodies reactive with solid phase NS3 protein were subcultured at
12-50 cells/well in U-bottom wells with 3000 R- irradiated allogeneic PBMC as feeder cells. B cell lines were subcultured at least twice before further characterization.
IgG heavy and light (L) chains were determined as described in Cerino A et al. J.Tmiminol., 147, p2692, 1991. Briefly, B cell line supernatants were incubated on NS3 coated wells for 1 hour at 37 °C and, after washing, human IgG subclass-specific murine mAb were added for 1 hour followed by a peroxidase-conjugated rabbit anti-mouse Ig for another hour. To determine IgG L chains, peroxidase-conjugated rabbit anti-human Ig kappa- or lambda-chain were used in a direct assay. In both cases the reaction was developed with orthophenylen- diamine/2 HCl as substrate.
Results:
Significant anti NS3 production was obtained after in vitro stimulation with 25 and 125 ng/ml of rNS3, whereas no increase in Ab production, compared with control cultures, was found using the highest Ag concentration. A total of 480 wells were seeded with cells derived from cultures at 25 ad 125 ng/ml of NS3: 3 of these cell lines secreted significant levels (A492>1) of anti-NS3 and were stable for a time sufficient to allow further characterization. One cell line, HCVHU.OT3 (deposited at the ECACC under deposit No. 92121609), satisfied the criteria for monoclonality. Indeed such line has been subcultured twice yielding clones secreting identical amounts of specific antibody and has been kept in continuous culture for > 6 months. Furthermore, clone HCVHU.OT3 produced IgGl (k) exclusively.
EXAMPLE 2: Testing on anti NS3 production of B-cell lines.
The specificity of oligoclonal and monoclonal IgG-containing supernatants was further tested with the following reagents:
1) A recombinant purified HCV nucleoprotein expressed in E.coli (Organon Teknika).
2) A recombinant purified NS3 protein expressed in E.coli (Organon Teknika).
3) A recombinant purified NS-5 protein expressed in E.coli.
4) Recombinant immunoblot assay (RIBA II generation, Ortho Diagnostics). This is a nitrocellulose-based assay that includes 4 recombinant HCV antigens:
- c100-3, derived from NS-4 protein,
- 5-1-1, a 42-aminoacid fragment of c100-3,
- c33c, derived from the NS3 protein,
- c22-3, derived from the viral nucleoprotein .
Human superoxide dismutase (SOD) is also present on nitrocellulose strips as a control. Results:
Sueernatant from clone HCVHU.OT3 was analyzed for specificity in the above described way.
Figure 1 illustrates the specific binding capacity of antibodies according to the invention. The binding of the antibodies according to the invention (HCVHU.OT3) to different HCV derived proteins is compared with the binding of antibodies specific for HCV core and NS4 proteins respectively. As can be seen from Figure 1, the monoclonal antibodies according to the invention (HCVHU.OT3) recognized
a recombinant NS3 prctein preparation and gave a clear positive reaction in Ortho II generation assay, whereas no binding to recombinant HCV core and NS-5 proteins could be documented. Analysis of HCVHU.OT3 supernatant by recombinant immunoblot (RIBA II generation) revealed clear binding to the c33c polypeptide only, further attesting to the specificity of the mAb as can be seen from figure 2, where lane 2 represents an antibody according to the invention (HCVHU.OT3).
EXAMPLE 3: Production of anti-idiotypic antibodies
Monoclonal human IgG (HCVHU.OT3) was purified from the culture supernatant of the cell-line deposited at the European Collection of Animal Cell Cultures, Porton Down (UK) under deposit No. 92121609. This IgG was conjugated to KLH (Oosterlaken T.A.M. et al., (1990) Scandinavian Journal of Immunology, 31, 159) mixed with Complete Freunds adjuvant or Quil A and injected into 4 mice and 4 rats. Immunizations were performed according to the scheme in table 1. After 2 immunizations the animal with the highest serum titer, one mouse and one rat, was used for making monoclonal antibodies, essentially as described in
Steenbakkers et al. (Journal Of Immunological
Methods, 163, 33, 1993 and Journal of Immunological Methods, 152, 59, 1992). Clones secreting putative anti-idiotypic antibodies were also detected with the assay described in figure 3. In this way 16 clones were obtained that reacted well in this assay. The clones secreting putative anti-idiotypic antibodies were cultured and subcloned to 100% clonality according to standard procedures (Köhler, G. and Milstein, C.
