EP0736087A1 - Monoclonal antibodies against hev orf-2 and methods for using same - Google Patents

Monoclonal antibodies against hev orf-2 and methods for using same

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Publication number
EP0736087A1
EP0736087A1 EP95905967A EP95905967A EP0736087A1 EP 0736087 A1 EP0736087 A1 EP 0736087A1 EP 95905967 A EP95905967 A EP 95905967A EP 95905967 A EP95905967 A EP 95905967A EP 0736087 A1 EP0736087 A1 EP 0736087A1
Authority
EP
European Patent Office
Prior art keywords
hev
monoclonal antibody
antibody
orf
antigen
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Withdrawn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
EP95905967A
Other languages
German (de)
English (en)
French (fr)
Inventor
Deborah A. Paul
Mark F. Knigge
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 EP0736087A1 publication Critical patent/EP0736087A1/en
Withdrawn legal-status Critical Current

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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
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • This invention relates generally to antibodies which specifically bind to
  • HEV Hepatitis E Virus
  • HEV orf-2 antigen a novel hybridoma cell line which secretes monoclonal antibodies to HEV orf-2 antigen, and methods for using these monoclonal antibodies.
  • HEV variously referred to as waterborne, epidemic or enterically l o transmitted non-A, non-B hepatitis (ET-NANBH)
  • ET-NANBH enterically l o transmitted non-A, non-B hepatitis
  • HEV genome has been cloned and expressed in E. coli as fusion proteins with glutathione-S-transferase (GST). See, for example, S. 5 J. Skidmore et al., supra.
  • GST glutathione-S-transferase
  • B Burmese
  • M Mexican strain
  • the two antigens from the Mexican strain correspond to l o amino acid sequences at the carboxy-terminus of the second open reading frame (ORF-2) and the third open reading frame (orf-2), respectively.
  • the two antigens from the Burmese strain, B 3-2 and B 4-2, correspond to amino acid sequences at the carboxy-terminus of orf-2 and orf-2, respectively.
  • the M 3-2 and B 3-2 recombinant antigens both comprise 42 amino acids from the carboxy terminus of
  • the degree of amino acid homology between these sequences of 42 amino acids is 90.5%.
  • the M 4-2 and B 4-2 recombinant antigens each comprise 33 amino acids from the carboxy terminus of orf-2; the degree of homology between these two sequences of 33 amino acids is 73.5%. Id.
  • Tests developed for detection of HEV must contain reagents which are
  • the present invention provides a highly specific and novel monoclonal antibody or fragment thereof that can be employed for the detection of HEV orf-2 antigen.
  • the monoclonal antibody specifically binds to the HEV orf-2 protein.
  • the hybridoma which secretes the monoclonal antibody is identified as: Hybridoma cell line H1C119 (A.T.C.C. deposit No. HB11521, secreting monoclonal antibody (H1C119).
  • H1C119 secreting monoclonal antibody
  • the monoclonal antibody of the present invention can be used in an immunoassay for the detection of HEV antigen or antibody.
  • Many such immunoassay formats are known in the art and can be modified by adding a known amount of a monoclonal antibody which specifically binds to HEV orf-2 antigen.
  • a particularly preferred assay format is a competitive assay for determining the presence and amount of HEV antibody which may be present in a test sample.
  • the assay involves contacting a test sample suspected of containing HEV antibodies with a solid phase coated with HEV antigens and an indicator reagent comprising a signal generating compound and a monoclonal antibody which specifically binds to HEV orf-2 antigen, for a time and under conditions sufficient to form antigen/antibody complexes of the test sample and solid phase and/or indicator reagent and solid phase; and determining the presence of HEV antibody present in the test sample by detecting the reduction in binding of the indicator reagent to the solid phase as compared to the signal generated from a negative test sample to indicate the presence of HEV antibody in the test sample
  • Figure 1 is a schematic representation of the plasmid used for production of orf-2 CKS/HEV SG-2 antigen from plasmids pJO201 and pGEX-HEV-ORF2- SG-3.
  • the monoclonal antibody of the present invention can be employed in various assay systems to determine the presence, if any, of HEV antigens or HEV antibodies in a test sample. Fragments of the monoclonal antibody provided also may be used.
  • the monoclonal antibody of the present invention may be screened for as follows.
  • Recombinant or synthetic HEV orf-2 antigen is used on a solid phase 5 (preferably, polystyrene beads).
