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WO1992014155A1 - Anticorps monoclonaux diriges contre des antigenes mycobacteriens - Google Patents

Anticorps monoclonaux diriges contre des antigenes mycobacteriens Download PDF

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Publication number
WO1992014155A1
WO1992014155A1 PCT/US1992/001159 US9201159W WO9214155A1 WO 1992014155 A1 WO1992014155 A1 WO 1992014155A1 US 9201159 W US9201159 W US 9201159W WO 9214155 A1 WO9214155 A1 WO 9214155A1
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Prior art keywords
monoclonal antibody
antibody
lam
cells
antibodies
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PCT/US1992/001159
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English (en)
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Patrick J. Brennan
Becky L. Rivoire
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Dynagen, Inc.
Colorado State University Research Foundation
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Application filed by Dynagen, Inc., Colorado State University Research Foundation filed Critical Dynagen, Inc.
Publication of WO1992014155A1 publication Critical patent/WO1992014155A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • C07K16/1267—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

  • This invention pertains to the field of monoclonal antibodies, particularly monoclonal antibodies useful as diagnostic tools for diseases associated with mycobacteria, e.g. tuberculosis.
  • the mycobacteria are a diverse assemblage of acid-fast, Gram-positive bacteria, some of which are important disease-causing agents in humans and animals. Bloom et a . , Rev. Infect. Pis. , 5 ⁇ :765-780 (1983); Chaparas, CRC Rev. Microbiol. , 9_:139-197 (1982). In man the two most common diseases caused by mycobacteria are tuberculosis and leprosy, the causative agents being Mycobacterium tuberculosis and Mycobacterium leprae, respectively.
  • mycobacterial species are capable of causing tuberculosis or tuberculosis-like disease. Wallace, R.J., et al., Review of Infectious Diseases, 5:657-679 (1984).
  • Mycobacterium avium for example, causes tuberculosis in fowl and in other birds. Members of the M. avium-intracellulare
  • MAI immuno-deficiency syndrome
  • AIDS immuno-deficiency syndrome
  • the members of the MAI-complex are resistant to standard anti-tuberculosis drugs. Pitchenik, A.E. , et al., Annals of Internal Medicine, lJU:641-645 (1984).
  • tuberculosis results from respiratory infection with M. tuberculosis. Infection may often be asymptomatic, but could result in disease, producing pulmonary or other lesions which lead to severe debilitation or death.
  • tuberculosis remains a significant health problem especially in developing countries.
  • an estimated 11 million people are affected by the disease and about 3.5 million new cases occur each year.
  • U.S. Congress, OTA "Status of Biomedical Research and Related Technology for Tropical Diseases", OTA-H-258, Washington, D.C. 1985.
  • certain groups of individuals such as those who are HIV positive have a markedly increased incidence of tuberculosis. Early diagnosis of TB is particularly important because the disease is preventable, treatable and curable.
  • immunoassays Due to the disadvantages of using a diagnostic method reliant on microscopic observations, immunoassays have been studied for their applicability in diagnosing TB.
  • Several immunological methods for detecting mycobacterial antigens such as enzyme linked immunosorbent assay (ELISA) and radioimmunoassay (RIA) have been mentioned as possible alternatives to microscopy (Daniel, Reviews of Infectious Diseases Vol II Supplement 2, March-April, 1989, pp.S471-S478) .
  • ELISA enzyme linked immunosorbent assay
  • RIA radioimmunoassay
  • This invention relates to monoclonal antibodies useful as diagnostic tools for diseases associated with mycobacteria, e.g. tuberculosis and leprosy.
  • the monoclonal antibodies also may be useful therapeutically for treating individuals having or suspected of having such diseases.
  • the monoclonal antibodies bind to all species of mycobacteria, and are capable of being used in a variety of diagnostic immunoassays.
  • This invention in one aspect pertains to a solution containing a monoclonal antibody reactive with a mycobacterial antigen.
  • the monoclonal antibody is at sufficient titre and has sufficient binding capability such that the solution is capable of being used, for example, to detect mycobacteria in an agglutination assay such as that described in Example 7, below.
  • This assay permits detection of bacteria in samples obtained from biological fluids containing less than 10,000 bacteria/ml.
  • the antibody titre is at least 70K, and most preferably is 100K [approximately 3 to 5 mg of antibody per ml] .
  • This invention in another aspect pertains to a monoclonal antibody capable of reacting with a surface-exposed, mycobacterial antigen, e.g. lipoarabinomannan (LAM).
  • LAM lipoarabinomannan
  • the preferred monoclonal antibody of this invention is designated ML9D3, which is the antibody product of the ML9D3 hybridoma cell line.
