WO1986003498A1 - Monoclonal antibodies and their use - Google Patents

Monoclonal antibodies and their use Download PDF

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Publication number
WO1986003498A1
WO1986003498A1 PCT/GB1985/000566 GB8500566W WO8603498A1 WO 1986003498 A1 WO1986003498 A1 WO 1986003498A1 GB 8500566 W GB8500566 W GB 8500566W WO 8603498 A1 WO8603498 A1 WO 8603498A1
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WIPO (PCT)
Prior art keywords
monoclonal antibody
hepatitis
antigen
immunoassay
labeled
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PCT/GB1985/000566
Other languages
French (fr)
Inventor
Bruce William Wright
Peter John Cox
Alice Margaret Noyes
Danny Widdows
Simon James Yvon Saxby
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Technology Licence Company Limited
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Publication of WO1986003498A1 publication Critical patent/WO1986003498A1/en

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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • Hepatitis viruses There are two types of hepatitis viruses, A and B. Hepatitis A is transmitted through the faeces and urine and thus occurs under conditions of poor sanitation. Hepatitis B is transmitted by contact with the serum or blood of a person who has, or has had, the disease.
  • Hepatitis A virion consists of a number of as yet incompletely understood viral protein antigens.
  • Hepatitis B is divided into serotypes based on its antigens, in particular the Hepatitis B antigens known as surface antigen (HBsAg) , e antigen (HBeAg) , delta antigen and core antigen.
  • Hepatitis has been the subject of extensive study, to the point where the entire genome of the Hepatitis B virus has been gene-sequenced, and the corresponding amino-acid sequences of the core, e and surface antigens at least have been determined.
  • the surface antigen is of primary importance because its appearance and subsequent disappearance from the serum or plasma of infected individuals is a reliable indicator of the presence of the disease state and its subsequent remission.
  • the surface antigen consists of a peptide of 226 amino-acid residues. For reference, they have been numbered 1-226, beginning from the amine terminal.
  • the immunogenic or hydrophilic antibody binding region is known to span residues 110-160 and is referred to generally as the "a" determinant.
  • the group "a” determinant is really composed of at least three overlapping sequences with residues 110-137 specifying the major "d” and "y” subtype system.
  • Residue 139 to 147 specify the biological "a” determinant and at present no subtypes or substitutions in this sequence are believed to occur.
  • the Hepatitis B surface antigen has been categorised into four major subtypes, i.e.
  • adw, adr, ayw and ayr having the type specific "a" determinant in common.
  • the amino and substitutions responsible for subtype specifically occur in the region from 110-137 and the type specific biological "a" determinant has been localised to a sequence of eight amino-acids running from 139-147.
  • Hepatitis B screening is important in blood transfusion and blood products and also in vaccination programs.
  • Hepatitis A and B antigens are currently diagnosed by immunological techniques for the detection of circulating antigen in the blood.
  • the present methods require expensive, slow, labour-intensive cultures and other tests.
  • agglutination immuno-fluorescent, chemilum- inescent or fluorescent im unoassay, immuno- electron microscopy, radiometric assay systems, radio immunoassays, and enzyme-linked immunoassays are the most common techniques used with the monoclonal antibodies. Other techniques include bioluminescent, fluorescence polarization, and photon-counting immunoassays.
  • EIA enzyme-linked immunoassay procedure
  • the enzyme-linked monoclonal antibody can then be used in the known enzyme-linked immunosor- bent assay procedure to determine the presence of an antigenic substance.
  • the serotype of the infecting virus can be deter ⁇ mined, and appropriate treatment can then be initiated to rapidly and efficiently eliminate the disease.
  • the present invention provides novel mono ⁇ clonal antibodies for use in accurately and rapidly diagnosing samples for the ,presence of Hepatitis antigens and/or viruses.
  • the present invention com ⁇ prises high affinity monoclonal antibodies spe ⁇ cific for an antigen or species of Hepatitis; in particular, the antigens or species of Hepati ⁇ tis A virus, as well as a monoclonal antibody broadly cross-reactive with an antigen for each species of the Hepatitis virus.
  • the invention also comprises labeled mono ⁇ clonal antibodies for use in diagnosing the presence of the Hepatitis antigens, each com ⁇ prising a monoclonal antibody against one of the above-mentioned antigens to Hepatitis or to a particular species thereof and linked thereto an appropriate label.
  • the label can be chosen from the group consisting of a radioactive iso ⁇ tope, enzyme, fluorescent compound, chemilumines- cent compound, bioluminescent compound, ferromag ⁇ netic atom, or particle, or any other label.
  • the invention further comprises the process for diagnosing the presence of Hepatitis anti ⁇ gens or viruses in a specimen comprising con ⁇ tacting said specimen with the labeled monoclonal antibody in an appropriate immunoassay procedure.
  • the invention is also directed to a therapeutic composition
  • a therapeutic composition comprising a mono ⁇ clonal antibody for .
  • an antigen of Hepatitis and a carrier or diluent as well as kits contain ⁇ ing at least one labeled monoclonal antibody to an antigen of a Hepatitis virus.
  • the monoclonal antibodies of the present invention are prepared by fusing spleen cells, from a mammal which has been immunized against the particular Hepatitis antigen, with an appro ⁇ priate myeloma cell line, preferably NSO (unclon- ed), P3NS1-Ag4/1, or Sp2/0 Agl4.
  • the resultant product is then cultured in a standard HAT (hypox- anthine, aminopterin, and thymidine) medium. Screening tests for the specific monoclonal antibodies are employed utilizing immunoassay techniques which will be described below.
  • the immunized spleen cells may be derived from any mammal, such as primates, humans, rodents (i.e., mice, rats, and rabbits), bovine, ovine, canine, or the like, but the present invention will be described in connection with mice.
  • the mouse is first immunized by injection of the particular Hepatitis antigen chosen generally for a period of approximately eleven weeks. When the mouse shows sufficient antibody produc ⁇ tion against the antigen, as determined by conven ⁇ tional assay, it is given a booster injection of the appropriate Hepatitis antigen, and then killed so that the immunized spleen may be remov ⁇ ed. The fusion can then be carried out utilizing immunized spleen cells and an appropriate myeloma cell line.
  • the fused cells yielding an antibody which give a positive response to the presence of the particular Hepatitis antigen are removed and cloned utilizing any of the standard methods.
