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
  • C07K16/1289—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria from Mycobacteriaceae (F)
• • 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 Mycobac ⁇ terium are desired which when used will rapidly diagnose the presence of such organisms in speci ⁇ mens.
• Mycobac ⁇ terium species Divisions have been made among the Mycobac ⁇ terium species. Some of the representative members include Mycobacterium tuberculosis, M. butyricum, M. chelonei, M. marinum, M. phlei, M. smegirtetis, and M. leprae.
• M. tuberculosis is the causative agent in tuberculosis, which is one of the most common and important infectious diseases of mankind. Tuberculosis attacks the lung in the primary infection in human-human transmission where it produces a chronic pneumonia indistinguishable from many other chronic pneu ⁇ monias. It may then progress in the lungs to
• SUBSTITUTE SHEET cavities which are distinctive but still require differentiation from many of the other fungal - infections. Tuberculosis diagnosis is costly and time consuming because of the technical skill required and the very slow growth character ⁇ istics of the organism. Diagnosis may often be delayed and sometimes missed.
• Tuberculosis can, in some patients, ravage the body with abcesses in every organ. It can produce meningitis, bone involvement, and it can involve the spleen and liver and the genital tracts of males and females. Extensive control programs and treatment of active cases have caused the disease to dramatically decline in the United States in the last twenty-five years. This has led to lessened skills in the routine microbiology lab in its diagnosis in many areas of the country where the disease is not common. Since it is such a highly infectious disease, and since so many cases have been imported into the United States from Asia, and to the United Kingdom from Asia and Africa, the expection is that a rise in frequency of disease-bearing patients could lead to a flare up in the United States and elsewhere.
• Applicant's invention will assist in the immediate detection of Mycobac- terium tuberculosis in tissue, sputum, and other excretions of humans and animals for the rapid diagnosis of tuberculosis.
• the frequency with which chronic pneumonias are evaluated for tuberc ⁇ ulosis is very high in the United States and elsewhere and the evaluation is costly, time consuming, expensive, and insensitive.
• the invention can be used as a part of a system ⁇ atic kit to exclude pneumonia, fungal, and/or tuberculin infections.
• a wide variety of 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, .che ilum- 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 inescent, 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 Mycobacterium antigens and/or organisms.
• the present invention com- prises monoclonal antibodies specific for an antigen or species of Mycobacterium; in partic ⁇ ular, the antigens or species of Mycobacterium tuberculosis (designated as M. tuberculosis I, II, III, IV, V, VI, VII, or VIII), and the antigens or species of M. butyricum, M. chelonei, M. marinum, M. phlei, M. smegmetis, _M. leprae as well as a monoclonal antibody broadly cross- reactive with an antigen for each species of the genus Mycobacterium.
• M. tuberculosis I, II, III, IV, V, VI, VII, or VIII the antigens or species of M. butyricum
• M. chelonei M. marinum
• M. phlei M. smegmetis
• _M. leprae as well as a monoclonal antibody broadly cross- reactive with an antigen
• the invention also comprises labeled mono ⁇ clonal antibodies for use in diagnosing the presence of the Mycobacterium antigens, each comprising a monoclonal antibody against one of the above-mentioned antigens to Mycobacterium or to a particular species thereof and linked thereto an appropriate label.
• the label can be chosen from the group consisting of a radio ⁇ active isotope, enzyme, fluorescent compound, che iluminescent compound, bioluminescent com ⁇ pound, ferromagnetic atom, or particle, or any other label.
• the invention further comprises the process for diagnosing the presence of Mycobacterium antigens or organisms in a specimen comprising contacting said specimen with the labeled mono- clonal 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 Mycobacterium and a carrier or diluent, as well as kits contain ⁇ ing at least one labeled monoclonal antibody to an antigen of a Mycobacteriu .
• the monoclonal antibodies of the present invention are prepared by fusing spleen cells, from a mammal which has been immunized against the particular Mycobacterium 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 Mycobacterium 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 Mycobacterium 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 Mycobacterium 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 Mycobacterium antigen.
• the monoclonal antibody selected, which is specific for the particular Mycobacterium 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 im unosorbent 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 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 Mycobacterium 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 Mycobacterium 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 Mycobacterium in various specimens. It is also possible to use the broadly cross-reactive monoclonal antibody which can identify the genus Mycobacterium alone or as part of a kit containing antibodies that can identify other bacterial genera or species of Mycobacterium 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 costlyj therapy in a sick patient. A test that would refurn 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 Mycobacterium.
• kits comprising at least one labeled monoclonal antibody against a particular Mycobacterium antigen or species, as well as any appropriate stains, counterstains, or reagents.
• kits containing at least one control sample of a Mycobacterium antigen and/or a cross-reactive labeled monoclonal antibody which would detect the presence of any of the Mycobacterium organisms in a ' particular sample.