  • This invention also pertains to immortalized, stable antibody producing cell lines, e.g. hybridoma cell lines, capable of producing the above-described antibodies, as well as immunoassays using the antibodies, and therapeutic compositions having the antibodies as a component.
  • the invention also relates to kits useful in aiding in the diagnosis of mycobacterial disease.
  • This invention in one aspect relates to a solution containing a monoclonal antibody reactive with a mycobacterial antigen.
  • the monoclonal antibody is at a sufficient titer and has sufficient binding capability such that the solution is capable of being used in a visually detectable agglutination assay.
  • the solution may be any solution capable of containing the monoclonal antibody in its active form.
  • the solution may contain other components as long as the components do not detrimentally affect the binding capabilities of the monoclonal antibody to such an extent that the antibody cannot perform its intended function.
  • Examples of such solutions include purified or non-purified monoclonal antibodies from mouse ascites or hybridoma tissue culture supernatant.
  • the monoclonal antibodies of this invention are reactive with antigens from mycobacteria, including those mycobacteria that are the causative agents of tuberculosis.
  • Mycobacterium is a genus of microorganism occurring as Gram positive slender rods and distinguished by acid-fast staining. Examples of mycobacteria which may be detected include Mycobacterium tuberculosis, Mycobacterium leprae, and Mycobacterium avium intracellulare etc.
  • the antigen being detected is lipoarabinomannan (LAM) .
  • LAM is a highly immunogenic lipopolysaccharide. LAM is a prominent component of the cell walls of mycobacteria, including both M. tuberculosis and M. leprae and has been implicated as a major B cell stimulant in tuberculosis and leprosy. Portions of LAM are exposed on the surface of intact mycobacteria.
  • monoclonal antibodies is intended to include whole antibodies, antibody fragments capable of binding to the appropriate antigen, chimeric antibodies containing portions from two different species, e.g. human and murine, and synthetic peptides identical to or analogous to the monoclonal antibody. It should be understood that more than one type of monoclonal antibody may be combined with each other to achieve the same, similar or improved results.
  • the preferred form of monoclonal antibodies is whole antibodies, and more preferably • s neat ascites containing whole antibodies.
  • the preferred monoclonal antibodies are designated ML9D3, obtained from the ML9D3 monoclonal-antibody producing hybridoma cell line deposited at the ATCC, Accession Number HB 10684, Rockville, Md.
  • Antibody fragments such as F(ab') 2 , Fab and may be produced by standard techniques of enzyme digestion.
  • synthetic peptides representing Fab and F analogues can be produced by genetic engineering techniques. See e.g.. Better, M. et al. (1988) Science 240:1041; Huston, J.S. et al. (1988) Proc. N tl. Acad. Sci. USA .85:5879-5883.
  • the chimeric antibodies may be produced by preparing a DNA construct which encodes each of the light and heavy chain components of the chimeric
  • the construct includes a fused gene having a first DNA segment which encodes at least the functional portion of the murine variable region (e.g. functionally rearranged variable regions with joining segment) linked to a second DNA segment encoding at least a part of a human constant region.
  • Each fused gene is assembled in or inserted into an expression vector.
  • Recipient cells capable of expressing the gene products are transfected with the genes.
  • the transfected recipient cells are cultured and the expressed antibodies are recovered.
  • the monoclonal antibody preferably is at sufficient titer and has a binding capability such that the solution containing the antibody may be used in a visually detectable agglutination assay as described in Example 7, below, and in copending U.S. application serial no. 07/654,256 entitled “Agglutination Test for Mycobacterial Antigens in Biological Samples", filed February 12, 1991, the entire disclosure of which is incorporated herein by reference.
  • a selection process such as an ELISA assay may be used to determine whether a particular lot or batch of monoclonal antibodies would be useful in such an immunoassayN n an EL SA assay, 1 ⁇ g/ml of LAM antigen can be coated on a solid phase, e.g. multiwell plate.
  • LAM may be prepared as described in Example 6, below and in copending U.S.
  • the coated solid phase then is incubated with various dilutions of monoclonal antibodies at 37°C for approximately one hour.
  • the unbound monoclonal antibodies are separated from the solid phase.
  • the solid phase with attached antibodies is subsequently incubated with a second antibody directed towards the monoclonal antibody and conjugated to an enzyme (Incubation is for 1 hour at room temperature) .
  • the solid phase-antibody-secondary antibody conjugate is incubated for an additional hour at 37°C in the presence of the substrate for the enzyme.
