EP0552297A1 - Antikörper gegen hepatitis-c antigene - Google Patents

Antikörper gegen hepatitis-c antigene

Info

Publication number
EP0552297A1
EP0552297A1 EP91920333A EP91920333A EP0552297A1 EP 0552297 A1 EP0552297 A1 EP 0552297A1 EP 91920333 A EP91920333 A EP 91920333A EP 91920333 A EP91920333 A EP 91920333A EP 0552297 A1 EP0552297 A1 EP 0552297A1
Authority
EP
European Patent Office
Prior art keywords
antibody
virus
plasma
hepatitis
hcv
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP91920333A
Other languages
English (en)
French (fr)
Other versions
EP0552297A4 (de
Inventor
Mahlon F. Miller
Richard R. Lesniewski
David A. Peterson
Warren Hugh Bryce
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Abbott Laboratories
Original Assignee
Abbott Laboratories
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Abbott Laboratories filed Critical Abbott Laboratories
Publication of EP0552297A1 publication Critical patent/EP0552297A1/de
Publication of EP0552297A4 publication Critical patent/EP0552297A4/xx
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1081—Togaviridae, e.g. flavivirus, rubella virus, hog cholera virus
    • C07K16/109—Hepatitis C virus; Hepatitis G virus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C12N5/12—Fused cells, e.g. hybridomas
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies

