EP0836708A1 - Nachwisverfahren für antikörper gegen das zweite hüllprotein des hepatitis c virus - Google Patents

Nachwisverfahren für antikörper gegen das zweite hüllprotein des hepatitis c virus

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Publication number
EP0836708A1
EP0836708A1 EP96917969A EP96917969A EP0836708A1 EP 0836708 A1 EP0836708 A1 EP 0836708A1 EP 96917969 A EP96917969 A EP 96917969A EP 96917969 A EP96917969 A EP 96917969A EP 0836708 A1 EP0836708 A1 EP 0836708A1
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EP
European Patent Office
Prior art keywords
hcv
antigen
antibody
dna sequence
specific binding
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Withdrawn
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EP96917969A
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English (en)
French (fr)
Inventor
Gregory F. Okasinski
Verlyn G. Schaefer
Thomas S. Suhar
Richard R. Lesniewski
James W. Scheffel
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Abbott Laboratories
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Abbott Laboratories
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Publication date
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Publication of EP0836708A1 publication Critical patent/EP0836708A1/de
Withdrawn legal-status Critical Current

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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
    • 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

  • the present invention relates generally to a method for detecting antibodies to hepatitis C virus (HCV), and more particularly, relates to a method for detecting antibodies to an additional marker for HCV infection, the second envelope glycoprotein (E2) of HCV.
  • HCV hepatitis C virus
  • E2 the second envelope glycoprotein
  • HCV 1.0 EIAs first generation hepatitis C virus enzyme immunoassays
  • HCV 2.0 EIAs second generation HCV enzyme immunoassays
  • HCV anti-HCV
  • NS3 viral protease
  • NS4 unknown function genes of the virus.
  • Third generation screening assays HCV 3.0 EIAs, which include an additional antigen from the NS5 region (viral polymerase and a second unknown function), are now available and are being used in Europe [Lavanchy etal., J. Clin. Microbiol. 32:2272-75 (1994)].
  • Additional unlicensed immunoblot assays such as an immunodot assay or a strip immunoblot assay (SLA) ' can be used for the detection of antibodies to individual HCV proteins, including core, NS3, NS4 and NS5. These assays are intended for use as an additional, more particular test for those human serum or plasma test samples found repeatedly reactive using a licensed anti-HCV screening assay, e.g., HCV 2.0 EIA. These assays are used as a means to confirm reactivity in a HCV 2.0 screening assay. According to the conventional interpretation of results for these assays, reactivity to at least two HCV proteins corresponding to antigens encoded by different parts of the HCV genome is interpreted as positive. Reactivity to only a single HCV protein is interpreted as mdeterminate.
  • SLA strip immunoblot assay
  • Any donor demonstrating antibodies specific for 2 or more HCV gene products should be regarded as having seroconverted to HCV subsequent to infection with this virus [Alter, 1994; Bresters et al., Transfusion 33:634-38 (1993); Sayers & Gretch, Transfusion 33:809-13 (1993)].
  • these donors should be considered potentially infectious.
  • blood banks have a significant concern with respect to how to handle mdeterminate cases and the added expense of follow- , up testing.
  • the E2 protein has been shown to contain a hypervariable region with mutation characteristics similar to that observed in the hypervariable V3 loop of gpl20 of the human immunodeficiency virus [Kato et al. , Molecular and Biological Medicine 7:495-501 (1990); Houghton et al., Hepatology 14:381-388 (1991); Weiner et al., Proc. Natl. Aca. Sci. USA 89:3468-72 (1992)].
  • envelope proteins El and E2 are likely to be located partially or entirely at the surface of the virion particle. This association with the structural surface of the virus would seem to make these proteins prime targets for humoral immune responses to infection with HCV.
  • HCV E2 hypervariable region of HCV E2 appears to mutate in response to humoral immune selective pressure [Inchauspe et al. , Proc. Natl. Acad. Sci. USA 88:10292-96 (1991); Ogata et al., Proc. Natl. Acad. Sci. USA 88:3392-96 (1991); Lesniewski et al., J. Med. Virol. 40:150-56 (1993)].
