EP0199754A1 - Anticorps monoclonaux et leur utilisation - Google Patents

Anticorps monoclonaux et leur utilisation

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Publication number
EP0199754A1
EP0199754A1 EP85905081A EP85905081A EP0199754A1 EP 0199754 A1 EP0199754 A1 EP 0199754A1 EP 85905081 A EP85905081 A EP 85905081A EP 85905081 A EP85905081 A EP 85905081A EP 0199754 A1 EP0199754 A1 EP 0199754A1
Authority
EP
European Patent Office
Prior art keywords
monoclonal antibody
giardia
antigen
antibody
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Withdrawn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
EP85905081A
Other languages
German (de)
English (en)
Inventor
Bruce William Wright
Peter John Church Cottage Church Road COX
Alice Margaret Noyes
Danny Widdows
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 EP0199754A1 publication Critical patent/EP0199754A1/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • monoclonal antibodies specific for the antigens or species of Giardia are desired which when used will rapidly diagnose the presence of such organisms in specimens.
  • -OBSTITUTH SHEET spec es. ome o e represen a ve mem ers t include Giardia lamblia and Giardia intestinalis. Giardia will be described with particular refer ⁇ ence to Giardia lamblia, the organism causing giardiasis. Giardiasis is very widespread in tropical countries but has spread extensively into temperature countries. Thousands of cases have been reported from mountainous areas such as Aspen, Colorado, where outbreaks of the disease involving hundreds of people due to contaminated water supplies have been reported. Travelers to Leningrad in Russia have suffered high percent ⁇ age attack rates of the disease.
  • the organism appears to produce diarrhea and then a chronic diarrhea which may lead to a severe malfunction of the bowel with weight loss, wasting and compli ⁇ cations from the malabsorption of food.
  • Treatment is potentially toxic, and diagnosis is time consuming, difficult and often insensitive, requiring skilled technicians to make a positive diagnosis. Travelers to other countries may be exposed to Giardia lamblia from contaminated food, particularly vegetables and fruit, and it is a frequent travelers 1 disease which leads to chronic diarrhea which may be inappropriately treated without a specific diagnosis.
  • the ability to antigens of Giardia can provide many opportuni ⁇ ties for diagnosis and 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 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 Giardia antigens and/or organisms.
  • the present invention com ⁇ prises monoclonal antibodies specific for an antigen or species of Giardia; in particular, the antigens or species of Giardia lamblia (desig ⁇ nated as Giardia lamblia I, II, III, IV, V, VI, VII, or VIII), and Giardia intestinalis, as well as a monoclonal antibody broadly cross- reactive with an antigen for each species of the genus Giardia.
  • the antigens or species of Giardia lamblia desig ⁇ nated as Giardia lamblia I, II, III, IV, V, VI, VII, or VIII
  • Giardia intestinalis as well as a monoclonal antibody broadly cross- reactive with an antigen for each species of the genus Giardia.
  • the invention also comprises labeled mono ⁇ clonal antibodies for use in diagnosing the presence of the Giardia antigens, each com ⁇ prising a monoclonal antibody against one of the above-mentioned antigens to Giardia 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 Giardia 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 Giardia and a carrier or diluent, as well as kits contain ⁇ ing at least one labeled monoclonal antibody to an antigen of a Giardia.
  • the monoclonal antibodies of the present invention are prepared by fusing spleen cells, from a mammal which has been immunized against the particular Giardia antigen, with an appropri ⁇ ate myeloma cell line, preferably NSO (uncloned), P3NS1-Ag4/1, or Sp2/0 Agl4.
  • the resultant product is then cultured in a standard HAT (hypoxanthine, 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 Giardia 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 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 Giardia 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 Giardia antigen.
  • the monoclonal antibody selected, which is specific for the particular Giardia 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 immunosorbent assay
  • Fluorescent-immunoassay is based on the labeling of antigen or antibody with fluorescent 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 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 chemilu inescent 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 Giardia 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 Giardia infections 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 species of Giardia in various speci ⁇ mens. It is also possible to use the broadly cross-reactive monoclonal antibody which can identify the genus Giardia alone or as part of a kit containing antibodies that can identify other bacterial genera or species of Giardia 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 Giardia.
  • One preferred embodiment of the present invention is a diagnostic kit comprising at least one labeled monoclonal antibody against a particular Giardia antigen or species, as well as any appropriate stains, counterstains, or reagents. Further embodiments include kits containing at least one control sample of a Giardia antigen and/or a cross-reactive labeled monoclonal antibody which would detect the pres ⁇ ence of any of the Giardia organisms in a partic ⁇ ular sample.
  • Specific antigens to be detected in this kit include the antigens of Giardia lamblia (applicant has further divided this species into eight subgroups: Giardia lamblia I, II, III, IV, V, VI, VII) and/or Giardia intestinalis.
  • Monoclonal diagnostics which detect the presence of Giardia antigens can also be used in periodic testing of water sources, food supplies and food processing operations.
  • the present invention describes the use of the labelled 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 and milk contain the particular Giardia antigen. More particularly, the invention could be utilised as a public health and safety diagnostic aid, whereby specimens such as water or food could be tested for possible contamination.
  • API Analytical Profile Index (ref. Ayerst Labs)
  • 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. , (1975) 292.
  • mice are injected with prepared Giardia lamblia antigen. They are given intraperitoneal and/or intravenous 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.
  • 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 NSO (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 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
  • the wells are kept undisturbed, and cultured at 37°C in 9% CO_ air at approximately 100% humidity.
  • the wells are kept undisturbed, and cultured at 37°C in 9% CO_ air at approximately 100% humidity.
  • 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.
  • the cells are then centrifuged at 1200 g for approximately 10 minutes, the cells discarded, and the antibody-rich ascites fluid collected.
  • the fluid is titrated, as noted above, to establish presence and level of antibody, and purified.
  • Sepharose method More particularly, about 10 ml of the ascites fluid are filtered through glass wool and centrifuged at 30,000 g for 10 minutes. The ascites is then diluted with twice its own volume of cold phosphate buffer (0.1 M sodium phosphate, pH 8.2). The diluted ascites is loaded on to a 2 ml column of protein A - Sepharose which has previously been equilibrated with phosphate buffer. The column is washed with 40 ml phosphate buffer, and the monoclonal antibody is eluted with citrate buffer (0.1 M sodium citrate, pH 3.5) into sufficient 1M tris buffer, pH 9.0, to raise the pH immediately to about 7.5. The eluate is dialysed in 2 x 1000 ml PBS at +4°C.
  • citrate buffer 0.1 M sodium citrate, pH 3.5
  • SP-Sephadex is allowed to settle and the supernatant is decanted.
  • the SP-Sephadex is packed in a column, washed with 60 ml of 0.1M acetate buffer, pH 5.0, and eluted with 60 ml of the same buffer plus 1M sodium chloride.
  • the eluate is stirred at +4°C, and an equal volume of saturated ammonium sulphate added slowly.
  • the suspension is stirred for a further 30 minutes.
  • the precipitate is then harvested by centrifugation at 10,000 g for 10 minutes.
  • the precipitate is dissolved in a minimum volume of either cold phosphate/EDTA buffer (20mM sodium phosphate, lOmM EDTA, pH 7.5, + 0.02% sodium azide) for
  • 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 Giardia.
  • 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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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Engineering & Computer Science (AREA)
  • Urology & Nephrology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Hematology (AREA)
  • Organic Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Genetics & Genomics (AREA)
  • Biophysics (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Biotechnology (AREA)
  • Cell Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Microbiology (AREA)
  • Virology (AREA)
  • Food Science & Technology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Peptides Or Proteins (AREA)