(1975) Nature, 256, 495), (Kόhler. G. et al.,
(1976) European Journal of Immunology, 6, 292) and (Eshar, Z. ( 1985) In: T.A. Springer (Editor),
Hybridoma Technology in the Biosciences and Medicine p3).
Twelve of the monoclonal antibodies were of mouse origin, 6 were of rat origin. Isotypes of the different antibodies are listed in table 2.
Table 1
Immunization of mice and rats.
* Adjuvant: - FCA = Freund's complete adjuvant
- FIA = Freund's incomplete adjuvant
** Mice and rats were immunized with 50 μg of HCVHU.OT3 or 50 μg of HCVHu.OT3 coupled to Keyhole Limpet Haemocyanin (KLH) by the procedure described in ref. 1.
***
- Immunizations with FCA and FIA were given intraperitoneally (ip)
- Immunizations with Quil A were given intracutaneously (ic)
Table 2
List of monoclonal anti-idiotypic antibodies directed to HCV
Hu. OT3
The cell lines were grown in higher cell densities and cultured for 5 days in serumfree medium. Antibodies were purified from the culture supernatant using a Protein A-SepharoseR column. Purification was performed according to the manufacturers instructions (Pharmacia-LKB).
From two of the rat monoclonal antibodies no pure anti-idiotypic antibody could be obtained (HCVHU.OT3H and HCVHU.OT3E).
The anti-idiotypic antibodies were further analysed for their ability to mimic the NS-3 epitope recognized by the human monoclonal antibody HCVHU.OT3. For that purpose the purified monoclonal antibodies were conjugated to HRP with the Actizyme-peroxidase kit (Zymed laboratories). The procedure was performed according to the manufacturers instructions.
The procedure to analyse the ability to mimic the NS-3 epitope is further illustrated hereunder.
C33-β-galactosidase (containing amino acids 1192 to 1457 of the HCV genome) was immobilized on a 96-well micro ELISA plate according to standard procedures (1 μg/ml in 0.05M carbonate buffer, pH=9.6, incubated overnight at 4 °C). Serum samples of mice or rats or culture supernatant of the cells to be screened for the production of anti-idiotypic antibodies was mixed with HCVHU.OT3-HRP and incubated at 37 °C for 1 hour. Thereafter the mixture was brought to the well coated with the C33-β-galactosidase. Binding of the anti-idiotypic antibodies to HCVHU.OT3 was detected after extensive washing with PBS supplemented with 0.05 % Tween 20R. If antiidiotypic antibodies had bound to HCVHU.OT3-HRP, the signal decreased to background value.
With this material a sandwich assay was developed consisting at one hand of one of the monoclonal anti-idiotypic antibodies immobilized in a well of a 96-well micro ELISA plate and on the other hand of the same monoclonal antibody labelled with HRP in solution. (Figure 4).
The assay procedure to validate putative anti-idiotypic antibodies is further illustrated hereunder.
Putative anti-idiotypic antibody was immobilized on a 96-well micro ELISA plate according to standard procedures (10 μg/ml, 0.05M carbonate buffer, pH=9.6, incubation overnight at
4 °C).
Serum dilutions or dilutions of HCVHU.OT3 were added to the wells followed by 1 hour incubation at 37 °C.
After extensive washing with PBS supplemented with 0.05% Tween 20R (4 times), HRP labelled putative anti-idiotypic antibody was added.
Thereafter the plate was incubated for 1 hour at 37 °C, followed by washing (4 times) with PBS with 0.05% Tween 20R.
Thereafter substrate for HRP was added.
Using the assay described in figure 4 , further evidence was obtained for the specificity of the monoclonal anti-idiotypic antibodies. The anti-idiotypic antibodies were able to discriminate between HCVHU.OT3 and an irrelevant human monoclonal antibody of the same isotype as HCVHU.OT3. In figure 5 representative examples of this specificity are shown.
In the same assay (figure 5) it was established whether the anti-idiotyic antibodies were able to specifically bind to antibodies in serum from human individuals infected with HCV. For that purpose a panel of human sera was selected, known to contain antibodies against the HCV NS-3 antigen as determined by the commercial RIBA assay (Chiron Corporation Emmeryville).