  • CKS CMP-KDO synthetase
  • SG-3 CMP-KDO synthetase
  • EIA immunoassay which uses the synthetic peptide spB3-2 (obtained as described in copending application U.S. Serial Number 08/089,877 l o filed July 9, 1993) on the solid phase is utilized.
  • Detection of non-specific binding is accomplished by coating the solid phase with CKS alone or with a non-HEV CKS recombinant protein.
  • Test samples (mouse serum, tissue culture supernatant, or mouse ascites fluid) are serially diluted in a specimen diluent and a portion is incubated with each solid phase for 1-2 hours at 40°C. Beads are then
  • the monoclonal antibody of the invention can be employed as a competitive probe for the detection of antibodies to HEV antigen.
  • HEV antigens coated on a solid phase are contacted
  • a polyclonal or monoclonal anti-HEV antibody or a fragment thereof, which has been coated on a solid phase is contacted with a test sample which may contain HEV antigens, to form a mixture.
  • a test sample which may contain HEV antigens
  • This mixture is incubated for a time and under conditions sufficient to form antigen/antibody complexes.
  • an indicator reagent comprising a monoclonal or a polyclonal antibody or a fragment thereof, which specifically binds to HEV antigen, to which a signal generating compound which generates a measurable signal has been attached, is contacted with the
  • HEV antigen/antibody complexes to form a second mixture.
  • This second mixture then is incubated for a time and under conditions sufficient to form antibody/antigen/indicator reagent complexes.
  • the presence of HEV antigen present in the test sample and captured on the solid phase, if any, is determined by detecting the measurable signal generated by the signal generating compound.
  • the l o amount of HEV antigen present in the test sample is proportional to the signal generated.
  • a polyclonal or monoclonal anti-HEV antibody or fragment thereof which is bound to a solid support, the test sample and an indicator reagent comprising a monoclonal or polyclonal antibody or fragments thereof, which
  • HEV antigen 15 specifically binds to HEV antigen to which a signal generating compound which generates a measurable signal is attached, are contacted simultaneously to form a mixture. This mixture is incubated for a time and under conditions sufficient to form antibody/antigen/indicator reagent complexes. The presence, if any, of HEV antigen present in the test sample and captured on the solid phase is determined by
  • the monoclonal antibody of the invention can be employed either as the capture phase or as part of the indicator reagent.
  • the monoclonal antibody of the present invention can be employed in the detection of HEV antigen in fixed tissue sections, as well as fixed cells by immunohistochemical analysis, by standard methods well- known to those skilled in the art.
  • the monoclonal antibody can be bound to matrices similar to
  • the monoclonal antibody of the invention also can be used for the generation of chimeric antibodies for therapeutic use, or other similar applications.
  • the monoclonal antibody or fragment thereof can be provided individually
  • Combinations of the monoclonal antibody (and fragments thereof) of the present invention provided herein also may be used in combination with other monoclonal antibodies that have differing specificities for HEV as components in a mixture or "cocktail" of HEV antibodies, each having different binding specificities.
  • this cocktail can include the monoclonal antibody of the invention directed to orf-2 protein from the HEV genome, along with different monoclonal antibodies directed to other HEV regions, such as the orf-3 HEV protein or other binding sites on orf-2 HEV protein.
  • This cocktail of monoclonal 5 antibodies would then be used in place of the single monoclonal antibody as described in the assay formats herein.
  • the polyclonal antibody or fragment thereof which can be used in the assay formats should specifically bind to HEV antigen.
  • the polyclonal antibody used preferably is of mammalian origin and includes but is not limited to human, l o goat, rabbit or sheep anti-HEV polyclonal antibody.
  • the polyclonal antibodies used in the assays can be used either alone or as a cocktail of polyclonal antibodies. Since the cocktails used in the assay formats are comprised of either monoclonal antibodies or polyclonal antibodies having different HEV specificity, they would be useful for diagnosis, evaluation and prognosis of HEV infection, as
  • Test samples which can be tested by the methods of the present invention described herein include human and animal body fluids such as whole blood, serum, plasma, cerebrospinal fluid, urine, biological fluids such as cell culture supernatants, fixed tissue specimens and fixed cell specimens.
  • the "solid phase” is not critical and can be selected by one skilled in the art.
  • latex particles, microparticles, magnetic or non-magnetic beads, membranes, plastic tubes, walls of microtiter wells, glass or silicon chips and tanned sheep red blood cells are all suitable examples.
