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WO1994024565A1 - Antigenes et anticorps utiles dans l'immunodiagnostic du virus de l'hepatite c - Google Patents

Antigenes et anticorps utiles dans l'immunodiagnostic du virus de l'hepatite c Download PDF

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Publication number
WO1994024565A1
WO1994024565A1 PCT/US1994/004174 US9404174W WO9424565A1 WO 1994024565 A1 WO1994024565 A1 WO 1994024565A1 US 9404174 W US9404174 W US 9404174W WO 9424565 A1 WO9424565 A1 WO 9424565A1
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Prior art keywords
seq
antigen
val
ser
leu
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PCT/US1994/004174
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English (en)
Inventor
Gregory R. Reyes
Jungsuh P. Kim
Randolph Moeckli
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Genelabs Technologies, Inc.
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Priority to AU67695/94A priority Critical patent/AU6769594A/en
Publication of WO1994024565A1 publication Critical patent/WO1994024565A1/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
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/576—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis
    • G01N33/5767—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis non-A, non-B hepatitis
    • 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

  • This invention relates to specific peptide viral antigens which are immunoreactive with sera from patients infected with parenterally trans ⁇ mitted non-A, non-B hepatitis (PT-NANBH) virus, to antibodies immunoreactive with these antigens, and to methods of using the antibodies for detecting PT-NANBH infection in human sera.
  • PT-NANBH non-A, non-B hepatitis
  • NANBH non-A, non-B hepatitis
  • HAV hepatitis E virus
  • the second NANB virus type known as parenterally transmitted NANBH, or PT-NANBH
  • PT-NANBH is transmitted by parenteral routes, typically by exposure to blood or blood products.
  • RNA isolated from infected chimpanzee sera has been used to construct cDNA libraries in an expression vector for immunoscreening with chronic-state human PT- NANBH serum. This procedure identified a PT-NANBH specific cDNA clone and the viral sequence was then used as a probe to identify fragments making up 7,300 contiguous basepairs of a PT-NANBH viral agent (Houghton, et al . , (A), EP Pub. No. 0318216; Houghton, et al . , (B) , EP Pub. No. 0388232).
  • the present invention provides a method for detecting the presence of hepatitis C virus (HCV) antigens in a sample.
  • the sample is contacted with at least one antibody which is reactive with an HCV antigen.
  • the antibody is usually attached to a solid support and is immunoreactive with a portion of the polypeptide presented as SEQ ID NO:6.
  • the support-bound antibody is then examined for the presence of bound HCV antigen. This examining typically involves reacting the solid support with an antigen-reporter complex, where the HCV antigen competes with binding of the antigen-reporter complex to the antibody.
  • the level of antigen- reporter complex, which is bound to the solid support is then determined.
  • the method can be used to analyze samples from a variety of sources including, but not limited to, the following: tissue culture medium, chimpanzee serum, and human serum.
  • a number of solid supports are useful in the method of the present invention, including microtiter plates.
  • two or more antibodies, with their corresponding antigen-reporter complexes can also be used.
  • Antigens useful in generating antibodies for the method of the present invention include the polypeptides selected from the group of sequences presented as SEQ ID NO:6, SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:19, and derivatives thereof. These antigens are also useful in antibody-capture assays, where the antigen polypeptide is attached to a solid support and serum is screened for antibodies capable of binding to the antigens.
  • a number of reporters can be employed in the present method.
  • the antigen-reporter complexes can contain one or more of the following: enzymatic reporters, radioactive reporters, and fluorescent reporters.
  • An exemplary enzymatic reporter is horse radish peroxidase.
  • kits for use in the screening samples for the presence of hepatitis C virus (HCV) antigen, by the method of the present invention.
  • kits typically include (i) at least one antibody which is immunoreactive with a portion of the polypeptide presented as SEQ ID NO:6, and (ii) an antigen-reporter complex, where the HCV antigen competes with binding of the antigen-reporter complex to the antibody. Further, the antibody can be attached to a solid support.
  • the antigens and antibodies are as described above.
  • the present invention includes purified antibodies that are immunoreactive with a polypeptide consisting essentially of a sequence selected from the following: SEQ ID NO:6, SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:19. These antibodies can be polyclonal or monoclonal antibodies.
  • Another aspect of the present invention includes polypeptides consisting essentially of the following sequences: SEQ ID NO:6, SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:19. These polypeptides, and polypeptides containing other epitopes included in the clone 36 sequence, are useful in antibody-capture assays, the generation of antibodies for the diagnostic method of the present invention, and the generation of antibodies for use in immunoprophylaxis. Further, these polypeptides may be useful as polypeptide antigen vaccines effective against HCV.
  • Figure 1 illustrates the construction of a modified clone 36 insert lacking internal Ncol and BamHI sites.
  • Figure 2 schematically illustrates the basis design of an antibody based competition immunoassay.
  • PT-NANBH hepatitis viral agent
  • HCV HCV
  • HCV PT-NANBH viral agent whose polynucleotide sequence includes the sequence of the 7,300 basepair region of HCV (Houghton, et al . , (A), EP Pub. No. 0318216; Houghton, et al . , (B) , EP Pub. No. 0388232), and variations of the sequence, such as degenerate codons, or variations which may be present in different isolates or strains of HCV.
  • Two nucleic acid fragments are "homologous" if they are capable of hybridizing to one another under hybridization conditions described in Maniatis et al., op. cit.. pp. 320- 323, using the following wash conditions: 2 x SCC, 0.1% SDS, room temperature twice, 30 minutes each; then 2 x SCC, 0.1% SDS, 50°C once, 30 minutes; then 2 x SCC, room temperature twice, 10 minutes each, homologous sequences can be identified that contain at most about 25-30% basepair mismatches. More preferably, homologous nucleic acid strands contain 15-25% basepair mismatches, even more preferably 5-15% basepair mismatches. These degrees of homology can be selected by using more stringent wash or hybridization conditions for identification of clones from gene libraries (or other sources of genetic material) , as is well known in the art.
  • a DNA fragment is “derived from” HCV if it has substantially the same basepair sequence as a region of the HCV viral genome which was defined in (2) above.
  • a protein is "derived from” a PT-NANBH or HCV viral agent if it is encoded by an open reading frame of a cDNA or RNA fragment derived from a PT-NANBH or HCV viral agent, respectively.
  • the inserts were radioactively labelled and used as probes against Hindlll / EcoRI doubly-digested human peripheral lymphocyte (PBL) DNA (Example 3) . Approximately 46% (43/93) of the inserts hybridized with normal human PBL DNA and were therefore not pursued. Inserts from 11 PT-NANBH-immunopositive clones derived from chimpanzee #771 sera were characterized as exogenous to normal human PBL DNA (Example 3) . Of these 11 clones 2 PT-NANBH clones were identified having the following characteristics.
  • clone 40 was clearly exogenous by repeated hybridization tests against normal human PBL DNA, had a relatively small insert size (approximately 0.5 kilobases) , and was quite unreactive with negative control serum.