  • the enzymatic reaction is measured using spectrophotometric means, e.g. absorbance, as an indication of the amount of monoclonal antibody bound to the antigen.
  • a curve may be established by plotting the absorbance vs. dilution and the "titer" of the monoclonal antibodies may be determined.
  • the quantitation of specific activity or titer is expressed as the reciprocal of the dilution of monoclonal antibody which exhibits 50% of maximum absorbance. This can be determined from a curve established by plotting the % of maximum absorbance of particular dilutions of monoclonal antibodies versus the dilution of the monoclonal antibodies
  • the readout absorbance (B) is 0.783(B), and the nonspecific absorbance (NSB) is 0.110, and the maximum absorbance (B0) is always considered 1.000 (100% absorbance, the % of maximum absorbance of MAb at this dilution then is calculated as follows:
  • the titer is 85,000 Ab units/unit volume (85K).
  • the solution has a titer of antibodies against LAM of at least 70K, and more preferably 10OK, at 50% absorbance, then the lot or batch used in preparing the dilutions may be used in immunoassays according to the invention.
  • This invention also relates to immortalized, stable, antibody producing cell lines capable of
  • antibody-producing cell lines include hybridoma cell lines, myeloma cell lines, or viral or oncogenically transformed lymphoid cells.
  • the preferred cell line of this invention is an enhanced or expanded hybridoma cell line.
  • Hybridoma cells which can produce the specific antibodies of this invention may be made by the standard somatic cell hybridization technique of Kohler and Milstein, Nature 256:495 (1975) or similar procedures employing different fusing agents. Briefly, the procedure is as follows: the hybridoma which secretes the monoclonal antibodies are produced by immunizing an animal with an antigen derived from a mycobacterium. Lymphoid cells (e.g. splenic lymphocytes) are then obtained from the immunized animal and fused with immortalizing cells (e.g. myeloma or heteromyeloma) to produce hybrid cells.
  • Lymphoid cells e.g. splenic lymphocytes
  • immortalizing cells e.g. myeloma or heteromyeloma
  • the hybrid cells are screened to identify those which produce the desired antibody and then cloned and tested to prove that the cells produce only monoclonal antibodies.
  • the hybridoma cells producing the desired antibody can be subsequently expanded.
  • the hybridomas are expanded by injecting them intraperitoneally into mice under conditions which allow ascites fluid to develop. The ascites fluid is collected from the mice, pooled together
  • Human hybridomas which secrete human antibody may also be produced by the Kohler and Milstein technique. Although human antibodies are especially preferred for treatment of humans, in general, the generation of stable human-human hybridomas for long-term production of human monoclonal antibody may be difficult. Hybridoma production in rodents, especially mice, is a very well-established procedure. Stable murine hybridomas provide an unlimited source of antibody of select characteristics. Murine antibodies, however, may have limited use in the treatment of humans because they are highly immunogenic and may themselves induce undesirable immunogenic reactions in the patient.
  • the antibodies of this invention also may be produced in large quantities in large-scale tissue culture systems such as various continuous perfusion systems, hollow fiber systems, static maintenance culture systems or other systems.
  • Genetically engineered human antibodies or antibody fragments may also be produced by engineering gene sequences of human antibodies which encode the hypervariable (complementarity determining) regions to provide appropriate
  • the immunoassays of this invention may be any immunoassay in which a monoclonal antibody as described above may be useful.
  • a biological sample is contacted with the monoclonal antibody reactive with a mycobacterial antigen.
  • types of immunoassays include agglutination assays, enzyme linked immunosorbent assays (ELISA), radioimmunoassays, and immunofluorescent assays.
  • the format of the assay may also be varied, e.g. forward, reverse, sandwich, direct, indirect, or competitive binding. Examples of such assays are described in Example 7, below and in U.S. serial no. 07/654,381, filed Februay 12, 1991 and entitled "Immunofluorescent Test for Mycobacterial Antigens in Biological Fluids", the entire disclosure of which is incorporated herein by reference.
  • compositions of this invention contain an effective amount of the monoclonal antibodies described above in combination with a pharmaceutically acceptable carrier.
  • the therapeutic compositions may be useful in treating individuals having mycobacterial diseases, e.g. tuberculosis or leprosy.
  • the compositions also may
  • the antibodies of this invention may inhibit that effect.
  • An effective amount is the amount sufficient or necessary to reduce, eliminate or prevent symptoms associated with a mycobacterial disease, or the amount sufficient or necessary to reduce, eliminate or prevent the biological effects of having free LAM in an individual's system.