  • the monoclonal antibodies from the clones are then tested against standard antigens to determine their specificity for the particular Hepatitis antigen.
  • the monoclonal antibody selected. which is specific for the particular Hepat t s antigen or virus, is then bound to an appropri ⁇ ate label.
  • Amounts of antibody sufficient for labeling and subsequent commercial production are produced by the known techniques, such as by batch or continuous tissue culture or culture in vivo in mammals, such as mice.
  • the monoclonal antibodies may be labeled with a multitude of different labels, such as enzymes, fluorescent compounds, luminescent compounds, radioactive compounds, ferromagnetic labels, and the like.
  • labels such as enzymes, fluorescent compounds, luminescent compounds, radioactive compounds, ferromagnetic labels, and the like.
  • the present invention will be described with reference to the use of an enzyme labeled monoclonal antibody.
  • Some of the enzymes utilized as labels are alkaline phosphatase, glucose oxidase, ' galactosidase, peroxidase, or urease, and the like.
  • Such linkage with enzymes can be accomplished by any one of the conventional and known methods, such as the Staphylococcal Protein A method, the glutaraldehyde method, the benzoquinone method, or the periodate method.
  • EIA enzyme- linked immunosorbent assay
  • Fluorescent-immunoassay is based on the labeling of antigen or antibody with fluorescent probes. A nonlabeled antigen and a specific antibody are combined with identical fluorescently labeled antigen. Both labeled and unlabeled antigen compete for antibody binding sites. The amount of labeled antigen bound to the antibody is dependent upon, and therefore a measurement of, the concentration of nonlabeled antigen. Examples of this particular type of fluorescent- immunoassay would include heterogenous systems such as Enzyme-Linked Fluorescent Immunoassay, or homogeneous systems such as the Substrate Labeled Fluorescent Immunoassay. The most suit ⁇ able fluorescent probe, and the one most widely - ⁇ used is fluorescein. While fluorescein can be subject to considerable interference from scattering, sensitivity can be increased by the use of a fluorometer optimized for the probe utilized in the particular assay and in which the effect of scattering can be minimized.
  • Fluorescence polarization In fluorescence polarization, a labeled sample is excited with polarized light and the degree of polarization of the emitted light is measured. As the antigen binds to the antibody its rotation slows down and the degree of polari ⁇ zation increases. Fluorescence polarization is simple, quick, and precise. However, at the present time its sensitivity is limited to the micromole per liter range and upper nano- mole per liter range with respect to antigens in biological samples.
  • Luminescence is the emission of light by an atom or molecule ' as an electron is transferred to the ground state from a higher energy state.
  • the free energy of a chemical reaction provides the energy required to produce an inter ⁇ mediate reaction or product in an electronically excited state. Subsequent decay back to the ground state is accompanied by emission of light.
  • Bioluminescence is the name given to a special form of chemiluminescence found in biological systems, in which a catalytic protein or enzyme, such as luciferase, increases the efficiency of the luminescent reaction. The best known chemiluminescent substance is luminol.
  • a further aspect of the present invention is a therapeutic composition
  • a therapeutic composition comprising one or more of the monoclonal antibodies to the particular Hepatitis antigen or virus, as well as a pharmacologically acceptable carrier or diluent.
  • Such compositions can be used to treat humans and/or animals afflicted with some form of Hepatitis virus and they are used in amounts effective to cure; an amount which will vary widely dependent upon the individual being treated and the severity of the infection.
  • One or more of the monoclonal antibodies can be assembled into a diagnostic kit for use in diagnosing for the presence of an antigen, antigens, or viruses of Hepatitis in various specimens. It is also possible to use the broadly cross-reactive monoclonal antibody which can identify the Hepatitis virus alone or as part of a kit containing antibodies that can identify other viral or bacterial genera or species. In the past there have been difficulties . in developing rapid kits because of undesirable cross-reactions of specimens with antiserum. The use of monoclonal antibodies can eliminate these problems and provide highly specific and rapid tests for diagnosis. A rapid and precise kit could replace or augment existing tests and permit early direct therapy. A rapid diag ⁇ nosis of a viral infection could represent sub ⁇ stantial patient and hospital savings.
  • a kit can be used on an out-patient basis.
  • the lack of a rapid test giving "same day” answers may delay the initiation of treatment until the patient has developed more severe symptoms or may require the initiation of more costly therapy in a sick patient.
  • a test that wouid return results within an hour or two would be a substantial convenience to patients.
  • kit could be included as a component in a comprehensive line of compatible immunoassay reagents sold to reference laboratories to detect the species and serotypes of Hepatitis.
  • kits comprising at least one labeled monoclonal antibody against a particular Hepatitis antigen or species, as well as any appropriate stains, counterstains, or reagents.
  • Specific antigens to be detected in this kit include the antigens of Hepatitis A and Hepatitis B; in particular, the Hepatitis B surface antigen (HBsAg).
  • Monoclonal diagnostics which detect the presence of Hepatitis antigens can also be used in periodic testing of water sources, food sup ⁇ plies and food processing operations.
  • the present invention describes the use of the labeled monoclonal antibodies to determine the presence of a standard antigen
  • the invention can have many applications in diagnosing the presence of antigens by determining whether specimens such as urine, blood, stool, water, milk, and the like contain the particular Hepati ⁇ tis antigen.
  • the invention could be utiliz ⁇ ed as a public health and safety diagnostic aid, whereby specimens such as water or food could be tested for possible contamination by Hepatitis A.
  • a monoclonal antibody was prepared generally according to the method of Kohler and Milstein (Eur. J. Immunol. ⁇ _, 292 (1975)).
  • HBsAg Hepati ⁇ tis B virus
  • mice Six Balb/c mice were injected with the prepared antigen. They were given one intraperi- toneal injection per week (5 ug/injection) of Hepatitis B virus surface antigen for a total of three weeks followed by two weekly intravenous injections, plus one in Freunds Complete Adjuvant (F.C.A.) after one week. The mice were then be bled approximately six days after the last injection and the serum tested for antibodies by assay.
  • the conventional assay used for this serum titer testing was the enzyme-linked immuno- sorbent assay system.
  • mice showed antibody production after this regimen, generally a positive titer of at least 10,000, a mouse was selected as a fusion donor and given a booster injection (0.02 ml of 10-*' cells/ml) intravenously three days prior to splenectomy.