  • the second clone (clone 36) was shown to be reactive with multiple PT-NANBH antisera, had a relatively large insert size (approximately 1.5 kilobases) , and was exogenous by hybridization testing against normal human PBL DNA.
  • the immunoreactive characteristics of clones 36 and 40 are summarized in Table 1 (Example 3) .
  • Clone 36 was immunoreactive with chimpanzee #771 sera and two HCV-positive human sera, AG and BV.
  • the clone 36 antigen did not immunoreact with the negative control serum SKF.
  • Clone 40 was immunoreactive with chimpanzee #771 sera and was cleanly nonreactive when the negative control sera was used for screening.
  • the DNA sequences of clones 36 and 40 were determined and are presented as SEQ ID NO:5 and SEQ ID N0:l, respectively.
  • the clone 36 sequence corresponds to nucleotides 5010 to 6516 of the HCV sequence given in Houghton, et al . , (A), EP Pub. No. 0318216.
  • the clone 40 sequence is homologous to the HCV sequence (Houghton, et al . , (A), EP Pub. No. 0318216) in the region of approximately nucleotides 6515 to 7070.
  • the sequences for clones 36 and 40 are contiguous sequences, with the clone 36 sequences being located 5' of the clone 40 sequences as presented in Houghton, et al . , (A), EP Pub. No. 0318216, and Houghton, et al . , (B) , EP Pub. No. 0388232. Accordingly, these two clones represent isolation of a significant block of the HCV genome by the above-described immunoscreening methods.
  • the inserts of two other chimpanzee #771 clones, clones 44 and 45, were found to be homologous to clone 40 by hybridization and sequence analysis (Example 4) . Isolation and characterization of the four lambda gtll clones 36, 40, 44, and 45 were previously described in PCT International Application PCT/US91/02370, Publication No. WO 91/15516, Publication Date 17 October 1991.
  • Table 2 presents the data for preliminary immunoscreening of clone 36 and 45 antigens (the insert of clone 45 is essentially the same as the insert of clone 40) against well- documented PT-NANBH chronic sera which showed strong immunoreactivity to the 5-1-1 HCV peptide antigen (Kuo) .
  • the 5-1-1 HCV peptide antigen has previously been identified as immunoreactive against a high percentage of human PT-NANBH chronic sera.
  • the 5-1-1 antigen is encoded by the sequence between basepairs 3731 and 3857 in the HCV genome (Houghton, et al. , (A), EP Pub. No. 0318216; Houghton, et al .
  • the antigens produced by clones 36 and 40 yield HCV-specific immunopositive signals with selected samples. Additionally, subfragments of clone 36, when expressed in a lambda gtll expression system, were immunopositive with HCV- positive serum samples.
  • the data presented in Table 3 suggest that clone 36 identified all HCV-positive sera (within a selected sample) that were identified by any other single HCV antigen or combination of HCV antigens, including the C100 antigen which was employed in a commercial HCV diagnostic assay (the C-100 antigen corresponds essentially to clone 5-1-1, Table 3) .
  • the results show that the clone 36 antigen detected six unique positive sera samples.
  • the clone 36 antigen tested negative with all negative control sera.
  • the results presented in Table 2 and 3 support the use of the clone 36 antigen in immunodiagnostic assays directed to the detection of HCV.
  • the amplified subfragments were cloned into lambda gtll vectors (gtll-36-1, gtll-36-2, and gtll-36-3) for expression of the clone 36 polypeptide fragments encoded by each subfragment. These clones were used to test for expression of polypeptides capable of reacting with HCV-positive antisera in an immunoscreening assay.
  • the antigens were immunoscreened for reactivity with a number of human anti-sera including well- characterized HCV positive sera (Example 9) .
  • Each of the three sub-cloned clone 36 fragments tested immunopositive with at least one of the human anti-sera and tested negative with known HCV- negative control sera. These results indicate that each of the three portions contained at least one epitope immunoreactive with HCV-positive sera.
  • antigen containing portions of the clone 36 coding sequence can be identified by the methods described above.
  • the recombinant peptides of the present invention can be purified by standard protein purification procedures which may include differential precipitation, molecular sieve chromatography, ion-exchange chromatography, isoelectric focusing, gel electrophoresis and affinity chromatography.
  • a fused protein such as the beta-galactosidase fused proteins prepared as above
  • the fused protein can be isolated readily by affinity chromatography, by passing cell lysis material over a solid support having surface-bound anti-beta-galactosidase antibody.
  • affinity chromatography purification of a beta- galactosidase/fusion protein, derived from clone 36 coding sequences, by affinity chromatography is described in Examples 6.
  • a fused protein pontaining the clone 36 peptide fused with glutathione-S-transferase (Sj26) protein has also been expressed using the pGEX vector system in E. coli KM392 cells (Smith) .
  • This expression system has the advantage that the fused protein is generally soluble and therefore can be isolated under non-denaturing conditions.
  • the fused Sj26 protein can be isolated readily by glutathione substrate affinity chromatography
  • Example 7 This method of expressing this fusion protein is given in Example 7 and is applicable to any of the other antigen coding sequences described by the present invention.
  • Example 11 Yet another protein isolation method is described in Example 11 for the isolation of the pET-clone 36 antigen. Briefly, production of the pET-clone 36-encoded HCV antigen is induced in bacterial cells. The cells are lysed and the pelleted proteins are subjected to repeated rounds of suspension and re-pelleting. Then the protein pellet is suspended, filtered and subjected to size fractionation. Size fractionation can be achieved by any standard method including FPLC. Fractions resulting from the size fractionation are then assayed for the presence of the protein.
  • Fractions containing the antigenic protein of interest can be identified by a number of means including, immunoreactivity (e.g., ELISA assays) or separation on SDS polyacrylamide gels (identification by molecular weight) .
  • the polypeptide antigens of the present invention have a number of uses including, but not limited to, the following: antigens in immunoassays, such as ELISA (Example 10) ; vaccine compositions; and the generation of monoclonal and polyclonal antibodies.
  • the present invention includes antibodies specific against the recombinant antigens described above, including the clone 36 and clone 40 antigens, and further derivatives of either.
  • polyclonal antibodies typically, a host animal, such as a rabbit, is immunized with the purified antigen or fused protein antigen.
  • the host serum or plasma is collected following an appropriate time interval, and this serum is tested for antibodies specific against the antigen.
  • Example 7 describes the production of rabbit serum antibodies which are specific against the clone 36 antigens in the Sj26/clone 36 fusion protein. These techniques are equally applicable to the other antigens of the present invention.
  • the gamma globulin fraction or the IgG antibodies of immunized animals can be obtained, for example, by use of saturated ammonium sulfate or DEAE Sephadex, or other techniques known to those skilled in the art for producing polyclonal antibodies.
  • Hybridomas Alternatively, the purified antigen or fused antigen protein may be used for producing monoclonal antibodies.
