WO1992014155A1

WO1992014155A1 - Monoclonal antibodies directed towards mycobacterial antigens

Info

Publication number: WO1992014155A1
Application number: PCT/US1992/001159
Authority: WO
Inventors: Patrick J. Brennan; Becky L. Rivoire
Original assignee: Dynagen, Inc.; Colorado State University Research Foundation
Priority date: 1991-02-12
Filing date: 1992-02-12
Publication date: 1992-08-20
Also published as: AU1423892A

Abstract

A solution containing a monoclonal antibody reactive with a mycobacterial antigen is provided. The monoclonal antibody titre is at least 70K, and preferably is at least 100K. Most preferably the monoclonal antibody is capable of binding LAM and in particular is capable of binding to all strains of M. tuberculosis. Immunoassays, therapeutic compositions and kits containing the foregoing monoclonal antibodies also are provided.

Description

MONOCLONA ANTIBODIES DIRECTED TOWARDS MYCOBACTERIAL ANTIGENS

Field of the Invention This invention pertains to the field of monoclonal antibodies, particularly monoclonal antibodies useful as diagnostic tools for diseases associated with mycobacteria, e.g. tuberculosis.

Background

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.

Other 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) complex are pathogenic among individuals with acquired immuno-deficiency syndrome (AIDS), as well as among other individuals having a compromised immune system. 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).

At present, nearly all 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. Today, tuberculosis remains a significant health problem especially in developing countries. Worldwide, 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. Further, 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.

Current diagnostic measures for these mycobacterial diseases are barely adequate. Efficient patient management and control of

transmission are compromised by current inadequacies in techniques for the rapid identification of the etiologic agent in the laboratory. Although bacilli may be detected by microscopy, Shoemaker, S.A., et al. , Am. Rev. Respir. Pis. , 131:760-763 (1985), intact bacilli are required and sensitivity is low. Samples are therefore cultured to allow for more accurate diagnosis as well as to permit definitive species identification. However, M. tuberculosis is difficult to culture and has a generation time of 15-20 hours. Wayne, L.G., Am. Rev. Respir. Pis. , 125 (Suppl.) 31-41 (1982). A delay of up to 6 weeks before results of laboratory tests are available is not unusual.

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) . However, the use of immunoassays in diagnosing TB has been restricted by the lack of availability of monoclonal antibodies to TB having adequate titre and specificity.

Summary 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. Preferably 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). The monoclonal antibody

reacts in particular with the virulent Erdman strain of M. tuberculosis, which is important because this strain is believed to be the predominant isolate among diseased patients. 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.

Brief Description of the Drawing Figure 1 is a graph used in selecting the monoclonal antibodies of this invention.

Detailed Description 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. Preferably, 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.

The term 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. In addition, 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

antibody. 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 μg/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.

application serial no. 07/654,321, filed February 12, 1991, and entitled "Purified LAM and Synthetic Analogs Thereof". 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) . Subsequently, 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

(MAb) as shown in Figure 1. The % of maximum absorbance of each dilution can be calculated by using the following formula:

B- SB Bo-NSB^{X ■LUυ}

B = read out absorbance of each dilution of antibody

Bo = maximum absorbance which is considered

1.000

NSB ~ read out absorbance of nonspecific binding

For example, if at dilution of 1:30,000 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:

^1:30'⁰⁰⁰ " 1.000-^.l fe^{100 = 75}-^6%

Following the procedure of the above ELISA and using the monoclonal antibody derived from hybridoma cell line ML9D3, the dilution which shows 50% of maximum absorbance was 1:85,000, (Figure 1) which is inversely proportional to the titer of that particular batch of monoclonal antibodies. Thus, - li ¬

the titer is 85,000 Ab units/unit volume (85K).

If 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.

Other methods may be used to determine the suitability of various lots or batches of antibodies and also to improve lots or batches of antibodies such that they are useful within this invention. For example, even if the titer of antibodies is less than 70K at a 50% absorbance measured using the method described above, the solution still may be useful in certain of the immunoassays of the invention after various manipulations, e.g. purification and/or concentration processing steps. Problems may be encountered if attempting solely concentration of the antibodies because the other components of the ascites, enzymes etc., also become concentrated. Concentration alone thus may lead to nonspecific binding and a decrease in stability. However, concentration coupled with purification may be desirable not only to attain adequate titer, but also to increase the stability of the monoclonal antibodies.

This invention also relates to immortalized, stable, antibody producing cell lines capable of

producing the monoclonal antibodies as described above. Examples 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. 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

and centrifuged. The supernatant fluid from this process is termed neat ascites.

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

anti-mycobacterial specificity. See e.g., Robert, S. et al.. Nature 328:731-733 (1987); Better, M. et al. , (1988) Science 240:1041.