  • Suitable methods for immobilizing peptides on solid phases include ionic, hydrophobic, covalent
  • 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 0 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 immobilized upon (attached to) the solid phase and which has the ability to immobilize the capture reagent through a specific binding reaction.
  • 35 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 thus can be a plastic, derivatized plastic, magnetic or non-magnetic metal, glass or silicon surface of a test tube, microtiter well, sheet, bead, microparticle, chip, and other configurations known to those of ordinary skill in the art.
  • the solid phase also can comprise any suitable porous material with sufficient porosity to allow 5 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, l o 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
  • vinyl polymers including polyethylene, polypropylene, polystyrene, polyvinylchloride, 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 sulfates or
  • alkaline earth metals and magnesium including barium sulfate, calcium sulfate, calcium carbonate, silicates of alkali and alkaline earth metals, aluminum and magnesium; and aluminum or silicon oxides or hydrates, such as clays, alumina, talc, kaolin, zeolite, silica gel, or glass (these materials may be used as filters with the above polymeric materials); and mixtures or copolymers of
  • graft copolymers obtained by initializing polymerization of synthetic polymers on a pre-existing natural polymer. All of these materials may be used in suitable shapes, such as films, sheets, or plates, or they may be coated onto or bonded or laminated to appropriate inert carriers, such as paper, glass, plastic films, or fabrics.
  • the porous structure of nitrocellulose has excellent absorption and adsorption qualities for a wide variety of reagents including monoclonal antibodies.
  • Nylon also possesses similar characteristics and also is suitable.
  • porous solid supports described hereinabove are preferably in the form of sheets of thickness from about 0.01 to 0.5 mm,
  • the pore size may vary within wide limits, and is preferably from about 0.025 to 15 microns, especially from about 0.15 to 15 microns.
  • a charged substance can be coated directly to the material or onto microparticles which then are retained by a solid phase support material.
  • microparticles can serve as the solid phase, by being retained in a column or being suspended in the 5 mixture of soluble reagents and test sample, or the particles themselves can be retained and immobilized by a solid phase support material.
  • retained and immobilized is meant that the particles on or in the support material are not capable of substantial movement to positions elsewhere within the support material.
  • the particles can be selected by one skilled in the art from any suitable l o type of particulate material and include those composed of polystyrene, polymethylacrylate, polypropylene, latex, polytetrafluoroethylene, polyacrylonitrile, polycarbonate, or similar materials.
  • the size of the particles is not critical, although it is preferred that the average diameter of the particles be smaller than the average pore size of the support material being used.
  • the methods of the present invention can be adapted for use in systems which utilize microparticle technology including automated and semi-
  • the indicator reagent comprises a signal generating compound (label) which is capable of generating a measurable signal detectable by external means conjugated (attached) to a specific binding member for HEV.
  • label a signal generating compound
  • Specific binding member means a member of a specific binding pair. That is, two different molecules where one of the molecules through chemical or physical means specifically binds to the second molecule.
  • the indicator reagent also can be a member of any specific binding pair, including either hapten-anti-hapten systems 5 such as biotin or anti-biotin, avidin or biotin, a carbohydrate or a lectin, a complementary nucleotide sequence, an effector or a receptor molecule, an enzyme cofactor and an enzyme, an enzyme inhibitor or an enzyme, and the like.
  • An immunoreactive specific binding member can be an antibody, an antigen, or an antibody/antigen complex that is capable of binding either to HEV as in a sandwich l o assay, to the capture reagent as in a competitive assay, or to the ancillary specific binding member as in an indirect assay.
  • the various signal generating compounds (labels) contemplated include chromogens, catalysts such as enzymes, luminescent compounds such as fluorescein and rhodamine, chemiluminescent compounds, radioactive elements,
  • enzymes include alkaline phosphatase, horseradish peroxidase, beta-galactosidase, and the like.
  • the selection of a particular label is not critical, but it will be capable of producing a signal either by itself or in conjunction with one or more additional substances.
  • An immobilizable immune complex is separated from the rest of the reaction mixture by ionic interactions between the negatively charged poly-anion/immune complex and the previously treated, positively charged porous matrix and detected by using
  • 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.
  • Such systems include those described in pending U. S. Patent Applications 425,651 and 425,643, which correspond to published EPO applications Nos. EP 0425 633 and EP 0424 634, respectively, which are incorporated herein by reference.