• Specific antigens to be detected in this kit include the antigens of Mycobacterium tuberculosis (applicant has further divided this species into eight subgroups: M. tuberculosis I, II, III, IV, V, VI, VII, or VIII), M. butyricum, M. chelonei, M. marinum, M. phlei, M. smegmetis, and M. leprae.
• Monoclonal diagnostics which detect the presence of Mycobacterium 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 labeled 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, milk, and the like contain the particular Mycobacterium antigen. More particularly, 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 contamination.
• the invention will be further illustrated in connection with the following Examples which are set forth for the purposes of illustration only and not by way of limitation.
• API Analytical Profile Index (ref. Ayerst Labs)
• DMEM Dulbecco's Modified Eagles Medium
• FCS Foetal Calf Serum
• PBS phosphate-buffered saline % T refers to vaccine concentration measured in a 1 cm light path
• Monoclonal antibodies of the present invention are prepared generally according to the method of Koehler and Milstein, Eur. J. Immunol. ⁇ _, (1975) 292.
• the cells were harvested and disrupted by milling with glass beads.
• the resulting slurry was centrifuged at 5°C (300 rpm for 60 minutes, equivalent to 2800 x g) .
• the supernatant was drawn off and centrifuged at 50,000 x g for 1 hour at 5°C.
• the supernatant was passed through a train of filters (5.0, 0.45 and 0.22 ⁇ m) .
• the sterile filtrate was stored at -20°C.
• the final protein concentration was 10.00 mg/ml.
• mice are first given 100 ⁇ l 80%T vaccine intraperitoneally and, 14 days later, 50 ⁇ l vaccine intravenously.
• 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.
• a positive titer of at least 10,000 a mouse is selected as a fusion donor and given a booster injection (50 ⁇ l 80% T vaccine) intravenously, three days prior to splenectomy.
• Spleen cells from the immune mice are harvested three days after boosting, by conventional techniques.
• the donor mouse selected is killed and surface-sterilised by immersion in 70% ethyl alcohol.
• the spleen is then removed and immersed in approximately 2.5 ml DMEM to which has been added 3% FCS.
• the spleen is then gently homogenised in a LUX homogenising tube until all cells have been released from the membrane, and the cells are washed in 5 ml 3% FCS-DMEM.
• the cellular debris is then allowed to settle and the spleen cell suspension placed in a 10 ml centrifuge tube. The debris is then rewashed in 5 ml 3% FCS-DMEM. 50 ml suspension are then made in 3% FCS-DMEM.
• the myeloma cell line used is NSO (uncloned) , obtained from the MRC Laboratory of Molecular Biology in Cambridge, England.
• the myeloma cells are in the log growth phase, and rapidly dividing.
• Each cell line is washed using, as tissue culture medium, DMEM containing 3% FCS.
• the spleen cells are then spun down at the same time that a relevant volume of myeloma cells are spun down (room temperature for 7 minutes at 600 g) , and each resultant pellet is then separately resuspended in 10 ml 3% FCS-DMEM.
• 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 108 Spleen cells are 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 is placed in a 37°C water-bath. 1 ml of a 50 w/v solution of polyethylene glycol 1500
• each well contains 1.0 ml of the standard HAT medium (hypoxanthine, aminopterin and thy idine) 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.
• 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 and utilised as a template to define the specificity of the parent group. Clones reacting to antigens present in other genera, e.g. E ⁇ _ coli are discarded. The EIA immunoassay noted above may be used.
• Balb/c 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 buffer.
• cold phosphate buffer 0.1M sodium phosphate, pH 8.2
• 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 stored at -20 C.
• the monoclonal antibody specific against the antigen, prepared as above, is linked to an enzyme, viz. highly-purified alkaline phosphatase.
• Monoclonal antibody was dialysed with alkaline phosphatase (Sigma Type VII-T) , against 2 x 1000 ml of phosphate buffered saline (PBS), pH 7.4 at +4 C. After dialysis the volume was made up to 2.5 ml with PBS and 25 ⁇ l of a 20% glutaraldehyde in PBS solution added. The conjugation mixture was left at room temperature for 1.5 hours.
• Example 1 The general procedure of Example 1 was followed, in most respects.
• the antigen was Mycobacterium tuberculosis obtained from the Royal Post-graduate Medical School.
• a pressate was obtained by milling viable organisms, centrifuging them for 1 hour, 5 C, 300 rpm, centrifuging the supernatant at 50,000 g for 1 hour at 5 C, and filtering the supernatant.
• the animal immunisation schedule comprised an ip injection followed by 3 iv injections after successive 2, 3 and 1 week intervals. Cloning was by limiting dilution.
• the monoclonal was specific to Mycobacterium tuberculosis and to a strain (H37RV) thereof, and negative to other species of Mycobacteria, E . coli, Shigella, Klebsiella, Pseudomonas, Enterobacter, Salmonella and Streptococcus.
• 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 Mycobacterium.
• 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.

Landscapes

• Chemical & Material Sciences (AREA)
• Organic Chemistry (AREA)
• Health & Medical Sciences (AREA)
• General Health & Medical Sciences (AREA)
• Biochemistry (AREA)
• Biophysics (AREA)
• Life Sciences & Earth Sciences (AREA)
• Genetics & Genomics (AREA)
• Medicinal Chemistry (AREA)
• Molecular Biology (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Immunology (AREA)
• Preparation Of Compounds By Using Micro-Organisms (AREA)
• Peptides Or Proteins (AREA)

Applications Claiming Priority (2)

Publications (1)

Family

ID=10568435

Family Applications (1)

Country Status (3)

Families Citing this family (2)

Family Cites Families (1)

• 1984
  • 1984-10-19 GB GB848426466A patent/GB8426466D0/en active Pending
• 1985
  • 1985-10-16 EP EP19850905082 patent/EP0198002A1/de not_active Withdrawn
  • 1985-10-16 WO PCT/GB1985/000468 patent/WO1986002357A1/en unknown

Non-Patent Citations (1)

Also Published As

Similar Documents

Legal Events