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WO1995019781A1 - Dosages permettant de rechercher le mycobacterium tuberculosis a l'aide d'anticorps monospecifiques - Google Patents

Dosages permettant de rechercher le mycobacterium tuberculosis a l'aide d'anticorps monospecifiques Download PDF

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Publication number
WO1995019781A1
WO1995019781A1 PCT/US1994/014685 US9414685W WO9519781A1 WO 1995019781 A1 WO1995019781 A1 WO 1995019781A1 US 9414685 W US9414685 W US 9414685W WO 9519781 A1 WO9519781 A1 WO 9519781A1
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WIPO (PCT)
Prior art keywords
antibody
tuberculosis
monoclonal antibody
binds
mtb
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Application number
PCT/US1994/014685
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English (en)
Inventor
Salman H. Siddiqi
Raisur A. F. Rahman
Richard Terry Root
Original Assignee
Rodrick, Richard, J.
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Filing date
Publication date
Application filed by Rodrick, Richard, J. filed Critical Rodrick, Richard, J.
Priority to AU15534/95A priority Critical patent/AU1553495A/en
Publication of WO1995019781A1 publication Critical patent/WO1995019781A1/fr

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G01N33/5695—Mycobacteria
    • 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)

Definitions

  • the present invention relates to antibodies which bind to Mycobacterium tuberculosis while showing little cross-reactivity with other Mycobacterium species, and to assays for the detection of Mycobacterium tuberculosis using these antibodies.
  • Tuberculosis is a necrotizing bacterial infection which can be caused in man by two species of tubercle bacilli: Mycobacterium tuberculosis and Mycobacterium bovis .
  • M. tuberculosis By far the greatest number of tuberculosis cases in the United States are caused by M. tuberculosis .
  • the lungs are most commonly affected, although infection may occur in the kidneys, bones, lymph nodes, meninges, or may be disseminated throughout the body. Confirmation of active M. tuberculosis infection requires the identification of the bacillus from tissue or body fluid.
  • a sustained course of chemotherapy is the primary form of treatment for M. tuberculosis infection.
  • mycobacteria may cause chronic infections, including M. avium/intracellulare and M. kansasii .
  • a first aspect of the present invention is a monospecific antibody selected from the group consisting of: (a) a monoclonal antibody produced by the cell line Mtb-b5.3.5; (b) a monoclonal antibody which binds to the 65 kilodalton protein of Mycobacterium tuberculosis bound by the monoclonal antibody of (a) ; (c) a monoclonal antibody which binds to the epitope bound by the monoclonal antibody of (a) ; and (d) a polyclonal antibody which binds to the epitope bound by the monoclonal antibody of (a) .
  • These monospecific antibodies exhibit essentially no binding to Mycobacterium avium under the same conditions where they bind to Mycobacterium tuberculosis .
  • a further aspect of the present invention is a monoclonal antibody selected from the group consisting of (a) a monoclonal antibody produced by the cell line Mtb- b5.3.5; (b) a monoclonal antibody which binds to the 65 kilodalton protein of Mycobacterium tuberculosis bound by the monoclonal antibody of (a) ; and (c) a monoclonal antibody which binds to the epitope bound by the monoclonal antibody of (a) .
  • These monoclonal antibodies exhibit essentially no binding to Mycobacterium avium under the same conditions where they bind to Mycobacterium tuber cul osi s .
  • FIG. 1 Further aspects of the present invention are a monoclonal antibody produced by the cell line Mtb-b5.3.5, the cell line Mtb-b5.3.5, and a cell line producing the monoclonal antibodies described above.
  • a further aspect of the present invention is a method of assaying the presence of Mycobacterium tuberculosis in a sample.
  • a sample is contacted with an antibody as described above under conditions permitting the antibody to bind to M. tuberculosis , and then the presence or absence of the antibody bound to the protein is detected.
  • the assay may be either heterogenous or homogenous, and is preferably homogenous.
  • a further aspect of the present invention is a kit for assaying Mycobacterium tuberculosis in a sample, using an antibody as described above conjugated to a detectable group.
  • a further aspect of the present invention is a kit for assaying Mycobacterium tuberculosis in a sample, including an antibody as described above and a specific binding partner for the antibody conjugated to a detectable group.
  • FIGURE 1 shows the results of specificity testing of candidate monoclonal antibody Mtb-b5 with a variety of Mycobacterium species adsorbed on the surface of a microtiter plate, by enzyme-linked immunosorbent assay (ELISA) .
  • FIGURE 2A shows specificity testing of clone Mtb-b5.3 obtained from spent media on a variety of A- cobacterium species and strains.
  • FIGURE 2B shows specificity testing of clone Mtb-b5.3 obtained from ascites on a variety of Mycobacterium species and strains.
  • FIGURE 2C shows specificity testing of clone Mtb- b5.3.5 obtained from ascites on a variety of Mycobacterium species and strains.