  • An effective amount may be determined on an individual basis and will be based, at least in part, in consideration of the size of the individual, the therapeutic goal and/or the severity of the symptoms to be treated, the specific antibody, etc.. Thus, an effective amount may be determined by one of ordinary skill in the art employing such factors and using no more than routine experimentation.
  • Administration of the monoclonal antibodies of this invention may be made by any method which allows the antibodies to reach their target site. Typical methods include oral, rectal, peritoneal, topical, intravenous and subcutaneous applications.
  • compositions may be in the form of dragees, tablets, syrups and ampules.
  • compositions When the compositions are administered rectally, the composition may be in the form of a suppository.
  • compositions When the compositions are administered by topical application, they may be in the form of a gel.
  • the pharmaceutically acceptable carrier is a carrier capable of containing and delivering the monoclonal antibody in a form which is active in vivo.
  • the carrier may be solid or liquid.
  • liquid carriers include water, clear aqueous solutions of non-toxic salts, or aqueous solutions containing organic solvents such as ethanol and suitable emulsions, such as oil-in-water emulsions.
  • Solid carriers include both nutritive carriers, such as sucrose or gelatin, and non-nutritive carriers, such as cellulose or talc.
  • kits useful in aiding in the diagnosis of mycobacterial disease contain a monoclonal antibody as described above and may further contain instructions directing the user how to use the monoclonal antibody in particular immunoassays. Special procedures may be desirable depending on the type of biological sample
  • the monoclonal antibody in the kit may also be labeled, depending on its intended use.
  • Murine monoclonal antibody ML9D3 reactive to LAM was produced by immunizing 8-week-old female Balb-C mice intravenously with 500 ⁇ g (dry weight) M. leprae intact cells combined with 50 ⁇ g purified Phenolic glycolipid-I (PGL-I) in 100 ⁇ l phosphate buffered saline (PBS), pH 7.4.
  • PBS phosphate buffered saline
  • the whole M. leprae used as the antigen was received as infected armadillo liver and spleen tissue from Eleanor Storrs, Medical Research Institute, Florida Institute of Technology and purified as indicated by Draper, P., et al. (Protocol 1/79: Purification of M.
  • the mouse selected was exsanguinated and sacrificed, and the spleen was used as a donor of immune lymphocytes in the fusion protocol.
  • the nonsecreting B-cell line SP2/oAgl4 Vector Borne Disease Center, Centers for Disease Control, Fort Collins, Colo.
  • the nonsecreting B-cell line SP2/oAgl4 was maintained at 37°C in either Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% fetal bovine serum (Flow Laboratories, McLean, Va.), L-glutamine, sodium pyruvate, nonessential amino acids, 50 ⁇ g of streptomycin per ml, and 50 IU of penicillin per ml (complete medium; all supplements were obtained from
  • the SP2/0 myelomas were diluted from log-phase cultures three days before fusion to yield 1 x 10 5 to 7 x 105 cells per ml in log-phase growth on the day of fusion.
  • Murine spleen cells were prepared in incomplete RPMI by teasing the cells into a single-cell suspension. These cells were used without lysis of red cells or enrichment for any lymphocyte population.
  • Splenocytes and SP2/0 cells at a 4:1 ratio were washed three times in incomplete RPMI 1640 medium (i.e. 50 IU of penicillin per ml, 50 ⁇ g of streptomycin per ml, and 0.29 mg of L-glutamine per ml).
  • incomplete RPMI 1640 medium i.e. 50 IU of penicillin per ml, 50 ⁇ g of streptomycin per ml, and 0.29 mg of L-glutamine per ml.
  • cells 1.0 ml per 1.6 x 10 splenocyte cells
  • 50% polyethylene glycol 4,000 molecular weight
  • Polyethylene glycol was added slowly while the solution was stirred gently over a 37°C water bath for a duration of one minute. Stirring continued for an additional minute and was followed by a 10% addition of a calculated volume (10 ml per o
  • Fused cells were plated at 0.1 ml per well into 96-well polystyrene plates.
  • Fusion cultures in wells were fed with 0.1 ml of complete medium containing hypoxanthine (1 x
  • Example 4 Screening for Antibodies Using An Enzyme Linked Immunosorbent Assay (ELISA) Assays for the monoclonal antibodies were performed in a variety of configurations on polystyrene microtiter plates (Dynatech Laboratories, Chantilly, Va.) or as nitrocellulose-based dot ELISA.