The immunoassays of this invention may be any immunoassay in which a monoclonal antibody as described above may be useful. In the immunoassays, a biological sample is contacted with the monoclonal antibody reactive with a mycobacterial antigen. Examples of 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.

The therapeutic 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

be used to treat the biological effects or conditions resulting from having free LAM in an individual's system. It is believed that some LAM antigen is released from mycobacteria in free form and the biological consequences of having the free antigen is not entirely understood. If the free LAM antigen has some adverse biological effect, 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.

The form in which the antibodies are administered may depend on the route of administration. For example, if administered orally, the compositions may be in the form of dragees, tablets, syrups and ampules. When the compositions are administered rectally, the composition may be in the form of a suppository. 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. Examples of 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.

This invention also pertains to kits useful in aiding in the diagnosis of mycobacterial disease. The kits 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

collected by the user or the types of conditions which allow antibody-antigen-binding to occur. The monoclonal antibody in the kit may also be labeled, depending on its intended use.

This invention will be further illustrated by the following non-limiting examples.

Example 1 - Production of Monoclonal Antibody ML9D3

Murine monoclonal antibody ML9D3 reactive to LAM was produced by immunizing 8-week-old female Balb-C mice intravenously with 500 μg (dry weight) M. leprae intact cells combined with 50 μg purified Phenolic glycolipid-I (PGL-I) in 100 μl phosphate buffered saline (PBS), pH 7.4. 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. leprae, Annex 1 to the Report of the Enlarged Steering Committee for Research on the Immunology of Leprosy (IMMLEP) Meeting of 7-8 February 1979. Geneva: World Health Organization, 1979, p. 4). The mixture was briefly probe sonicated to provide a suspension. The antigen suspension was administered intravenously to the mice six times, spaced three days apart. The murine sera were tested three days

after the sixth injection using a plate ELISA described below for reactivity to LAM and/or PGL-I. The mouse responding with the highest titer was given a final intravenous boost ten days following the sixth injection. Three days later, this mouse was used as an immune spleen-cell donor for polyethylene glycol mediated fusion with the myeloma line SP2/0. Specific details of hybridoma production, limiting dilution cloning and ELISA are described in the examples below and/or can be found in Rivoire et al. , Infect. Immun. , 1989, 57:3147-3158, the contents of which is expressly incorporated by reference.

Example 2 - Myeloma and Hybridoma Cell Culture

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.) 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 μg of streptomycin per ml, and 50 IU of penicillin per ml (complete medium; all supplements were obtained from

K. C. Biological). 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.

Example 3 - Cell fusion and Selection of Hybrids

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 μg of streptomycin per ml, and 0.29 mg of L-glutamine per ml). After being washed, cells (1.0 ml per 1.6 x 10 splenocyte cells) were fused with 50% polyethylene glycol (4,000 molecular weight) (VWR Scientific, Denver, Colo.) in incomplete RPMI medium. 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

1.6 x 10 spleen cells) of completed RPMI medium while being stirred gently for one minute. A further 10% of the volume was added over the next minute, followed by 30 and 50% volumetric additions

in subsequent minutes, This gradual, gentle addition is important to the success of healthy hybridomas. Cells were centrifuged for five minutes at low speed. The pellet was loosened with gentle tapping and gentle pipetting with a precalculated volume of complete RPMI medium (22 ml per 1.6 x a

10 spleen cells). 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

10 —4 mM), aminopterin (4 x 10—7 mM) and thymidine (1.6 x 10 mM) on days 2, 3, 4, and 5 after fusion. Screening for antibody generally commenced on day ten. Cloning of positive cultures was done on 24-well plates in complete medium containing hypoxanthine and thymidine only. Limiting dilution of cells onto 96-well thymocyte feeder layers (5 x 10 thymocyte cells per 100 μl of complete medium) was monitored by growth and rescreening for antibody production. 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. For polystyrene

plates, 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. 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. After washing, peroxidase was detected by addition of 0.4 mg of o-phenylenediamine (Sigma) per ml and 0.012% H₂0₂ in citrate phosphate buffer, pH 5. Color development was stopped by addition of 50 μl of 2.5 N H₂S0₄. Optical density at 490 nm was read by using a spectrophotometer (Dynatech Industries, Inc., McLean, Va.) for microtiter plates.

Example 5 - Protocol for Expansion of Hybridomas In Vivo

Balb/c 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

(tetramethylpentadecane) seven to twenty-eight days before inoculation with hybridoma cells.

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.

Hybridoma cells were used at a ratio of 5 x 10 to

1 x 10 cells/mouse to ensure good production of antibody in murine ascites. The hybridoma cells in growth medium were centrifuged for ten minutes at

400 X g and the pellet (which contains 5 x 10 to

1 x 10 cells/mouse) was resuspended in 1 ml of prewarmed (at 37°C) saline. The cells were inoculated into the mouse by intraperitoneal

6 7 injection of 1 ml saline with 5 x 10 to 1 x 10 cells.