Definitions

  • the present invention relates to antibodies which selectively bind to antigens associated with hepatitis C viral particles isolated from infected patients and to processes for isolating hepatitis C virus from plasma including a procedure by which HCV is partially purified.
  • hepatitis C virus or HCV HCV has historically been referred to as non-A non-B hepatitis or NANBH
  • HCV hepatitis C virus
  • NANBH non-A non-B hepatitis
  • the prevention of the transmission of this form of hepatitis by blood and blood products requires reliable, sensitive and specific diagnostic and prognostic assays which may be used to identify HCV carriers as well as contaminated blood and blood products.
  • the present invention relates to an antibody capable of selectively binding with one or more HCV antigen which are associated with the whole virus particle and which produce an immune response in a patient.
  • HCV antigens associated with the virus particle are isolated from a mammal selected from the group consisting of chimpanzees and humans.
  • Both monoclonal and polyclonal antibodies are provided by this invention. Both of these types of antibodies may be prepared according to processes known in the art for generating hybrido a cells from immuno-challenged mammals. Specifically, these antibodies may be isolated from cell lines selected from the group consisting of the hybridoma cell lines H18 C27 and H18 C68 which were deposited on August 15, 1990 with the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Maryland, 20852 under ATCC Nos. HB 10529 and HB 10530, respectively.
  • ATCC American Type Culture Collection
  • This invention also provides a process for isolating an infectious HCV particle in a plasma sample taken from an infected patient which includes clarifying the plasma sample by centrifugation; filtering the clarified plasma through a series of filters having sequentially smaller pore sizes, wherein the pore size of the filters is 3.0, 1.0, 0.8, 0.6, 0.4, and 0.2 microns, respectively; pelleting the filtered plasma through a 10% sucrose solution; and resuspending the pelleted material.
  • the present process provides resuspended material having infectivity titers up to and about 10 7 CID/ml even though this material contains less than about 1%, and preferably less than 0.1%, of the total pelletable protein of the original plasma sample.
  • the antibodies of the present invention are useful in an immunological assay for detecting an antigen specifically associated with HCV.
  • the present invention provides a method to isolate and visualize the agent(s) known to cause human and chimpanzee HCV.
  • Past efforts to immune aggregate a putative virus, raise hyperimmune sera, or clone this illusive agent have been thwarted by the lack of availability of adequately purified virus from blood plasma. Little was known about the physical properties of this infectious agent except that it is probably a chloroform and B-propiolactone sensitive virus capable of passing through about a 100 n filter. Attempts by others to determine the bouyant density of the virus have yielded ambiguous results and, until now, there has not been a reliable means for purifying and, at the same time, concentrating the virus.
  • the present invention describes a scheme for purifying and enriching whole, infectious blood plasma and using the isolate to generate antibodies.
  • polycarbonate filters are preferred because, unlike nitrocellulose, polyester or other types of filters ; -rf ⁇ ich are made of a relatively thick meshwork of interwoven fibers, polycarbonate filters are thin and contain precisely sized, straight-bore holes. These features limit the loss of particles caused by non-specific absorption or entrapment in the filter matrix associated with nitrocellulose, polyester or other types of filters.
  • Table 1 illustrates a preferred scheme to isolate HCV particles.
  • Whole blood plasma was first clarified by low speed centrifugation and then sequentially pressure filtered through 3.0, 1.0, 0.8, 0.6, 0.4 and 0.2 micron Nuclepore filters. The 0.2 micron filtrate was then layered on top of buffered 10% sucrose and pelleted for 4 hours at 105,000 x g. Following pelleting thru 10% sucrose, fine protein was removed. Pellets were resuspended in phosphate buffered saline to yield a 10OX stock concentrate and then further diluted as desired. The portion of the final concentrate to be used for infectivity studies was diluted in buffer containing 2% BSA as a cryroprotectant. TABLE 1
  • Step #4 0.8 Micron Filter
  • Step #9 Resuspend to 100 x
  • the partially purified plasma passed easily through the filters and at each step of the purification process 100 microliter aliquots of .plasma were prepared for electron microscopic viewing using a method developed by Miller (Miller, M.F., Electron Microscopy in Biology, Vol.2, J.D. Griffith, ed., John Wiley and Sons, Inc., New York, N.Y., 1982, pp. 305-339) which employs the commercially available Beckman Airfuge ultracentrifuge and Em-90 electron microscopy particle counting rotor.
  • the filtration procedure may be used to provide concentrated plasma needed to isolate the infectious agent associated with HCV.
  • One method to isolate HCV immune aggregation electron microscopy, may be performed by mixing appropriate concentrations of the plasma concentrate and a suitable antibody and visualizing resultant immune aggregates using a transmission electron microscope.
  • the antibody is generally pre-centrifuged at about 40,000 RPM for 10 minutes to remove undesirable background particulates found in most sera.
  • the preferred aggregation procedure to study a variety of hepatitis viruses includes: a) mixing 20 microliters of well dispersed plasma concentrate with 50 microliters of PBS, pH 7.2, and 20 microliters of antibody, b) incubating the mixture for one hour at room temperature and then overnight at 4°C, c) pelleting the immune aggregates for 30 minutes at 20 psi in a Beckman Airfuge Ultracentrifuge, d) discarding the supernatant and resupsending the pellet in 20 microliters of water, e) applying 5 microliter droplets of suspension to Formvar coated EM specimen grids, f) blotting away the droplet with absorbent paper, and g) negatively staining adherent particulates with 2% phosphotungstic acid. Specimen grids are then carefully surveyed for immune aggregates with a transmission electron microscope at magnifications ranging from 10 to 45,000 x.
  • the monoclonal antibody or other monoclonal antibodies derived using the described isolated HCV preparations may be used in diagnostic tests for antigens of HCV separately, in combination with each other or in combination with human, chimpanzee, rabbit or other mammalian polyclonal antibodies.
  • the antibody may be bound to a suitable solid support, contacted with a sample or aliquot containing HCV, and then contacted with a second labeled antibody which may be developed to provide a detectable signal.
  • a second labeled antibody which may be developed to provide a detectable signal.
  • An assay which uses antibody of the present invention which has been labeled with a signal generating moiety is an alternative procedure.
  • the labeled antibody may be used in a competitive antibody assay. In this type of assay, the labeled antibody is incubated with the sample or aliquot in the presence of a solid support which is coated with an antigen containing the immunodominant epitope of the antigen contained in the sample.
  • Antibodies to HCV present in the sample will compete for binding with the labeled antibody and with the bound antibody. Therefore, a sample containing an antibody to HCV will produce a detectably lower signal compared to the signal produced in an assay which has no added labeled antibody.