  • a positive titer of at least 10,000 a mouse was selected as a fusion donor and given a booster injection (0.02 ml of 10-*' cells/ml) intravenously three days prior to splenectomy.
  • Spleen cells from the immune mice were then harvested three days after boosting by conventional techniques.
  • First the donor mouse selected was killed and surface sterilized by immersion in 70% ethyl alcohol.
  • the spleen was then removed and immersed in approximately 2.5 ml of Dulbeccos Modified Eagles Medium (DMEM) to which had been added 3% fetal calf serum (FCS).
  • DMEM Dulbeccos Modified Eagles Medium
  • FCS fetal calf serum
  • the spleen was then gently homogenized in a LUX homogenizing tube until all cells have been released from the membrane and the cells washed in 5 ml 3% FCS DMEM.
  • the cellular debris was then allowed to settle and the spleen cell suspension placed in a 10 ml centrifuge tube.
  • the debris was then rewashed in 5 ml 3% FCS DMEM. Fifty mis of suspension were then made in 3% FCS DMEM.
  • the myeloma cell line used was NS0 (unclon- ed), obtained from the MRC Laboratory of Molecular - - Biology in Cambridge, England. The myeloma cells were in the log growth phase, and rapidly dividing. Each cell line was washed using a tissue culture medium DMEM containing 3% fetal calf serum.
  • the spleen cells were then spun down at the same time that a relevant volume of myeloma cells were spun down (room 'temperature for 7 minutes at 600 g) , and each resultant pellet was then separately resuspended in 10 ml 3% FCS DMEM.
  • 0.1 ml of the suspension was diluted to 1 ml and used with a haemacytometer with phase micro ⁇ scope to make the count.
  • 0.1 ml .of the suspension was diluted to 1 ml with Methyl Violet and used with a haemacytometer and light microscope and the stained nuclei of the cells counted.
  • lO* ⁇ spleen cells were then mixed with 5x10? myeloma cells, the mixture washed in serum- free DMEM high in glucose and centrifuged and all the liquid removed. The resultant cell pellet was placed in a 37° C. water bath and there was added over the period of one minute 1 ml of a 50% wt. vol. solution of polyethylene glycol 1500 (PEG) in saline Hepes; pH of approxi ⁇ mately 7.5, and the mixture gently stirred for approximately one and one-half minutes.
  • PEG polyethylene glycol 1500
  • Each well contained 1.0 ml of the standard HAT medium (hypoxanthine, amino- pterin, and thymidine) and a feeder layer of Balb/c macrophages at a concentration of 5xl0 4 macrophages/well.
  • HAT medium hyperxanthine, amino- pterin, and thymidine
  • the wells were kept undisturbed and cultured at 37° C. in 9% CO2 air at approximately 100% humidity. The wells were then analyzed for growth utilizing the conventional inverted micro ⁇ scope procedure after about 5 to 10 days. In those wells in which growth was present in the inhibiting HAT medium, screening tests for the specific monoclonal antibody were made utilizing the conventional enzyme immunoassay screening method described below. Somewhere around 10 days to 14 days after fusion, sufficient antibody against the antigen to the Hepatitis B virus had been developed in at least one well.
  • the clones were assayed by the enzyme immunoassay method to determine antibody production.
  • a positive clone may be recloned.
  • the monoclonal antibodies from the clones are screened by the standard techniques for binding to Hepatitis B virus surface antigen, prepared as in the immunization, and for specificity in a test battery of Hepatitis virus antigens and known trace contaminant antigens.
  • mice were primed with pristane and injected
  • the ascites fluid was harvested when the mice were swollen with fluid but still alive. The fluid was centrifuged at 1200 g for approximately 10 minutes, the cells discarded, and the antibody-rich ascites fluid collected, and stored at -20 C. 5 ml of ascites fluid were filtered through glass wool and centrifuged at 30,000 g for 10 minutes. The ascites was then stirred at +4°C and an equal volume of cold, saturated ammonium sulphate added slowly. The mixture was stirred for a further 30 minutes after addition is complete. The precipitate was harvested by centrifugation at 10,000 g for 10 minutes.
  • the precipi ⁇ tate was dissolved in a minimum volume of cold phosphate/EDTA buffer (20 mM sodium phosphate, 10 mM EDTA, pH 7.5, + 0.02% sodium azide) .
  • the solution is dialyzed versus 2x1000 ml of the same buffer at +4°C.
  • the dialyzed, redissolved precipitate was centrifuged at 30,000 g for 10 minutes and applied to a 10 ml column of DEAE-cellulose, previously equilibrated in phosphate/EDTA buffer.
  • the monoclonal antibody was eluted with phosphate/EDTA buffer.
  • the purified antibody was titrated by solid-phase EIA.
  • the monoclonal antibody specific against the antigen, prepared and screened as described above, is then bound to an appropriate enzyme such as horseradish peroxidase or a highly-purified alkaline phosphatase.
  • an appropriate enzyme such as horseradish peroxidase or a highly-purified alkaline phosphatase. This can be accomplished by any known method, e.g. the one-step glutaraldehyde method, the periodate coupling method or benzoquinone conjugation.
  • the conjugate is eluted with 3.5 ml PBS and then dialyzed against 2x2000 -ml of TRIS buffer (50 mM TRIS, 1 mM magnesium chloride, pH 8.0 plus 0.02% sodium azide) at +4°C.
  • TRIS buffer 50 mM TRIS, 1 mM magnesium chloride, pH 8.0 plus 0.02% sodium azide
  • To the dialyzed conjugate is added 1/lOth its own volume of 10% BSA in TRIS buffer.
  • the conjugate is then sterile filtered through a 0.22 ⁇ m membrane filter into a sterile amber vial and stored at +4°C.
  • alkaline phosphatase (Sigma Type VII-T) are dialysed against 2 x 500 ml of 0.25M sodium phosphate buffer, pH 6.0, at +4°C.
  • Para-benzoquinone 18 mg, is dissolved in warm AR ethanol, 0.6 ml, and added to the dialysed alkaline phosphatase.
  • the benzoquinone/alkaline phosphatase mixture is left in the dark at room temperature for 1 hour.