  • the present invention provides a heretofore unrealized confirmatory marker for dete ⁇ nimng true HCV infection.
  • the present invention provides an improved method for detecting the presence of antibodies to HCV antigen which may be present in a test sample comprising contacting said sample with HCV antigen and dete ⁇ nining whether antibodies are bound to said HCV antigen, wherein the improvement comprises employing as said HCV antigen at least one recombinant HCV protein comprising a recombinant polypeptide that is the expression product of mammalian cells transformed by a heterologous expression vector comprising a DNA sequence encoding an E2 truncated protein, a DNA sequence encoding a rabbit heavy chain signal sequence and a DNA sequence encoding an amino- terminal sequence of human pro-urokinase, wherein said HCV antigen DNA sequence is located downstream to said other DNA sequences.
  • the present invention further provides a test kit for detecting the presence of antibodies to HCV antigen which may be present in a test sample, comprising a container containing a recombinant polypeptide that is the expression product of mammalian cells transformed by a heterologous expression vector comprising a DNA sequence encoding an E2 truncated protein, a DNA sequence encoding a rabbit heavy chain signal sequence and a DNA sequence encoding an ammo-terminal sequence of human pro-urokinase, wherein said HCV antigen DNA sequence is located downstream to said other DNA sequences.
  • Fig. 1 shows the immunofluorescent staining pattern of HCV E2 antigen in transfected CHO cells using (A) rabbit anti-peptide serum (amino acids 509-551); and (B) HCV positive human serum.
  • Fig. 2 shows the results of a RIP A analysis of HCV E2 antigen produced in CHO cells.
  • 35 S-labeled E2 antigen was i munoprecipitated with: pre-immune rabbit sera (Lane 2), hyperimmune serum from a rabbit immunized with synthetic peptide aa 509-551 (Lane 3), normal human plasma (Lane 4), and three different HCV antibody positive human plasma (Lanes 5-7).
  • Lane 1 contains radioactive molecular weight markers.
  • Fig. 3 is a SDS-PAGE gel of purified HCV E2 antigen produced in CHO cells. Lane 1: Molecular weight markers. Lanes 2 and 3: Purified E2 antigen. DETAILED DESCRIPTION OF THE INVENTION
  • the present invention provides an improved method for detecting the presence of antibodies to HCV antigen which may be present in a test sample comprising contacting said sample with HCV antigen and determining whether antibodies are bound to said HCV antigen, wherein the improvement comprises employing as said HCV antigen at least one recombinant HCV protein comprising a recombinant polypeptide that is the expression product of mammalian cells transformed by a heterologous expression vector comprising a DNA sequence encoding an E2 truncated protein, a DNA sequence encoding a rabbit heavy chain signal sequence and a DNA sequence encoding an amino- terminal sequence of human pro-urokinase, wherein said HCV antigen DNA sequence is located downstream to said other DNA sequences.
  • Recombinant polypeptides produced as described herein in mammalian expression systems provide antigens for diagnostic assays which can be used to determine the viremia of a patient based on a strong correlation between the presence of E2 antigen and patients found to be viremic using reverse transcriptase polymerase chain reaction (RT-PCR) amplification.
  • the antigens also are useful as an early marker of seroconversion, and provide another means for determining true HCV exposure in indeterminate test samples tested by commercially available confirmatory tests.
  • the antigens also provide a means for resolving discrepant results between commercially ⁇ : available second generation and third generation HCV screening assays.
  • the present invention confers several technical advantages over the prior art.
  • the presence of antibodies to HCV E2 in specimens already positive for HCV antibody provides additional, and therefore, more compelling evidence of true HCV infection.
  • the presence of antibodies to HCV E2 along with reactivity in a licensed HCV 2.0 screening assay as well as reactivity to the HCV core or NS3 protein in an immunoblot assay, point to a consistent interpretation that individuals with these serologic profiles have had previous, or have ongoing, HCV infection.