  • the purified virus prepared by the process of this invention may also be used to characterize unknown HCV antigens.
  • the purified virus used to generate an antibody may be labeled with radioactive iodine using both Chloramine T and Bolton Hunter reaction conditions according to established procedures before and after solubilization of the virus with detergents.
  • the antibodies may be used in an immunoprecipitation experiment where the antibody is allowed to react in solution with the solubilized labeled virus. After this incubation period, several micrograms of fixed Staphalococcus aureus (Staph A) cells are incubated with the antigen-antibody mixture. Immunoglobulin receptors on these cells will bind the antigen-antibody complexes as well as free immunoglubulins.
  • the bound cells are pelleted by centrifugation and washed repeatedly to remove unadsorbed labeled antigen.
  • the Staph A cells with the adsorbed complexes are then dissociated by heating to 100°C in a solution containing 2- mercaptoethanol, detergents and buffer and then electrophoresed on polyacrylamide slab gels. After electrophoresis the gels are fixed, treated with chemicals known to enhance detection of radioactivity, dried and exposed to x-ray film at -70°C for an extended period of days, weeks or even months.
  • the x-ray film bands will appear on the film at positions where the radiolabeied antigens migrated in the gel based on their molecular size. By comparing the positions of these bands to those of radioactive standards of known molecular weight, the molecular size of the precipitated viral antigen can be determined. The information may be valuable in the determination of the size of the actual gene products of HCV which are unknown at this time.
  • Variations of this technique may also be utilized to identify labeled viral antigens produced in cell lines capable of supporting viral replication. In essence, this technique can be used to screen various cell lines for the presence of viral replication and therefore is useful as a tool for establishing a tissue culture system for hepatitis C diagnosis.
  • monoclonal antibodies according to the invention will be useful in assays for detecting HCV infection and infectious agents in bodily fluids, tissues, experimental reagents and fluids.
  • the monoclonal antibodies of the invention may also be used to isolate antigens specifically associated with HCV by affinity chromatography. Such immunopurified antigen may be used to immunize non-primate species such as goats and rabbits, in order to obtain antibodies having* multiple epitope specificities. Alternatively, the isolated antigen may be used to immunize mice or rats allowing for preparation of hybridoma cell lines capable of producing additional monoclonal antibodies.
  • Antibodies generated according to the present inventions will allow the characterization of antigens specifically associated with HCV infection and infectious agents.
  • standard methods such as analytical and preparative gel electrophoresis may be employed to determine the molecular weight of antigens reactive with the antibodies of this invention.
  • Reactive isolates so obtained may be subjected to further characterization through use of various selective agents including reducing agents, proteases, upases, glycosidases, and the like.
  • Antibodies which react with ' HCV viral antigens may be utilized in a variety of immunoassays including competitive and non-competitive immunoassays, radioimmunoassays, ELISA, EIA and the like.
  • Example 1 describes a process to purify HCV from an infected chimpanzee
  • Example 2 describes the production of a monoclonal antibody
  • Example 3 describes a diagnostic test.
  • Chimpanzee blood plasma having an infectivity titer of 10 5 CID/ml (chimp infectious doses/ illiliter) of HCV was purified as follows.
  • Raw plasma was first clarified by low speed centrifugation for 20 minutes at lOOOXg in a tabletop centrifuge to remove large cellular debris and then sequentially pressure filtered through polycarbonate membrane filters having pore sizes of 3.0, 1.0, 0.8, 0.6, 0.4 and 0.2 micron diameter. Particulates remaining in the 0.2 um filtrate were then pelleted by ultracentrifugation through phosphate buffered 10% sucrose for 4 hours at 105,000 X g. This step removed fine proteins and served to concentrate the virus.
  • Pelleted virus was resuspended in phosphate buffered saline to yield a final 100X concentrate.
  • the portion of the concentrate to be used for infectivity studies was diluted with buffer containing 0.2% Bovine Serum Albumin as a cryoprotectant.
  • Electron micrographs showing sedimented and thin-sectioned particulates derived from the purified chimpanzee plasma were compared to the similarly prepared raw starting plasma. Using a sensitive spectrophotometric assay it was found that total pelletable protein was decreased more than 250 fold. When purified virus product was inoculated into chimpanzees it was found to be infectious (100% of titer recovered as evidenced by the characteristic elevation of serum enzymes and appearance of hepatocyte ultrastructural alterations). The purified product was used to generate monoclonal antibodies.
  • mice Six female Balb c/J mice were selected on the basis of low reactivity of their sera by immunofluorescent microscopy on liver sections from hepatitis C infected chimpanzees. These selected mice were used for inoculation with purified HCV. Pre-inoculation bleeds were collected from all mice prior to inoculation with virus. Purified virus obtained by the procedure described in Example 1 was used as the inoculum.
  • IP intraperitoneally
  • mice Two mice each were inoculated according to the following regimen: intraperitoneally (IP) with 0.1 ml of the purified virus stock, 0.2 ml of a 1/10 dilution of the virus stock in normal saline, or 0.2 ml of a 1/100 dilution of the virus stock in normal saline.
  • Blood samples were collected by eye bleeds at day 21.
  • all mice were boosted (IP) with the same inocula and dose as their primary immunization regimen.
  • Blood samples were again collected by eye bleeds on day 49.
  • IV intravenously
  • the IV boosted mice were sacrificed and their spleen lymphocyte cells were isolated and fused to SP 2/0 mouse myeloma cells following the procedure of Kohler and Milstein (Kohler G., and Milstein C, Nature, 256:495-497, 1975).
  • the remaining 3 mice were boosted IV as previously described, and sacrificed three days later and their spleen lymphocytes were isolated and fused to SP 2/0 cells.
  • the monoclonal antibody prepared according to the procedure of example 2 is used as an antigen capture reagent bound to a solid phase polystyrene bead support.
  • An aliquot of bodily fluid such as serum, plasma or cerebral spinal fluid from a patient is incubated with the antibody bound to the bead.
  • an antigen or whole virus binds to the solid phase.
  • unbound antigens are washed away from the solid support.
  • the same antibody, or another monoclonal antibody binding to a different epitope of an HCV antigen, or a polyclonal antibody, coupled to horseradish peroxidase is incubated with the bead.
  • This labeled antibody will bind to antigen trapped on the solid phase forming an antibody-antigen-antibody sandwich. Excess peroxidase labeled antibody is washed away and the bead is exposed to a suitable enzyme substrate such as OPD (orthophenylene diamine) . The resulting colored product is detected spectrophotometrically in amounts directly proportional to the amount of antigen which was present in the original aliquot.
  • OPD orthophenylene diamine
EP91920333A 1990-10-12 1991-10-09 Antikörper gegen hepatitis-c antigene Withdrawn EP0552297A1 (de)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US59681190A 1990-10-12 1990-10-12
US596811 1990-10-12

