EP0199756A1 - Monoclonal antibodies and their use - Google Patents

Monoclonal antibodies and their use

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Publication number
EP0199756A1
EP0199756A1 EP19850905086 EP85905086A EP0199756A1 EP 0199756 A1 EP0199756 A1 EP 0199756A1 EP 19850905086 EP19850905086 EP 19850905086 EP 85905086 A EP85905086 A EP 85905086A EP 0199756 A1 EP0199756 A1 EP 0199756A1
Authority
EP
European Patent Office
Prior art keywords
monoclonal antibody
antigen
entamoeba
immunoassay
labeled
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Withdrawn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
EP19850905086
Other languages
German (de)
French (fr)
Inventor
Bruce William Wright
Peter John Church Cottage Church Road COX
Alice Margaret Noyes
Danny Widdows
Simon James Yvon Saxby
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.)
TECHNOLOGY LICENCE Co Ltd
Original Assignee
TECHNOLOGY LICENCE Co Ltd
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 TECHNOLOGY LICENCE Co Ltd filed Critical TECHNOLOGY LICENCE Co Ltd
Publication of EP0199756A1 publication Critical patent/EP0199756A1/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Withdrawn legal-status Critical Current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
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/20—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans from protozoa
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56905—Protozoa
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • monoclonal antibodies specific for the antigens or species of Entamoeba are desired which when used will rapidly diagnose the presence of such organisms in specimens.
  • Entamoeba histolytica the major causative agent
  • Amebiasis occurs in every country of the world. In some populations 50 percent of the persons may be carrying the organ ⁇ ism Entamoeba histolytica at one time in their bowel. A smaller number of persons will suffer severe, bloody dysentery or diarrhea from the organism. A certain percentage, as many as 1-5 percent, in some populations, may suffer widespread abcesses that can be fatal, involving the liver, the lung lining, or the brain. Because of this potentially fatal complication, amebiasis is a disease much dreaded by travelers. It has become more common in the United States
  • SUBSTITUTESHEET because of widespread sexual promiscuity, which has led in certain populations to the sexual transmission of amebiasis, particularly among homosexual males. Under this circumstance, the need to diagnose and treat the organism has risen sharply in the United States.
  • the present methods of detection are cumbersome, time consuming, and insensitive with multiple stool cultures and microscopic examination being required.
  • the ability of monoclonal antibodies specifically to bind to antigens of Entamoeba can provide many opportunities for diagnosis and treatment. Such specificity is a most impor ⁇ tant requirement for proper and accurate analysis and/or diagnosis, particularly in diagnosing the presence of diseases which require prompt treatment.
  • isotopic and nonisotopic immunoassays have been utilized in conjunction with monoclonal antibodies to test for the pres ⁇ ence of an antigenic substance.
  • agglutination, immuno-fluorescent, chemilum- inescent or fluorescent immunoassay, 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 organism can be determined, 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 Entamoeba antigens and/or organisms.
  • the present invention com ⁇ prises monoclonal antibodies specific for an antigen or species of Entamoeba; in particular, the antigens or species of Entamoeba histolytica (designated as Entamoeba histolytica I, II, III, IV, V, VI, VII, or VIII), as well as a monoclonal antibody broadly cross-reactive with an antigen for ' each species of the genus Entamoe ⁇ ba.
  • the invention also comprises labeled mono ⁇ clonal antibodies for use in diagnosing the presence of the Entamoeba antigens, each com ⁇ prising a monoclonal antibody against one of the above-mentioned antigens to Entamoeba 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 Entamoeba anti ⁇ gens or organisms 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 Entamoeba and a carrier or diluent, as well as kits contain ⁇ ing at least one labeled monoclonal antibody to an antigen of an Entamoeba.
  • the monoclonal antibodies of the present invention are prepared by fusing spleen cells, from a mammal which has been immunized against the particular Entamoeba 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 (hy- poxanthine, 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 Entamoeba 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 Entamoeba 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 Entamoeba 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 Entamoeba antigen.
  • the monoclonal antibody selected, which is specific for the particular Entamoeba antigen or species, 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 im unosorbent 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 Entamoeba antigen or species, 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 Entamoeba infections and they are used in amounts effective to cure; an amount which will vary widely dependent upon the individ ⁇ ual being treated and the severity of the infec ⁇ tion.