  • 3 mg monoclonal antibody are dialysed against 2 x 500 ml of 0.15M sodium chloride at +4°C. Dialysed antibody is added to 8 mg of benzoquinone-activated alkaline phosphatase, immediately followed by sufficient 1M sodium bicarbonate to give a final concentration of 0.1M. The conjugation mixture is left in the dark at +4°C for 48 hours. After this time, sufficient 1M lysine is added to give a final concentration of 0.1M. After 2 hours in the dark at room temperature, the conjugate is dialysed against 2 x 1000 ml of phosphate buffered saline + 0.02% sodium azide at +4°C. An equal volume of glycerol is added. The conjugate is sterile-filtered through a 0.22 ⁇ membrane filter into a sterile amber vial and stored at +4°C.
  • the enzyme immunoassay method is used for testing.
  • This assay method comprises coating the wells of a standard polyvinyl chloride microtiter tray with the antigen, followed by addition of monoclonal antibody enzyme conjugate, and finally addition of the enzyme substrate, p-nitrophenyl phosphate.
  • the monoclonal antibody found to be specific for the antigen, can then be tested.
  • Four monoclonal antibodies have been tested; three of the class IgGl and one of the class IgG2a have been found, and have been numbered as 80, 192, 329 and 341.
  • Antibody 80, of class IgG2a can detect antigen at 5-10 ng/ml.
  • the particular epitopic site to which the antibody attaches to the antigen can also be " determined.
  • the same enzyme immunoassay method can also be used to determine whether diagnostic specimens such as urine, blood, stool, water or milk contain the antigen. In such cases, the antibody can first be bound to the plate.

Abstract

Monoclonal antibodies to the genus Hepatitis, the labelled antibodies, compositions and kits containing them, and their use in diagnosis of antigen and treatment.

Description

ONOCLONAL ANTIBODIES AND THEIR USE This invention relates to monoclonal antibodies and their use.
BACKGROUND OF THE INVENTION Of current interest in the fields of analysis and diagnosis is the use of monoclonal antibodies to determine the presence of antigens or viruses in specimens such as urine, blood, water, milk, and the like. There is also interest in utilising such antibodies to treat infectious diseases.
There are two types of hepatitis viruses, A and B. Hepatitis A is transmitted through the faeces and urine and thus occurs under conditions of poor sanitation. Hepatitis B is transmitted by contact with the serum or blood of a person who has, or has had, the disease.
These viruses are not similar, except for the fact they affect the liver during the disease process.
Hepatitis A virion consists of a number of as yet incompletely understood viral protein antigens. Hepatitis B is divided into serotypes based on its antigens, in particular the Hepatitis B antigens known as surface antigen (HBsAg) , e antigen (HBeAg) , delta antigen and core antigen.
Hepatitis has been the subject of extensive study, to the point where the entire genome of the Hepatitis B virus has been gene-sequenced, and the corresponding amino-acid sequences of the core, e and surface antigens at least have been determined. The surface antigen is of primary importance because its appearance and subsequent disappearance from the serum or plasma of infected individuals is a reliable indicator of the presence of the disease state and its subsequent remission.
It has been shown that the surface antigen consists of a peptide of 226 amino-acid residues. For reference, they have been numbered 1-226, beginning from the amine terminal. The immunogenic or hydrophilic antibody binding region is known to span residues 110-160 and is referred to generally as the "a" determinant. In fact, it is presently believed that the group "a" determinant is really composed of at least three overlapping sequences with residues 110-137 specifying the major "d" and "y" subtype system. Residue 139 to 147 specify the biological "a" determinant and at present no subtypes or substitutions in this sequence are believed to occur. The Hepatitis B surface antigen has been categorised into four major subtypes, i.e. adw, adr, ayw and ayr, having the type specific "a" determinant in common. The amino and substitutions responsible for subtype specifically occur in the region from 110-137 and the type specific biological "a" determinant has been localised to a sequence of eight amino-acids running from 139-147.
Chemically synthesised peptides corresponding to sequences 139 to 147 and 124 to 137 have been made and these are used to determine where a particular antibody will bind with the antigen.
It has now become clear that the specificity of a monoclonal antibody for a specific epitope or amino-acid sequence of the Hepatitis B surface antigen is important particularly because it is known that some epitopes are instrumental in inducing the formation of antibodies in the affected host, while other epitopes play no role in inducing antibody formation, yet serve as reliable indicators of Hepatitis B infection. This is particularly important in developing vaccines against Hepatitis B infection, as well as detecting and predicting antibody response.
For pregnant mothers, tests may be required at the time of delivery, where the infant is exposed to Hepatitis B. Hepatitis B screening is important in blood transfusion and blood products and also in vaccination programs.
Hepatitis A and B antigens are currently diagnosed by immunological techniques for the detection of circulating antigen in the blood. The present methods require expensive, slow, labour-intensive cultures and other tests.
The ability of monoclonal antibodies specifically to bind to antigens can provide many opportunities for diagnosis and treatment. Such specificity is a most important requirement for proper and accurate analysis and/or diagnosis, particularly in diagnosing the presence of diseases which require prompt treatment.
A wide variety of isotopic and non-isotopic immunoassays have been utilised in conjunction
with monoclonal antibodies to test for the pres¬ ence of an antigenic substance. At the present time, agglutination, immuno-fluorescent, chemilum- inescent or fluorescent im unoassay, immuno- electron microscopy, radiometric assay systems, radio immunoassays, and enzyme-linked immunoassays are the most common techniques used with the monoclonal antibodies. Other techniques include bioluminescent, fluorescence polarization, and photon-counting immunoassays.
When utilizing the enzyme-linked immunoassay procedure (EIA), it is necessary to bind, or conjugate, the monoclonal antibody with an enzyme capable of functioning in such assay; such as alkaline phosphatase.
The enzyme-linked monoclonal antibody can then be used in the known enzyme-linked immunosor- bent assay procedure to determine the presence of an antigenic substance.
After the specific antigen is identified, the serotype of the infecting virus can be deter¬ mined, and appropriate treatment can then be initiated to rapidly and efficiently eliminate the disease.
The production of monoclonal antibodies is now a well-known procedure first described by Kohler and Milstein (Eur. J. Immunol. 6_, 292 (1975)). While the general technique of preparing hybridomas and the resultant monoclonal antibodies is understood, it has been found that preparing a specific monoclonal antibody to a specific antigen is difficult, mainly due to the degree of specificity and variations required in producing a particular hybridoma. SUMMARY OF THE INVENTION
The present invention provides novel mono¬ clonal antibodies for use in accurately and rapidly diagnosing samples for the ,presence of Hepatitis antigens and/or viruses.
