WO1986002360A1 - Anticorps monoclonaux et leur utilisation - Google Patents

Anticorps monoclonaux et leur utilisation Download PDF

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Publication number
WO1986002360A1
WO1986002360A1 PCT/GB1985/000471 GB8500471W WO8602360A1 WO 1986002360 A1 WO1986002360 A1 WO 1986002360A1 GB 8500471 W GB8500471 W GB 8500471W WO 8602360 A1 WO8602360 A1 WO 8602360A1
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WIPO (PCT)
Prior art keywords
monoclonal antibody
hemophilus
antigen
immunoassay
labeled
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PCT/GB1985/000471
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English (en)
Inventor
Bruce William Wright
Peter John Cox
Alice Margaret Noyes
Danny Widdows
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Technology Licence Company Limited
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Publication date
Application filed by Technology Licence Company Limited filed Critical Technology Licence Company Limited
Publication of WO1986002360A1 publication Critical patent/WO1986002360A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • C07K16/1203—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria
    • C07K16/1242—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria from Pasteurellaceae (F), e.g. Haemophilus influenza
    • 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 Hemophilus are desired which when used will rapidly diagnose the presence of such organisms in specimens.
  • Hemophilus Divisions have been made among the Hemophilus species. Some of the representative members include Hemophilus influenzae and Hemophilus Cor Gardnerella vaginalis.
  • SUBSTITUTESHEET to antigens of Hemophilus can provide many oppor ⁇ tunities 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.
  • 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 biolu inescen , 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 Hemophilus antigens and/or organisms.
  • the present invention com ⁇ prises monoclonal antibodies specific for an antigen or species of Hemophilus; in particular, the antigens or species of Hemophilus vaginalis (designated as H_. vaginalis I, II, III, or IV), and the antigens or species of Hemophilus influ- enzae, as well as a monoclonal antibody broadly cross-reactive with an antigen for each species of the genus Hemophilus.
  • H_. vaginalis I, II, III, or IV the antigens or species of Hemophilus influ- enzae
  • the invention also comprises labeled mono ⁇ clonal antibodies for use in diagnosing the presence of the Hemophilus antigens, each com ⁇ prising a monoclonal antibody against one of the above-mentioned antigens to Hemophilus 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 Hemophilus 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 Hemophilus and a carrier or diluent, as well as kits contain ⁇ ing at least one labeled monoclonal antibody to an antigen of a Hemophilus.
  • the monoclonal antibodies of the present invention are prepared by fusing spleen cells, from a mammal which has been immunized against the particular Hemophilus 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 Hemophilus antigen chosen gener ⁇ ally 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 Hemophilus 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 Hemophilus 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 Hemophilus antigen.
  • the monoclonal antibody selected, which is specific for the particular Hemophilus 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 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 Hemophilus 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 Hemophilus 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 Hemophilus in various specimens. It is also possible to use the broadly cross-reactive monoclonal antibody which can identify the genus Hemophilus alone or as part of a kit containing antibodies that can identify other bacterial genera or species of Hemophilus 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 Hemophilus.
  • kits comprising at least one labeled monoclonal antibody against a particular Hemophilus antigen or species, as well as any appropriate stains, counterstains, or reagents.
  • Specific antigens to be detected in .this kit include the antigens of Hemophilus vaginalis (applicant has further divided this species into four subgroups: Hemo ⁇ philus vaginalis I, II, III, or IV), and Hemophi- lus influenzae.
  • Monoclonal diagnostics which detect the presence of Hemophilus 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 Hemophilus 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.
  • References to Hemophilus herein should also be read as references to Gardnerella, in general, and specifically in connection with Hemophilus vaginalis.
  • 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. 6_ (1975) 292.
  • EXAMPLE 1 A. Antigen Preparation
  • Gardnerella vaginalis is obtained from the National Collection of Type Culture (NCTC accession No. 10915) and tested by standard biochemical methods of microbial identification to confirm its identity (using API profiles) .
  • the antigen is removed from the lyophile, grown on blood agar, and tested by API to confirm its identity and purity. It is then transferred for growth into Todd-Hewitt broth in 10% C0 2 . After incubation, the cells are harvested by centrifugation, and washed three times in saline. They are then resuspended in 1% formol saline.
  • mice Six balb/c mice are injected with the prepared antigen. They are given one intraperitoneal injection per week for 3 weeks and then, after 1, 1 and 2 week intervals, an intravenous injection (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 (0.05 ml 80% T vaccine) intravenously, three days prior to splenectomy. c. Cell Fusion
  • 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.
  • 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.
  • 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.
  • PEG polyethylene glycol 1500
  • 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% CO. air at approximately 100% humidity.
  • the wells are analysed for growth, utilising the conventional inverted microscope procedure, after about 5 to 10 days.
  • 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 monoclonal is specific to Gardnerella vaginalis strains NCTC 10915, 17.4 and Corpus, and negative to E. 5 coli, Pseudomonas, Klebsiella, Hemophilus and Shigella. F. Antibody Production and Purification
  • 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 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 0 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 5 stored at -20 C.
  • citrate buffer 0.1M sodium citrate, pH 3.5
  • 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 0 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 monoclonal antibody specific against the antigen, prepared as above, is linked to an enzyme, viz. highly-purified alkaline phosphatase.
  • alkaline phosphatase (Sigma Type VII-T) were dialysed against 2 x 500 ml of 0.25 M sodium phosphate buffer, pH 6.0, at +4 C. 18 mg p-benzoquinone were dissolved in 0.6 ml warm AR ethanol, and added to the dialysed alkaline phosphatase. The benzoquinone/alkaline phosphatase mixture was left in the dark at room temperature for 1 hour. Unreacted benzoquinone and reaction by-products were then removed and the buffer exchanged by gel filtration on a Pharmacia PD-10
  • Example 1 The general procedure of Example 1 was followed, but with various differences.
  • the antigen was Hemophilus influenzae type b, NCTC 7279.
  • the bacteria were grown in chocolate agar and harvested.
  • the organisms were suspended in formol saline.
  • the animals were immunised intramuscularly (in Complete Freunds Adjuvant) and, 4 weeks later, intravenously.
  • the monoclonal was specific to the H. influenzae type b antigen, and negative to other strains of Hemophilus, Neisseria, E coli, Salmonella, Pseudomonas, Serratia, Enterobacter and Gardnerella.
  • cells of the monoclonal antibody-producing cell line were grown in batch tissue culture.
  • DMEM-10% FCS was used to support growth in mid-log phase, to 1 litre volume.
  • the culture was then allowed to overgrow, to allow maximum antibody production.
  • the culture was then centrifuged at 1200 g for approximately 10 minutes, the cells discarded and the antibody-rich supernatant collected.
  • Example 3 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 Gardnerella (or Hemophilus) . 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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  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
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  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
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  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
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Abstract