  • 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 species of Entamoeba in various specimens. It is also possible to use the broadly cross-reactive monoclonal antibody which can identify the genus Entamoeba alone or as part of a kit containing antibodies that can identify other bacterial genera or species of Entamoeba and/or other bacteria.
  • kits 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 using precise antibiotics. Avoiding multiple antibiotics or more expensive or hazardous antibiotics would represent substantial patient and hospital sav ⁇ ings.
  • 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 would 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 Entamoeba.
  • One preferred embodiment of the present invention is a diagnostic kit comprising at least one labeled monoclonal antibody against a particular Entamoeba antigen or species, as well- as any appropriate stains, counterstains, or reagents. Further embodiments include kits containing at least one control sample of an Entamoeba antigen and/or a cross-reactive labeled monoclonal antibody which would detect the pres ⁇ ence of any of the Entamoeba organisms in a particular sample.
  • Specific antigens to be detected in this kit include the antigens of Entamoeba histolytica (applicant has further divided this species into eight subgroups: Entamoeba histolytica I, II, III, IV, V, VI, VII, or VIII) .
  • Monoclonal diagnostics which detect the presence of Entamoeba 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 Entamoe ⁇ ba antigen. More particularly, the invention could be utilized as a public health and safety diagnostic aid, whereby specimens such as water or food could be tested for possible contamination.
  • DMEM Dulbecco's Modified Eagles Medium
  • FCS Foetal Calf Serum
  • PBS phosphate-buffered saline % T refers to vaccine concentration measured in a 1 cm light path
  • Monoclonal antibodies of the present invention are prepared generally according to the method of Koehler and Milstein, Eur. J. Immunol. 6_, (1975) 292. EXAMPLE 1
  • mice are injected with prepared Entamoeba histolytica antigen obtained in the form of a pellet from the Liverpool School of Tropical Medicine, Strain Title 200 N1H. Immunisation is intramuscular (in Complete Freunds Adjuvant) and, 3 weeks later, intravenous (in PBS) , using injections (0.05 ml 80% T vaccine) of vaccine prepared as above. The mice are bled approximately six days after the last injection and the serum tested for antibodies by assay. A conventional assay used for this serum titer testing is the enzyme-linked immunosorbent assay system.
  • mice show antibody production after this regimen, generally a positive titer of at least 10,000, a mouse is selected as a fusion donor and given a booster injection (0.02 ml 80% T vaccine) intravenously, three days prior to splenectomy.
  • Spleen cells from the immune mice are harvested three days after boosting, by conventional techniques.
  • the donor mouse selected is killed and surface-sterilised by immersion in 70% ethyl alcohol.
  • the spleen is then removed and immersed in approximately 2.5 ml DMEM to which has been added 3% FCS.
  • the spleen is then gently homogenised in a LUX homogenising tube until all cells have been released from the membrane, and the cells are washed in 5 ml 3% FCS-DMEM.
  • the cellular debris is then allowed to settle and the spleen cell suspension placed in a 10 ml centrifuge tube.
  • the debris is then rewashed in 5 ml 3% FCS-DMEM. 50 ml suspension are then made in 3% FCS-DMEM.
  • the myeloma cell line used is NS0 (uncloned) , obtained from the MRC Laboratory of Molecular Biology in Cambridge, England.
  • the myeloma cells are in the log growth phase, and rapidly dividing.
  • Each cell line is washed using, as tissue culture medium, DMEM containing 3% FCS.
  • the spleen cells are then spun down at the same time that a relevant volume of myeloma cells are spun down (room temperature for 7 minutes at 600 g) , and each resultant pellet is then separately resuspended in 10 ml 3% FCS-DMEM.
  • a relevant volume of myeloma cells are spun down (room temperature for 7 minutes at 600 g)
  • each resultant pellet is then separately resuspended in 10 ml 3% FCS-DMEM.
  • 0.1 ml of the suspension is diluted to 1 ml and a haemacytometer with phase microscope is used.
  • 0.1 ml of the suspension is diluted to 1 ml with Methyl Violet-citric acid solution, and a haemacytometer and light microscope are used to count the stained nuclei of the cells.
  • Spleen cells are then mixed with 5 x 10 myeloma cells, the mixture washed in serum-free DMEM high in glucose, and centrifuged, and all the liquid removed.
  • the resultant cell pellet is placed in a 37°C water-bath.