  • SPM scanning probe microscopy
  • the capture phase for example, at least one of the monoclonal antibodies of the invention
  • a scanning probe microscope is utilized to detect antigen/antibody complexes which may be present on the surface of the solid phase.
  • the use of scanning tunneling microscopy l o eliminates the need for labels which normally must be utilized in many immunoassay systems to detect antigen/antibody complexes. Such a system is described in pending U. S. patent application Serial No. 662,147, which enjoys common ownership and is incorporated herein by reference.
  • one member of a specific binding partner is attached to a surface suitable for scanning.
  • the attachment of the analyte specific substance may be by adsorption to a test piece which comprises a solid phase of a plastic or metal surface, following methods known to those of ordinary skill in the art.
  • a specific binding partner analyte specific substance
  • Covalent attachment methods are known to those skilled in the art and include a variety of means to irreversibly link specific binding partners to the test piece. If the test piece is silicon or glass, the surface must be
  • Activated silane compounds such as triethoxy amino propyl silane (available from Sigma Chemical Co., St. Louis, MO), triethoxy vinyl silane (Aldrich Chemical Co., Milwaukee, WI), and (3-mercapto-propyl)-trimethoxy silane (Sigma Chemical Co., St. Louis, MO) can be used to introduce reactive groups such as amino-, vinyl, and thiol,
  • Such activated surfaces can be used to link the binding partner directly (in the cases of amino or thiol) or the activated surface can be further reacted with linkers such as glutaraldehyde, bis (succinimidyl) suberate, SPPD 9 succinimidyl 3-[2-pyridyldithio] propionate), SMCC (succinimidyl-4-[N- maleimidomethyl] cyclohexane-1-carboxylate), SLAB (succinimidyl [4-iodoacetyl]
  • linkers such as glutaraldehyde, bis (succinimidyl) suberate, SPPD 9 succinimidyl 3-[2-pyridyldithio] propionate), SMCC (succinimidyl-4-[N- maleimidomethyl] cyclohexane-1-carboxylate), SLAB (succinimidyl [4-iodoacet
  • the vinyl group can be oxidized to provide a means for covalent attachment. It also can be used as an anchor for the polymerization of various polymers such as poly acrylic acid, which can provide multiple attachment points for specific binding partners.
  • the amino surface can be reacted with oxidized dextrans of various molecular weights to provide hydrophilic linkers of different size and capacity.
  • oxidizable dextrans examples include Dextran T-40 (molecular weight 40,000 daltons), Dextran T-l 10 (molecular 5 weight 110,000 daltons), Dextran T-500 (molecular weight 500,000 daltons), Dextran T-2M (molecular weight 2,000,000 daltons) (all of which are available from Pharmacia, Piscataway, NJ), or Ficoll (molecular weight 70,000 daltons (available from Sigma Chemical Co., St. Louis, MO). Also, polyelectrolyte interactions may be used to immobilize a specific binding partner on a surface of a l o test piece by using techniques and chemistries described by pending U. S. Patent applications Serial No.
  • the surface 15 may be further treated with materials such as serum, proteins, or other blocking agents to minimize non-specific binding.
  • the surface also may be scanned either at the site of manufacture or point of use to verify its suitability for assay purposes. The scanning process is not anticipated to alter the specific binding properties of the test piece.
  • the monoclonal antibody of the invention can be used as a positive control in an assay which is designed to detect the presence of HEV antibody.
  • HEV antigens would be used as a capture phase. These HEV antigens could be prepared by various means from viral lysates, synthetic peptides of various
  • the reagent employed for the assay 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 monoclonal antibody, or a cocktail of monoclonal antibodies, employed in the assay.
  • kits also could 5 contain vials or containers of other reagents needed for performing the assay, such as washing, processing and indicator reagents.
  • a recombinant DNA clone was constructed to contain the gene encoding the full length orf-2 of HEV as a fusion protein with CKS.
  • This antigen (designated SG-3 protein) expressed by this clone was used to immunize a mouse from which an immune splenocyte was fused to a SP2/0-Agl4 myeloma cell to produce a hybridoma cell line that secretes a monoclonal antibody
  • immunoglobulin (Ig) class Gi (IgGi) reactive with HEV orf-2.
  • the resultant immunoglobulin was produced in mouse ascites fluid and was purified by affinity chromatogr aphy .