  • FIGURE 3 shows the interpolation of molecular weight standards on Western Blot to determine the molecular weight of the protein antigen bound by Mtb-b5.3.5.
  • FIGURE 4 Results of a dipstick format for the detection of M. tuberculosis using monoclonal antibody Mtb- b5.3.5 and dilutions of heat-killed M. tuberculosis (strain H37Rv) (striped bar) and M. kansasii (strain 714) (solid bar) cells, with goat ⁇ anti-Mouse IgG liposomes containing sulforhodamine red for staining.
  • MAbs Monoclonal antibodies
  • Such MAbs have been used to identify cultured M. avium and
  • an assay To be useful in the clinical diagnosis of M. tuberculosis , an assay must reliably detect the presence of ATycoJbacterium tuberculosis while not cross reacting significantly with M. kansasii , M. scrofulaceum, M. gordonae, M. avium, M. intracellulare, M. fortuitum or M. chelonae, as these are organisms commonly isolated in the clinical environment and are similar to M. tuberculosis .
  • sensitivity refers to the ability of an assay to reliably detect the presence of Mycobacterium tuberculosis (i.e., to give a high percentage of true positive reactions and a low percentage of false negative reactions) .
  • specificity refers to the ability of an assay to reliably not react with mycobacterial species other than M. tuberculosis (i.e., to give a low percentage of false positive reactions and a high percentage of true negative reactions) .
  • Monospecific antibodies of the present invention preferably bind strongly to one or more M. tuberculosis strains with little binding, under the same conditions, to any M-. avium. More preferably, monospecific antibodies of the present invention preferably bind strongly to one or more M. tuberculosis strains with little binding, under the same conditions, to anyone of M. avium, M. intracellulare . M. gordonae, M. fortui tum, M. smegmatis , M. szulgai , M. xenopi , M. phlei , M. terrae, M. gastri , M. scrofulaceum, M. kansasii , and M.
  • monospecific antibodies of the present invention preferably bind strongly to one or more M. tuberculosis strains with little binding, under the same conditions, to any combination of M. avium, M. intracellulare . M. gordonae, M. fortui tum, M. smegmatis, M. szulgai , M. xenopi , M. phlei , M. terrae, M. gastri , M. scrofulaceum, M. kansasii , and M. chelonae .
  • monospecific antibodies of the present invention bind strongly to one or more M. tuberculosis strains with essentially no binding, under the same conditions, to M. avium. More preferably, monospecific antibodies of the present invention bind strongly to one or more M. tuberculosis strains with essentially no binding, under the same conditions, to any one of M. avium, M. intracellulare, M. gordonae, M. fortui tum, M. smegmatis, M. szulgai , M. xenopi , M. phlei , M. terrae, M. gastri , M. scrofulaceum, M. kansasii and M.
  • monospecific antibodies of the present invention bind strongly to one or more M. tuberculosis strains with essentially no binding, under the same conditions, to any combination of M. avium, M. intracellulare . M. gordonae, M. fortui tum, M. smegmatis, M. szulgai , M. xenopi , M. phlei , M. terrae, M. gastri , M. scrofulaceum, M. kansasii and M. chelonae.
  • the term 'essentially no binding' means that, in ELISA assays as described in Example 4 herein, the optical density (OD) reading at 450nM is OD ⁇ . 100 milli-A.
  • the term 'little binding' means that, in ELISA assays as described in Example 4 herein, the optical density (OD) reading at 450nM is 100 milli-A ⁇ . OD ⁇ . 300 milli-A.
  • the term 'bind strongly to' means that, in ELISA assays as described in Example 4 herein, the optical density (OD) reading at 450nM is OD _> 300 milli-A.
  • Antibodies which may be used to carry out the present invention include (a) monoclonal antibody Mtb-b5.3.5, (b) monoclonal antibodies which bind to the 65 kilodalton protein (or more preferably the epitope) bound by monoclonal antibody Mtb-b5.3.5, and (c) fragments of (a) or
  • monoclonal antibody Mtb-b5.3.5 binds to the antigen (or more preferably the epitope) bound by monoclonal antibody Mtb-b5.3.5.
  • monoclonal antibody Mtb-b5.3.5 was developed by Salman H. Siddiqi, A.F. Raisur Rahman, and Richard T. Root.
  • antibodies refers to all types of immunoglobulins, including IgG, IgM, IgA, IgD, and IgE. Of these, IgG is particularly preferred.
  • the antibodies may be of any species of origin, including (for example) , mouse, rat, rabbit, horse, or human, or may be chimeric antibodies. See, e . g. , M. Walker et al. , Molec . Immunol . , 26, 403 (1989) .
  • the antibodies may be recombinant monoclonal antibodies produced according to the methods disclosed in Reading, U.S. Patent No. 4,474,893, or Cabilly et al. , U.S. Patent No. 4,816,567.
  • the antibodies may also be chemically constructed by specific antibodies made according to the method disclosed in Segel et al. , U.S. Patent No. 4,676,980.