  • 10 ml serum-free tissue culture medium DMEM are then slowly added, followed by up to 50 ml of such culture medium, centrifugation and removal of all the supernatant, and resuspension of the cell pellet in 10 ml of DMEM containing 18% by weight FCS.
  • each well contains 1.0 ml of the standard HAT medium (hypoxanthine, aminopterin and thymidine) and a feeder layer of Balb/c
  • the wells are kept undisturbed, and cultured at 37°C in 9% C0_ air at approximately 100% humidity.
  • the wells are analysed for growth, utilising the conventional inverted microscope procedure, after about 5 to 10 days.
  • screening tests for the specific monoclonal antibody are made utilising the conventional enzyme immunoassay screening method described below. Somewhere around 10 days to 14 days after fusion, sufficient antibody against the antigen may develop in at least one well.
  • cells are removed and cloned using the dilution method.
  • dilution method dilutions of cells suspensions in 18% FCS-DMEM + Balb/c mouse macrophages were made to achieve 1 cell/well and half cell/well in a 96-well microtitre plate. The plates were incubated for 7-14 days at 37 C, 95% RH, 7-9% CO until semi-confluent. The supernatants were then assayed for specific antibody by the standard enzyme immunos ⁇ rbent assay. The clones may be assayed by the enzyme immunoassay method to determine antibody production.
  • the monoclonal antibodies from the clones are screened by the standard techniques for binding to the antigen, prepared as in the immunisation, and for specificity in a test battery of the class bearing different antigens. Specifically, a grid of microtiter plates containing a representative selective of organisms is prepared, boiled, and utilised as a template to define the specificity of the parent group.
  • the EIA immunoassay noted above may be used.
  • Balb/c mice were primed with pristane for at least 7 days, and were then injected with 10 cells of the monoclonal antibody-producing cell line. Ascitic 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 collected and stored at -20 C.
  • Ascites fluid was 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 the addition was complete. The precipitate was harvested by centrifugation at 10,000 g for 10 minutes. The precipitate 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 was dialysed vs 2 x 1000 ml of the same buffer at +4 C.
  • the dialysed, 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.
  • ascites fluid was filtered through glass wool and centrifuged at 30,000 g for 10 minutes.
  • the ascites was then diluted with twice its own volume of cold phosphate buffer (0.1M sodium phosphate, pH 8.2).
  • the diluted ascites was applied to a 2 ml column of Protein A-Sepharose, previously equilibrated with phosphate buffer.
  • the column was washed with 40 ml of phosphate buffer.
  • the monoclonal antibody was eluted with citrate buffer (0.1M sodium citrate, pH 3.5) into sufficient 1M TRIS buffer, pH 9.0 to raise the pH immediately to about 7.5.
  • the eluate was dialysed in PBS, pH 7.4, at 4 C and stored at -20 C.
  • the monoclonal antibody specific against the antigen, prepared as above, is linked to an enzyme, viz. highly-purified alkaline phosphatase.
  • the one-step glutaraldehyde method or benzoquinone conjugation is used.
  • the conjugate is eluted with 3.5 ml PBS and then dialysed against 2 x 2000 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 dialysed 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.
  • Example 1 The general procedure of Example 1 may be followed to produce a monoclonal antibody broadly cross-reactive with an antigen of all species of the genus Entamoeba.
  • Tests using the present invention are superior to existing tests, based on the following advantages: (i) greater accuracy; (ii) same day results, within an hour or two; (iii) reduction in amount of skilled labour required to administer laboratory procedures, resulting in reduced labour costs; (iv) reduction in laboratory time and space used in connection with tests, resulting in reduced overhead expenses; and (v) improved therapy based upon early, precise diagnosis.

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Abstract

Anticorps monoclonaux pour le genre Entamoeba, anticorps marqués, compositions et kits les contenant, et leur utilisation pour le diagnostic et le traitement d'antigènes.Monoclonal antibodies for the genus Entamoeba, labeled antibodies, compositions and kits containing them, and their use for the diagnosis and treatment of antigens.

Description

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 species in specimens such as urine, blood, water, milk, and the like.
More particularly, monoclonal antibodies specific for the antigens or species of Entamoeba are desired which when used will rapidly diagnose the presence of such organisms in specimens.