  • the recombinant polypeptides produced can be provided in the form of a kit with one or more containers such as vials or bottles, with each container containing a separate reagent such as a recombinant polypeptide, packaged as test kits for the convenience of performing assays.
  • Other aspects of the present invention include a recombinant polypeptide comprising an HCV E2 epitope attached to a solid phase.
  • the present invention provides assays which utilize the recombinant proteins produced as described herein in various formats, any of which may employ a signal generating compound which generates a measurable signal in the assay. All of the assays described generally detect antibody, and include contacting a test sample with at least one HCV antigen provided herein to form at least one antigen-antibody complex and detecting the presence of the complex so formed. These assays are described in detail herein.
  • test sample refers to any component of an individual's body which can be a source of the antibodies of interest. These components are well known in the art.
  • biological samples which can be tested by the methods of the present invention described herein and include human and animal body fluids such as whole blood, serum, plasma, cerebrospinal fluid, urine, lymph fluids, and various external sections of the respiratory, intestinal and genitourinary tracts, tears, saliva, milk, white blood cells, myelomas and the like, biological fluids such as cell culture supematants, fixed tissue specimens and fixed cell specimens.
  • Solid phases are known to those in the art but not critical and include the walls of wells of a reaction tray, test tubes, polystyrene beads, magnetic or non-magnetic beads, nitrocellulose strips, membranes, microparticles such as latex particles, plastic tubes, glass or silicon chips and sheep red blood cells are all suitable examples and others. Suitable methods for immobilizing peptides on solid phases include ionic, hydrophobic, covalent interactions and the like.
  • a “solid phase”, as used herein, refers to any material which is insoluble, or can be made insoluble by a subsequent reaction. The solid phase can be chosen for its intrinsic ability to attract and immobilize the capture reagent.
  • the solid phase can retain an additional receptor which has the ability to attract and immobilize the capture reagent.
  • the additional receptor can include a charged substance that is oppositely charged with respect to the capture reagent itself or to a charged substance conjugated to the capture reagent.
  • the receptor molecule can be any specific binding member which is attached to the solid phase and which has the ability to immobilize the capture reagent through a specific binding reaction. The receptor molecule enables the indirect binding of the capture reagent to a solid phase material before the performance of the assay or during the performance of the assay.
  • the solid phase also can comprise any suitable porous material with sufficient porosity to allow access by detection antibodies and a suitable surface affinity to bind antigens.
  • Microporous structures are generally preferred, but materials with gel structure in the hydrated state may be used as well.
  • Such useful solid supports include: natural polymeric carbohydrates and their synthetically modified, cross-linked or substituted derivatives, such as agar, agarose, cross ⁇ linked alginic acid, substituted and cross-linked guar gums, cellulose esters, especially with nitric acid and carboxylic acids, mixed cellulose esters, and cellulose ethers; natural polymers containing nitrogen, such as proteins and derivatives, including cross-linked or modified gelatins; natural hydrocarbon polymers, such as latex and rubber; synthetic polymers which may be prepared with suitably porous structures, such as vinyl polymers, including polyethylene, polypropylene, polystyrene, polyvmylchloride, polyvinylacetate and its partially hydrolyzed derivatives, polyacrylamides, polymethacrylates, copolymers and terpolymers of the above polycondensates, such as polyesters, polyamides, and other polymers, such as polyurethanes or polyepoxides; porous inorganic materials such as sulf
  • the porous structure of nitrocellulose has excellent absorption and adsorption qualities for a wide variety of reagents.
  • Nylon also possesses similar characteristics and also is suitable.
  • porous solid supports described herein above are preferably in the form of sheets of thickness from about 0.01 to about 0.5 mm, preferably about 0.1 mm.
  • the pore size may vary within wide limits, and is preferably from about 0.025 to about 15 microns, especially from about 0.15 to about 15 microns.
  • the surfaces of such supports may be activated by chemical processes which cause covalent linkage of the antigen to the support. The irreversible binding of the antigen is obtained, however, in general, by adsorption on the porous material by poorly understood hydrophobic forces.