  • Monoclonal antibodies other than Mtb-b5.3.5 which bind the antigen (or more preferably the epitope) bound by the Mtb-b5.3.5 antibody may be obtained in accordance with known techniques. For example, cells identified as carrying the antigen bound by Mtb-b5.3.5 can be washed with an aqueous solution containing a detergent to remove the antigen therefrom, the various fractions in the solution separated by chromatography (e.g., high performance liquid chromatography) , and the fraction containing the antigen identified by its ability to bind the Mtb-b5.3.5 antibody.
  • chromatography e.g., high performance liquid chromatography
  • the Mtb-b5.3.5 antibody may be immobilized on a solid support to provide an affinity chromatography column, a sample of proteins extracted from M. tuberculosis cell walls passed through the column, antigen bound to the Mtb-b5.3.5 antibody eluted from the column, and the eluted antigen used to produce an antibody.
  • Antibodies which bind to the epitope (i.e., the specific binding site) bound by Mtb-b5.3.5 can be identified in accordance with known techniques, such as their ability to compete with labelled Mtb-b5.3.5 antibody in a competitive binding assay.
  • antibody includes antibody fragments such as, for example, Fab, F(ab') 2 , and Fv fragments, and the corresponding fragments obtained from antibodies other than IgG.
  • antibody fragments can be produced by known techniques.
  • Monoclonal Fab fragments may be produced in Esherichia coli by recombinant techniques known to those skilled in the art. See, e . g. , Huse, Science 246, 1275-81 (1989) .
  • Monoclonal antibodies of the present invention may be produced in a hybridoma cell line according to the technique of Kohler and Milstein, Nature, 265, 495-97
  • a solution containing the appropriate antigen may be injected into a mouse and, after a sufficient time, the mouse sacrificed and spleen cells obtained.
  • the spleen cells are then immortalized by fusing them with myeloma cells or with lymphoma cells, typically in the presence of polyethylene glycol, to produce hybridoma cells.
  • the hybridoma cells are then grown in a suitable medium and the supernatant screened for monoclonal antibodies having the desired specificity as given herein. Accordingly, a further aspect of the present invention is to provide hybridomas which produce antibodies against an antigen found on M. tuberculosis cell surface.
  • antibodies includes monospecific polyclonal antibodies (e.g., antibodies of polyclonal origin which bind to a single epitope) , the monospecific polyclonal antibodies directed to the same epitope as is bound by Mtb-b5.3.5.
  • Polyclonal antibodies to the 65 kilodalton protein bound by Mtb-b5.3.5 may be produced in accordance with techniques known in the art.
  • polyclonal antibodies to the 65 kilodalton protein may be screened using a peptide consisting essentially of the epitope bound by Mtb-b5.3.5.
  • Monospecific polyclonal antibodies may also be obtained by screening polyclonal antibodies using said 65 kilodalton protein; the 65 kilodalton protein is immobilized and saturated with Mtb-b5.3.5 (or another monoclonal antibody which binds the epitope bound by Mtb-b5.3.5), and then saturated with the polyclonal antibody, thus leaving unbound only the polyclonal antibodies specific for the epitope of Mtb-b5.3.5.
  • the hybridoma cell line Mtb-b5.3.5 which produces monoclonal antibody Mtb-b5.3.5, was deposited with the American Type Culture Collection, 123Ql Parklawn Drive, Rockville, Maryland 20852, USA, in accordance with the provisions of the Budapest Treaty on 14 December 1993, and has been assigned ATCC Accession Number HB 11500.
  • the method disclosed herein may be used with specimens from subjects suspected of carrying the M. tuberculosis organism and specimens from subjects who have not been previously diagnosed as having an M. tuberculosis infection.
  • the methods disclosed herein are applicable to testing for pulmonary tuberculosis, miliary (disseminated) tuberculosis, tuberculous meningitis, genitourinary tuberculosis (including renal tuberculosis) , tuberculosis of bones and joints, and tuberculosis of other organs such as the pericardium, gastrointestinal tract, and adrenal glands. Specimens taken from the subject are used to initiate culture growth, and the culture growth is assayed for the presence of M. tuberculosis .
  • Specimens taken from human subjects for use in preparing cultures for use in the methods disclosed herein are generally biological fluids such as sputum, blood plasma, bronchial secretions, gastric secretions, urine, cerebrospinal fluid, serous effusion, ascites fluid or pus from an abscess or sinus.
  • the specimen may be a tissue biopsy sample, such as from kidney, lymph node, or other organ. The appropriate selection of the biological specimen and appropriate culture techniques will be readily apparent to one skilled in the art. Specimens taken from subjects may also be assayed directly (i.e., assaying the specimen itself for the presence of M. tuberculosis, rather than assaying the growth cultured from the specimen) .
  • the subject from whom the specimen is taken has not been previously diagnosed as harboring an M. tuberculosis infection.
  • the subject from which the specimen is taken has been previously diagnosed as carrying M. tuberculosis and possibly has already undergone treatment thereof.