Divisions have been made among the Entamoeba species. One of the best known species is Ent¬ amoeba histolytica, the major causative agent
SUBSTITUTE SHEET of amebic dysentery. Amebiasis occurs in every country of the world. In some populations 50 percent of the persons may be carrying the organ¬ ism Entamoeba histolytica at one time in their bowel. A smaller number of persons will suffer severe, bloody dysentery or diarrhea from the organism. A certain percentage, as many as 1-5 percent, in some populations, may suffer widespread abcesses that can be fatal, involving the liver, the lung lining, or the brain. Because of this potentially fatal complication, amebiasis is a disease much dreaded by travelers. It has become more common in the United States
SUBSTITUTESHEET because of widespread sexual promiscuity, which has led in certain populations to the sexual transmission of amebiasis, particularly among homosexual males. Under this circumstance, the need to diagnose and treat the organism has risen sharply in the United States. The present methods of detection are cumbersome, time consuming, and insensitive with multiple stool cultures and microscopic examination being required. The ability of monoclonal antibodies specifically to bind to antigens of Entamoeba can provide many opportunities for diagnosis and treatment. Such specificity is a most impor¬ tant 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 nonisotopic immunoassays have been utilized 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 immunoassay, 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 organism can be determined, 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. _, 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 requlred 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 Entamoeba antigens and/or organisms.
Briefly stated, the present invention com¬ prises monoclonal antibodies specific for an antigen or species of Entamoeba; in particular, the antigens or species of Entamoeba histolytica (designated as Entamoeba histolytica I, II, III, IV, V, VI, VII, or VIII), as well as a monoclonal antibody broadly cross-reactive with an antigen for' each species of the genus Entamoe¬ ba.
The invention also comprises labeled mono¬ clonal antibodies for use in diagnosing the presence of the Entamoeba antigens, each com¬ prising a monoclonal antibody against one of the above-mentioned antigens to Entamoeba 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 Entamoeba anti¬ gens or organisms 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 Entamoeba and a carrier or diluent, as well as kits contain¬ ing at least one labeled monoclonal antibody to an antigen of an Entamoeba.
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 Entamoeba 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 (hy- poxanthine, 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 Entamoeba 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 Entamoeba 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 Entamoeba 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 Entamoeba antigen. The monoclonal antibody selected, which is specific for the particular Entamoeba antigen or species, 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 methods. The particular method chosen will 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, sa,fety, sensitivity, and ease of procedure. One example is enzyme- linked im unosorbent 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 Entamoeba antigen or species, 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 Entamoeba infections and they are used in amounts effective to cure; an amount which will vary widely dependent upon the individ¬ ual being treated and the severity of the infec¬ tion.
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 species of Entamoeba in various specimens. It is also possible to use the broadly cross-reactive monoclonal antibody which can identify the genus Entamoeba alone or as part of a kit containing antibodies that can identify other bacterial genera or species of Entamoeba and/or other bacteria.
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 using precise antibiotics. Avoiding multiple antibiotics or more expensive or hazardous antibiotics would represent substantial patient and hospital sav¬ ings. Additionally, 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 would 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 Entamoeba.
One preferred embodiment of the present invention is a diagnostic kit comprising at least one labeled monoclonal antibody against a particular Entamoeba antigen or species, as well- as any appropriate stains, counterstains, or reagents. Further embodiments include kits containing at least one control sample of an Entamoeba antigen and/or a cross-reactive labeled monoclonal antibody which would detect the pres¬ ence of any of the Entamoeba organisms in a particular sample. Specific antigens to be detected in this kit include the antigens of Entamoeba histolytica (applicant has further divided this species into eight subgroups: Entamoeba histolytica I, II, III, IV, V, VI, VII, or VIII) .
Monoclonal diagnostics which detect the presence of Entamoeba 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 Entamoe¬ ba antigen. More particularly, the invention could be utilized as a public health and safety diagnostic aid, whereby specimens such as water or food could be tested for possible contamination.
The invention will be further illustrated in connection with the following Examples which are set forth for the purposes of illustration only and not by way of limitation.