Publications (2)

Publication Number Publication Date
EP0552297A1 true EP0552297A1 (de) 1993-07-28
EP0552297A4 EP0552297A4 (de) 1994-02-09

Family

ID=24388816

Family Applications (1)

Application Number Title Priority Date Filing Date
EP91920333A Withdrawn EP0552297A1 (de) 1990-10-12 1991-10-09 Antikörper gegen hepatitis-c antigene

Country Status (7)

Country Link
EP (1) EP0552297A1 (de)
JP (1) JPH06502075A (de)
KR (1) KR930702506A (de)
AU (1) AU663064B2 (de)
CA (1) CA2093919A1 (de)
TW (1) TW216797B (de)
WO (1) WO1992007001A1 (de)

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP3242402B2 (ja) * 1990-11-07 2001-12-25 アボット・ラボラトリーズ Ｃ型肝炎ウイルスに対するモノクローナル抗体およびその使用法
JP3217600B2 (ja) * 1994-07-12 2001-10-09 株式会社先端生命科学研究所 非ａ非ｂ型肝炎ウイルス関連抗原のイムノアッセイ、それに使用するモノクローナル抗体、およびこの抗体を産生するハイブリドーマ
ES2377968T3 (es) 2003-04-01 2012-04-03 Institut National De La Sante Et De La Recherche Medicale (Inserm) Anticuerpos dirigidos contra el complejo E1E2 del virus de la hepatitis C y composiciones farmacéuticas
RS53258B (en) 2007-02-21 2014-08-29 University Of Massachusetts HUMAN ANTIBODIES AGAINST HEPATITIS C (HCV) VIRUS AND USE

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ES2012739T5 (es) * 1987-11-18 2001-12-01 Chiron Corp Diagnosticos para nanbv.
AU632271B2 (en) * 1988-03-30 1992-12-24 Abbott Laboratories Production of monoclonal antibodies specific for non-a, non-b hepatitis infected liver

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
No further relevant documents disclosed *
See also references of WO9207001A1 *

Also Published As

Publication number Publication date
CA2093919A1 (en) 1992-04-13
EP0552297A4 (de) 1994-02-09
TW216797B (de) 1993-12-01
WO1992007001A1 (en) 1992-04-30
AU8945791A (en) 1992-05-20
JPH06502075A (ja) 1994-03-10
KR930702506A (ko) 1993-09-09
AU663064B2 (en) 1995-09-28

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