Briefly stated, the present invention com¬ prises high affinity monoclonal antibodies spe¬ cific for an antigen or species of Hepatitis; in particular, the antigens or species of Hepati¬ tis A virus, as well as a monoclonal antibody broadly cross-reactive with an antigen for each species of the Hepatitis virus.
The invention also comprises labeled mono¬ clonal antibodies for use in diagnosing the presence of the Hepatitis antigens, each com¬ prising a monoclonal antibody against one of the above-mentioned antigens to Hepatitis or to a particular species thereof and linked thereto an appropriate label. The label can be chosen from the group consisting of a radioactive iso¬ tope, enzyme, fluorescent compound, chemilumines- cent compound, bioluminescent compound, ferromag¬ netic atom, or particle, or any other label.
The invention further comprises the process for diagnosing the presence of Hepatitis anti¬ gens or viruses in a specimen comprising con¬ tacting said specimen with the labeled monoclonal antibody in an appropriate immunoassay procedure.
Additionally, the invention is also directed to a therapeutic composition comprising a mono¬ clonal antibody for . an antigen of Hepatitis and a carrier or diluent, as well as kits contain¬ ing at least one labeled monoclonal antibody to an antigen of a Hepatitis virus.
DETAILED DESCRIPTION
The monoclonal antibodies of the present invention are prepared by fusing spleen cells, from a mammal which has been immunized against the particular Hepatitis antigen, with an appro¬ priate myeloma cell line, preferably NSO (unclon- ed), P3NS1-Ag4/1, or Sp2/0 Agl4. The resultant product is then cultured in a standard HAT (hypox- anthine, aminopterin, and thymidine) medium. Screening tests for the specific monoclonal antibodies are employed utilizing immunoassay techniques which will be described below.
The immunized spleen cells may be derived from any mammal, such as primates, humans, rodents (i.e., mice, rats, and rabbits), bovine, ovine, canine, or the like, but the present invention will be described in connection with mice. The mouse is first immunized by injection of the particular Hepatitis antigen chosen generally for a period of approximately eleven weeks. When the mouse shows sufficient antibody produc¬ tion against the antigen, as determined by conven¬ tional assay, it is given a booster injection of the appropriate Hepatitis antigen, and then killed so that the immunized spleen may be remov¬ ed. The fusion can then be carried out utilizing immunized spleen cells and an appropriate myeloma cell line.
The fused cells yielding an antibody which give a positive response to the presence of the particular Hepatitis antigen are removed and cloned utilizing any of the standard methods. The monoclonal antibodies from the clones are then tested against standard antigens to determine their specificity for the particular Hepatitis antigen. The monoclonal antibody selected. which is specific for the particular Hepat t s antigen or virus, is then bound to an appropri¬ ate label.
Amounts of antibody sufficient for labeling and subsequent commercial production are produced by the known techniques, such as by batch or continuous tissue culture or culture in vivo in mammals, such as mice.
The monoclonal antibodies may be labeled with a multitude of different labels, such as enzymes, fluorescent compounds, luminescent compounds, radioactive compounds, ferromagnetic labels, and the like. The present invention will be described with reference to the use of an enzyme labeled monoclonal antibody. Some of the enzymes utilized as labels are alkaline phosphatase, glucose oxidase, ' galactosidase, peroxidase, or urease, and the like.
Such linkage with enzymes can be accomplished by any one of the conventional and known methods, such as the Staphylococcal Protein A method, the glutaraldehyde method, the benzoquinone method, or the periodate method.
Once the labeled monoclonal antibody is formed, testing is carried out employing one of a wide variety of conventional immunoassay vary according to the monoclonal antibody and the label chosen. At the present time, enzyme immunoassays are preferred due to their low cost, reagent stability, safety, sensitivity, and ease of procedure. One example is enzyme- linked immunosorbent assay (EIA) . EIA is a solid phase assay system which is similar in design to the radiometric assay, but which util¬ izes an enzyme in place of a radioactive isotope as the immunoglobulin marker.
Fluorescent-immunoassay is based on the labeling of antigen or antibody with fluorescent probes. A nonlabeled antigen and a specific antibody are combined with identical fluorescently labeled antigen. Both labeled and unlabeled antigen compete for antibody binding sites. The amount of labeled antigen bound to the antibody is dependent upon, and therefore a measurement of, the concentration of nonlabeled antigen. Examples of this particular type of fluorescent- immunoassay would include heterogenous systems such as Enzyme-Linked Fluorescent Immunoassay, or homogeneous systems such as the Substrate Labeled Fluorescent Immunoassay. The most suit¬ able fluorescent probe, and the one most widely - ¬ used is fluorescein. While fluorescein can be subject to considerable interference from scattering, sensitivity can be increased by the use of a fluorometer optimized for the probe utilized in the particular assay and in which the effect of scattering can be minimized.
In fluorescence polarization, a labeled sample is excited with polarized light and the degree of polarization of the emitted light is measured. As the antigen binds to the antibody its rotation slows down and the degree of polari¬ zation increases. Fluorescence polarization is simple, quick, and precise. However, at the present time its sensitivity is limited to the micromole per liter range and upper nano- mole per liter range with respect to antigens in biological samples.
Luminescence is the emission of light by an atom or molecule' as an electron is transferred to the ground state from a higher energy state. In both chemiluminescent and bioluminescent reactions, the free energy of a chemical reaction provides the energy required to produce an inter¬ mediate reaction or product in an electronically excited state. Subsequent decay back to the ground state is accompanied by emission of light. Bioluminescence is the name given to a special form of chemiluminescence found in biological systems, in which a catalytic protein or enzyme, such as luciferase, increases the efficiency of the luminescent reaction. The best known chemiluminescent substance is luminol.
A further aspect of the present invention is a therapeutic composition comprising one or more of the monoclonal antibodies to the particular Hepatitis antigen or virus, as well as a pharmacologically acceptable carrier or diluent. Such compositions can be used to treat humans and/or animals afflicted with some form of Hepatitis virus and they are used in amounts effective to cure; an amount which will vary widely dependent upon the individual being treated and the severity of the infection.