In the Examples:
DMEM = Dulbecco's Modified Eagles Medium
FCS = Foetal Calf Serum
PBS = phosphate-buffered saline % T refers to vaccine concentration measured in a 1 cm light path
Monoclonal antibodies of the present invention are prepared generally according to the method of Koehler and Milstein, Eur. J. Immunol. 6_, (1975) 292. EXAMPLE 1
A. Animal Immunisation
Balb/c mice are injected with prepared Entamoeba histolytica antigen obtained in the form of a pellet from the Liverpool School of Tropical Medicine, Strain Title 200 N1H. Immunisation is intramuscular (in Complete Freunds Adjuvant) and, 3 weeks later, intravenous (in PBS) , using injections (0.05 ml 80% T vaccine) of vaccine prepared as above. The mice are bled approximately six days after the last injection and the serum tested for antibodies by assay. A conventional assay used for this serum titer testing is the enzyme-linked immunosorbent assay system. When the mice show antibody production after this regimen, generally a positive titer of at least 10,000, a mouse is selected as a fusion donor and given a booster injection (0.02 ml 80% T vaccine) intravenously, three days prior to splenectomy.
B. Cell Fusion
Spleen cells from the immune mice are harvested three days after boosting, by conventional techniques. First,' the donor mouse selected is killed and surface-sterilised by immersion in 70% ethyl alcohol. The spleen is then removed and immersed in approximately 2.5 ml DMEM to which has been added 3% FCS. The spleen is then gently homogenised in a LUX homogenising tube until all cells have been released from the membrane, and the cells are washed in 5 ml 3% FCS-DMEM. The cellular debris is then allowed to settle and the spleen cell suspension placed in a 10 ml centrifuge tube. The debris is then rewashed in 5 ml 3% FCS-DMEM. 50 ml suspension are then made in 3% FCS-DMEM.
The myeloma cell line used is NS0 (uncloned) , obtained from the MRC Laboratory of Molecular Biology in Cambridge, England. The myeloma cells are in the log growth phase, and rapidly dividing. Each cell line is washed using, as tissue culture medium, DMEM containing 3% FCS.
The spleen cells are then spun down at the same time that a relevant volume of myeloma cells are spun down (room temperature for 7 minutes at 600 g) , and each resultant pellet is then separately resuspended in 10 ml 3% FCS-DMEM. In order to count the myeloma cells, 0.1 ml of the suspension is diluted to 1 ml and a haemacytometer with phase microscope is used. In order to count the spleen cells, 0.1 ml of the suspension is diluted to 1 ml with Methyl Violet-citric acid solution, and a haemacytometer and light microscope are used to count the stained nuclei of the cells.
Spleen cells are then mixed with 5 x 10 myeloma cells, the mixture washed in serum-free DMEM high in glucose, and centrifuged, and all the liquid removed. The resultant cell pellet is placed in a 37°C water-bath. 1 ml of a 50 w/v solution of polyethylene glycol 1500 (PEG) in saline Hepes, pH approximately 7.5, is added, and the mixture gently stirred for approximately 1.5 minutes. 10 ml serum-free tissue culture medium DMEM are then slowly added, followed by up to 50 ml of such culture medium, centrifugation and removal of all the supernatant, and resuspension of the cell pellet in 10 ml of DMEM containing 18% by weight FCS.
10 μl of the mixture are placed in each of 480 wells of standard multiwell tissue culture plates. Each well contains 1.0 ml of the standard HAT medium (hypoxanthine, aminopterin and thymidine) and a feeder layer of Balb/c
4 macrophages at a concentration of 5 x 10 macrophages/well.
The wells are kept undisturbed, and cultured at 37°C in 9% C0_ air at approximately 100% humidity. The wells are analysed for growth, utilising the conventional inverted microscope procedure, after about 5 to 10 days. In those wells in which growth is present in the inhibiting HAT medium, screening tests for the specific monoclonal antibody are made utilising the conventional enzyme immunoassay screening method described below. Somewhere around 10 days to 14 days after fusion, sufficient antibody against the antigen may develop in at least one well. c. Cloning
From those wells which yielded antibody against the antigen, cells are removed and cloned using the dilution method. In limiting dilution, dilutions of cells suspensions in 18% FCS-DMEM + Balb/c mouse macrophages were made to achieve 1 cell/well and half cell/well in a 96-well microtitre plate. The plates were incubated for 7-14 days at 37 C, 95% RH, 7-9% CO until semi-confluent. The supernatants were then assayed for specific antibody by the standard enzyme immunosσrbent assay. The clones may be assayed by the enzyme immunoassay method to determine antibody production.