  • ELISA Enzyme Linked Immunosorbent Assay
  • LAM was coated in the wells by evaporation of a sonicated suspension in absolute ethanol. Unbound sites on the polystyrene were blocked with 0.05% polyoxyethylene sorbitan monolaurate (Tween 80; Sigma Chemical Co., St. Louis, Mo.) in PBS (PBS-Tween) for two minutes or with 1% bovine serum albumin (BSA) (Fraction V; Miles Scientific, Naperville, 111.) in PBS-Tween for one hour. Antibody was incubated in the well for at least one hour after the blocking step. Hybridoma supernatant fluids were diluted in SP2/0 culture supernatant.
  • BSA bovine serum albumin
  • Unbound antibody was removed with PBS, and the second antibody of horseradish peroxidase-conjugated goat anti-mouse immunoglobulin, reactive with immunoglobulin G (igG), IgM, and IgA classes of immunoglobulin (Cappel Laboratories, West Chester, Pa.) was added.
  • the second antibody was diluted in PBS-Tween or 10% normal goat serum in PBS-Tween; the incubation time was twenty minutes.
  • peroxidase was detected by addition of 0.4 mg of o-phenylenediamine (Sigma) per ml and 0.012% H 2 0 2 in citrate phosphate buffer, pH 5. Color development was stopped by addition of 50 ⁇ l of 2.5 N H 2 S0 4 .
  • Optical density at 490 nm was read by using a spectrophotometer (Dynatech Industries, Inc., McLean, Va.) for microtiter plates.
  • mice or Fl hybrid mice (preferably CAF1, Jackson Labs, Maine) were primed with mineral oil to be more susceptible to hybridoma growth and secretion.
  • the mice were primed by intraperitoneal injection of 0.5 ml pristane
  • Hybridoma cells were maintained in a logarithmic phase of multiplication in a T_ 5 flask with 10% fetal calf serum in Dulbecco's Modification of Eagle Medium (DMEM) with l mM L-glutamine.
  • DMEM Dulbecco's Modification of Eagle Medium
  • Hybridoma cells were used at a ratio of 5 x 10 to
  • mice developed ascites in two to four weeks after cell inoculation and when their abdomen was fat (full with ascites fluid), the mice were tapped for ascites collection.
  • the fluid ascites from the mice was pooled (the harvest of one day constitutes one batch) , centrifuged at 400 X g for ten minutes, and the supernatant fluid was saved as neat ascites.
  • the fluid ascites was stored at -20°C and assayed for antibody titer using an ELISA test with 1 ⁇ g/ml LAM antigen coated plates and 1 hour incubation periods each with various dilutions of ascites (room temperature), with conjugate (room temperature), and with substrate (at 37°C) as described earlier in this application.
  • the monoclonal antibody (ascites) was assayed for suitability in a latex agglutination assay based on the dilution that gives 50% of the maximum of absorbance.
  • Mycobacterium tuberculosis strain TMC 107 (Erdman) is grown for eight weeks in a glycerol-alanine-salts medium as a shaken culture.
  • the Erdman stain was obtained from the Trudeau Mycobacterium Culture Collection, Trudeau Institute, Saranac Lake, N.Y. USA, 12983, culture number TMC 107 and also is availabel at the ATCC, No. 35801, Rockville, Md., U.S.A.
  • Other strains may be
  • LAM also may be isolated from nonturbculosis mycobacteria e.g. M. ⁇ megmatis.
  • the cultures were autoclaved at 80°C for 1 h, cooled and filtered using sterile 0.22 micron filtration system (Nalge Co., Rochester, NY). The harvested cells were washed several times with distilled water and stored frozen (-20°C) until ready for breakage. Harvested cells ( ⁇ 130 g wet weight) were resuspended in PBS containing 0.5% Triton X100 and 0.02% NaNg (200 ml). A thick suspension is desirable in order to achieve complete breakage of cells. LAM, LM and PIM have a great affinity for detergent. Use of Triton X100 when breaking the cells helped to keep most of these amphipathic molecules in solution, thereby giving a maximum yield during acetone precipitation.
  • the suspension was sonicated while cooling in an ice bath for 10 min with a W-385 Sonicator Ultrasonic Liquid Processor (Heat
  • the sonicate was passed four times through a French pressure cell (Model SA073; American Instruments Co., Urbana, IL) at 20,000 lb per sq. in.
  • the sonicate pressate was centrifuged at 27,000 x g for 45 min, two times.
  • the pellet was washed twice with the above buffer (50 ml each time) and recentrifuged.
  • the supernatant fluids were combined and recentrifuged (at 27,000 x g) in order to remove most of the cell wall. (The supernatant fluid appeared translucent after centrifugation.)