  • the spleen or lymphocytes from an immunized animal are removed and immortalized or used to prepare hybridomas by methods known to those skilled in the art (Mishell) .
  • mice are immunized by intraperitoneal injection of the purified or partially purified antigen derived from clone 36 containing phage infected cells.
  • the antigen is mixed with an adjuvant, such as killed Bordatella pertussis organisms.
  • Hybridomas are formed by fusion of splenic B-lymphocytes and stable myeloma fusion partner cells.
  • Hybridomas are selected by growth in selective medium, then are tested for production of antibodies reactive with the specific im unogen, in one or more assays, such as a solid phase (ELISA) assay, plaque immunoscreening, or Western blot analysis (Ausubel, et al . ) .
  • assays such as a solid phase (ELISA) assay, plaque immunoscreening, or Western blot analysis (Ausubel, et al . ) .
  • Hybridomas found to produce reactive antibodies are further clone purified by limiting dilution.
  • Example 10 describes the generation of monoclonal antibodies directed against the pET36 antigen.
  • Hybrido a cells were formed by fusion of splenic B-lymphocytes from seropositive mice and stable myeloma fusion partner cells. Hybridomas were selected by growth in selective medium and tested for production of anti-pET-36 antibodies in a solid phase assay (ELISA) . Forty-nine positive clones were identified and isolated in one such selection. Hybridomas found to produce antibodies to pET 36 were further clone purified by limiting dilution. From the 49 positive clone, 5 were subcloned by limiting dilution.
  • Human hybridomas can also be produced by fusing a human lymphocyte with an appropriate immortalized fusion partner.
  • a donor known to be infected with an HCV virus may serve as a suitable lymphocyte donor.
  • Lymphocytes can be isolated from a peripheral blood sample or from spleen cells, if the donor is subject to splenectomy.
  • antibody- secreting B-lymphocytes are activated prior to selection using a transforming virus, such as Epstein-Barr virus, or can alternatively be achieved by exposure of the cells to other B-cell activators known in the art, such as pokeweed mitogen, or to the specific antigen recognized by the cells.
  • cells are grown in culture, then examined for production of specific anti-HCV antigen activity, using an appropriate antibody detection assay.
  • Cells showing activity in such an assay are selected for immortalization, by fusion with a heteromyeloma fusion partner.
  • Formation of a stable hybridoma that secretes a human anti-HCV antibody is achieved by fusing an activated B-lymphocyte with a heteromyeloma cell such as the K6H6-B5 cell line (Carroll, et al . ) or the H73C11 cell line (Perkins, et al.), originally produced by fusing activated human lymphocytes with a mouse myeloma fusion partner.
  • Such fusion can be achieved by a number of methods known in the art (Harlow, et al . ) including exposure of mixed cells to polyethylene glycol or exposure of cells to strong electric field (electrofusion) .
  • Hybridomas are selected by growth in selective medium, then are tested for HCV and antigen specificity in one or more i munoassays.
  • Primary in vitro immunization with peptide or protein antigens of hybridomas in culture can also be used in the generation of monoclonal antibodies.
  • Antibodies secreted by the immortalized cells are screened to determine the clones that secrete antibodies of the desired specificity.
  • FIG. 2 The basic design of the immunoassay is illustrated in Figure 2.
  • One example of the antigen detection assay is described in Example 12.
  • a rabbit anti-clone 36 antibody coated well and a clone 36 antigen-horse radish peroxidase (HRPO) conjugate are used in the assay.
  • Microwells were coated with the above- described monoclonal antibodies.
  • the antibody coated wells is then incubated with the test samples, such as, clone 36 polypeptide, HCV infected tissue culture media, human sera or chimpanzee sera. After incubation, clone 36-HRPO conjugate is added to each well.
  • Combination of the conjugate to the solid phase antibody coat was detected by the addition of the substrate-2 , 2 ' - azino-bis(3-ethylbenzthiazoline-6-sulfonic acid (ABTS) .
  • An alternative substrate which can be used is 5-aminosalicylic acid (5AS) .
  • the presence of a clone 36 HCV-associated viral antigen is identified by color diminution due to successful inhibition of binding clone 36-HRPO.
  • reporter labels other than HRPO can be used in the method of the present invention, including the following: enzymatic reporter systems, such as HRPO alkaline phosphatase, ⁇ -galactosidase, and glucose oxidase (Pierce, Rockford IL) ; fluorochrome reporters, such as fluorescein, R-phycoerythrin, rhodamine, rhodamine 600, and "TEXAS RED" (Pierce); biotin and avidin (Pierce) ; radioactive labelling, such as 125 I or synthesis of antigen polypeptides containing 3 H or 14 C; light emitting reporters, such as luciferase (de Wet, et al . ) ; and chromophors, such as heme (Sigma, St. Louis MO) . Reporter labels are conjugated to antigen peptides by appropriate standard methods in the art.
  • fluorochrome reporters such as fluorescein, R-phycoery
  • the antigen detection assay of the present invention can also be tested by addition of increasing quantities of free clone 36 polypeptide.
  • the addition of the clone 36 polypeptide over a linear range of concentrations results in a linear inhibition curve.
  • a method to confirm the specificity of the antigen detection reaction is described in Example 13. This confirmation assay is based on blocking the inhibition of clone 36-HRPO binding to its cognate antibody, which usually occurs by binding of a clone 36-based antigen to the same antibody.
  • the blocking of inhibition is accomplished by addition of anti-clone 36 positive sera to the antigen positive plasma before testing in the antibody coated plates.
  • the antigens obtained by the methods of the present invention are advantageous for use as diagnostic agents for anti-HCV antibodies present in HCV-infected sera; particularly, clone 36 antigen and related antigens 36-1, 36-2 and 36-3.
  • the clone 36 antigen provides an advantage over known HCV antigen reagents 5-1-1 and C-100 in that clone 36 is immunoreactive with a wider range of PT-NANBH infected sera.
  • test serum is reacted with a solid phase reagent having a surface-bound HCV antigen obtained by the methods of the present invention, e.g., the clone 36 antigen.
  • a solid phase reagent having a surface-bound HCV antigen obtained by the methods of the present invention e.g., the clone 36 antigen.
  • the reagent is reacted with reporter- labeled anti-human antibody to bind reporter to the reagent in proportion to the amount of bound anti-PT-NANBH antibody on the solid support.
  • the reagent is again washed to remove unbound labeled antibody, and the amount of reporter associated with the reagent is determined.
  • the reporter is an enzyme which is detected by incubating the solid phase in the presence of a suitable fluorometric or colorimetric substrate (Harlow, et al . ) .
  • a suitable fluorometric or colorimetric substrate Hard, et al .
  • the solid surface reagent in the above assay is prepared by known techniques for attaching protein material to solid support material, such as polymeric beads, dip sticks, 96-well plate or filter material. These attachment methods generally include non-specific adsorption of the protein to the support or covalent attachment of the protein, typically through a free amine group, to a chemically reactive group on the solid support, such as an activated carboxyl, hydroxyl, or aldehyde group.
