WO1986000643A1 - Anticorps monoclonaux et leur utilisation - Google Patents

Anticorps monoclonaux et leur utilisation Download PDF

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Publication number
WO1986000643A1
WO1986000643A1 PCT/GB1985/000293 GB8500293W WO8600643A1 WO 1986000643 A1 WO1986000643 A1 WO 1986000643A1 GB 8500293 W GB8500293 W GB 8500293W WO 8600643 A1 WO8600643 A1 WO 8600643A1
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WIPO (PCT)
Prior art keywords
monoclonal antibody
immunoassay
enzyme
labeled
morganella
Prior art date
Application number
PCT/GB1985/000293
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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
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 Company Limited filed Critical Technology Licence Company Limited
Publication of WO1986000643A1 publication Critical patent/WO1986000643A1/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
    • C07K16/1228—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • 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 AND THEIR USE This invention relates to monoclonal antibodies and their use.
  • a single organism described as Morganella morganii has been distinguished from other Proteus species. It is often an extremely difficult organism to treat. It is commonly seen as a cause of urinary tract and blood infection in man. Morganella is also known to cause gram-negative sepsis which is a blood stream infection. It is one of the major infectious disease problems encountered in modern medical centers. While it can be transient and self-limited, severe gram-negative sepsis constitutes a medical emergency.
  • test for gram-negative sepsis involves processing blood and urine- cultures and other procedures on occasion.
  • blood culture tests are cumbersome. They require expert laboratory skills because of the complex nature of human blood, which tends to interact nonspecifically with many of the test reagents.
  • a microscopic examination is made, to determine the presence of micro- organisms as a preliminary screening.
  • the microscopic examination cannot distinguish among gram-negative bacteria.
  • a second step is a urine culture to identify the organism isolated in the urine sample. A delay in diagnosis and initiation of treatment can result in serious complications.
  • the present invention comprises monoclonal antibodies specific for an antigen of
  • Morganella in particular, the antigens of Morganella morganii, as well as a monoclonal antibody broadly cross-reactive with, an. antigen for each species of the genus Morganella.
  • the invention also comprises labelled monoclonal antibodies for use in diagnosing the presence of the Morganella antigens, each comprising a monoclonal antibody against one of the above-mentioned antigens to
  • the label can be chosen from the group consisting of a radioactive isotope, enzyme, fluorescent compound, chemiluminescent compound, bioluminescent compound, ferromagnetic atom, or particle, or any other label.
  • the invention further comprises the process for diagnosing the presence of Morganella antigens or organisms in a specimen comprising contacting said specimen with the labelled monoclonal antibody in an appropriate i munoassay procedure.
  • the invention is also directed to a therapeutic composition
  • a therapeutic composition comprising a monoclonal antibody for.an antigen of Morganella and a carrier or diluent, as well as kits containing at least one labelled monoclonal antibody to an antigen of a Morganella.
  • the monoclonal antibodies of the present inventio ⁇ are prepared by fusing spleen cells, from a mammal which has been immunized against the particular Morganella antigen, with an appropriate 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.
  • HAT hyperxanthine, aminopterin, and thymidine
  • the immunized spleen cells may be derived from any mammal, such as primates, humans, rodents (i.e.., mice, rats, and rabbits) ,. bovines, ovines, canines, but the present invention will be described in connection with mice.
  • the mouse is first immunized by injection of the particular Morganella antigen chosen generally for a period of approximately eleven weeks. When the mouse shows sufficient antibody production against the antigen, as determined by conventional assay, it is given a booster injection of the appropriate Morganella antigen, and then killed so that the immunized spleen may be removed. 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 Morganella 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 Morganella antigen.
  • the monoclonal antibody selected, which is specific for the particular Morganella antigen or species, is then bound to an appropriate label.
  • Amounts of antibody sufficient for labelling 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 labelled 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 labelled 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.
  • the labelled 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.
  • enzyme immunoassays are preferred due to their low cost, reagent stability, safety, sensitivity, and ease of procedure.
  • EIA enzyme-linked immunosorbent assay
  • EIA is a solid phase assay system which is similar in design to the radiometric assay, but which utilizes an enzyme in place of a radioactive isotope as the immunoglobulin marker.
  • Fluorescent-immunoassay is based on the labelling of antigen or antibody with fluorescent probes. A nonlabeled antigen and a specific antibody are combined with identical fluorescently labelled antigen. Both labelled and unlabeled antigen compete for antibody binding sites. The amount of labelled antigen bound to the antibody is dependent upon, and therefore a measurement of, the concentration of nonlabeled antigen.
  • fluorescent- immunoassay examples include heterogenous systems such as Enzyme-Linked Fluorescent Immunoassay, or homogeneous systems such as the Substrate Labeled Fluorescent Immunoassay.
  • the most suitable 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 a labelled- 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 polarization 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 intermediate 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.
  • a catalytic protein or enzyme such as luciferase
  • 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 Morganella 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 Morganella 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 Morganella in various specimens- It is also possible to use the broadly cross-reactive monoclonal antibody which can identify the genus Morganella alone or as part of a kit containing antibodies that can identify other bacterial genera or species of Morganella and/or other bacteria.
  • kits can be used in pathology laboratories for the rapid detection of gram-negative bacteria in urine, or on an out-patient basis.
  • conjugated or labelled monoclonal antibodies for antigens and/or species of Morganella and other gram-negative bacteria can be utilized in a kit to identify such antigens and organisms in blood samples taken from patients for the diagnosis of possible
  • the monoclonal test is an advance over existing procedures in that it is more accurate than existing tests; it gives "same day” results, provides convenience to the patient and improves therapy as a result of early, accurate diagnosis; and it reduces labor costs and laboratory time required for administration of the tests.