  • GST glutathione S-transferase
  • the CKS/HEV-SG-3 protein was expressed in E. coli strain XL 1 -Blue at greater than 20% of total cell protein after induction with isopropyl b-D thiogalactoside (IPTG).
  • IPTG isopropyl b-D thiogalactoside
  • the clones were propagated in 10 liter fermentors, yielding 170-260 grams of wet cell paste per fermentation.
  • the E. coli cells expressing the SG-3 protein were lysed at pH 10 in the presence of various protease inhibitors.
  • the CKS fusion protein was insoluble 5 and remained in the pellet after centrifugation. Pellets were washed with various detergents to remove non-specific proteins. Following solubilization in Tris, pH 8.5 containing 0.5% SDS, the CKS fusion protein was found to represent 50 to 60% of the total protein as evaluated by SDS-PAGE and gel densitometry.
  • the solubilized protein was further purified by Sephacryl S-300HR column l o chromatography. Fractions were analyzed by SDS-PAGE, pooled and evaluated for purity by gel densitometry. Final purified protein was greater than 90% pure.
  • the immunization regimen (4 mice) consisted of an initial immunization
  • mice 15 with additional immunizations at six and nine weeks.
  • mice (Balb/c) were inoculated intraperitoneally with this emulsion.
  • mouse serum was screened for 0 enzyme immunoassay (EIA) immunoreactivity as described below.
  • EIA enzyme immunoassay
  • the serum anti-HEV titer tested against beads coated with HEV SG-3 was 2 x 10 ** * in specimen diluent containing E. coli and CKS lysate and 10 3 against beads coated with CKS alone.
  • mice Four weeks after the first immunization, three mice were boosted intraperitoneally with 10 ⁇ g of the immunizing protein in incomplete Freund's adjuvant.
  • post-immunization anti-HEV titers from boosted mouse No. 4 against beads coated with protein HEV SG-3 described above were 1.5 x 0 l ⁇ 6 in specimen diluent with no additives to block out unwanted reactivity, 2 x 10 ⁇ in specimen diluent with additives and 5.0 x 10 ⁇ against spB3-2.
  • This mouse was then boosted intravenously (via the tail vein) with 30 ⁇ g of the immunizing protein and used for fusion 3 days later.
  • Normal mouse splenocytes were prepared by aseptically removing the spleen from a non-immunized mouse and crushing it using the plunger of a syringe through a screen that fits into a 60 x 15 mm Petri dish containing a small amount of Dulbecco's' Minimum Essential (DME) medium.
  • DME Dulbecco's' Minimum Essential
  • the splenocyte solution was washed with DME medium, and the red blood cells were lysed by adding 1 ml of 0.83% NH4CI in lOmM Tris to the cell pellet for 1 minute. The cells were again washed and resuspended in 40 mis of DME medium containing 20% Fetal Bovine Serum (FBS).
  • DME Dulbecco's' Minimum Essential
  • the immunized mouse was sacrificed, and the spleen was removed and processed as described herein for the preparation of normal splenocytes except that the splenocytes were resuspended in 10 mis DME medium and counted.
  • Splenocytes were fused in a 1 : 1 ratio with the SP2/0 myeloma cell line using the described modification of conventional methods
  • Hybrid #1 had an optical density (O.D). of 1.155 when tested at a 1: 10 dilution against SG-3 antigen- coated beads and was negative against CKS-only coated beads, and HEV spB3-2 beads. HI was then cloned at one cell per well by limiting dilution technique as described in Goding,
  • 96 multiwell tissue culture tray an aliquot containing 100 cells was added to 20 mis of DME media containing 20% Fetal Bovine Serum (FBS) and 5x10 ⁇ normal mouse splenocytes. An eight channel pipetman adjusted to 0.2 ml was used to plant the cell suspension. The trays were incubated at 37°C in a humidified 5% CO2 incubator and refed on days 5 and 7 or as needed because of evaporation.
  • FBS Fetal Bovine Serum
  • HlCl 19 Monoclonal Antibody Clones selected for further evaluation were scaled up in tissue culture T- flasks and 10" cells were injected into the peritoneal cavity of pre-pristaned BALB/c mice (Charles River Biotechnical Services, Inc., Wilmington, MA) (see Hurrell, supra).
  • the resulting ascites fluid was harvested 7-10 days after injection, centrifuged and frozen.
  • the antibody was affinity purified on a Protein A Sepharose CL-4B (Pharmacia-LKB Biotechnologies, Piscataway, NJ). Bound monoclonal antibody was eluted from the column at a pH of 5.5, indicating its subtype as IgG] .