In figure 5, reaction with HCVHU.OT3 is shown as black bars, reaction with an irrelevant human monoclonal antibody with the same subclass as HCVHU.OT3 is shown as shaded bars. Other monoclonal anti-idiotypic antibodies gave comparable results.
The ability of the anti-idiotypic antibodies prepared as described above were indeed able to mimic the immunodominant NS-3 epitope as defined by the human monoclonal HCVHU.OT3, is exemplified in table 3.
Table 3 shows the reactivity of human sera from HCV infected patients and normal controls with anti-idiotypic antibodies HCVID.OT2A and HCVID.OT2I.
Table 3
Reactivity of human sera from HCV infected patients and normal controls with anti-idiotypic antibodies HCVID. OT2A and HCVID. OT2I.
OD = optical density at 450 nm
N.R. = normalised response, calculated as OD450 of serum sample divided by cut off value.
Cut off value is average normal serum (n=20) + 3 times standard deviation.
Figures:
Figure 1 is a graph illustrating the binding specificity of monoclonal antibody HCVHU.OT3 for the NS3 protein.
Figure 2 is a photograph of a recombinant immunoblot assay. The characters A-G represent the following proteins;
A: High Ig control
B: 5-1-1 (NS-4)
C: c100-3 (NS-4)
D: C33c (NS-3)
E: c22-3 (core)
F: superoxide dismutase
G: low Ig control.
Lane 1 represents negative control serum,
lane 2 represents an anti NS-3 monoclonal antibody (HCVHU.OT3),
lane 3 represents an anti-core monoclonal (HCVHU.OT2), and
lane 4 represents polyclonal serum from a HCV- infected patient.
Figure 3 represents a screening procedure used for detection of anti-idiotypic antibodies against human monoclonal antibody HCVHU.OT3 in serum and in culture supernatant of monoclonal antibody producing cells.
Figure 4 represents an assay procedure used to validate putative anti-idiotypic antibodies which emerged from the screening procedure described in figure 3.
Figure 5 represents a sandwich assay (as shown in figure 4) to determine the specificity of 6 different anti-idiotypic antibodies (2A, 2B, 2I, 2P, 2Q and 3A) for HCVHU.OT3.
Reaction with HCVHU.OT3 is shown as black bars, reaction with an irrelevant human monoclonal antibody with the same subclass as HCVHU.OT3 is shown as shaded bars. Other monoclonal anti-idiotypic antibodies gave comparable results.
Claims
1. Monoclonal antibody which binds to an epitope of the NS3 protein of hepatitis C virus, which epitope is recognized by monoclonal antibodies secreted by the Epstein-Barr virus- transformed human lymphocyte-B cell line, deposited with the European Collection of Animal Cell Cultures (ECACC), Porton Down (UK), under deposit No. 92121609.
2. Monoclonal antibody according to claim 1, secreted by the Epstein-Barr virus-transformed human lymphocyte-B cell line deposited with the European Collection oi Animal Cell Cultures (ECACC), Porton Down (UK), under deposit No. 92121609.
3. Immortalized cell line capable of excreting antibodies according to claim 1.
4. Cell line deposited at the ECACC under deposit No. 92121609.
5. Method for the detection of hepatitis C virus in a sample comprising contacting the sample with a monoclonal antibody according to claim 1 or 2, and detecting immune complexes formed between the monoclonal antibody and a hepatitis C antigen.
6. Immunodiagnostic reagent comprising a monoclonal antibody according to claim 1 or 2.
7. Test kit for the detection of hepatitis C virus in a sample, comprising an immunodiagnostic reagent according to claim 6.
8. Anti-idiotypic antibody reactive with the antibody according to claim 1.
9. Anti-idiotypic antibody according to claim 8, secreted by the hybridoma cell line deposited with the European Collection of Animal Cell Cultures (ECACC), Porton Down (UK), under deposit NO. 93122307, No. 93122308, No. 93122309, No. 93122310, No. 93122311, No. 93122312, No. 93122313, No. 93122314, No. 93122315, No. 93122316, No. 93122317, No. 93122318, No. 93122319, No. 93122320, No. 93122321 or No. 93122322.