One or more of the monoclonal antibodies can be assembled into a diagnostic kit for use in diagnosing for the presence of an antigen, antigens, or viruses of Hepatitis in various specimens. It is also possible to use the broadly cross-reactive monoclonal antibody which can identify the Hepatitis virus alone or as part of a kit containing antibodies that can identify other viral or bacterial genera or species. In the past there have been difficulties . in developing rapid kits because of undesirable cross-reactions of specimens with antiserum. The use of monoclonal antibodies can eliminate these problems and provide highly specific and rapid tests for diagnosis. A rapid and precise kit could replace or augment existing tests and permit early direct therapy. A rapid diag¬ nosis of a viral infection could represent sub¬ stantial patient and hospital savings. Addi¬ tionally, a kit can be used on an out-patient basis. At present the lack of a rapid test giving "same day" answers may delay the initiation of treatment until the patient has developed more severe symptoms or may require the initiation of more costly therapy in a sick patient. A test that wouid return results within an hour or two would be a substantial convenience to patients.
In addition to being sold individually, the kit could be included as a component in a comprehensive line of compatible immunoassay reagents sold to reference laboratories to detect the species and serotypes of Hepatitis.
One preferred embodiment of the present invention is a diagnostic kit comprising at least one labeled monoclonal antibody against a particular Hepatitis antigen or species, as well as any appropriate stains, counterstains, or reagents. Further embodiments include kits containing at least one control sample of a Hepatitis antigen and/or a cross-reactive labeled monoclonal antibody which would detect the pres¬ ence of any of the Hepatitis viruses in a partic¬ ular sample. Specific antigens to be detected in this kit include the antigens of Hepatitis A and Hepatitis B; in particular, the Hepatitis B surface antigen (HBsAg).
Monoclonal diagnostics which detect the presence of Hepatitis antigens can also be used in periodic testing of water sources, food sup¬ plies and food processing operations. Thus, while the present invention describes the use of the labeled monoclonal antibodies to determine the presence of a standard antigen, the invention can have many applications in diagnosing the presence of antigens by determining whether specimens such as urine, blood, stool, water, milk, and the like contain the particular Hepati¬ tis antigen. Also, the invention could be utiliz¬ ed as a public health and safety diagnostic aid, whereby specimens such as water or food could be tested for possible contamination by Hepatitis A.
The invention will be further illustrated in connection with the following example which is set forth for purposes of illustration only and not by way of limitation.
EXAMPLE
A monoclonal antibody was prepared generally according to the method of Kohler and Milstein (Eur. J. Immunol. §_, 292 (1975)).
A. Antigen Preparation
Surface (HBsAg) antigen of the Hepati¬ tis B virus was used and it was prepared by Dr. Colin Howard of the London School of Hygiene and Tropical Medicine.
B. Animal Immunization
Six Balb/c mice were injected with the prepared antigen. They were given one intraperi- toneal injection per week (5 ug/injection) of Hepatitis B virus surface antigen for a total of three weeks followed by two weekly intravenous injections, plus one in Freunds Complete Adjuvant (F.C.A.) after one week. The mice were then be bled approximately six days after the last injection and the serum tested for antibodies by assay. The conventional assay used for this serum titer testing was the enzyme-linked immuno- sorbent assay system. When the mice showed antibody production after this regimen, generally a positive titer of at least 10,000, a mouse was selected as a fusion donor and given a booster injection (0.02 ml of 10-*' cells/ml) intravenously three days prior to splenectomy. C. Cell Fusion
Spleen cells from the immune mice were then harvested three days after boosting by conventional techniques. First the donor mouse selected was killed and surface sterilized by immersion in 70% ethyl alcohol. The spleen was then removed and immersed in approximately 2.5 ml of Dulbeccos Modified Eagles Medium (DMEM) to which had been added 3% fetal calf serum (FCS). The spleen was then gently homogenized in a LUX homogenizing tube until all cells have been released from the membrane and the cells washed in 5 ml 3% FCS DMEM. The cellular debris was then allowed to settle and the spleen cell suspension placed in a 10 ml centrifuge tube. The debris was then rewashed in 5 ml 3% FCS DMEM. Fifty mis of suspension were then made in 3% FCS DMEM.
The myeloma cell line used was NS0 (unclon- ed), obtained from the MRC Laboratory of Molecular - - Biology in Cambridge, England. The myeloma cells were in the log growth phase, and rapidly dividing. Each cell line was washed using a tissue culture medium DMEM containing 3% fetal calf serum.
The spleen cells were then spun down at the same time that a relevant volume of myeloma cells were spun down (room 'temperature for 7 minutes at 600 g) , and each resultant pellet was then separately resuspended in 10 ml 3% FCS DMEM. In order to count the myeloma cells, 0.1 ml of the suspension was diluted to 1 ml and used with a haemacytometer with phase micro¬ scope to make the count. In order to count the spleen cells, 0.1 ml .of the suspension was diluted to 1 ml with Methyl Violet and used with a haemacytometer and light microscope and the stained nuclei of the cells counted. lO*^ spleen cells were then mixed with 5x10? myeloma cells, the mixture washed in serum- free DMEM high in glucose and centrifuged and all the liquid removed. The resultant cell pellet was placed in a 37° C. water bath and there was added over the period of one minute 1 ml of a 50% wt. vol. solution of polyethylene glycol 1500 (PEG) in saline Hepes; pH of approxi¬ mately 7.5, and the mixture gently stirred for approximately one and one-half minutes. There was then slowly added 10 ml of serum-free tissue culture medium DMEM, followed by the addition of up to 50 ml of such culture medium, centrifu¬ gation and removal of all the supernatant, and resuspension of the cell pellet in 10 ml of DMEM containing 18% by weight fetal calf serum.
Ten microliters of the mixture was placed in each of 500 wells of standard multiwell tissue culture plates. Each well contained 1.0 ml of the standard HAT medium (hypoxanthine, amino- pterin, and thymidine) and a feeder layer of Balb/c macrophages at a concentration of 5xl04 macrophages/well.
The wells were kept undisturbed and cultured at 37° C. in 9% CO2 air at approximately 100% humidity. The wells were then analyzed for growth utilizing the conventional inverted micro¬ scope procedure after about 5 to 10 days. In those wells in which growth was present in the inhibiting HAT medium, screening tests for the specific monoclonal antibody were made utilizing the conventional enzyme immunoassay screening method described below. Somewhere around 10 days to 14 days after fusion, sufficient antibody against the antigen to the Hepatitis B virus had been developed in at least one well.