  • homogeneous assay In a second diagnostic configuration, known as a homogeneous assay, antibody binding to a solid support produces some change in the reaction medium which can be directly detected in the medium.
  • Known general types of homogeneous assays proposed heretofore include (a) spin-labeled reporters, where antibody binding to the antigen is detected by a change in reported mobility (broadening of the spin splitting peaks) , (b) fluorescent reporters, where binding is detected by a change in fluorescence efficiency, (c) enzyme reporters, where antibody binding effects enzyme/substrate interactions, and (d) liposome- bound reporters, where binding leads to liposome lysis and release of encapsulated reporter.
  • the assay method involves reacting the serum from a test individual with the protein antigen and examining the antigen for the presence of bound antibody.
  • the examining may involve attaching a labeled anti-human antibody to the antibody being examined, either IgM (acute phase) or IgG (convalescent or chronic phase) , and measuring the amount of reporter bound to the solid support, as in the first method, or may involve observing the effect of antibody binding on a homogeneous assay reagent, as in the second method.
  • an assay system or kit for carrying out the assay method just described is also forming part of the invention.
  • the kit generally includes a support with surface-bound recombinant HCV antigen (e.g., the clone 36 antigens), and a reporter labeled reporter-labeled anti-human antibody for detecting surface-bound anti-PT- NANBH-antigen antibody.
  • HCV antigen e.g., the clone 36 antigens
  • reporter labeled reporter-labeled anti-human antibody for detecting surface-bound anti-PT- NANBH-antigen antibody.
  • HCV antigens identified by the methods of the present invention can be formulated for use in a HCV vaccine.
  • the vaccine can be formulated by standard methods, for example, in a suitable diluent such as water, saline, buffered salines, complete or incomplete adjuvants, and the like.
  • the immunogen is administered using standard techniques for antibody induction, such as by subcutaneous administration of physiologically compatible, sterile solutions containing inactivated or attenuated virus particles or antigens.
  • An immune response producing amount of virus particles is typically administered per vaccinizing injection, typically in a volume of one milliliter or less.
  • a specific example of a vaccine composition includes, in a pharmacologically acceptable adjuvant, a recombinant clone 36 peptide.
  • the vaccine is administered at periodic intervals until a significant titer of anti-HCV antibody is detected in the serum.
  • test serum is reacted with a solid phase reagent having a surface-bound anti-HCV-antigen antibody, either polyclonal or monoclonal, obtained by the methods of the present invention: for example, an anti-clone 36 antigen antibody.
  • a solid phase reagent having a surface-bound anti-HCV-antigen antibody either polyclonal or monoclonal, obtained by the methods of the present invention: for example, an anti-clone 36 antigen antibody.
  • the solid phase is washed and then contacted with a reporter labeled antigen containing the epitope corresponding to the surface-bound anti-HCV-antigen antibody.
  • the level of reporter is then quantitated and the serum-antigen levels are determined based on the percent inhibition of antigen-reporter binding obtained in the presence of the antigen-containing serum: typically by comparison to a standard curve.
  • the reporter is an enzyme which is detected by incubating the solid phase in the presence of a suitable fluorometric or colorimetric substrate.
  • the solid surface reagent in the above assay is prepared by known techniques for attaching protein material to solid support material, such as polymeric beads, dip sticks, 96-well plate or filter material (such as nitrocellulose) (Harlow, et al . ) .
  • These attachment methods generally include non-specific adsorption of the protein to the support or covalent attachment of the protein, typically through a free amine group, to a chemically reactive group on the solid support, such as an activated carboxyl, hydroxyl, or aldehyde group.
  • the assay method involves reacting the serum from a test individual with a support bound anti-HCV antibody and examining the antibody for the presence of bound antigen.
  • kits for carrying out the assay method just described.
  • the kit generally includes a support with surface-bound anti-HCV antibody and a reporter-labeled cognate antigen (e.g. , clone 36-HRPO) for detecting antibody bound HCV-antigen.
  • a reporter-labeled cognate antigen e.g. , clone 36-HRPO
  • the more antigen, from a test sample, bound to the antibody the more inhibition of reporter-antigen binding: accordingly, lower levels of detectable reporter.
  • Polyclonal and monoclonal antibodies for use in the present invention, can be prepared as described above utilizing the peptides of the present invention.
  • the antibodies can be purified by standard methods to provide antibody preparations which are substantially free of serum proteins that may affect reactivity (e.g., affinity purification (Harlow et al . ) ) .
  • a number of the antigenic peptides (the 409- 1-1 series, clone 40, clone 36, and clone 36 subclones, e .g . , 36-1, 36-2, and 36-3) of the present invention can be used singly or in combination in the antigen-inhibition assay of the present invention.
  • Antibodies generated against these peptides can be combined with each other and/or anti-clone 36 antibodies for immunological detection of HCV infected sera.
  • the multiple antigen-reporter molecules can be similarly or differentially labeled.
  • microtiter plate wells i.e., multiwell plates (Corning)
  • the probe is then a mixture of, for example, 36-1-HRPO and 36-3-HRPO.
  • the results of this assay are then read as a single-specificity.
  • the two antigen-reporter molecules can be differentially labeled by, for example, labeling one antigen with HRPO and the second antigen with alkaline phosphatase.
  • differential labeling is the use of two fluorescent reporters having different emission wavelengths: for example, phycoerythrin (PE) and fluorescein isothiocyanate (FICT) .
  • PE phycoerythrin
  • FICT fluorescein isothiocyanate
  • Multi-well microtiter plates can then be scanned (Dynatech Corp. , Cambridge MA) and the relative levels of antigens determined based on the emission spectra.
  • the present invention also includes kits containing multiple antibodies and cognate antigen-reporter complexes, as well as antibodies generated against the antigens of the present invention.
  • the anti-HCV antibodies of the invention can be used as a means of enhancing an anti-HCV immune response since antibody-virus complexes are recognized by macrophages and other effector cells.
  • the antibodies can be administered in amounts similar to those used for other therapeutic administrations of antibody. For example, pooled gamma globulin is administered at 0.02-0.1 ml/lb body weight during the early incubation of other viral diseases such as rabies, measles and hepatitis B to interfere with viral entry into cells.
  • antibodies reactive with, for example, the 409-1-1(c-a) antigen can be passively administered alone in a "cocktail" with other anti-viral antibodies or in conjunction with another anti-viral agent to a host infected with an PT-NANBH virus to enhance the immune response and/or the effectiveness of an antiviral drug.
  • E. coli DNA polymerase I (Klenow fragment) was obtained from Boehringer Mannheim Biochemicals (Indianapolis, IN) .
  • T4 DNA ligase and T4 DNA polymerase were obtained from New England Biolabs (Beverly, MA) ; Nitrocellulose filters were obtained from Schleicher and Schuell (Keene, NH) .