  • specimens may be collected from subjects prior to initiating therapy and at points subsequent thereto to assess the effects of the therapy
  • the monoclonal antibody of the present invention may be utilized in any immunoassay format known in the art as suitable for use with monoclonal antibodies.
  • assays include, but are not limited to, latex agglutination assays, solid phase immunoassays, enzyme linked immunosorbent assays (ELISA), and dip stick assays.
  • a preferred immunoassay format is a "flow through device" utilizing the antibody of the present invention.
  • a flow through device refers to immunoassay devices in which the monoclonal antibody is placed on a physical support able to trap bacteria while allowing liquid phases to pass therethrough.
  • Such physical supports include filtering means, such as filter paper, which allows liquid phases of the assay to pass through the filter while retaining M. tuberculosis .
  • Immunoassays carried out in accordance with the present invention may be homogenous assays or heterogenous immunological assays.
  • the immunological reaction usually involves the specific antibody (e.g., Mtb-b5.3.5), a labeled analyte, and the sample of interest.
  • the signal arising from the label is modified, directly or indirectly, upon the binding of the binding pair consisting of the monoclonal antibody and the labeled analyte.
  • Both the immunological binding of the monoclonal antibody and the analyte of interest in the sample and the detection of the extent thereof, are carried out in a homogenous solution.
  • Immunochemical labels which may be employed to detect the binding pair include free radicals, radioisotopes, fluorescent dyes, enzymes, bacteriophages, coenzymes, and so forth.
  • the reagents are usually the specimen, the antibody of the invention (e.g., Mtb-b5.3.5), and means for producing a detectable signal indicating the binding of the binding pair consisting of the monoclonal antibody and the analyte of interest contained in the sample. Similar specimens as described above may be used.
  • the antibody is generally placed or immobilized on a support (such as a bead, plate, filter or slide) , and contacted with the specimen suspected of containing the antigen in a liquid phase. The support is then separated from the liquid phase and the support phase is examined for a detectable signal employing means for producing such signal.
  • the signal is related to the binding of the monoclonal antibody with analyte present in the specimen.
  • Means for producing a detectable signal include the use of detectable groups such as radioactive labels, fluorescent labels, enzyme labels, and so forth.
  • detectable groups such as radioactive labels, fluorescent labels, enzyme labels, and so forth.
  • an antibody which binds to that site can be conjugated to a detectable group and added to the liquid phase reaction solution before the separation step.
  • the presence of the detectable group on the solid support indicates the presence of the antigen of interest in the test sample.
  • Exemplary of heterogenous immunoassays are the radioimmunoassay, immunofluorescence methods, enzyme- linked immunoassays, luminescent immunoassays and the like.
  • Monoclonal antibodies as described herein may be used in a "two-site" or “sandwich” assay. While a single cell line may serve as a source for both the labeled monoclonal antibody and the bound monoclonal antibody in a sandwich assay, it is understood by those skilled in the art that such labeled and bound monoclonal antibodies used in a sandwich assay must bind to the analyte at separate sites, such that the binding of the labeled monoclonal antibody does not interfere with the binding of the analyte and the bound monoclonal antibody. Exemplary assays are described in U.S. Patent No. 4,376,110.
  • Antibodies described herein may be supported on a solid support suitable for a diagnostic assay (such as beads, plates, slides or wells formed from materials such as latex, polystyrene; or filters, sheets, membranes, test strips, dipsticks, cards or the like formed from materials such as nitrocellulose) in accordance with known techniques.
  • Antibodies as described herein may be conjugated to detectable groups such as radiolabels (e.g., 35 S, 125 I, 131 I) , enzyme labels (e.g., horseradish peroxidase, alkaline phosphates), and fluorescent labels (e.g., fluorescein) in accordance with known techniques.
  • radiolabels e.g., 35 S, 125 I, 131 I
  • enzyme labels e.g., horseradish peroxidase, alkaline phosphates
  • fluorescent labels e.g., fluorescein
  • Detectable groups for use in carrying out the methods of the present invention include those detectable groups which are visible without further treatment when bound to the monoclonal antibody, the analyte, or the binding pair consisting of the monoclonal antibody and the analyte.
  • Such visible detectable groups include, for example, sacks containing a dye or some other material which is visible without lysing of the sack; such sacks include liposomes (single walled or multi-lamellar) and microcapsules (for example polymer microcapsules); (see, e . g. , U.S. Patent Nos. 4,703,017, 4,920,046, incorporated herein by reference in their entirety) .
  • the diagnostic kit comprises (a) an antibody of the invention (e.g., Mtb-b5.3.5) and (b) a second antibody conjugated to a detectable group.
  • the reagents may also include ancillary agents such as buffering agents and protein stabilizing agents, e.g., polysaccharides and the like.
  • the diagnostic kit may further include, where necessary, other members of the signal-producing system of which system the detectable group is a member (e.g., enzyme substrates) , agents for reducing background interference in a test, control reagents, apparatus for conducting a test, and the like.