  • 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 Morganella.
  • One preferred embodiment of the present invention is a diagnostic kit comprising at least one labelled monoclonal antibody against a particular Morganella antigen or species, as well as any appropriate stains, counterstains, or reagents. Further embodiments include kits containing at least one control sample of a Morganella antigen and/or a cross-reactive labelled monoclonal antibody which would detect the presence of any of the Morganella organisms in a particular sample. Specific antigens to be detected in this kit include the antigen of Morganella morganii.
  • Monoclonal diagnostics which detect the presence of Morganella 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 Morganella antigen. More particularly, products of 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 contamina ⁇ tion.
  • the monoclonal antibody of the present invention was prepared generally according to the method of Kohler and Milstein (Eur. J. Immunol. 6_,_ 292 (1975)). In the Examples:
  • API Analytical Profile Index (ref. Ayerst Laboratories)
  • DMEM - Dulbeccos Modified Eagles Medium FCS Foetal Calf Serum % T refers to vaccine concentrations measured in a 1 cm light path
  • mice Six Balb/c mice were injected with the prepared antigen. They were given one intraperitoneal injection per week for three weeks (0.05 ml 80% T vaccine) followed by one intravenous injections per week for a further 3 weeks of boiled killed Morganella morganii prepared as above. The mice were rested for 7 weeks before being given a further intravenous dose of vaccine. The mice were bled approximately six days after the last injection and the serum tested for antibodies by assay. The conventional assay used for this serum titer testing was the enzyme-linked immunosorbent assay system.
  • mice showed antibody production after this regimen, generally a positive titer of at least 10,000, a mouse was selected as a fusion donor and given a booster injection (0.02 ml of 80% T vaccine) intravenously, three days prior to splenectomy.
  • the selected donor mouse was killed and surface sterilized by immersion in 70% ethyl alcohol.
  • the spleen was then removed and immersed in approximately 2.5 ml of DMEM to which had been added 3% FCS.
  • the spleen was then gently homogenized in a LUX homogenizing tube until all cells had been released from the membrane, and the cells were washed in 5 ml 3% FCS DMEM.
  • the cellular debris was then allowed to settle and the spleen cell suspension placed in a 10 ml centrifuge tube. The debris was then rewashed in 5 ml 3% FCS DMEM. Fifty mis of suspension were then made in 3% FCS DMEM.
  • the myeloma cell line used was NS0 (uncloned) , obtained from the MRC Laboratory of Molecular Biology in Cambridge, England. The myeloma cells were in the log growth phase, and rapidly dividing. Each cell line was washed using a tissue culture medium DMEM containing 3% FCS. The spleen cells were then spun down at the same time that a relevant volume of myeloma cells were spun down (room temperature for 7 minutes at 600 g) , and each resultant pellet was then separately resuspended in 10 ml 3% FCS DMEM.
  • 0.1 ml of the suspension was diluted to 1 ml and a haemacytometer with phase microscope was used.
  • 0.1 ml of the suspension was diluted to 1 ml with Methyl Violet-citric acid solution, and a haemacytometer and light microscope were used to count the stained nuclei of the cells.
  • 1 xlO 8 spleen cells were then mixed with 5 x 107 myeloma cells, the mixture washed in serum-free DMEM high in glucose and centrifuged, and all the liquid removed. The resultant cell pellet was placed in a 37°C water bath.
  • cells were removed and cloned using the limiting dilution method.
  • limiting dilution dilutions of cell suspensions in 18% FC-DMEM + Balb/c mouse acrophages 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 immunosorbent assay.
  • the monoclonal antibodies from the clones were screened by the standard techniques for binding to Morganella morganii NCTC 235 prepared as in the immunisation, and for specificity in a test battery of Morganella species and related genera bearing different antigens. Specifically, a grid of microtiter plates containing a representative selection of O-serotype organisms, i.e. Morganella, Serratia, Proteus and
  • mice were primed with pristane for at least 7
  • the fluid was titrated, as noted above, to establish presence and level of antibody, and purified. Purification is accomplished using the Protein
  • A-Sepharose method in which about 10 ml of the ascites fluid are 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 (0.1 M sodium phosphate, pH 8.2). The diluted ascites was applied to a 2 ml column of Protein A-Sepharose which had previously been equilibrated with phosphate buffer. The column was washed with 40 ml of phosphate buffer. The monoclonal antibody was eluted with citrate buffer (0.1 M sodium citrate, pH 3.5) into sufficient IM Tris buffer, pH 9.0, to raise the pH immediately to about 7.5.
  • citrate buffer 0.1 M sodium citrate, pH 3.5
  • the eluate was dialysed in 2 x 1000 ml PBS, pH 7.4 at +4°C, and stored at -20 ⁇ C.
  • G. Enzyme-Monoclonal Linkage The monoclonal antibody specific against Morganella morganii antigen, prepared and screened as described above, was then bound to an appropriate enzyme (in this case, a highly purified alkaline phosphatase) , using the one-step glutaraldehyde method. Monoclonal antibody was dialysed with alkaline phosphatase (Sigma Type VII-T) against 2 x 1000 ml of PBS pH 7.4, at +4°C.
  • the volume was made up to 2.5 ml with PBS and 25 ⁇ l of a 20% solution of glutaraldehyde in PBS was added.
  • the conjugation mixture was left at room temperature for 1.5 hours.
  • glutaraldehyde was removed by gel filtration on a Pharmacia PD-10 (Sephadex G-25M) column, previously equilibrated in PBS.
  • the conjugate was 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.
  • the enzyme immunoassay method was used for testing. This assay method comprises coating the wells of a standard polyvinyl chloride microtiter tray with the antigen, followed by addition of monoclonal antibody enzyme conjugate, and finally addition of the enzyme substrate, para-nitrophenol phosphate.
  • the monoclonal antibody was found to be specific for the antigen of Morganella morganii.
  • the monoclonal antibody was also tested and shown to be of the Class IgG2b.
  • Example 2 The same procedure as in Example 1 may be utilized in preparing a monoclonal antibody broadly cross-reactive with an antigen of many or all species of the genus Morganella, but using another Morganella obtained from the National Collection of Type Cultures.
  • Tests using the present invention are superior to the 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 labor required to administer laboratory procedures, resulting in reduced labor costs; (iv) reduction in laboratory time and space used in connection with tests, resulting in reduced overhead expense; and (v) improved therapy based upon early, precise diagnosis.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
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  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
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Abstract

