Classifications

• • 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
  • C07K16/1267—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria
  • C07K16/1282—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria from Clostridium (G)
• • 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 Clostrid ⁇ ium are desired which when used will rapidly diagnose the presence of such organisms in speci ⁇ mens.
• Clostrid ⁇ ium species Some of the representative members include Clostridium botulinum, C_. perfringens, C. tetani, and C. difficile.
• SUBSTITUTE SHEET In general, the pathogenic Clostridium are normal soil inhabitants, with little or no invasive power; the diseases they produce result from the production of a variety of highly toxic proteins or exotoxins. Tetanus, caused by Clostridium tetani, and gas gangrene are caused by wound infections; tissue damage leads to the development of an anaerobic environment which permits localized growth and toxin formation by these organisms. Presently, tetanus is pre ⁇ vented by the use of- tetanus toxoid through
• Botulism caused by Clostridium botulinum, and other less serious types of clostridial food poisoning, caused by Clostridium perfringens, generally result from the ingestion of foods in which these organisms have previously developed and formed toxins.
• Clostridium botulinum and other less serious types of clostridial food poisoning, caused by Clostridium perfringens, generally result from the ingestion of foods in which these organisms have previously developed and formed toxins.
• the ability of monoclonal antibodies specifically to bind to antigens of Clostridium or Clostridium toxin can provide many opportuni ⁇ ties for diagnosis and treatment. Such speci ⁇ ficity is a most important requirement for proper and accurate analysis and/or diagnosis, particu ⁇ larly 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- ele ⁇ tron 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 Clostridium antigens and/or organisms.
• the present invention com ⁇ prises monoclonal antibodies specific for an antigen or species of Clostridium; in particular, the antigens or- species of Clostridium botulinum, and the antigens or species of Clostridium per- fringens, Clostridium tetani, and Clostridium difficile, the antigen or antigens for the toxins of C_. botulinum, C_. perfringens, C_. tetani , and C_. difficile, as well as a monoclonal anti ⁇ body broadly cross-reactive with an antigen for each species of the genus Clostridium.
• the invention also comprises labeled mono ⁇ clonal antibodies for use in diagnosing the presence of the Clostridium antigens, each com ⁇ prising a monoclonal antibody against one of *
• the label can be chosen from the group consisting of a radioactive isotope, enzyme, fluorescent compound, chemiluminescent compound, biolumines ⁇ cent compound, ferromagnetic atom, or particle, or any other label.
• the invention further comprises the process for diagnosing the presence of Clostridium anti ⁇ gens, organisms, or toxins in a specimen compris ⁇ ing contacting 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 or toxin of Clos ⁇ tridium and a carrier or diluent, as well as kits containing at least one labeled monoclonal antibody to an antigen or toxin of Clostridium.
• the monoclonal antibodies of the present invention are prepared by fusing spleen cells, from a mammal which has been immunized against the particular Clostridium antigen, with an appropriate myeloma cell line, preferably NSO (uncloned), P3NS1-Ag4/1, or Sp2/0 Agl4.
• 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. Screening tests for the specific mono ⁇ clonal antibodies are employed utilizing immuno ⁇ assay 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 Clostridium 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 Clostridium 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 Clostridium 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 Clostridium antigen.
• the monoclonal antibody selected, which is specific for the particular Clostridium antigen, species, or toxin, is then bound to an appropriate 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 Clostridium antigen, species, or toxin, 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 Clostridium 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 antigens, toxins, or species of Clostridium in various specimens. It is also possible to use the broadly cross-reactive monoclonal antibody which can identify the genus Clostridium alone or as part of a kit containing antibodies that can identify other bacterial genera or species of Clostridium and/or other toxins.
• 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.
• 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 Clostridium.
• kits comprising at least one labeled monoclonal antibody against a particular Clostridium antigen, toxin, or species, as well as any appropriate stains, counterstains, or reagents.
• Specific antigens to be detected in this kit include the antigens of Clostridium botulinum, C_. perfringens, C_. tetani, and C_. difficile, as well as the antigen or antigens for the toxins of . botullinum, C_. perfringens, C_. tetani, and C_. difficile.
• Monoclonal diagnostics which detect the presence of Clostridium 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 or milk contain the particular Clostridium 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. 6_, (1975) 292.
• EXAMPLE 1 A. Antigen Preparation
• Antigen (Clostridium botulinum) is obtained (samples are available from the National Collection of Type Cultures) 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 DMEM. After incubation, the cells are held at 100°C for 1 hour, harvested by centrifugation, and washed three ti es in saline. They are then resuspended in 1% formol saline.
• mice are injected with the prepared antigen. 5 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
• 1° 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
• Spleen cells from the immune mice are harvested three days after boosting, by conventional techniques. First, the donor mouse selected is killed 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 35 3% FCS.
• Th ' e 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.
• 1 x 10 8 Spleen cells are then mixed with 6 xlO7 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.
• 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
• cells are removed and cloned using the standard agar or dilution method.
• 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.
• Purification is accomplished using the protein A - 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 el ate is dialysed in 2 x 1000 ml PBS at +4°C.
• the fluid may then be titrated, as noted above, to establish presence and level of antibody, and purified by a combination of batch ion-exchange chromatography, ammonium sulphate precipitation and column ion-exchange (a possible alternative would be protein A - Sepharose) chromatograph .
• 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 DEAE-cellulose chromatograph , or phosphate buffer (0.1M sodium phosphate, pH 8.2 + 0.02% sodium azide) for protein A-Sepharose chromatography.
• the dissolved precipitate is dialysed versus 2 x 1000 ml of the dissolution buffer at +4°C, and the appropriate chromatography step carried out as previously described.
• 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 2 The procedure of Example 1, in most respects, was followed.
• the antigen was Clostridium difficile toxin A supplied by Birmingham University.
• the prepared toxin was neutralised by polyclonal antibody and immunised sub-cutaneously with Complete Freunds Adjuvant twice, with an interval of one week; this was followed by two ip injections of PBS + neutralised toxin, after 2 and one week intervals, followed after a further week by challenge with toxin iv; fusion followed 3 days later. Limiting dilution was used for cloning.
• 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 Clostridium.
• 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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• 1984
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