  • distilled acetone was added (to a final concentration of 90% acetone) to precipitate mainly polysaccharides. Some proteins were also precipitated during this procedure. (Considering that very large volumes of solvents were used, it was more efficient when the supernatants were divided into two 1000 ml Erlenmeyer flasks.) The acetone precipitate was stored at 4°C for 48 h.
  • the precipitate was collected by centrifugation at 10,000 x g and air dried. Dry precipitate (1 g) was suspended in 6 ml of 6 M guanidine HC1 in lOmM Tris HC1, pH 7.4 by pansonication. (The insoluble material remaining is removed by low speed centrifugation (2000xg) prior to application to the column.) The soluble material is applied to a sephacryl S-400 column (1.5 x 150 cm) in the same buffer. Fractions (2 ml) are collected arr. monitored by PAGE. Fractions are pooled according to enrichment with LAM, LM and PIM and dialyzed extensively against water (5 to 6 changes of
  • LAM LAM, LM, PIM enriched fraction obtained from the S-400 column above to a Sephacryl S-200 column with a buffer containing lOmM Tris, 0.2 M NaCl, lmM EDTA 0.02% sodium azide and 0.25% deoxycholate.
  • About 150-180 mg of crude material was applied to a column size of (2.5 x 120 cm) and 4 ml fractions were collected and monitored by PAGE.
  • Use of deoxycholate as a detergent on a simple sizing column keeps LAM, LM and PIM from aggregating; therefore, they purify rapidly as separate entities. This method of purification replaced several laborious and tedious ion exchange chromatography steps, as well as purification.
  • LAM has many LPS-like biological activities. To ensure that LPS contamination was not present in
  • lyophilized LAM was redissolved in pyrogen-free water, filtered through 0.45 ⁇ m PTFE filtration unit and passed through 2.0 ml of Detoxi-Gel column (Pierce Chemical, Rockford, IL), refiltered through a second 0.20 ⁇ m sterile filter and the filtrate collected into a sterile, pyrogen free vial using sterile pyrogen-free water to elute it off the gel.
  • caseine 5% fetal calf serum in coating buffer (20 mM Tris-HCl, 0.15 M NaCl, pH 7.5)] to the tube and incubating for one hour at room temperature while mixing in a rotator.
  • the blocking reagent was removed and the tube was washed twice with six ml of saline.
  • Two ml of a solution of 1% latex red beads (0.2 micron average diameter, Rhone Poulenc, France) in coating buffer was then delivered to the tube.
  • An additional four ml of coating buffer was delivered to the tube and the mixture was centrifuged in the cold (4°C) at 27,000xg for fifteen minutes.
  • the supernatant fluid was discarded using vacuum aspiration and the pellet formed during centrifugation was resuspended in two ml of coating buffer.
  • the suspension was vortexed vigorously (and/or pipetted back and forth) to disperse the latex particles. An additional four ml of coating buffer was added and the suspension was centrifuged in the cold again at 27,000xg for fifteen minutes. The supernatant fluid was discarded and the pellet was washed once more as described above.
  • the washed pellet was resuspended in two ml of coating buffer and the latex beads were dispersed in the coating buffer using a pansonicator. The homogeneity of the latex beads was confirmed by microscopic examination.
  • the suspension was
  • the latex beads are ready to be examined for the presence of antibody on their surface and for the specific activity of these antibodies.
  • the neat ascites may be purified prior to sensitizing the beads. This may be desirable because it
  • Enhancer An enhancer (PEG) is dissolved in saline (0.8% NaCl in deionized water) at concentrations ranging from 7% to 0.5%. These various concentrations of enhancer are run in a standardized agglutination assay to determine the minimum concentration of enhancer which causes agglutination. Then, the concentration below that which results in agglutination visible to the naked eye is selected as that for use in the assay of Example 5.
  • the enhancer concentration selected for use in the agglutination assay of the invention is chosen as that concentration below, but close to, the minimum concentration of enhancer that shows autoagglutination. In this example, that concentration either is 4.0% enhancer or 3.5% enhancer. Four % enhancer would provide a slightly more sensitive assay than one using an enhancer concentration of 3.5%.
  • M. tuberculosis for use as a positive control in the latex agglutination test was prepared.
  • Mycobacterium tuberculosis strain TMC 107 (Erdman) or H37Ra is grown for eight weeks in a glycerol-alanine-salts medium as a shaker culture.
  • the Erdman strain was obtained from the Trudeau Mycobacterium Culture Collection, Trudeau Institute, Sarenac Lake, NY, culture number TMC 107.
  • the H37Ra strain was obtained from K. Takayama, Madison, WI, and is described by Takayama, K., et al. (1975), J. Lipid Res., 16, 308-317.