10. Anti-idiotypic antibody according to claim 8 or 9, capable of competing with an epitope for the binding to the monoclonal antibody according to claim 1, where the epitope is the epitope recognized by the monoclonal antibodies secreted by the Epstein-Barr virus- transformed human lymphocyte-B cell line deposited with the European Collection of Animal Cell Cultures (ECACC), Porton Down (UK), under deposit No. 92121609.
11. Immortalized cell line capable of secreting anti-idiotypic antibodies according to any of the claims 8-10.
12. Cell line deposited with the European Collection of Animal Cell Cultures (ECACC),
Porton Down (UK), under deposit No. 93122307, No.
93122308, No. 93122309, No. 93122310, No. 93122311, No. 93122312, No. 93122313, No. 93122314, No. 93122315, No. 93122316, No. 93122317, No. 93122318, No. 93122319, No. 93122320, No. 93122321 or No. 93122322.
13. Method for the detection of anti-HCV antibodies in a sample, comprising contacting the sample with an anti-idiotypic antibody according to any of the claims 8-10, and detecting immune complexes formed between the anti-idiotypic antibody and anti-HCV antibodies.
14. Immunodiagnostic reagent comprising an anti-idiotypic antibody according to any of the claims 8-10.
15. Testkit for the detection of anti-HCV antibodies in a sample comprising an immunodiagnostic reagent according to claim 14.
Priority Applications (5)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1019940703025A KR950700423A (en) | 1992-12-29 | 1993-12-27 | MONOCONAL ANTIBODIES AND ANTI-IDIOTYPIC ANTIBODIES TO HEPATITIS C VIRUS |
| JP6507580A JPH07504333A (en) | 1992-12-29 | 1993-12-27 | Monoclonal antibodies and anti-idiotypic antibodies against hepatitis C virus |
| EP94904191A EP0628084A1 (en) | 1992-12-29 | 1993-12-27 | Monoclonal antibodies and anti-idiotypic antibodies to hepatitis c virus |
| AU58346/94A AU682335B2 (en) | 1992-12-29 | 1993-12-27 | Monoclonal antibodies and anti-idiotypic antibodies to hepatitis C virus |
| FI943524A FI943524A0 (en) | 1992-12-29 | 1994-07-27 | Monoclonal antibodies and anti-idiotype antibodies against hepatitis C virus |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| EP92204107 | 1992-12-29 | ||
| EP92204107.4 | 1992-12-29 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO1994014974A1 true WO1994014974A1 (en) | 1994-07-07 |
Family
ID=8211185
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/EP1993/003707 WO1994014974A1 (en) | 1992-12-29 | 1993-12-27 | Monoclonal antibodies and anti-idiotypic antibodies to hepatitis c virus |
Country Status (8)
| Country | Link |
|---|---|
| EP (1) | EP0628084A1 (en) |
| JP (1) | JPH07504333A (en) |
| KR (1) | KR950700423A (en) |
| AU (1) | AU682335B2 (en) |
| CA (1) | CA2127797A1 (en) |
| FI (1) | FI943524A0 (en) |
| WO (1) | WO1994014974A1 (en) |
| ZA (1) | ZA939756B (en) |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1998016647A1 (en) * | 1996-10-14 | 1998-04-23 | Juridical Foundation The Chemo-Sero-Therapeutic Research Institute | Hepatitis c virus epitope |
| US5871904A (en) * | 1994-07-12 | 1999-02-16 | The Tokyo Metropolitan Institute Of Medical Science | Immunassay of non-A, non-B hepatitis virus-related antigens, monoclonal antibodies for use therein, and hybridomas producing the antibodies |
| WO2002072126A2 (en) * | 2001-03-14 | 2002-09-19 | TransMIT Gesellschaft für Technologietransfer mbH | Invention relating to hcv diseases |
| US7374876B2 (en) * | 2001-04-03 | 2008-05-20 | Rapid Medical Diagnostic Corporation | Anti-idiotypic antibody and its use in diagnosis and therapy of Hepatitis C Virus related diseases |