  • a second embodiment of a test kit comprises (a) an antibody as described herein, and (b) a specific binding partner for the antibody conjugated to a detectable group.
  • Ancillary agents as described above may likewise be included.
  • the test kit may be packaged in any suitable manner, typically with all elements in a single container along with a sheet of printed instructions for carrying out the test.
  • the test device used in carrying out the assay of the present invention is a flow through solid phase assay device as described in U.S. Patent Nos. 4,920,046, 5,073,340, 5,185,127 and 5,204,061 (applicants intend the disclosures of all U.S. patents cited herein to be incorporated herein in their entirety) .
  • Such assay devices may be used in any flow through immunoassay procedure including competitive and sandwich assays; the end point may be color development visible to the naked eye such that no optical instruments are required to ascertain the test results.
  • Such color development may be achieved using a monoclonal antibody supported on a test area, the monoclonal antibody capable of forming a binding pair with the analyte of interest contained in the sample, and a tracer which binds to the binding pair, wherein the bound tracer is visible to the naked eye without further treatment.
  • tracers may include, for example, liposomes and microcapsules containing a visible dye (see, e. g. , U.S. Patent No. 4,703,017) .
  • the assays of the present invention are suited to being carried out using samples of cultures grown on solid culture media.
  • Suitable solid media includes egg-based media as well as agar-based media such as, for example, Lowenstein-Jensen (LJ media, available from BBL and Diffco) , Middlebrook 7H 10 (BBL) , Middlebrook 7H 11 (BBL) , and Ogawa medium.
  • LJ media Lowenstein-Jensen
  • BBL Middlebrook 7H 10
  • BBL Middlebrook 7H 11
  • Ogawa medium Ogawa medium.
  • Assays of the present invention may also be carried out using clinical specimens directly (i.e., assaying the specimen itself for the presence of M. tuberculosis, rather than assaying the growth cultured from the specimen) .
  • Clinical specimens assayed directly may include, but are not limited to, sputum, bronchial secretions, blood plasma, gastric secretions, urine, cere.brOspinal fluid, serous effusion, ascites fluid or pus from an abscess or sinus.
  • the sample to be tested may be prepared from a tissue biopsy specimen, such as from a kidney, lymph node, or other organ. Any suitable assay device or format may be used.
  • the specimens When clinical specimens are used in a flow- through assay device, the specimens must have physical characteristics such that the specimen can be washed through the physical support. Treatment of clinical specimens (e.g., to reduce the viscosity thereof) may be required to achieve proper flow characteristics. Such treatments may include liquefying, diluting, centrifuging, and extracting steps performed on the specimen, and may further include additional steps as are known in the art.
  • Mtb-b5, Mtb-b5.3 and Mtb-b5.3.5 designate the same antibody produced by different subclones of cells. The designations indicate the subclone origin of the monoclonal antibody.
  • ml means milliliter
  • ⁇ l means microliter
  • ⁇ g means microgram
  • ⁇ means micron
  • °C means degrees Centigrade
  • OD optical density
  • M means molar
  • nM means nanometer
  • ELISA enzyme linked immunosorbent assay
  • PBS phosphate buffered saline
  • HRP horseradish peroxidase
  • Filtrate proteins for use in the following Examples were produced by growing bacteria in protein-free medium (Proskauer-Beck) for six weeks, then filtering to remove cells. The filtrate was sterilized by treating at 60°C for four hours. Proteins were quantitated using the Pierce BCA assay (see U.S. Patent No. 4,839,295) . Thimerosal was added as a preservative to 0.02%.
  • Bacterial lysate for use in the following Examples was produced by grinding whole, heat killed bacteria in a tissue grinder for 4-5 minutes in phosphate buffered saline. Thimerosal was added as a preservative to 0.02%.
  • the bacterial strains used for hybridoma production were jM coJbacterium tuberculosis strains Va3 , Va7, 351, and H37Rv.
  • M. tuberculosis H37Rv is available from the American Type Culture Collection (ATCC) , 12301 Parklawn Drive, Rockville, Maryland 20852, ATCC catalog number 25618. Additional bacterial strains used in testing the sensitivity and specificity of monoclonal antibodies were: M. avium (strains 33, 61, 39); M. kansasii (strains 714, 7, 711); M. scrofulaceum (1004, 78, 9); M. gordonae (strains 1104, 1102, 10); M.
  • strains 2801, 16, 2803 M. chelonae (strains 3001, 18, 2904) ; M. intracellulare (strains 1708, 40, 1701); M. smegmatis (strains 17, 2401, 2403); M. szulgai (strains 11, 1203, 1205); M. xenopi (strains 1902, 1901, 1905); M. phlei (strains 2101, 2104, 15) ; M. terrae (strains 13, 1504, 1505) ; and M. gastri (strains 1305, 1306, 1312) .
  • Antibody-producing hybridomas were generated according to a modification of the original method of Kohler and Milstein, Nature, 256, 495-96 (1975) .