  • the cultures are autoclaved at 80°C for 1 h, cooled and filtered.
  • the harvested cells are washed several times with distilled water and stored frozen (-20°C) until ready for breakage.
  • the suspension was sonicated while cooling in an ice bath for 10 min with a W-385 Sonicator Ultrasonic Liquid Processor (Heat
  • the sonicate was passed four times through a French pressure cell (Model SA073; American Instruments Co., Urbana, IL) at 20,000 lb sq. in.
  • the sonicate pressate was centrifuged twice at 27,000 x g for 45 min.
  • the pellet was washed twice with the above buffer (50 ml each time) and recentrifuged.
  • the supernatant fluids were combined and recentrifuged (at 27,000x g) in order to remove most of the cell wall. (The supernatant fluid appeared translucent after centrifugation.)
  • distilled acetone was added (to a final concentration of 90% acetone) to precipitate mainly polysaccharides.
  • the precipitate was collected by centrifugation at 10,000 x g and air dried.
  • N-acetyl-L-cysteine (NALC powder) is added to an equal volume of a sputum sample in a 50 ml centrifuge tube and vortexed thoroughly. Preferable at least 1 ml of sputum is present. The mixture is allowed to stand for fifteen minutes. The tube is filled up to the 45 ml mark with distilled water and centrifuged at 2800 x g to 3000 x g for fifteen minutes. Subsequently, the supernatant fluid is carefully decanted and 100-200 ⁇ l of PO ⁇ buffer (pH 6.8) is added to the pellet.
  • PO ⁇ buffer pH 6.8
  • the processed sputum is neutralized, if necessary, with neutralizing reagent (1 N HCl).
  • neutralizing reagent (1 N HCl).
  • the pH of the sputum to be used in the agglutination assay must be approximately pH 7 to allow appropriate physiological conditions for antibody-antigen binding to occur.
  • agglutination assay 20 ⁇ l enhancer, 40 ⁇ l processed sputum sample, and 20 ⁇ l sensitized beads is delivered to a slide.
  • the various reagents are mixed with a mixing stick and the slide is placed on a mechanical rotator for five minutes. The presence of agglutination is then detected.
  • a negative control latex is comprised of 20 ⁇ l enhancer, 40 ⁇ l of the same processed sputum,
  • the negative latex beads are prepared as set forth in Example I, using neat ascites to coat the beads with the exception that the sensitized beads are then stored for at least 7 days at 37°C prior to use. This results in beads sensitized with monoclonal antibody lacking specific activity i.e. the beads will agglutinate with anti-mouse antibody, but not with a sample of positive control antigen or a sample containing mycobacteria.
  • the negative control latex may also be obtained by sensitization of the latex beads with purified monoclonal antibody, but without the use of BSA as a coadsorbent. In this case also the beads bind to anti-mouse antibody, but lack specific activity.

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Abstract

L'invention concerne une solution contenant un anticorps monoclonal réagissant avec un antigène mycobactérien. Le titrage de l'anitcorps monoclonal est d'au moins 70K, et de préférence 100K. Plus préférablement, l'anticorps monoclonal est capable de se lier à LAM et en particulier peut se lier à toutes les souches de M. tuberculosis. Des agents d'immunoanalyse, des compositions thérapeutiques et des kits contenant les anticorps monoclonaux en question sont également décrits.