Anticorps monoclonaux pour le genre Hemophilus, anticorps marqués, compositions et kits les contenant, et leur utilisation pour le diagnostic et le traitement d'antigènes.
PCT/GB1985/000471 1984-10-19 1985-10-16 Anticorps monoclonaux et leur utilisation WO1986002360A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB848426469A GB8426469D0 (en) 1984-10-19 1984-10-19 Monoclonal antibodies
GB8426469 1984-10-19

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WO1986002360A1 true WO1986002360A1 (fr) 1986-04-24

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EP (1) EP0201519A1 (fr)
JP (1) JPS62500585A (fr)
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WO (1) WO1986002360A1 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0269388A2 (fr) * 1986-11-20 1988-06-01 Minnesota Mining And Manufacturing Company Procédé pour l'évaluation des microbes oraux
WO1988004932A1 (fr) * 1986-12-31 1988-07-14 Praxis Biologics, Inc. Vaccins et analyses diagnostiques pour l'haemophilus influenzae
FR2747387A1 (fr) * 1996-04-12 1997-10-17 Conseil General De L Orne Moyens pour la detection de bacteries du genre taylorella et applications biologiques

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4455296A (en) * 1982-02-02 1984-06-19 Board Of Regents, The University Of Texas System Hybrid cell lines producing monoclonal antibodies directed against Haemophilus influenzae
US4461829A (en) * 1981-09-14 1984-07-24 Miles Laboratories, Inc. Homogeneous specific binding assay element and lyophilization production method

Patent Citations (2)

* 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
US4455296A (en) * 1982-02-02 1984-06-19 Board Of Regents, The University Of Texas System Hybrid cell lines producing monoclonal antibodies directed against Haemophilus influenzae

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
Infection and Immunity, Volume 18, No. 2, November 1977, Washingtong, D.C., (US) M.F. SMARON et al.: "Immunological and Chemical Characterization of the Extra-Cellular Antigens from Corynebacterium Vaginale", see page 356, Abstract; page 359, left-hand column, lines 10-20; page 362, left-hand column, lines 12-22 *
R.E. BUCHANAN et al., Co-Editors, "Bergey's Manual of Determinative Bacteriology", 8th Edition, published 1975, The Williams & Wilkins Company, Baltimore, (US) pages 368-370, see page 368, lines 33-39 *

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0269388A2 (fr) * 1986-11-20 1988-06-01 Minnesota Mining And Manufacturing Company Procédé pour l'évaluation des microbes oraux
EP0269388A3 (fr) * 1986-11-20 1989-12-13 Minnesota Mining And Manufacturing Company Procédé pour l'évaluation des microbes oraux
WO1988004932A1 (fr) * 1986-12-31 1988-07-14 Praxis Biologics, Inc. Vaccins et analyses diagnostiques pour l'haemophilus influenzae
AU615429B2 (en) * 1986-12-31 1991-10-03 Wyeth Holdings Corporation Vaccines and diagnostic assays for haemophilus influenzae
EP0786472A1 (fr) * 1986-12-31 1997-07-30 Praxis Biologics, Inc. Vaccins et analyses diagnostiques pour l'haemophilus influenzae
FR2747387A1 (fr) * 1996-04-12 1997-10-17 Conseil General De L Orne Moyens pour la detection de bacteries du genre taylorella et applications biologiques
WO1997039034A1 (fr) * 1996-04-12 1997-10-23 Conseil General De L'orne Moyens pour la detection de bacteries de l'espece taylorella equigenitalis et applications biologiques
US6858209B2 (en) 1996-04-12 2005-02-22 Conseil General De L'orne Means for detecting bacteria of the taylorella equigenitalis species and their biological applications

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GB8426469D0 (en) 1984-11-28
EP0201519A1 (fr) 1986-11-20
JPS62500585A (ja) 1987-03-12

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