  • Suitable solid supports also are described in U.S. Patent Application Serial No. 227,272.
  • the "indicator reagent" comprises a "signal generating compound"
  • Specific binding member means a member of a specific binding pair, that is, two different molecules where each of the molecules through chemical or physical means specifically binds to the other molecule.
  • An immunoreactive specific binding member can be an antibody, an antigen, or an antibody /antigen complex that is capable of binding either to HCV as in a sandwich assay, to the capture reagent as in a competitive assay, or to the ancillary specific binding member as in an indirect assay.
  • the indicator reagent in addition to being an antibody member of a specific binding pair for HCV, the indicator reagent also can be a member of other specific binding pairs, including hapten-anti-hapten systems such as biotin or anti-biotin and avidin or biotin, a carbohydrate or a lectin, a complementary nucleotide sequence, an effector or a receptor molecule, an enzyme cofactor or an enzyme, an enzyme inhibitor or an enzyme, and the like.
  • hapten-anti-hapten systems such as biotin or anti-biotin and avidin or biotin, a carbohydrate or a lectin, a complementary nucleotide sequence, an effector or a receptor molecule, an enzyme cofactor or an enzyme, an enzyme inhibitor or an enzyme, and the like.
  • labels include chromogens, catalysts such as enzymes, luminescent compounds such as fluorescein and rhodamine, chemiluminescent compounds such as acridinium, phenanthridinium and dioxetane compounds including those described in co ⁇ pending U.S. Patent Application Serial No. 0_/921,979 corresponding to EP Publication No. 0 273,115, which enjoys common ownership and which is incorporated herein by reference, radioactive elements, and direct visual labels.
  • enzymes include alkaline phosphatase, horseradish peroxidase, B- galactosidase, and the like.
  • the methods of the present invention can be adapted for use in systems which utilize microparticle technology including in automated and semi-automated systems wherein the solid phase comprises a microparticle.
  • these recombinant proteins can be used to develop unique assays as described by the present invention to detect the presence of anti-HCV in test samples.
  • a test sample is contacted with a solid phase to which at least one recombinant HCV protein comprising E2 antigen is attached.
  • the test sample and solid phase are incubated for a time and under conditions sufficient to form antigen-antibody complexes.
  • the antigen-antibody complexes are detected.
  • Indicator reagents may be used to facilitate detection, depending upon the assay system chosen.
  • a test sample is contacted with a solid phase to which at least one recombinant HCV protein comprising E2 antigen produced as described herein is attached and also is contacted with a monoclonal or polyclonal antibody specific for the HCV protein(s), which preferably has been labeled with an indicator reagent.
  • a monoclonal or polyclonal antibody specific for the HCV protein(s) which preferably has been labeled with an indicator reagent.
  • the solid phase is separated from the free phase, and the label is detected in either the solid or free phase as an indication of the presence of anti-HCV.
  • test sample with a solid phase to which at least one recombinant HCV protein comprising E2 antigen produced as described herein is attached, incubating the solid phase and test sample for a time and under conditions sufficient to form antigen-antibody complexes, and then contacting the solid phase with a labeled recombinant antigen to form antigen-antibody-antigen sandwiches.
  • Assays such as this and others are described in U.S. Patent No. 5,254,458 which enjoys common ownership and is incorporated herein by reference.
  • HCV-H strain plasma [Ogata et al., supra, 1991] from a chimpanzee which represents a Type la genotype of HCV [Mishiro & Bradley, in Viral Hepatitis and Liver Disease 283-85 (Nishioka et al. eds., 1994)].
  • the envelope gene cDNA was isolated by RT-PCR amplification.
  • a truncated E2 sequence was obtained using PCR amplification of the region coding for amino acids 388-664 of the large open reading frame of HCV.
  • E2 complementary DNA was inserted into a plasmid vector downstream of both a rabbit heavy chain signal sequence and a human pro-urokinase amino terminal sequence to enhance signal protease processing, efficient secretion and final product stability in cell culture fluids.