Anticorps monoclonaux du genre Morganella, anticorps étiquetés, compositions et kits les contenant, et leur utilisation dans le diagnostic d'antigènes et dans le traitement.
PCT/GB1985/000293 1984-07-03 1985-07-02 Anticorps monoclonaux et leur utilisation WO1986000643A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB8416842 1984-07-03
GB848416842A GB8416842D0 (en) 1984-07-03 1984-07-03 Monoclonal antibodies

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WO1986000643A1 true WO1986000643A1 (fr) 1986-01-30

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EP (1) EP0187802A1 (fr)
JP (1) JPS61502629A (fr)
GB (1) GB8416842D0 (fr)
WO (1) WO1986000643A1 (fr)

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0077734A2 (fr) * 1981-10-19 1983-04-27 Mgi Pharma, Inc. Production d'anticorps monoclonaux contre des adhésines bactériennes
WO1983001739A1 (fr) * 1981-11-17 1983-05-26 Brigham & Womens Hospital Anticorps monoclonaux contre le brugia malayi
EP0101039A2 (fr) * 1982-08-10 1984-02-22 Meiji Seika Kabushiki Kaisha Anticorps monoclonal, méthode pour sa production et son utilisation
EP0105714A1 (fr) * 1982-09-29 1984-04-18 Serono Diagnostics Limited Immunoessai pour antigènes
EP0111762A1 (fr) * 1980-06-20 1984-06-27 Unilever Plc Procédés et appareil pour l'exécution d'essais de liaisons spécifiques

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0111762A1 (fr) * 1980-06-20 1984-06-27 Unilever Plc Procédés et appareil pour l'exécution d'essais de liaisons spécifiques
EP0077734A2 (fr) * 1981-10-19 1983-04-27 Mgi Pharma, Inc. Production d'anticorps monoclonaux contre des adhésines bactériennes
WO1983001739A1 (fr) * 1981-11-17 1983-05-26 Brigham & Womens Hospital Anticorps monoclonaux contre le brugia malayi
EP0101039A2 (fr) * 1982-08-10 1984-02-22 Meiji Seika Kabushiki Kaisha Anticorps monoclonal, méthode pour sa production et son utilisation
EP0105714A1 (fr) * 1982-09-29 1984-04-18 Serono Diagnostics Limited Immunoessai pour antigènes

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GB8416842D0 (en) 1984-08-08
JPS61502629A (ja) 1986-11-13
EP0187802A1 (fr) 1986-07-23

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