D. Monoclonal Selection
The monoclonal antibodies from the clones are screened by the standard techniques for binding to the antigen, prepared as in the immunisation, and for specificity in a test battery of the class bearing different antigens. Specifically, a grid of microtiter plates containing a representative selective of organisms is prepared, boiled, and utilised as a template to define the specificity of the parent group. The EIA immunoassay noted above may be used.
E. Antibody Production and Purification
Balb/c mice were primed with pristane for at least 7 days, and were then injected with 10 cells of the monoclonal antibody-producing cell line. Ascitic 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 collected and stored at -20 C.
Ascites fluid was 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 the addition was complete. The precipitate was harvested by centrifugation at 10,000 g for 10 minutes. The precipitate 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 was dialysed vs 2 x 1000 ml of the same buffer at +4 C. The dialysed, 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.
Alternatively, ascites fluid was filtered through glass wool and centrifuged at 30,000 g for 10 minutes. The ascites was then diluted with twice its own volume of cold phosphate buffer (0.1M sodium phosphate, pH 8.2). The diluted ascites was applied to a 2 ml column of Protein A-Sepharose, previously equilibrated with phosphate buffer. The column was washed with 40 ml of phosphate buffer. The monoclonal antibody was eluted with citrate buffer (0.1M sodium citrate, pH 3.5) into sufficient 1M TRIS buffer, pH 9.0 to raise the pH immediately to about 7.5. The eluate was dialysed in PBS, pH 7.4, at 4 C and stored at -20 C. F. Enzyme-Monoclonal Linkage
The monoclonal antibody specific against the antigen, prepared as above, is linked to an enzyme, viz. highly-purified alkaline phosphatase. The one-step glutaraldehyde method or benzoquinone conjugation is used.
In the one-step glutaraldehyde method, 3 mg monoclonal antibody (in about 1 ml of solution) are dialysed with 10 mg alkaline phosphatase (Sigma Type VII-T) against 2 x 1000 ml of PBS, pH 7.4, 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 are added. The conjugation mixture is left at room temperature for 1.5 hours. After this time, glutaraldehyde is removed by gel filtration on a Pharmacia PH-10 (Sephadex G-25 M) column, previously equilibrated in PBS. The conjugate is eluted with 3.5 ml PBS and then dialysed against 2 x 2000 ml of TRIS buffer (50 mM TRIS, 1 mM magnesium chloride, pH 8.0, plus 0.02% sodium azide) at +4°C. To the dialysed 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.
The sub-class is IgG2a. EXAMPLE 2
The general procedure of Example 1 may be followed to produce a monoclonal antibody broadly cross-reactive with an antigen of all species of the genus Entamoeba. Tests using the present invention are superior to existing tests, based on the following advantages: (i) greater accuracy; (ii) same day results, within an hour or two; (iii) reduction in amount of skilled labour required to administer laboratory procedures, resulting in reduced labour costs; (iv) reduction in laboratory time and space used in connection with tests, resulting in reduced overhead expenses; and (v) improved therapy based upon early, precise diagnosis.
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 monoclonal antibody specific for an antigen or species of Entamoeba.
2. The antibody of Claim 1 specific to the antigen or antigens of Entamoeba histolytica.
3. The antibody of Claim 1 specific to the antigen or antigens of Entamoeba histolytica I.
4. The antibody of Claim 1 specific to the antigen or antigens of Entamoeba histolytica II.
5. The antibody of Claim 1 specific to the antigen or antigens of Entamoeba histolytica III.
6. The antibody of Claim 1 specific to the antigen or antigens of Entamoeba histolytica IV.
7. The antibody of Claim 1 specific to the antigen or antigens of Entamoeba histolytica V. 8. The antibody of Claim 1 specific to the antigen or antigens of Entamoeba histolytica VI.
9. The antibody of Claim 1 specific to the antigen or antigens of Entamoeba histolytica VII.
10. The antibody of Claim 1 specific to the antigen or antigens of Entamoeba histolytica VIII.
11. A monoclonal antibody broadly cross- reactive with an antigen of all species of the genus Entamoeba.
12. A labeled monoclonal antibody consisting essentially of a monoclonal antibody of Claims 1-11 and an appropriate label.