D. Cloning
From those wells which yielded antibody against the antigen, cells are removed and cloned using the standard dilution or agar method.
The clones were assayed by the enzyme immunoassay method to determine antibody production. A positive clone may be recloned.
E. Monoclonal Selection The monoclonal antibodies from the clones are screened by the standard techniques for binding to Hepatitis B virus surface antigen, prepared as in the immunization, and for specificity in a test battery of Hepatitis virus antigens and known trace contaminant antigens.
F. Antibody Production
Balb/c mice were primed with pristane and injected
7 intraperitoneally with 10 cells of monoclonal antibody specific against the antigen. The ascites fluid was harvested when the mice were swollen with fluid but still alive. The fluid was centrifuged at 1200 g for approximately 10 minutes, the cells discarded, and the antibody-rich ascites fluid collected, and stored at -20 C. 5 ml of ascites fluid were filtered through glass wool and centrifuged at 30,000 g for 10 minutes. The ascites was then stirred at +4°C and an equal volume of cold, saturated ammonium sulphate added slowly. The mixture was stirred for a further 30 minutes after addition is complete. The precipitate was harvested by centrifugation at 10,000 g for 10 minutes. The precipi¬ tate was dissolved in a minimum volume of cold phosphate/EDTA buffer (20 mM sodium phosphate, 10 mM EDTA, pH 7.5, + 0.02% sodium azide) . The solution is dialyzed versus 2x1000 ml of the same buffer at +4°C. The dialyzed, redissolved precipitate was centrifuged at 30,000 g for 10 minutes and applied to a 10 ml column of DEAE-cellulose, previously equilibrated in phosphate/EDTA buffer. The monoclonal antibody was eluted with phosphate/EDTA buffer.
The purified antibody was titrated by solid-phase EIA. G. Enzyme-Monoclonal Linkage
The monoclonal antibody specific against the antigen, prepared and screened as described above, is then bound to an appropriate enzyme such as horseradish peroxidase or a highly-purified alkaline phosphatase. This can be accomplished by any known method, e.g. the one-step glutaraldehyde method, the periodate coupling method or benzoquinone conjugation.
In the glutaraldehyde method, monoclonal antibody (3 mg in about 1 ml of solution) is dialyzed with 10 mg of alkaline phosphatase (Sigma Type VII-T) against 2x1000 ml of phosphate buffered saline, pH 7.4 (PBS) at +4°C After dialysis the volume is made up to 2.5 ml with PBS and 25 μl of a 20% glutaraldehyde in PBS solution added. The conjugation mixture is left at room temperature for 1.5 hours. After this time, gluteraldehyde is removed by gel filtration on a Pharmacia PD-10 (Sephadex G-25M) column, previously equilibrated in PBS. The conjugate is eluted with 3.5 ml PBS and then dialyzed against 2x2000 -ml of TRIS buffer (50 mM TRIS, 1 mM magnesium chloride, pH 8.0 plus 0.02% sodium azide) at +4°C. To the dialyzed conjugate is added 1/lOth its own volume of 10% BSA in TRIS buffer. The conjugate is then sterile filtered through a 0.22 μm membrane filter into a sterile amber vial and stored at +4°C.
In the benzoquinone method, 24 mg alkaline phosphatase (Sigma Type VII-T) are dialysed against 2 x 500 ml of 0.25M sodium phosphate buffer, pH 6.0, at +4°C. Para-benzoquinone, 18 mg, is dissolved in warm AR ethanol, 0.6 ml, and added to the dialysed alkaline phosphatase. The benzoquinone/alkaline phosphatase mixture is left in the dark at room temperature for 1 hour. After this time, unreacted benzoquinone and reaction by-products are removed and the buffer exchanged, by gel filtration on a Pharmacia PD-10 (Sephadex G-25M) column, previously equilibrated, in 0.15M sodium chloride. The benzoquinone-activated alkaline phosphatase thus produced is sufficent for three 3 mg antibody conjugations.
3 mg monoclonal antibody are dialysed against 2 x 500 ml of 0.15M sodium chloride at +4°C. Dialysed antibody is added to 8 mg of benzoquinone-activated alkaline phosphatase, immediately followed by sufficient 1M sodium bicarbonate to give a final concentration of 0.1M. The conjugation mixture is left in the dark at +4°C for 48 hours. After this time, sufficient 1M lysine is added to give a final concentration of 0.1M. After 2 hours in the dark at room temperature, the conjugate is dialysed against 2 x 1000 ml of phosphate buffered saline + 0.02% sodium azide at +4°C. An equal volume of glycerol is added. The conjugate is sterile-filtered through a 0.22 μ membrane filter into a sterile amber vial and stored at +4°C.
H. Monoclonal Antibody Conjugate Testing
The enzyme immunoassay method is used for testing. This assay method comprises coating the wells of a standard polyvinyl chloride microtiter tray with the antigen, followed by addition of monoclonal antibody enzyme conjugate, and finally addition of the enzyme substrate, p-nitrophenyl phosphate. The monoclonal antibody, found to be specific for the antigen, can then be tested. Four monoclonal antibodies have been tested; three of the class IgGl and one of the class IgG2a have been found, and have been numbered as 80, 192, 329 and 341. Antibody 80, of class IgG2a, can detect antigen at 5-10 ng/ml. 80 did not bind with the synthetic peptides corresponding to amino-acid sequences 124 to 137 and 139 to 147 of the Hepatitis B surface antigen. In like manner, 192 and 341 did not bind with sequence 139 to 147, although 192 did bind with sequence 124 to 137. Reference to these antibodies and their common properties (IgG, sequence binding etc.) may also be found in the copending Patent Applications claiming priority from British Patent Application No. 8431170.
If deemed necessary, the particular epitopic site to which the antibody attaches to the antigen can also be " determined. The same enzyme immunoassay method can also be used to determine whether diagnostic specimens such as urine, blood, stool, water or milk contain the antigen. In such cases, the antibody can first be bound to the plate.
While the invention has been described in connection with certain preferred embodiments, it is not intended to limit the scope of the invention to the particular form set forth, but, on the contrary, it is intended to cover such alternatives, modifications, and equivalents as may be included within the spirit and scope of the invention as defined by the appended claims.