  • Synthetic oligonucleotide linkers and primers were prepared using commercially available automated oligonucleotide synthesizers. Alternatively, custom designed synthetic oligo- nucleotides may be purchased, for example, from Synthetic Genetics (San Diego, CA) .
  • cDNA synthesis kit and random priming labeling kits were obtained from Boehringer-Mannheim Biochemical (BMB, Indianapolis, IN) . Standard manipulations of molecular biology have been carried out as previously described (Ausubel, et al . ; Sambrook, et al . ; Maniatis, et al . ) . Methods for antibody preparation and standard diagnostic applications are described in Harlow, et al .
  • PLC Polystyrene 96 well plates Immulon II
  • Antisera are typically diluted in 0.1 M PBS, pH 7.2. The desired dilution(s) of antisera (0.1 mL) are added to each well and the plate incubated 1 hours at 37°C. The plates are then washed 5 times with PBS 0.5% "TWEEN-20 M . A detection antibody is then used to detect the binding of antibodies present in the anti- serum, to the support bound antigen. For example, if rabbit anti-sera is used, horseradish peroxidase (HRP) conjugated goat anti-rabbit antiserum (Cappel) is diluted 1/5,000 in PBS. 0.1 L of this solution is added to each well. The plate is incubated 30 min at 37°C, then washed 5 times with PBS.
  • HRP horseradish peroxidase
  • Cappel horseradish peroxidase conjugated goat anti-rabbit antiserum
  • Sigma ABTS (substrate) is prepared just prior to addition to the plate.
  • the reagent consists of 50 mL 0.05 M citric acid, pH 4.2, 0.078 mL 30% hydrogen peroxide solution and 15 mg ABTS. 0.1 mL of the substrate is added to each well, then incubated for 30 min at room temperature. The reaction is stopped with the addition of 0.050 mL 5% SDS (w/v) . The relative absorbance for each sample well is determined at 410 nm.
  • RNA pellet was recovered by centrifugation in a microfuge at 12,000 x g for 15 minutes at 5°C. The pellet was washed in 70% ethanol and dried under vacuum.
  • SAM S-adenosyl-methionine
  • the reaction mixture was extracted with an equal volume of phenol/chloroform (1:1) and then with an equal volume of chloroform/isoamyl alcohol (24:1) .
  • the cDNA was precipitated with two volumes of ethanol. To maximize the number of blunt ends for the addition of linkers (Maniatis et al , 1982) the cDNA was then treated with the Klenow fragment of DNA polymerase I. The pelleted cDNA was resuspended in 11.5 ⁇ l of distilled water.
  • the reaction mixture was incubated at room temperature for 30 minutes.
  • the reaction mixture was extracted with phenol/chloroform and chloroform isoamyl alcohol as described above, and then precipitated with two volumes of ethanol.
  • the linkers used in the construction of the cDNAs contained an EcoRI site which allowed for direct insertion of the amplified cDNAs into lambda gtlO and gtll vectors (Promega, Madison WI) .
  • Lambda vectors were purchased from the manufacturer (Promega) which were already digested with EcoRI and treated with bacterial alkaline phosphatase, to remove the 5' phosphate and prevent self-ligation of the vector.
  • the _E.coi?J-linkered cDNA preparations were ligated into both lambda gtlO and gtll (Promega) .
  • the ligation reaction tubes were placed at 14°C overnight (12-18 hours).
  • the ligated cDNA was packaged the following morning by standard procedures using a lambda DNA packaging system (GIGAPAK, Stratagene, LaJolla, CA) , and then plated at various dilutions to determine the titer and recombinant frequency of the libraries.
  • a standard X-gal blue/white assay was used to screen the lambda gtll libraries (Miller; Maniatis et al . ) .
  • E. coli HG415 (from Howard Gersenfeld, Dept.of Pathology, Stanford School of Medicine) plating bacteria, which allows only plaque formation by recombinant clones, was used for plating the lambda gtlO libraries.
  • the standard strain, E. coli C600Hfl may be used as an alternative to E . coli HG415.
  • the five lambda gtll libraries generated in Example 1 were screened for specific HCV encoded viral antigens by immunoscreening.
  • the phage were plated for plaque formation using the Escherichia coli bacterial plating strain E. coli KM392 (Kevin Moore, DNax, Palo Alto, CA) .
  • E. coli Y1088 may be used.
  • the fusion proteins expressed by the lambda gtll clones were screened with serum antibodies (Young et al . ) from the following sources: chimpanzee #771 and various human PT-NANBH sera (including EGM, BV, WEH and AG) .
  • Human peripheral blood lymphocyte (PBL) DNA was restriction digested with Hindlll and EcoRI, loaded on a 0.7% agarose gel (as above, except 10 ⁇ g of DNA was loaded per lane) and the fragments separated electrophoretically.
  • the DNA fragments in the agarose gels were transferred to nitrocellulose filters (Southern) and the genomic DNA probed with the nick-translated lambda gtll inserts which were prepared above.
  • the filters were washed (Southern; Maniatis et al . ) and exposed to X-ray film. Forty-three of the 93 lambda clone inserts displayed a positive hybridization reaction with the human PBL DNA.
  • Clone 40 was clearly exogenous, i.e., not derived from normal human DNA, as evidenced by repeated hybridization tests against normal human PBL DNA, and a second clone, designated clone 36, was not only exogenous but also reactive with multiple PT- NANBH antisera.
  • Commercially available sequencing primers New England Biolabs
  • homologous to flanking lambda sequences at the 5' and 3' ends of the inserts were initially used for sequencing.
  • sequencing progressed primers were constructed to correspond to newly discovered sequences.
  • Synthetic oligonucleotide primers were prepared using commercially available automated oligonucleotide synthesizers. Alternatively, custom designed synthetic oligonucleotides may be purchased, for example, from Synthetic Genetics (San Diego, CA) .
  • DNA sequences were determined for the complete insert of clones 36 and 40 (SEQ ID NO:5 and SEQ ID N0:1, respectively).
  • the clone 36 sequence corresponds to nucleotides 5010 to 6515 of the HCV genome (Houghton, et al . , (A), EP Pub. No. 0318216; Houghton, et al . , (B) , EP Pub. No. 0388232) .
  • the clone 40 sequence corresponds to nucleotides 6516 to 7070 of the HCV genome.
  • the screening sera were as follows: GLI-1 sera was a human chronic PT-NANBH sera; BV, community acquired NANBH; SKF, PT-NANBH negative; and FEC, PT-NANBH positive.
  • the numbered sera samples correspond to human clinical serum samples which were PT-NANBH positive: these samples were obtained from Dr. Francoise Fabiani-Lunel, Hospital La Pitie Salpetriere, Paris, France.
  • the antigens produced by clones 36 and 40 yield HCV-specific immunopositive signals.
  • Clone 36 was also used for sera immunoscreening which employed an ELISA format instead of plaque screening.