  • BALBc/AnCrl mice were immunized intraperitoneally with a single dose of 100 ⁇ l of complete Freund's adjuvant, then at four weeks with a single dose of 22 ⁇ g total protein of filtrate proteins and bacterial lysate (M. tuberculosis H37Rv) in water, prepared as described in Example 1, above.
  • mice were injected intraperitoneally with 50 ⁇ g of bacterial lysate ⁇ M. tuberculosis H37Rv) .
  • mice were injected intraperitoneally with 50 ⁇ g heat killed mixed tuberculosis cells in water, the mixed tuberculosis cells consisting of equal quantities of the following Mycobacterium tuberculosis strains: Va3, Va7, 351, and H37Rv.
  • mice were injected intraperitoneally and in the foot pads with 50 ⁇ g of M. tuberculosis strain H37Rv culture filtrate and 50 ⁇ g of H37Rv lysate in water. Animals were sacrificed four days after the last injection and the spleen and popliteal and inguinal lymph nodes were removed for fusion.
  • Fusion was accomplished using standard polyethylene glycol (PEG) technique. See Fazekas et al. , J. Immunol . Meth. 35, 1-21 (1980) .
  • PEG polyethylene glycol
  • To produce hybridoma cells the spleen cells and lymph node cells were fused with murine BALB/C line P3/X63-Ag8 myeloma cells (ATCC CRL 1580) in the presence of polyethylene glycol in accordance with the method described in Fazekas et al.
  • DMEM medium with 10% fetal calf serum, HMT additive (Sigma #H8016) and Ewing Sarcoma Growth Factor (ICN Biochemicals) at a 1:40 dilution.
  • HMT additive Sigma #H8016
  • Ewing Sarcoma Growth Factor ICN Biochemicals
  • the screening process consisted of transferring 50 ⁇ l of media from a growing well to a cell-coated well, and 50 ⁇ l of media to a filtrate-coated well. The wells were incubated for 1 hour at 37°C. The wells were then washed with lOO ⁇ l of PBS-Tween, and 50 ⁇ l of goat anti-mouse IgGAM-HRP (Cappel, 55556) diluted 1:6,000 in PBS-Tween was added. Wells were incubated for 1 hour at 37°C. The plates were washed six times with 100 ⁇ l of PBS-Tween, and 50 ⁇ l of TMB substrate solution (Moss, TMBE-1000) was added for 10 minutes.
  • TMB substrate solution Moss, TMBE-1000
  • EXAMPLE 6 Specificity of Monoclonal Antibody Mtb-b5.3
  • the 69 candidate monoclonals were subjected to specificity testing by the ELISA assay (as described in Example 4, above, using adsorbed mycobacterium) using a variety of mycobacteria species and strains.
  • Mtb-b5 was found to bind to M. tuberculosis with little binding to non-tuberculosis strains as compared to other monoclonal antibody candidates. Results are shown in FIG. 1, where OD means optical density (milli-A units) ;
  • M.tb H37Rv means Mycobacterium tuberculosis strain H37Rv;
  • M.av means M. avium;
  • M.sc means M.
  • M.go means M. gordonae
  • M.fo means M. fortui tum
  • M.ch means M. chelonae
  • M.in means M. intracellulare
  • M.ka means M. kansasii .
  • FIGS. 2A and 2B show specificity testing of clone Mtb-b5.3 obtained from spent media on a variety of A-yco acteriu ⁇ - species and strains;
  • Figure 2B shows specificity testing of clone Mtb-b5.3 obtained from ascites on a variety of Mycobacterium species and strains.
  • OD means optical density (milli-A units) .
  • Mycobacterial strains correlated to results as shown in Figures 2A, 2B, and 2C.
  • Clone Mtb-b5.3 was re-cloned to select for a high producing sub-clone, via the standard limiting dilution procedure.
  • the high-producing sub-clone designated Mtb- b5.3.5 was selected for further development.
  • Mycobacterium tuberculosis heat-killed bacteria were pelleted by centrifugation. Approximately 50 ⁇ l of packed cells were re-suspended in 1 ml PBS. This solution was subjected to ultra-sonic disruption at 100 watts for 3 minutes, then 5 minutes, with a 2 minute pause for cooling at 4°C. The resulting suspension was centrifuged for 10 minutes at 10,000 x G. The supernatant was transferred to another tube and centrifuged for 30 minutes at 30,000 x G. This pellet was re-suspended in 0.1 ml distilled water. Protein was quantitated via the Pierce BCA assay.
  • This suspension was diluted to 870 ⁇ g/ml in Laemmli sample buffer, and the sealed tube incubated at 100°C for 5 minutes.
  • One microliter samples were separated via SDS electrophoresis on a 12.5% acrylamide gel, using the Pharmacia PhastGel system. Biotinylated molecular weight standards (Pierce) were also separated on the gel.