Abstract

Anticorps monoclonaux pour le genre Giardia, anticorps marqués, compositions et kits les contenant, et leur utilisation pour le diagnostic et le traitement d'antigènes.
EP85905081A 1984-10-19 1985-10-16 Anticorps monoclonaux et leur utilisation Withdrawn EP0199754A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB8426460 1984-10-19
GB848426460A GB8426460D0 (en) 1984-10-19 1984-10-19 Monoclonal antibodies

Publications (1)

Publication Number Publication Date
EP0199754A1 true EP0199754A1 (fr) 1986-11-05

Family

ID=10568429

Family Applications (1)

Application Number Title Priority Date Filing Date
EP85905081A Withdrawn EP0199754A1 (fr) 1984-10-19 1985-10-16 Anticorps monoclonaux et leur utilisation

Country Status (4)

Country Link
EP (1) EP0199754A1 (fr)
JP (1) JPS62500584A (fr)
GB (1) GB8426460D0 (fr)
WO (1) WO1986002356A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2006232963B2 (en) * 2005-04-01 2009-12-03 Raytheon Company Integrated smart power switch

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2012745A1 (fr) * 1989-03-30 1990-09-30 John D. Rosoff Antigene specifique de giardia lamblia dans les selles, anticorps monospecifiques contre lesdits antigenes et methode de diagnostic des giardiases

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4461829A (en) * 1981-09-14 1984-07-24 Miles Laboratories, Inc. Homogeneous specific binding assay element and lyophilization production method

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO8602356A1 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2006232963B2 (en) * 2005-04-01 2009-12-03 Raytheon Company Integrated smart power switch
US7839201B2 (en) 2005-04-01 2010-11-23 Raytheon Company Integrated smart power switch
US8076967B2 (en) 2005-04-01 2011-12-13 Raytheon Company Integrated smart power switch

Also Published As

Publication number Publication date
WO1986002356A1 (fr) 1986-04-24
GB8426460D0 (en) 1984-11-28
JPS62500584A (ja) 1987-03-12

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