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
  • C07K16/1278—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria from Bacillus (G)
• • 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 Bacillus are desired which when used will rapidly diagnose the presence of such organisms in specimens.
• Bacillus cereus Some of the representative members which have been characterized as causing diseases in certain circumstances include Bacillus cereus, Bacillus megaterium, Bacillus subtilis, Bacillus anthracis and Bacillus licheniformis.
• Bacillus will be described with particular reference to Bacillus cereus.
• the Bacillus cereus spore is known to cause a special form of food poisoning associated with the ingestion of rice. This spore is resistant to heat and in routine cooking survives destruction. If rice is maintained in restaurants at a slightly warmer temperature to retain its fluffiness, the bacillus spore will germinate and produce a toxin which produces severe gastrointestinal
• Bacillus cereus is known to produce four toxins: cereolysin, diarrheagenic, emetic, and enterotoxins.
• Bacillus subtilis causes the subtilysin toxin, and Bacillus anthracis produces an anthracis toxin.
• isotopic and nonisotopic immunoassays have been utilized in conjunction with monoclonal antibodies to test for the pres ⁇ ence of an antigenic substance.
• agglutination, immuno-fluorescent, chemilum ⁇ inescent or fluorescent immunoassay, immuno- electron microscopy, radiometric assay systems,, radio immunoassays, and enzyme-linked immunoassays are the most common techniques used with the monoclonal antibodies. Other techniques include bioluminescent, fluorescence polarization, and photon-counting immunoassays.
• EIA enzyme-linked immunoassay procedure
• the enzyme-linked monoclonal antibody can then be used in the known enzyme-linked immunosor- , bent assay procedure to determine the presence of an antigenic substance.
• the serotype of the infecting organism can be determined, and appropriate treatment can then be initiated to rapidly and efficiently eliminate the disease.
• the present invention provides novel mono ⁇ clonal antibodies for use in accurately and rapidly diagnosing samples for the presence of Bacillus antigens and/or organisms.
• the present invention com ⁇ prises monoclonal antibodies specific for an antigen or species of Bacillus; in particular, the antigens or species of Bacillus cereus, and the antigens or species of Bacillus megaterium.
• Bacillus subtilis Bacillus anthracis, the antigen or antigens for the toxins of Bacillus cereus, B_. megaterium, ]_. subtilis, and B_. anthracis, as well as a monoclonal antibody broadly cross-reactive with an antigen for each species of the genus Bacillus.
• the invention also comprises labeled mono ⁇ clonal antibodies for use in diagnosing the presence of the Bacillus antigens, each com ⁇ prising a monoclonal antibody against one of the above-mentioned antigens to Bacillus or to a particular species or toxins thereof and linked thereto an appropriate label.
• the label can be chosen from the group consisting of a radioactive isotope, enzyme, fluorescent compound, chemiluminescent compound, bioluminescent com ⁇ pound, ferromagnetic atom, or particle, or any other label.
• the invention further comprises the process for diagnosing the presence of Bacillus anti ⁇ gens, organisms, or toxins in a specimen comprising 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 Bacillus and a carrier or diluent, as well as kits containing at least one labeled monoclonal antibody to an antigen or toxin of Bacillus.
• the monoclonal antibodies of the - present invention are prepared by fusing spleen cells, from a mammal which has been immunized against the particular Bacillus 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 Bacillus antigen chosen generally for a period of approximately eleven weeks. When the mouse shows sufficient antibody produc ⁇ tion against the antigen, as determined by conven ⁇ tional assay, it is given a booster injection of the appropriate Bacillus 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 Bacillus 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 *
• the monoclonal antibody selected which is specific for the particular Bacillus 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- ole 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 Bacillus 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 Bacillus 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 antigens, toxins, or species of Bacillus in various speci ⁇ mens.
• a rapid diagnostic method requiring limited technical skill could be utilized in the detection of Bacillus cereus toxin in public health analysis of restaurant practices, food handling in the food technology industry, and as an emergency diagnostic in the emergency care of patients with acute gastrointestinal symptoms.
• the broadly cross-reactive monoclonal antibody which can identify the genus Bacillus alone or as part of a kit containing antibodies that can identify other bacterial genera or species of Bacillus 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.
• 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 Bacillus.
• kits comprising at least one labeled monoclonal antibody against . a particular Bacillus antigen, toxin, or species, as well as any appropriate stains, counterstains, or reagents. Further embodiments include kits containing at least one control sample of a Bacillus antigen and/or a cross-reactive labeled monoclonal antibody which would detect the pres ⁇ ence of any of the Bacillus organisms or toxins in a particular sample. Specific antigens to be detected in this kit include the antigens of Bacillus cereus. Bacillus megaterium. Bacillus subtilis. Bacillus anthracis, as well as the antigen or antigens for the toxins of Bacillus cereus, B_. megaterium, B_. subtilis, and B_. anthracis.
• Monoclonal diagnostics which detect the presence of Bacillus 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 Bacillus 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 tes.ted 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
• Antigen (Bacillus cereus) 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 5 times in saline. They are then resuspended in 1% formol saline.
• Balb/c mice are injected with the prepared antigen. They are given intraperitoneal and/or intravenous
• mice are bled approximately six days after the last injection and the serum tested for antibodies by assay.
• a conventional assay used for this serum titer testing is the enzyme-linked immunosorbent assay system.
• mice show antibody production after this regimen, generally a positive titer of at least 10,000, a mouse is selected as a fusion donor and given a booster injection (0.02 ml 80% T vaccine) intravenously, three days prior to splenectomy.
• Spleen cells from the immune mice are harvested three days after boosting, by conventional techniques.
• the donor mouse selected is killed and surface-sterilised by immersion in 70% ethyl alcohol.
• the spleen is then removed and immersed in approximately 2.5 ml DMEM to which has been added 3% FCS.
• the spleen is then gently homogenised in a LUX homogenising tube until all cells have been released from the membrane, and the cells are washed in 5 ml 3% FCS-DMEM.
• the myeloma cell line used is NSO (uncloned) ,
• the myeloma cells are in the log growth phase, and rapidly dividing. Each cell line is washed using, as tissue culture medium, DMEM containing 3% FCS. The spleen cells are then spun down at the same time that a relevant volume of myeloma cells are spun down (room temperature for 7 minutes at 600 g) , and each resultant pellet is then separately resuspended in 10 ml 3% FCS-DMEM. In order to count the myeloma cells, 0.1 ml of the suspension is diluted to 1 ml and a haemacytometer with phase microscope is used.
• 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
• each well contains 1.0 ml of the standard HAT medium (hypoxanthine, aminopterin and thymidine) and a feeder layer of Balb/c
• the wells are kept undisturbed, and cultured at 37°C in 9% C0 2 air at approximately 100% humidity.
• the wells are analysed for growth, utilising the conventional inverted microscope procedure, after about 5 to 10 days.
• screening tests for the specific monoclonal antibody are made utilising the conventional enzyme immunoassay screening method described below.
• 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 IM 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
• 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) chromatography.
• 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 IM 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 2 The procedure of Example 1 may be followed to produce a monoclonal antibody against Bacillus cererus diarrheal toxin.
• the antigen is prepared by brain-heart infusion (1% glucose) , and the supernatant filtered.
• EXAMPLE 3 The procedure of Example 1 may be followed to produce a monoclonal antibody against Bacillus cererus diarrheal toxin.
• the antigen is prepared by brain-heart infusion (1% glucose) , and the supernatant filtered.
• 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 Bacillus.
• 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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