13. The labeled monoclonal antibody of Claim 12, wherein said label is a member of the group selected from a radioactive isotope, enzyme, fluorescent compound, bioluminescent compound, chemiluminescent compound, or ferro¬ magnetic atom, or particle. .14. The labeled monoclonal antibody of
Claim 13, wherein said label is an enzyme capable of conjugating with a monoclonal antibody and of being used in an enzyme-linked immunoassay procedure.
15. The labeled monoclonal antibody of Claim 14, wherein said enzyme is alkaline phos¬ phatase, glucose oxidase, galactosidase, or peroxidase.
16. The labeled monoclonal antibody of Claim 13, wherein said label is a fluorescent compound or probe capable of being used in an immuno-fluorescent or fluorescent immunoassay procedure, enzyme fluorescent immunoassay, or fluorescence polarization immunoassay, photon counting immunoassay, or the like procedure.
17. The labeled monoclonal antibody of Claim 16, wherein said fluorescent compound or probe is fluorescein.
18. The labeled monoclonal antibody of Claim 13, wherein said label is a chemiluminescent compound capable of being used in a luminescent or enzyme-linked luminescent immunoassay.
19. The labeled monoclonal antibody of Claim 18, wherein such chemiluminescent compound is luminol or a luminol derivative.
20. The labeled monoclonal antibody of Claim 13, wherein said label is a bioluminescent compound capable of being used in an appropriate bioluminescent immunoassay.
21. The labeled monoclonal antibody of Claim 20, wherein such bioluminescent compound is luciferase or a luciferase derivative.
22. A process for diagnosing for the pre¬ sence of an antigen of Entamoeba in a specimen comprising contacting at least a portion of said specimen with a labeled monoclonal antibody of Claim 12 in an immunoassay procedure appropri¬ ate for said label.
23- The process of Claim 22, 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.
24. The process of Claim 23, wherein said label is an enzyme capable of being used in an enzyme-linked immunoassay procedure.
25. The process of Claim 24, wherein said enzyme is selected from alkaline phosphatase, glucose oxidase, galactosidase, or peroxidase.
26. The process of Claim 23, 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.
27. The process of Claim 26, wherein said fluorescent compound or probe is fluorescein.
28. The process of Claim 23, wherein said label is a chemiluminescent compound capable of being used in a luminescent or enzyme-linked luminescent immunoassay.
29. The process of Claim 28, wherein said chemiluminescent compound is luminol or a luminol derivative.
30. The process of Claim 23, wherein said label is a bioluminescent compound capable of being used in a bioluminescent or enzyme-linked bioluminescent immunoassay.
31. The process of Claim 30, wherein said bioluminescent compound is luciferase or a lucif- erase derivative.
32. A therapeutic composition comprising one or more of the monoclonal antibodies in Claims 1-11 and a pharmaceutically acceptable carrier or diluent.
33. A therapeutic composition comprising one or more of the labeled monoclonal antibodies in Claim 12 and a pharmaceutically acceptable carrier or diluent. "34. A method of treating Entamoeba infec¬ tions comprising administering an effective amount of a monoclonal antibody of Claims 1-11.
35. A kit for diagnosing for the presence of an antigen or species of Entamoeba in a diag¬ nostic specimen comprising at least one monoclonal antibody of Claims 1-11.
36. The kit of Claim 35, wherein said at least one antibody is labeled.
37. The kit of Claim 36, wherein said at least one monoclonal antibody is labeled with a fluorescent compound.
38. The kit as in Claim 36, wherein said at least one monoclonal antibody is labeled with an enzyme.
39. The kit as in Claim 36, 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. 40. The kit of Claims 36, 37, 38, and 39 additionally containing at least one known Entamoeba antigen as a control.
41. The kit of Claims 36, 37, 38, 39, and 40 containing each known antigen of Entamoeba histolytica.
42. A kit for diagnosing for the presence of an antigen or species of Entamoeba in a diag¬ nostic specimen comprising at least one monoclonal antibody of Claims 1-11 and a control.
43. The kit of Claim 42, wherein said at least one antigen is labeled and said control is at least one known antigen of Entamoeba.
44. A kit for diagnosing for the presence of a Entamoeba infection comprising at least one monoclonal antibody of Claims 1-11.
45. The kit of Claim 44, wherein said at least one monoclonal antibody is labeled.
EP19850905086 1984-10-19 1985-10-16 Monoclonal antibodies and their use Withdrawn EP0199756A1 (en)

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