  • the gel was electroblotted onto PVDF membrane (Millipore, Inc.) using the manufacturer's method. The membrane was blocked with 1% non-fat dried milk in PBS-Tween (PBST) for 1 hour.
  • PBST PBS-Tween
  • the membrane was transferred to 5 ml of Monoclonal antibody Mtb-b5.3.5 diluted to 30 ⁇ g/ml in PBST and incubated at room temperature with shaking overnight. The membrane was washed three times in 5 ml of PBST for 15 minutes, then transferred to 5 ml of Goat anti-mouse IgG+IgA+IgM-HRP conjugate (Cappell) diluted 1:6000, and Avidin-HRP (Sigma) diluted to 1 ⁇ g/ml, for 2 hours at room temperature with shaking. The blot was washed 5 times with 5 ml PBST for 15 minutes each. Bands were visualized by developing with TMB membrane stain (KP Laboratories) . Molecular weight was determined from interpolation of a plot of the logarithm of the molecular weights of the Pierce standards versus the migration distance. FIG. 3.
  • the dipsticks were then dried for 10 minutes at 56°C, and blocked with 10 drops of a filtered solution of 3% non-fat dried milk and 0.5% ZONYLTM FSN-100 (Du-Pont) in PBS, for 1 minute, then rinsed with PBS-Tween.
  • the dipstick was immersed for 5 minutes in 1 ml monoclonal antibody Mtb-b5.3.5 diluted to 20 ⁇ g/ml in PBS-Tween.
  • the sticks were then rinsed three times in PBS-Tween and dried at 37°C, and staining using goat ⁇ anti-Mouse IgG liposomes containing the marker sulforhodamine red (visible to the eye without further treatment) was carried out.
  • the preparation of liposomes for use as markers in assays is known in the art; see, e. g. , U.S. Patents No. 4,342,826 and 4,703,017.
  • the commercially available flow-through solid phase detection test kit known as DirectigenTM Flu A Test (Becton, Dickinson and Co.) was modified for use with Mtb-b5.3.5 as the assay binder to detect M. tuberculosis .
  • DirectigenTM Flu A Test Becton, Dickinson and Co.
  • DirectigenTM Flu A Test uses a ColorPAKTM flow through assay device (Becton, Dickinson and Co.) .
  • Test procedure The mycobacterium solutions were applied to the test area and allowed to completely adsorb (using the ColorPACTM assay device, the test solutions are applied to a triangular shaped area in the center of the well) . Following a 1 minute wait after complete adsorption of the sample fluid, 250 ⁇ l of a filtered solution of 3% non-fat dried milk, and 0.5% ZONYLTM FSN-100 (Du-Pont) in PBS was added. Then 100 ⁇ l of Mtb-b5.3.5 diluted to 20 ⁇ g/ml was added and allowed to adsorb. Then 0.5 ml of PBS-Tween was added.
  • a flow-through solid phase assay device and test procedure for the detection of Mycobacterium tuberculosis was constructed using components of the DirectigenTM Flu A test kit (Becton, Dickinson and Company) and the
  • Test Procedure The assay used components of the DirectigenTM Flu A and DirectigenTM RSV tests, both of which use a ColorPAKTM flow through device. Monoclonal antibody Mtb-b5.3.5 was labelled with alkaline phosphatase in accordance with procedures known in the art. Buffers and reagents from the DirectigenTM RSV test were used.
  • Results A sample was prepared for each strain of mycobacteria and tested in a ColorPAKTM test device as described above. TABLE 2. All 40 strains of M. tuberculosis tested positive; .all other species were negative except for 3 strains of M. kansasii which gave false positive results. Slight cross-reactivity with M. kansasii is not considered a problem in the clinical setting, as M. kansasii colonies are pigmented and M. tuberculosis colonies are not.
  • Isolated cultures grown from fresh sputum specimens from subjects with known mycobacteria infections were also tested. Seventeen specimens processed at the Maryland State Department of Health were inoculated on L-J slants. When growth was observed for the first time, several colonies were picked and a suspension was made with turbidity equal to McFarland 0.5 - 1.0 standard and the resulting cultures were assayed. All 14 known M. tuberculosis infections tested positive; two known M. kansasii specimens and one known M. avium- intracellulare complex were negative. TABLE 4.
  • the device was also tested using M. tuberculosis colonies taken from the solid media contained on Septi-Chek AFBTM (Becton Dickinson Microbiology Systems) . Media was either Lowenstein-Jensen agar or 7H10 agar. Samples were taken when colonies were first visible with the naked eye. In all cases tested, positive reactions were noted on either the Lowenstein-Jensen agar, the 7H10 agar, or both. TABLE 5.
  • the device was also tested using mycobacterial growth on Middlebrook 7H11, Ogawa 1% and Ogawa 3% medium with similar results (data not shown) .
  • Mtb-b5.3.5 in a flow-through test device provides 100% sensitivity to the strains of M. tuberculosis tested, and high specificity among Mycobacteria tested. Cross reactivity is limited to a few strains of M. kansasii .