PCT/US1992/001159 1991-02-12 1992-02-12 Anticorps monoclonaux diriges contre des antigenes mycobacteriens WO1992014155A1 (fr)

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US65430291A 1991-02-12 1991-02-12

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WO1996012190A3 (fr) * 1994-10-13 1996-06-27 Brigham & Womens Hospital Presentation d'antigenes hydrophobes a des lymphocytes t par des molecules cd1
WO1997034149A1 (fr) * 1996-03-12 1997-09-18 Stefan Svenson Methode diagnostique de mycobacteriose et trousse d'essai immunologique
US5679347A (en) * 1992-12-10 1997-10-21 Brigham And Women's Hospital Methods of isolating CD1-presented antigens, vaccines comprising CD1-presented antigens, and cell lines for use in said methods
US5853737A (en) * 1992-12-10 1998-12-29 Brigham And Women's Hospital Method for inducing a CD1-restricted immune response
RU2130615C1 (ru) * 1998-05-18 1999-05-20 Гизатулина Надия Мансуровна Способ экспресс-диагностики туберкулеза на основе реакции латекс-агглютинации
US6214789B1 (en) * 1993-03-12 2001-04-10 Xoma Corporation Treatment of mycobacterial diseases by administration of bactericidal/permeability-increasing protein products
US6238676B1 (en) 1992-12-10 2001-05-29 Brigham And Women's Hospital Presentation of hydrophobic antigens to T-cells by CD1 molecules
US6383763B1 (en) 1996-07-26 2002-05-07 Case Western Reserve University Detection of mycobacteria
WO2006012413A1 (fr) * 2004-07-20 2006-02-02 Chemogen, Inc. Anticorps enrichi pour detecter une infection mycobacterienne, procedes pour l'utiliser et test diagnostique utilisant celui-ci
US7063844B2 (en) 1992-12-10 2006-06-20 The Brigham And Women's Hospital, Inc. Presentation of hydrophobic antigens to T-cells by CD1 molecules
US9315566B2 (en) 2011-01-24 2016-04-19 National University Of Singapore Pathogenic mycobacteria-derived mannose-capped lipoarabinomannan antigen binding proteins

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INFECTION AND IMMUNITY, Volume 55, No. 11, issued November 1987, GAYLORD et al., "Most Mycobacterium leprea Carbohydrate-Reactive Monoclonal Antibodies are Directed to Lipoarabinomannan", pages 2860-2863. *
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Cited By (19)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5679347A (en) * 1992-12-10 1997-10-21 Brigham And Women's Hospital Methods of isolating CD1-presented antigens, vaccines comprising CD1-presented antigens, and cell lines for use in said methods
US5853737A (en) * 1992-12-10 1998-12-29 Brigham And Women's Hospital Method for inducing a CD1-restricted immune response
US7063844B2 (en) 1992-12-10 2006-06-20 The Brigham And Women's Hospital, Inc. Presentation of hydrophobic antigens to T-cells by CD1 molecules
US6238676B1 (en) 1992-12-10 2001-05-29 Brigham And Women's Hospital Presentation of hydrophobic antigens to T-cells by CD1 molecules
US6620785B2 (en) 1993-03-12 2003-09-16 Xoma Corporation Treatment of Mycobacterial diseases by administration of bactericidal/permeability-increasing protein products
US6214789B1 (en) * 1993-03-12 2001-04-10 Xoma Corporation Treatment of mycobacterial diseases by administration of bactericidal/permeability-increasing protein products
AU694299B2 (en) * 1994-10-13 1998-07-16 Brigham And Women's Hospital Presentation of hydrophobic antigens to T-cells by CD1 molecules
WO1996012190A3 (fr) * 1994-10-13 1996-06-27 Brigham & Womens Hospital Presentation d'antigenes hydrophobes a des lymphocytes t par des molecules cd1
WO1997034149A1 (fr) * 1996-03-12 1997-09-18 Stefan Svenson Methode diagnostique de mycobacteriose et trousse d'essai immunologique
US6383763B1 (en) 1996-07-26 2002-05-07 Case Western Reserve University Detection of mycobacteria
RU2130615C1 (ru) * 1998-05-18 1999-05-20 Гизатулина Надия Мансуровна Способ экспресс-диагностики туберкулеза на основе реакции латекс-агглютинации
WO2006012413A1 (fr) * 2004-07-20 2006-02-02 Chemogen, Inc. Anticorps enrichi pour detecter une infection mycobacterienne, procedes pour l'utiliser et test diagnostique utilisant celui-ci
US7335480B2 (en) 2004-07-20 2008-02-26 Chemogen, Inc. Enriched antibody for detecting mycobacterial infection, methods of use and diagnostic test employing same
JP2008507544A (ja) * 2004-07-20 2008-03-13 キーモゲン インコーポレイテッド マイコバクテリア感染検出用濃縮抗体、使用方法、及びそれを使用する診断検査
US7615222B2 (en) 2004-07-20 2009-11-10 Chemogen, Inc. Enriched antibody for detecting mycobacterial infection, methods of use and diagnostic test employing same
EA013228B1 (ru) * 2004-07-20 2010-04-30 Чемоджен, Инк. Способ получения обогащённого поликлонального антитела, высокоспецифичного к антигену поверхностного полисахарида липоарабиноманнана микобактерий, и способы его применения
AU2005267111B2 (en) * 2004-07-20 2011-03-03 Chemogen, Inc. Enriched antibody for detecting mycobacterial infection, methods of use and diagnostic test employing same
US8057797B2 (en) 2004-07-20 2011-11-15 Chemogen, Inc. Method of preparing enriched antibodies for detecting mycobacterial infection
US9315566B2 (en) 2011-01-24 2016-04-19 National University Of Singapore Pathogenic mycobacteria-derived mannose-capped lipoarabinomannan antigen binding proteins

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