  • the following Table presents a comparison of the abilities of 5 different antigens to identify HCV positive sera among a number of HCV-positive and HCV-negative sera samples.
  • HCV antigens correspond to portions of the following HCV proteins: 409-1-1, a portion of the NS3 protein; 33CU, a portion of the NS3 protein; NC450, a portion of the capsid protein; and 5-1-1, a portion of the NS4 protein.
  • the sera samples in the above table were as follows: samples 1-27 (human) and 28-30 (animal) , correspond to sera where the HCV-status of each serum was not previously characterized (NC) ; samples 31-35 and 39-40 were sera previously characterized as HCV-positive; and samples 36-38 were HCV-negative control sera. Empty cells in Table 3 indicate a negative ELISA result. Cells containing a "+" indicate a positive ELISA result.
  • EXAMPLE 6 Isolation of Clone 36 Fusion Protein Sepharose 4B beads conjugated with anti-beta galactosidase were purchased from Promega. The beads are packed in a 2 ml column and washed successively with phosphate-buffered saline with 0.02% sodium azide and 10 ml TX buffer (10 mM Tris buffer, pH 7.4, 1% aprotinin) .
  • BNN103 lysogens infected with gtll/clone 36 are used to inoculate 500 ml of NZYDT broth.
  • the culture is incubated at 32°C with aeration to an O.D. of about .2 to .4, then brought to 43°C quickly in a 43°C water bath for 15 minutes to induce gtll peptide synthesis, and incubated further at 37°C for 1 hour.
  • the cells are pelleted by centrifugation, suspended in 10 ml of lysis buffer (10 mM Tris, pH 7.4 containing 2% "TRITON X-100" and 1% aprotinin added just before use.
  • the resuspended cells are frozen in liquid nitrogen, then thawed, resulting in substantially complete cell lysis.
  • the lysate is treated with DNasel to digest bacterial and phage DNA, as evidenced by a gradual loss of viscosity in the lysate.
  • Non-solubilized material is removed by centrifugation.
  • the clarified lysate material is loaded on the Sepharose column, the ends of the column are closed, and the column is placed on a rotary shaker for 2 hrs. at room temperature and 16 hours at 4°C. After the column settles, it is washed with 10 ml of TX buffer.
  • the fused protein is eluted with 0.1 M carbonate/bicarbonate buffer, pHlO.
  • the eluate from the affinity column can be concentrated by filtration, for example, using "CENTRICON-30" cartridges (Amicon, Danvers, Mass.).
  • the final protein concentrate is resuspended in PBS buffer. Protein purity can be analyzed by SDS-PAGE.
  • the clone 36 digest fragments from lambda gtll were released by EcoRI digestion of the phage and the insert purified by gel electrophoresis. The purified fragment is introduced into the pGEX expression vector (Smith) . Expression of gluta- thione S-transferase fused protein (Sj26 fused protein) containing the clone 36 polypeptide antigen can be achieved in E. coli strain KM392 (above) . The fusion protein is isolated from lysed bacteria, and isolated by affinity chro ⁇ matography on a column packed with glutathione- conjugated beads, according to published methods (Smith) .
  • the purified Sj26/clone 36 fused protein is injected subcutaneously in Freund's adjuvant in a rabbit. Typically, approximately 1 mg of fused protein is injected at days 0 and 21, and rabbit serum collected on days 42 and 56.
  • a control rabbit is typically immunized with purified Sj26 protein obtained from control bacterial lysate. Minilysates from the bacterial cultures are prepared are screened for the present of clone 36 and Sj26 antigens using the sera from the immunized rabbits.
  • the clone 36-insert was cloned into a pET3d vector (Novagen, Madison, WI) , essentially according to conventional methods.
  • the clone 36 insert was modified so that it lacked internal Ncol and BamHI sites. This modification was achieved as follows.
  • Three polymerase chain reaction primer pairs were selected from the sequence of the clone 36 insert that defined three regions of the insert ( Figure 1) : 36R, SEQ ID NO:11; 36F, SEQ ID NO:12; 36 Nco Block F, SEQ ID NO:13; 36 Nco Block R, SEQ ID NO:14; 36 Bam Block R, SEQ ID NO:15; and 36 Bam Block F, SEQ ID NO:16.
  • the primers were designed to eliminate internal Ncol and BamHI sites by modifying third position nucleotides in the nucleic acid sequence, where such modification did not affect the protein coding sequence.
  • the primers were used in three amplification reactions (Mullis; Mullis, et al . ; supplies from Perkin Elmer/Cetus) .
  • the reactions were overlap polymerase chain reactions designed to regenerate the full length clone 36 insert without the internal WcoJ and BamHI sites.
  • the amplification reactions were run for 20 cycles each (1 minute denaturation, 1 minute annealing at 55°C, and 2 minutes of extension) .
  • the amplification products were examined on agarose gels. If insufficient amplification had occurred the amplification reactions were continued for 10 more cycles.
  • the original template DNA (used in reaction 1, below) was typically the original clone 36 gtll phage.
  • the primers were used in the three amplification reactions shown in Table 4 to generate template DNAs.
  • the resulting insert was cloned into the pET vector and designated pET-36.
  • the resulting amplification products were gel purified by standard procedures.
  • the clone 36 subfragments, 36-1 (614 bp) , 36-2 (661 bp) , and 36-3 (497 bp) respectively span the length of the clone 36 coding sequence 5' to 3'.
  • Each fragment contained _E.co.RJ sites at each of its 5' and 3' ends.
  • the amplified subfragments contain
  • the amplified subfragments were cloned into lambda gtll vectors (gtll-36-1, gtll-36-2, and gtll-36-3) for expression of the clone 36 polypeptide fragments encoded by each subfragment
  • amplified DNA regions 36-1, 36-2 and 36-3 were ligated into a lambda gtll vector using standard procedures.
  • KM392 bacterial cells were infected to yield titers of 7.3 x 10 4 pfu/ml for 36-1, 7.3 x 10 4 for 36-2, and 7.3 x 10 4 pfu/ml for 36-3.
  • These phage were plated and nitrocellulose filter lifts prepared (Schleicher and Schuell, (Keene, NH) ) .
  • the filters were immunoscreened (Ausubel, et al.) for reactivity with the following human anti-sera DPII-C1, DPII-clO, FEC, SKF and AG described above.
  • DPII stands for the panel II received from Sanofi Diagnostics Pasteur.
  • C2 and CIO are well characterized HCV samples (i.e., the sera tested positive by immunoscreening for the following HCV antigens: C100, 5-1-1, 409- 1-1, and clone 36) .
  • Sera were pre-adsorbed, prior to testing, with semi-confluent gtll phage lysates.
  • Three positive plaques selected from each plate were amplified in bacterial culture. DNA isolated from the each selected plaque was amplified using the overlapping fragment primer pairs described in Example 8 (36-1F/36-1R, 36- 2F/36-2R, and 36-3F/36-3R) . Insert sizes were confirmed by size fractionation on agarose gels.