  • EIA's enzyme immunoassays
  • the CKS-HEV recombinant orf-2 (SG-3) (prepared as described in Example 1) was coated on the solid phase in either the unpurified (extracted and solubilized) form (ca. 50-60% pure HEV protein) or a purified protein (>90% pure)
  • another EIA used the synthetic peptide spB3-2 on the solid phase, and to detect non-specific binding the solid phase was coated with CKS alone or a non- HEV CKS recombinant protein.
  • mouse serum, tissue culture supernatant, mouse ascites fluid samples or monoclonal antibody (MAb) HlCl 19 obtained above were serially diluted 5 to 10-fold in specimen diluent containing phosphate-buff ered- saline/Tris/EDTA, pH 7.8 with 20% goat serum, 10% fetal calf serum, 1% bovine serum albumin, 0.2% Triton-X 100, 0.1% sodium azide, with or without 1% E. coli lysate and 0.01% CKS added, then 200 ⁇ l was incubated with each solid phase for 1-2 hours at 40°C. Beads were then washed 3 times with 5 ml distilled water.
  • MAb monoclonal antibody
  • Bound antibody was detected using 200 ⁇ l HRPO-labeled second antibody (Goat anti-mouse IgG H+L) from Kirkegaard and Perry Labs) at 0.3 ⁇ g/mL in conjugate diluent containing Tris-buffered-saline with 10% goat serum, 10% fetal calf serum and 0.15% Triton-X 100. Beads were incubated with conjugate for 1 hour at 40°C, and washed as before.
  • O-Phenylenediamine (OPD) substrate was freshly prepared by adding 1 OPD tablet (Abbott) per 5 mis of OPD Diluent (Abbott), and 300 ⁇ l of OPD substrate solution was then added to each washed bead and incubated in the dark at room temperature for 30 minutes.
  • One ml of sulfuric acid added to stop the reaction, and the amount of color generated was determined by measuring the absorbance of the substrate at 492 nm with 2 hours of sulfuric acid addition. Results given in Table 1 below indicate that the antibody was specific for the orf-2 region of HEV.
  • Solid phase was coated with HEV orf-2 (SG-3) or a combination of l o proteins coding for the orf-2 and orf-3 regions.
  • the solid phases were pre- incubated with a 1 :2 dilution of HEV negative human plasma (negative control) or human plasma known to contain HEV Ab (sample) in specimen diluent overnight at room temperature. Beads were then washed and monoclonal antibody (MAb) of the invention against HEV orf-2, diluted to give an O.D. of 1.000 - 2.000 when
  • the HEV Ab positive sample showed 20.0% inhibition of MAb HlCl 19 when the SG-3 bead was used, and 54.2 % inhibition when the HEV orf-2/orf-3 Combo bead was used.
  • the results are given in Table
  • Solid phase is coated with HEV orf-2 (SG-3) protein.
  • Monoclonal antibody (MAb) HlCl 19 at a concentration previously determined to give an O.D. of 1.000 - 2.000 in this assay format, is pre-incubated with sample at a time l o and temperature long enough to allow binding of HEV antigen in the sample to MAb (30-120 minutes, at room temperature to 40°C).
  • the solid phase is then added, and the mixture and bead are incubated at a time and temperature long enough to allow any remaining unbound MAb to bind to the solid phase (temperature range: room temperature to 40°C, time range: 1 - 16 hours). Binding
  • MAb 15 of MAb to the solid phase is then detected either using a labeled second antibody (e.g. horseradish peroxidase (HRPO)-labelled Goat anti-mouse IgG) with incubation of 1-2 hours at 40°C, or MAb itself could be labeled, thus making the assay potentially performable in one step.
  • a labeled second antibody e.g. horseradish peroxidase (HRPO)-labelled Goat anti-mouse IgG
  • HRPO horseradish peroxidase
  • HEV antigen containing sample will inhibit the MAb from binding to the solid phase and reduce the signal at least 50%.

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EP95905967A 1993-12-22 1994-12-16 Monoclonal antibodies against hev orf-2 and methods for using same Withdrawn EP0736087A1 (en)

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US6207416B1 (en) 1992-09-18 2001-03-27 The United States Of America As Represented By The Department Of Health And Human Services Recombinant proteins of a Pakistani strain of hepatitis E and their use in diagnostic methods and vaccines
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