  • cDNA E2 complementary DNA
  • the 3.5 kb E2 expression cassette contained a duplex synthetic oligonucleotide that had been digested with Spel and Xbal inserted at the Xbal cloning site of the cassette by sticky end ligation.
  • the synthetic oligonucleotide sequence contained a sequence derived from a rabbit immunoglobulin gamma heavy chain signal peptide and other sequences to create restriction sites for cloning purposes, inserted downstream of a promoter element and transcription start site.
  • the E2 expression cassette also contained the PCR-derived E2 fragment inserted as an Xbal fragment downstream of the rabbit sequence.
  • the sequence encoding the amino terminal sequence of human pro-urokinase ⁇ serine, asparagine, glutamic acid and leucine (SNEL) ⁇ followed.
  • the pro-urokinase sequence promoted signal protease processing, efficient secretion and product stability in culture fluids.
  • CHO cells lacking dhfr (dhfr-) (Dxb- 111) were transfected with the HCV E2 plasmid and stable cell lines were obtained after several rounds of methotrexate selection.
  • Uriacio et al., Proc.. Acad. Sci. 77:4451-4466 (1980). These cells are available from the American Type Culture Collection (A.T.C.C), 12301 Parklawn Drive, Rockville, MD 20852, under Accession number CRL 9096.
  • Ham's F12 custom minus formulation without glycine, hypoxanthine or thymidine supplemented with methotrexate hydrate, 5000 nm, G418 at an actual concentration of 300 ug/ml, dialyzed 10% fetal calf serum and HEPES buffer at a concentration of 8 ml per 500 ml of media (for non-CO2 incubation).
  • Ham's F12 Custom Minus Medium was overlayed onto just confluent monolayers for 12-24 hours at 37°C in 5% CO2.
  • VAS Custom Medium VAS Custom formulation with l-glutamine with HEPES without phenol red, available from JRH Bioscience, product number 52-08678P
  • VAS Custom formulation with l-glutamine with HEPES without phenol red available from JRH Bioscience, product number 52-08678P
  • test sample i.e., spiked standard or cell supernatant
  • E2 peptide-coated bead HCV synthetic peptide comprising amino acids 509-551
  • the amount of E2 antigen produced was measured by adding a gamma globulin specific goat anti- human-HRPO conjugate, incubating, adding OPD substrate and quenching the reaction with IN H 2 SO 4 .
  • the unknown cell culture supernatant was read off the standard curve to determine E2 concentration. It was determined that 6-10 milligrams of CHO-E2 were produced per liter of culture fluid using this method.
  • FIG. 1 shows the immunofluorescent staining pattern of HCV E2 antigen in transfected CHO cells using (A) rabbit anti-peptide serum (amino acids 509-551); and (B) HCV positive human serum. E2 expression also was detected by radioimmunoprecipitation analysis (RLPA) of lysed cell extracts from 35 S-labeled CHO cell cultures.
  • Figure 2 shows the results of a RLPA analysis of HCV E2 antigen produced in CHO cells. Both rabbit and human antisera specific for HCV E2 antigen precipitated a heterogeneous E2 protein.
  • E2 Purification of E2 was achieved by first concentrating the cell supernatants 50 fold followed by ion exchange and lectin chromatography.
  • the ion exchange chromatography consisted of two columns, S-Sepharose and DEAE-Sepharose. The harvests were clarified at 1500 x g for thirty minutes and the supematants were concentrated to 50x with an Amicon stirred-cell concentrator and employing an Amicon YM10 membrane (available from Amicon, Beverly, MA).
  • the 50x concentrate was 0.2 u final filtered and then extensively dialyzed against S-Sepharose running buffer comprising 0.02M sodium phosphate buffer (no salt), pH 6.5, conductivity approximately 2.0 mS.
  • the supernatant was loaded onto an equilibrated 200 ml S- Sepharose column at a flow rate of 5ml per minute.