  • EXAMPLE 10 Production of Monoclonal Antibodies A. Protein Isolation
  • a single bacterial colony containing pET-36 was inoculated into 500 ml LB + 100 ug/ml ampicillin and grown at 37°C, with vigorous aeration, until an O.D. of 0.7 at A600 was achieved. Production of the HCV antigen was induced by addition of 1.0 mM IPTG to the culture. After induction the culture was grown for approximately three hours.
  • the cells were pelleted by centrifugation at 4°C and held overnight at -21°C.
  • the frozen cells were resuspend cells in 25 ml PBS, 1 mM PMSF, 5 mM DTT, at room temperature. To this suspension,
  • the pellet was once again resuspended in 25 ml PBS, 1 mM PMSF, 1 mM DTT, and the suspension pelleted. The final supernatant was discarded and the pellet resuspended in 30 ml of 4M Urea, 20 Mm NaOAc, 5 mM DTT at pH 4.5 (Buffer A). This solution was spun-down in a Beckman JA20 rotor for 10 minutes at 10K rpm, 4°C. The resulting supernatant was filtered through a 0.45 ⁇ m "MILLEX" Filter (Millipore) .
  • Murine monoclonal antibodies were produced according to standard procedures (Mishell; Harlow, et al . ) . Mice were immunized .intraperitoneally two times, with a 21 day interval, using 200 ⁇ g pET clone 36 expression peptide (pET 36) . Blood was collected from the mice 1 week after the second injection. Sera prepared from the blood samples were tested at dilutions of 1/100, 1/1000, 1/2000 and 1/5000 in an ELISA assay for reactivity with purified pET-36 antigen.
  • the ELISA for detection of antibodies was performed essentially as follows. Polystyrene 96- 2311 plates (Falcon 3072, Becton Dickinson, Oxnard, CA) were coated with pET36 in 0.1 M bicarbonate, pH 9.0 (2 mg GSA/100 mL buffer) by adding 0.1 mL of this solution per well. Plates were incubated for 2 hrs, at 37°C, then washed 3 with PBS/0.05% "TWEEN-20". Plates were blocked by the addition of PBS/BSA (1%) for 1 hour at 37°C. Plates were washed 3x with PBS/0.05% "TWEEN-20", sealed and stored at 4°C. Antisera were diluted in 1% BSA in PBS.
  • the following dilutions of antisera were tested: 1/100, 1/1000, 1/2000 and 1/5000.
  • the desired dilution(s) of antisera (0.1 ml) were added to each well and the plate incubated 2 hours, at 37°C. The solution was then removed from the plate, and the wells washed 3 times with PBS/0.05% "TWEEN-20".
  • Alkaline phosphatase conjugated goat anti- mouse antiseru (affinity purified — Zymed, Berkeley, CA) was diluted 1/3000 in PBS/1% BSA. 0.1 mL of this solution was added to each well. This solution was left on the plate for 1 hour at room temperature. The plate was then washed twice with PBS/Tween-20 and three times with PBS, as above.
  • Color reagent ⁇ substrate was either BCIP (5- bromo-4-chloro-3-indolyl phosphate P-toluidine salt, 50 mg/ml in 100% dimethyl formamide) or NBT (Nitro blue tetrazoliu Grad. Ill, 50 mg/ml in 100% dimethyl formamide) , both reagents are available from Sigma ⁇ was prepared just prior to addition to the plate. 0.1 mL of the color reagent was added to each well, then incubated for 30 min at room temperature prior to determination of the absorbance, at 405 n , relative to the blank well, containing no mouse sera. Spleens were removed from mice exhibiting seroreactivity of at least 0.5 O.D.
  • Hybridoma cells were formed by fusion of splenic B-lymphocytes from seropositive mice and stable myeloma fusion partner cells, SP2/0 cells (SP2/0-Agl4; ATCC CRL 1581, American Type Culture Collection, Rockville, MD) , mixed at a ratio of about 1:1 (splenic cells:myeloma cells). Fusion of the cells was promoted by addition to the cells of polyethylene glycol (PEG-1500; Sigma Chemical Co., St. Louis, MO; final concentration: 50%).
  • PEG-1500 polyethylene glycol
  • Hybridomas were selected by growth in selective (HAT) medium, then were tested for production of anti-pET-36 antibodies in a solid phase assay (ELISA) as described above. Hybridomas found to produce antibodies to pET 36 were further clone purified by limiting dilution.
  • Example 8 The following clones were plated as described in Example 8: subfragment clones gtll-36-1, gtll- 36-2, and gtll-36-3; and lambda gtll. Each plate of phage-infected bacteria was overlaid with a nitrocellulose sheet. The expression products from the plaques transferred to filter paper. The plate and filter were indexed for matching corresponding plate and filter positions. The filter was removed after 6-12 hours, washed three times in TBS buffer (10 mM Tris, pH 8.0, 150 mM NaCl) , blocked with AIB (TBS buffer with 1% gelatin) , washed again in TBS, and incubated overnight with hybridoma supernatants from the 49 positive clones identified in Example 9. Each filter was washed twice in TBS, then incubated with alkaline-phosphatase-conjugated anti-human IgG to attach the labeled antibody at filter sites containing antigen recognized by the antiserum.
  • the filter was developed in a substrate medium containing 33 ⁇ l NBT (50 mg/ml stock solution maintained at 4°C) mixed with 16 ⁇ l BCIP (50 mg/ml stock solution maintained at 4°C) in 5 ml of alkaline phosphatase buffer (100 mM Tris, 9.5, 100 mM NaCl, 5 mM MgC12) . Reacted substrate precipitated at points of antigen production, as recognized by the antiserum.
  • NBT 50 mg/ml stock solution maintained at 4°C
  • BCIP 50 mg/ml stock solution maintained at 4°C
  • alkaline phosphatase buffer 100 mM Tris, 9.5, 100 mM NaCl, 5 mM MgC12
  • Results from immunoscreening experiments using selected hybridoma supernatants are shown in Tables 6 and 7.
  • the hybridoma supernatants used in Table 6 were strongly positive in the original screening against the entire clone 36 coding region (Example 10) .
  • the hybridoma supernatants used in Table 7 were less strongly positive than those used in Table 6.
  • the first column of each table presents the clone number used to produce the hybridoma supernatant. The presence of plaques showing antigen-positive reaction is indicated by "+" in these tables.
  • the results of overall immunoreactivity of each of the clone 36 coded sub-polypeptides, and the negative control (lambda gtll) are presented in the tables.
  • This example describes the use of an HCV non- structural protein antigen to design an antigen- based detection system for HCV.
  • sera containing polyclonal antibodies are initially fractionated by addition of ammonium sulfate.
  • the supernatant is then passed over a gel filtration column (Pierce) and the IgG containing fraction identified by absorbance (A 280 ) • IgG molecules can be isolated by a number of standard procedures (Garvey, et al . ) , including affinity chromatography, or by the use of commercially available kits (Pierce) .