| US9008369B2 (en) | 2004-04-15 | 2015-04-14 | Magna Electronics Inc. | Vision system for vehicle |
| US9436880B2 (en) | 1999-08-12 | 2016-09-06 | Magna Electronics Inc. | Vehicle vision system |
| US9555803B2 (en) | 2002-05-03 | 2017-01-31 | Magna Electronics Inc. | Driver assistance system for vehicle |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1990000597A1 (en) * | 1988-07-06 | 1990-01-25 | Genelabs Incorporated | Post-transfusion, non-a, non-b hepatitis virus and antigens |
-
1993
- 1993-12-27 JP JP6507580A patent/JPH07504333A/en active Pending
- 1993-12-27 AU AU58346/94A patent/AU682335B2/en not_active Expired - Fee Related
- 1993-12-27 WO PCT/EP1993/003707 patent/WO1994014974A1/en not_active Application Discontinuation
- 1993-12-27 KR KR1019940703025A patent/KR950700423A/en not_active Application Discontinuation
- 1993-12-27 EP EP94904191A patent/EP0628084A1/en not_active Withdrawn
- 1993-12-27 CA CA002127797A patent/CA2127797A1/en not_active Abandoned
- 1993-12-29 ZA ZA939756A patent/ZA939756B/en unknown
-
1994
- 1994-07-27 FI FI943524A patent/FI943524A0/en not_active Application Discontinuation
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1990000597A1 (en) * | 1988-07-06 | 1990-01-25 | Genelabs Incorporated | Post-transfusion, non-a, non-b hepatitis virus and antigens |
Non-Patent Citations (1)
| Title |
|---|
| N. HIRAMATSU ET AL.: "Immunohistochemical detection of hepatitis C virus-infected hepatocytes in chronic liver disease with monoclonal antibodies to core, envelope and NS3 regions of the hepatitis C virus genome.", HEPATOLOGY, vol. 16, no. 2, August 1992 (1992-08-01), BALTIMORE MD, USA, pages 306 - 311 * |
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5871904A (en) * | 1994-07-12 | 1999-02-16 | The Tokyo Metropolitan Institute Of Medical Science | Immunassay of non-A, non-B hepatitis virus-related antigens, monoclonal antibodies for use therein, and hybridomas producing the antibodies |
| WO1998016647A1 (en) * | 1996-10-14 | 1998-04-23 | Juridical Foundation The Chemo-Sero-Therapeutic Research Institute | Hepatitis c virus epitope |
| US9436880B2 (en) | 1999-08-12 | 2016-09-06 | Magna Electronics Inc. | Vehicle vision system |
| WO2002072126A2 (en) * | 2001-03-14 | 2002-09-19 | TransMIT Gesellschaft für Technologietransfer mbH | Invention relating to hcv diseases |
| WO2002072126A3 (en) * | 2001-03-14 | 2002-11-14 | Transmit Technologietransfer | Invention relating to hcv diseases |
| US7374876B2 (en) * | 2001-04-03 | 2008-05-20 | Rapid Medical Diagnostic Corporation | Anti-idiotypic antibody and its use in diagnosis and therapy of Hepatitis C Virus related diseases |
| US9555803B2 (en) | 2002-05-03 | 2017-01-31 | Magna Electronics Inc. | Driver assistance system for vehicle |
| US9643605B2 (en) | 2002-05-03 | 2017-05-09 | Magna Electronics Inc. | Vision system for vehicle |
| US9008369B2 (en) | 2004-04-15 | 2015-04-14 | Magna Electronics Inc. | Vision system for vehicle |
| US9191634B2 (en) | 2004-04-15 | 2015-11-17 | Magna Electronics Inc. | Vision system for vehicle |
| US9428192B2 (en) | 2004-04-15 | 2016-08-30 | Magna Electronics Inc. | Vision system for vehicle |
Also Published As
| Publication number | Publication date |
|---|---|
| FI943524A (en) | 1994-07-27 |
| EP0628084A1 (en) | 1994-12-14 |
| KR950700423A (en) | 1995-01-16 |
| AU5834694A (en) | 1994-07-19 |
| AU682335B2 (en) | 1997-10-02 |
| CA2127797A1 (en) | 1994-07-07 |
| ZA939756B (en) | 1994-08-18 |
| FI943524A0 (en) | 1994-07-27 |
| JPH07504333A (en) | 1995-05-18 |
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