  • the unbound flow was collected, concentrated to original volume and extensively dialyzed against DEAE-Sepharose running buffer comprising 0.2M Tris buffer, 0.1M NaCl, pH 8.5, conductivity approximately 12 mS.
  • the supernatant was loaded onto a 200 ml DEAE-Sepharose column at a flow rate of 5 ml per minute.
  • the unbound flow was collected, concentrated to original volume and extensively dialyzed against wheat germ agglutinin (WGA)-Sepharose 6MB running buffer comprising 0.01M sodium phosphate buffer, 0.13M NaCl, pH 7.0. After dialysis, the supernatant was loaded onto a 10 ml WGA-Sepharose 6MB column at a flow rate of 0.5 ml per minute. The unbound flow was collected and recirculated. The column flow was reversed and the purified CHO-E2 antigen was eluted using 10 mM N,N'-diacetylchitobiose in WGA- Sepharose running buffer.
  • FIG. 1 shows a SDS-PAGE gel of purified HCV E2 antigen produced in CHO cells.
  • the purified E2 antigen ran as a heterogeneous band of approximately 62-72 kDa on SDS-PAGE.
  • a similar heterogeneous band was confirmed to be E2 by RLPA analysis ( Figure 2).
  • the final purity was estimated to be greater than 90% using scanning densitometry of SDS-PAGE gels stained with Coomassie blue.
  • the CHO cells were grown in protein free media which greatly enhanced the efficiency of purification of this glycoprotein.
  • MATRIX HCV 1.0 and MATRIX HCV 2.0 were used to establish the specific antibody reactivity patterns.
  • RLBA HCV 2.0 and MATRIX HCV 1.0 assays were performed according to the manufacturer's instructions.
  • MATRIX HCV 2.0 is a second generation MATRIX immunoblot assay which contains an NS5 antigen in addition to core, NS3 and NS4. The assay procedure is the same as that for MATRIX HCV 1.0, as previously described [Vallari et al., Clin. Microbiol. 30: 552-56 (1992)]. Specimens Chronic and acute NANBH specimens were obtained from multiple U.S. sites.
  • a commercially available anti-HCV panel comprised of varying titer of anti-HCV (PHV203), well characterized with regard to HCV serological markers, was obtained from Boston Biomedica, Incorporated (BBI), West Bridgewater, Massachusetts.
  • HCV 2.0 reactive plasma specimens were obtained from North American Biologicals Incorporated (NABI) of which only samples concordantly reactive in both HCV 2.0 EIA's (Abbott and Ortho) were analyzed further.
  • Purified HCV E2 antigen prepared as described above, was coated onto polystyrene beads at a concentration of 1.0-2.0 ug/ml in a 0.1M borate, 0.15M NaCl buffer, pH 9.0.
  • the antigen coating buffer composition was adjusted with respect to pH and ionic strength to provide optimum sensitivity and specificity in the assay.
  • the assay procedure used was as follows. Specimens were diluted with specimen diluent and mixed. All specimens were tested at a 1:41 dilution in the assay.
  • the specimen diluent comprised a phosphate and TRIS- EDTA buffered saline solution containing bovine serum albumin, fetal bovine serum and goat serum with 0.002% Triton X-100 ® detergent.
  • the HCV E2 antigen-coated beads were added, one to each well of the tray, and incubated at 37° C for approximately 60 minutes in the rotation mode. The unbound materials were removed by washing the beads with water.
  • Anti-E2 remaining bound to the bead was detected by adding to each well containing a bead 200 ul of conjugate comprising goat anti-human IgG labeled with horseradish peroxidase as the label [Dawson et al., J. Clin. Microbiol. 29:1479-86 (1991)].
  • the beads were incubated at 37° C for approximately 30 minutes in the rotation mode.
  • the unbound materials were removed by washing the beads with water.
  • Color development was obtained by adding o-Phenylenediamine (OPD) solution containing hydrogen peroxide to the beads, and, after incubation for approximately 30 minutes, a yellow-orange color developed in proportion to the amount of anti-E2 which was bound to the bead.