  • microtiter plates (Dynatech) are coated with the antibody as follows (Harlow, et al . ) . Approximately 50 ⁇ l of purified antibody (approximately 20 ⁇ g/ml) , in phosphate buffered saline (PBS) (Maniatis, et al . ) is added to each well of polyvinylchloride plates. The plates are sealed and incubated for either 4 hours at room temperature or overnight at 4°C. Alternatively, polystyrene 96 well plates
  • IMMULON II PPC
  • suitable plastic plates Corning Biotechnology, Corning NY
  • 5 ug/ L (100 ⁇ L per well) antibodies in 0.1 M carb/bicarbonate buffer, pH 9.5, the plates sealed with parafilm and stored at 4°C overnight.
  • the wells can also be coated with an anti-mouse IgG antibody, followed by addition of the mouse monoclonal antibodies. After incubation the wells are washed twice with binding buffer (PBS or carb/bicarbonate buffer) .
  • binding buffer PBS or carb/bicarbonate buffer
  • BSA bovine serum albumin
  • the plates were then incubated for approximately 2 hours at room temperature and the liquid removed.
  • the antibody coated wells were then incubated with the test samples, such as, the clone 36- encoded polypeptide, HCV infected tissue culture media, human sera, or chimpanzee sera, for 1 hr. After incubation, clone 36 polypeptide-HRPO (horse radish peroxidase) conjugate was added to each well. Polypeptide-HRPO conjugates were formed using commercially available activated HRPO (Pierce) .
  • HRPO is coupled to peptides using one of the following techniques: (i) the glutaraldehyde technique that links through epsilon-amino acid groups on lysine residues, or other free amino groups (Pierce) ; or (ii) a two-step procedure using m-maleimidobenzoyl sulfosuccinimide ester (sulfo-MBS) to link through free disulfide linkages (Pierce) .
  • sulfo-MBS m-maleimidobenzoyl sulfosuccinimide ester
  • a cysteine residue or a lysine residue can be added to the N-terminus of the peptide.
  • Combination of the conjugate to the solid phase antibody coat was detected by the addition of the substrate-2,2'-azino-bis(3- ethylbenzthiazoline-6-sulfonic acid (ABTS) (Pierce) .
  • ABTS substrate-2,2'-azino-bis(3- ethylbenzthiazoline-6-sulfonic acid
  • the presence of a HCV-NS5 associated viral antigen was identified by color diminution due to successful inhibition of binding clone 36- HRPO.
  • the above protocol is first tested by addition of increasing quantities of free clone 36 polypeptide.
  • a linear inhibition curve is generally established, for example, over the range of 10 ⁇ g/ml to O.ooi ⁇ g/ml.
  • EXAMPLE 13 Antigen Confirmatory Assay A confirmatory assay was designed and tested to confirm positive antigen reactive plasma.
  • the basic design is a blocking of the clone 36-HRPO inhibition assay (Blocking of Inhibition) by addition of anti-clone 36 positive sera to antigen positive plasma before testing in the antibody coated plates.
  • the reagents for the confirmatory assay were evaluated by titration. HCV antibody positive sera are tested for their ability to block the inhibition reaction.
  • NAME Desjardins, Cathleen M.
  • ORGANISM Hepatitis C Virus
  • MOLECULE TYPE protein
  • ORGANISM Hepatitis HCV Virus
  • ORGANISM Hepatitis C Virus
  • GTA ACC CAC ATC AAC TCC GTG TGG AAA GAC CTT CTG GAA GAC AAT GTA 1296 Val Thr His He Asn Ser Val Trp Lys Asp Leu Leu Glu Asp Asn Val 420 425 430
  • ORGANISM Hepatitis C Virus
  • ORGANISM Hepatitis C Virus
  • MOLECULE TYPE protein
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE protein
  • HYPOTHETICAL NO
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)

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Abstract

L'invention concerne des compositions d'antigènes et d'anticorps ayant une immunoréaction avec des sérums infectés par le virus de l'hépatie C. La composition d'antigènes comprend un peptide correspondant à la protéine prédictive NS5 du VHC. La composition d'anticorps contient des anticorps monoclonaux ayant une immunoréaction avec plusieurs épitopes de cet antigène. Ces antigènes sont utiles pour produire des anticorps qui seront utilisés dans des procédés d'immunodiagnostic d'inhibition des antigènes afin de détecter la présence d'antigènes du VHC dans des échantillons testés.
PCT/US1994/004174 1993-04-22 1994-04-15 Antigenes et anticorps utiles dans l'immunodiagnostic du virus de l'hepatite c WO1994024565A1 (fr)

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GB2294690A (en) * 1994-11-01 1996-05-08 United Biomedical Inc Polypeptides for the diagnosis and therapy of hepatitis C virus(HCV)
FR2738827A1 (fr) * 1995-09-18 1997-03-21 Bio Merieux Detection des enterobacteries

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EP0318216A1 (fr) * 1987-11-18 1989-05-31 Chiron Corporation Diagnostics et vaccins de NANBV
EP0388232A1 (fr) * 1989-03-17 1990-09-19 Chiron Corporation Diagnostics et vaccins de NANBV
WO1991015516A2 (fr) * 1990-04-06 1991-10-17 Genelabs Incorporated Epitopes du virus de l'hepatite c
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EP0318216A1 (fr) * 1987-11-18 1989-05-31 Chiron Corporation Diagnostics et vaccins de NANBV
EP0388232A1 (fr) * 1989-03-17 1990-09-19 Chiron Corporation Diagnostics et vaccins de NANBV
WO1991015516A2 (fr) * 1990-04-06 1991-10-17 Genelabs Incorporated Epitopes du virus de l'hepatite c
WO1993006488A1 (fr) * 1991-09-16 1993-04-01 Genelabs Technologies, Inc. Dosages immunologiques virus de l'hepatite c, a base de peptides

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G.KUO, Q-.L. CHOO ET AL.: "An Assay for Circulating Antibodies to a Major Etiologic Virus of Human Non-A, Non-B Hepatitis", SCIENCE, vol. 244, no. 4902, 21 April 1989 (1989-04-21), LANCASTER,PA, US, pages 362 - 364 *
Q.-L. CHOO ET AL.: "Genetic organization and diversity of the hepatitis C virus", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA, vol. 88, no. 6, 15 March 1991 (1991-03-15), WASHINGTON US, pages 2451 - 2455 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2294690A (en) * 1994-11-01 1996-05-08 United Biomedical Inc Polypeptides for the diagnosis and therapy of hepatitis C virus(HCV)
GB2294690B (en) * 1994-11-01 1998-10-28 United Biomedical Inc Peptides effective for diagnosis and detection of hepatitis C infection
FR2738827A1 (fr) * 1995-09-18 1997-03-21 Bio Merieux Detection des enterobacteries
US5786147A (en) * 1995-09-18 1998-07-28 Bio Merieux Detection of enterobacteria

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