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Abstract

Cette invention concerne des anticorps monospécifiques qui se lient sélectivement au bacille Mycobactérium tuberculosis. Ces anticorps monospécifiques comprennent des anticorps monoclonaux et des anticorps polyclonaux monospécifiques. Des procédés de détection de la présence du Mycobactérium tuberculosis à l'aide d'anticorps monospécifiques sont décrits. Les procédés de dosage comprennent l'utilisation de dispositifs de test par immunodosage par filtration.
PCT/US1994/014685 1994-01-25 1994-12-20 Dosages permettant de rechercher le mycobacterium tuberculosis a l'aide d'anticorps monospecifiques WO1995019781A1 (fr)

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AU15534/95A AU1553495A (en) 1994-01-25 1994-12-20 Assays for (mycobacterium tuberculosis) using monospecific antibodies

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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0920622A4 (fr) * 1996-07-26 2004-12-01 Univ Case Western Reserve Detection de microbacteries
WO2005066631A1 (fr) * 2003-12-24 2005-07-21 3M Innovative Properties Company L'utilisation de tensioactifs non ioniques fluores pour reduire la fixation non specifique de molecules a une surface
US7322254B2 (en) 2003-12-12 2008-01-29 3M Innovative Properties Company Variable valve apparatus and methods
WO2007130846A3 (fr) * 2006-05-03 2008-12-31 Pritest Inc Compositions ameliorees et procedes de test de la tuberculose et d'une infection a mycobacterium
US7569186B2 (en) 2001-12-28 2009-08-04 3M Innovative Properties Company Systems for using sample processing devices
US8834792B2 (en) 2009-11-13 2014-09-16 3M Innovative Properties Company Systems for processing sample processing devices
US8931331B2 (en) 2011-05-18 2015-01-13 3M Innovative Properties Company Systems and methods for volumetric metering on a sample processing device
US9067205B2 (en) 2011-05-18 2015-06-30 3M Innovative Properties Company Systems and methods for valving on a sample processing device
US9168523B2 (en) 2011-05-18 2015-10-27 3M Innovative Properties Company Systems and methods for detecting the presence of a selected volume of material in a sample processing device
CN108918890A (zh) * 2018-07-23 2018-11-30 北京市结核病胸部肿瘤研究所 筛选特异性靶向结核分枝杆菌的人的配体蛋白的方法

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* Cited by examiner, † Cited by third party
Title
CLIN. EXP. IMMUNOL., Volume 89, issued 1992, HAJEER et al., "Monoclonal Antibody Epitopes of Mycobacterial 65-kD Heat-Shock Protein Defined by Epitope Scanning", pages 115-119. *
MACARIO et al., "Monoclonal Antibodies Against Bacteria", Vol. 1, Published 1985, by ACADEMIC PRESS INC. (FLORIDA), pages 59-90. *
MICROBIOL. IMMUNOL., Volume 35, Number 11, issued 1991, HARA et al., "Development of Monoclonal Antibodies Reacting Against Mycobacterial 65 kDa Heat Shock Protein by Using Recombinant Truncated Products", pages 995-1007. *
SCIENCE, Volume 246, issued 08 December 1989, HUSE et al., "Generation of a Large Combinatorial Library of the Immunoglobulin-Repertoire in Phage Lambda", pages 1275-1281. *

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0920622A4 (fr) * 1996-07-26 2004-12-01 Univ Case Western Reserve Detection de microbacteries
US7569186B2 (en) 2001-12-28 2009-08-04 3M Innovative Properties Company Systems for using sample processing devices
US7322254B2 (en) 2003-12-12 2008-01-29 3M Innovative Properties Company Variable valve apparatus and methods
WO2005066631A1 (fr) * 2003-12-24 2005-07-21 3M Innovative Properties Company L'utilisation de tensioactifs non ioniques fluores pour reduire la fixation non specifique de molecules a une surface
WO2007130846A3 (fr) * 2006-05-03 2008-12-31 Pritest Inc Compositions ameliorees et procedes de test de la tuberculose et d'une infection a mycobacterium
US8834792B2 (en) 2009-11-13 2014-09-16 3M Innovative Properties Company Systems for processing sample processing devices
US8931331B2 (en) 2011-05-18 2015-01-13 3M Innovative Properties Company Systems and methods for volumetric metering on a sample processing device
US9067205B2 (en) 2011-05-18 2015-06-30 3M Innovative Properties Company Systems and methods for valving on a sample processing device
US9168523B2 (en) 2011-05-18 2015-10-27 3M Innovative Properties Company Systems and methods for detecting the presence of a selected volume of material in a sample processing device
US9725762B2 (en) 2011-05-18 2017-08-08 Diasorin S.P.A. Systems and methods for detecting the presence of a selected volume of material in a sample processing device
CN108918890A (zh) * 2018-07-23 2018-11-30 北京市结核病胸部肿瘤研究所 筛选特异性靶向结核分枝杆菌的人的配体蛋白的方法

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