Claims

WHAT IS CLAIMED IS:
1. A high affinity monoclonal antibody specific for an antigen of a Hepatitis virus.
2. The antibody of Claim 1 specific to an antigen of Hepatitis B virus.
3. The antibody of Claim 1 specific to an antigen of Hepatitis A virus.
4. The antibody of Claim 1 specific to a surface antigen , core antigen, e antigen or delta antigen of Hepatitis B virus.
5. A monoclonal antibody broadly cross- reactive with antigens of Hepatitis virus.
6. A labeled monoclonal antibody consisting essentially of a monoclonal antibody of Claims 1-5 and an appropriate label.
- 7. The labeled monoclonal antibody of Claim 6, wherein said label is a member of the group selected from a radioactive isotope, enzyme, fluorescent compound, bioluminescent compound, chemiluminescent compound, or ferromagnetic atom, or particle.
8. The labeled monoclonal antibody of Claim 7, wherein said label is an enzyme capable of conjugating with a monoclonal antibody and of being used in an enzyme-linked immunoassay procedure.
9. The labeled monoclonal antibody of Claim 8, wherein said enzyme is alkaline phos¬ phatase, glucose oxidase, galactosidase, or peroxidase. ^
10. The labeled monoclonal antibody of
Claim 7, wherein said label is a fluorescent compound or probe capable of being used in an immuno-fluorescent or fluorescent immunoassay procedure, enzyme fluorescent immunoassay, δr fluorescence polarization immunoassay, photon counting immunoassay, or the like procedure.
11. The labeled monoclonal antibody of Claim 10, wherein said fluorescent compound or probe is fluorescein.
12. The labeled monoclonal antibody of Claim 7, wherein said label is a chemiluminescent compound capable of being used in a luminescent or enzyme-linked luminescent immunoassay.
13. The labeled monoclonal antibody of Claim 12, wherein such chemiluminescent compound is luminol or a luminol derivative.
14. The labeled monoclonal antibody of Claim 7, wherein said label is a bioluminescent compound capable of being used in an appropriate bioluminescent immunoassay.
15. The labeled monoclonal antibody of
Claim 14, wherein such bioluminescent compound is luciferase or a luciferase derivative.
16. A process for diagnosing for the pre¬ sence of an antigen of a Hepatitis virus in a specimen comprising contacting at least a portion of said specimen with a labeled monoclonal antibody of Claim 6 in an immunoassay procedure appropriate for said label.
17. The process of Claim 16, wherein the appropriately labeled immunoassay procedure is selected from immuno-fluorescent or fluorescent immunoassay, immuno-electron microscopy, radio- metric assay systems, enzyme-linked immunoassays, fluorescence polarization, photon-counting bio¬ luminescent, or chemiluminescent immunoassay.
18. The process of Claim 17, wherein said label is an enzyme capable of being used in an enzyme-linked immunoassay procedure.
19. The process of Claim 18, wherein said enzyme is selected from alkaline phosphatase, glucose oxidase, galactosidase, or peroxidase.
20. The process of Claim 17, wherein said label is a fluorescent compound or probe capable of being used in an immuno-fluorescent or fluores¬ cent immunoassay procedure, enzyme fluorescent immunoassay, or fluorescence polarization immuno¬ assay, or photon-counting immunoassay, or the like procedure.
21. The process of Claim 20, wherein said fluorescent compound or probe is fluorescein.
22. The process of Claim 17, wherein said label is a chemiluminescent compound capable luminescent immunoassay.
23. The process of Claim 22, wherein said chemiluminescent compound is luminol or a luminol derivative.
24. The process of Claim 17, wherein said label is a bioluminescent compound capable of being used in a bioluminescent or enzyme-linked bioluminescent immunoassay.
25. The process of Claim 24, wherein said bioluminescent compound is luciferase or a lucif- erase derivative.
26. A therapeutic composition comprising one or more of the monoclonal antibodies of Claims 1-5 and a pharmaceutically acceptable carrier or diluent.
27. A therapeutic composition comprising one or more of the labeled monoclonal antibodies in Claim 6 and a pharmaceutically acceptable carrier or diluent.
0)
28. A method of treating Hepatitis infec¬ tions comprising administering an effective amount of a monoclonal antibody of Claims 1-5.
29. A kit for diagnosing for the presence of an antigen or species of Hepatitis virus in a diagnostic specimen comprising at least one monoclonal antibody of Claims 1-5.
30. The kit of Claim 29, wherein said at least one antibody is labeled.
31. The kit of Claim 30, wherein said at least one monoclonal antibody is labeled with a fluorescent compound.
32. The kit as in Claim 30, wherein said at least one monoclonal antibody is labeled with an enzyme.
33. The kit as in Claim 30, wherein said at least one monoclonal antibody is labeled with a member of the group consisting of a radio¬ active isotope, chemiluminescent compound, bio- luminescent compound, ferromagnetic atom, or particle. — .to—
34. The kit of Claims 30, 31, 32, and 33 additionally containing at least one known antigen of Hepatitis virus as a control.
35. The kit of Claims 30, 31, 32, 33, and 34 containing each known antigen of Hepatitis
A and/or Hepatitis B virus.
36. The kit of Claims 30, 31, 32, 33, and 34 containing the antigens of Hepatitis A virus.
0 37. The kit of Claims 30, 31, 32, 33, and 34 containing the antigens of Hepatitis B virus.
38. The kit of Claims 30, 31, 32, 33, and 34 containing the surface antigens (HBsAG) 5 of Hepatitis B virus.
39. A kit for diagnosing for the presence of an antigen or species of Hepatitis virus in a diagnostic specimen comprising at least one monoclonal antibody of Claims 1-5 and a C control.
40. The kit of Claim 39, wherein said at least one antigen is labeled and said control is at least one known antigen of Hepatitis.
41. A kit for diagnosing for the presence of a Hepatitis infection comprising at least one monoclonal antibody of Claims 1-5.
42. The kit of Claim 41, wherein said at least one monoclonal antibody is labeled.
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WO1990006514A1 (en) * 1988-12-01 1990-06-14 Centocor, Inc. Immunometric assay for tumor necrosis factor-alpha
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EP0268987A3 (en) * 1986-11-21 1991-01-09 E.I. Du Pont De Nemours And Company Assays for antibody to hepatitis b core antigen
WO1990006514A1 (en) * 1988-12-01 1990-06-14 Centocor, Inc. Immunometric assay for tumor necrosis factor-alpha
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