  • OPD o-Phenylenediamine
  • the enzyme reaction was stopped by adding 1 ml of IN H 2 SO 4 .
  • the intensity of the color was measured using a spectrophotometer at a wavelength of 492 nm.
  • Example 1 The specificity of the anti-E2 EIA described in Example 1 was established by testing specimens from several populations of volunteer blood donors totaling 750 serum and plasma specimens. A cutoff value for the E2 antibody assay was established at a signal to negative control absorbance (S/N) ratio of 4.0. This cutoff represented a minimum of six (range of from six to ten) standard deviations from the mean of the absorbance distribution for any of these populations.
  • S/N signal to negative control absorbance
  • NS No seroconversion detected
  • Table I show that antibodies to E2 as detected by the anti-E2 EIA are a good index of HCV exposure by demonstrating that all five individuals who seroconverted to other HCV proteins also seroconverted to E2.
  • E2 antibodies appeared as the first serological marker of HCV infection in three of the five cases indicating that E2 antibodies are produced early after HCV infection in some individuals.
  • Example 5 RNA Positive Specimens
  • RNA positive plasma specimens identified in the section "Specimens" above, were tested using the anti-E2 EIA of Example 1 as well using the MATRIX HCV 2.0 assay to test for other individual HCV antibodies.
  • Table ⁇ 571 (97.3%) of these RNA positive specimens were shown to contain antibodies to E2 (Table LL), including 56 (94.9%) of the 59 specimens collected in Japan. All E2 positive samples contained other HCV antibodies as detected by MATRIX but no single antibody occurred with greater frequency than E2 antibody in this population.
  • E2 antibodies were found in 97.3% of these patients demonstrating that the presence of E2 antibody and HCV RNA, as detected by PCR, are very closely and positively correlated. Since 94.9% of 59 HCV patients from Japan (where Genotype lb predominates) were reactive for E2 antibodies, it appears that there must be conserved E2 epitopes among Type la and Type lb viruses. The close correlation between HCV RNA and E2 antibody suggests that the presence of E2 antibody alone is not sufficient for virus clearance and calls into question the role these antibodies may play in virus neutralization.
  • Example 6 HCV Populations Several HCV populations, identified in the section "Specimens" above, were tested in the anti-E2 EIA of Example 1.
  • Example 7 Samples with Discordant Results between HCV 2.0 and HCV 3.0 Assays
  • BBI Panel PHV203 specimen members were evaluated with the Abbott HCV 3.0 EIA and anti-E2 EIA of Example 1. Testing results from the commercially available assays Abbott HCV 2.0 EIA, Ortho HCV 2.0 EIA, Ortho HCV 3.0 EIA, MATRLX HCV 1.0 (Abbott) and RLBA HCV 2.0 (Chiron) were provided by BBI. Two panel members reported by BBI as HCV negative were also E2 antibody negative.
  • PHV203-01 1.4 1.9 1.6 0.6 Core, NS4 Core, NS3, 2.4 NS4
  • E.coli clOO antigens must occur in order to be considered reactive to NS4 on MATRIX 2.0 NR:
  • specimen numbers 5 and 8 which represent sequential donations from an individual donor are core and E2 antibody positive demonstrating the reproducibility and specificity of these assays.
  • Four specimens which reacted to core antigen on both RLBA 2.0 and MATRLX 2.0 also contained antibody to E2. These four specimens were reactive in the Abbott HCV 3.0 assay (S/CO values 1.99-3.01) but were negative in the Ortho HCV 3.0 assay (S/CO values 0.47-0.79).

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EP96917969A 1995-06-07 1996-06-04 Nachwisverfahren für antikörper gegen das zweite hüllprotein des hepatitis c virus Withdrawn EP0836708A1 (de)

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US481018 1990-02-16
US48101895A 1995-06-07 1995-06-07
PCT/US1996/008536 WO1996041196A1 (en) 1995-06-07 1996-06-04 Method for detection of antibody to hepatitis c virus second envelope glycoprotein

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CA2223277A1 (en) 1996-12-19
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