The 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 μg/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.

EXAMPLE 6

Preferred Method of Generation and Purification of LAM from Mycobacterium tuberculosis

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

employed, including the rapidly growing, attenuated strain H37Ra, obtained from K. Takayama, Madison, WI, described by Takayama, K. et al. (1915) J. Lipid Res. , 16, 308-317 and available at the ATCC,

No. . (LAM also may be isolated from nonturbculosis mycobacteria e.g. M. εmegmatis.)

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

Systems-Ultrasonic, Inc., Framingdale, NY) operating at optimal cavitation intensity. 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.)

To the precooled supernatant fluid, 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

water). There is no resolution between LAM, LM or PIM after this preliminary column fractionation. The dialyzed fractions were freeze dried to yield approximately 500 mg of impure material.

Final purification of LAM was achieved by applying the 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. Fractions containing pure LAM, LM and PIM (resolved at this stage) were pooled, dialyzed at 37°C for two days (48 hrs.) against the buffer without deoxycholate in order to remove detergent followed by dialysis against water at 4°C for two days (48 hrs.). Pure LAM, LM or PIM was stored as freeze-dried powder.

LAM has many LPS-like biological activities. To ensure that LPS contamination was not present in

preparations, lyophilized LAM was redissolved in pyrogen-free water, filtered through 0.45 μm PTFE filtration unit and passed through 2.0 ml of Detoxi-Gel column (Pierce Chemical, Rockford, IL), refiltered through a second 0.20 μm sterile filter and the filtrate collected into a sterile, pyrogen free vial using sterile pyrogen-free water to elute it off the gel.

As a means of quality control, all final preparations are subjected to 1) SDS-PAGE and silver stained with a periodate step to visualize the carbohydrates, 2) Western blot using the monoclonal antibody against LAM to verify its LAM content, and 3) Alditol acetate and GC analysis versus neutral sugar standards to estimate arabinose and mannose content.

Alternate Purification of LAM

The isolation of LAM-containing fractions from M tuberculosis and primary resolution on columns of DEAE-Sephacel in detergent-containing buffer, have been described (1,2). In addition to these steps, preparations of LAM, recovered from columns of DEAE-Sephacel and which were highly pure according to PAGE (1), were dialyzed, concentrated on an Amicon flow cell (10 kDa molecular weight cut-off membrane, Amicon model 8200; Danvers, MA),

precipitated with 85% ethanol, and redissolved in 0.01M Tris HCl (pH 7.4) containing 0.1% Triton X-100 and applied to a HYDROPORE Ax HPLC column (21.4 mm x 25 cm, Rainin, Woburn, MA) equilibrated in the same buffer. The column was eluted with the same buffer followed by a shallow gradient of 0 to 0.1M NaCl. Fractions (10 ml) were collected, analyzed for carbohydrate (2) and positive fractions re-examined by PAGE. Pure LM eluted with 0.01M NaCl, followed by PIM which eluted with 0.02M NaCl, and LAM which eluted with 0.05M NaCl. Fractions were pooled and subjected to a folch wash by treating with 6 parts Chloroform:methanol (2:1) and allowed to form a biphase. The aqueous layer was removed and dialyzed, concentrated and dried. LAM was reprecipitated with 85% ethanol at 0°C overnight and then centrifuged at 2000xg. To remove the last traces of detergent, a solution of pure LAM was passed through a column (2 ml) of Extracti Gel-D (Pierce Chemical Co., Rockford, IL) and eluted with H₂0.

Example 7 - Passive Coating of Latex Beads

(Adsorption) with Monoclonal Antibody Anti-LAM

A 15 ml polystyrene centrifuge tube was blocked by delivering six ml of a blocking reagent [3% milk

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

delivered to the previously blocked polystyrene tube and an additional 2 ml of coating buffer was added.

One hundred μl of neat ascites containing anti-LAM monoclonal antibody (neat murine ascites with a 100K titer of ML9D3 antibody as determined by ELISA assay, and stored at -20°C) was added to the latex suspension and vortexed for two seconds. The tube was mixed on a flying rotator for four hours at room temperature. The latex suspension then was transferred into a 6 ml centrifuge tube and was spun down at 27,000xg for fifteen minutes. The resulting pellet was resuspended in two ml of assay buffer (same as coating buffer) and vortexed to get a good dispersion. Then 4 ml of assay buffer was added, followed by centrifugation at 27,000xg for fifteen minutes. The pellet was resuspended in 2 ml of a second blocking reagent (1% BSA, 5% glycerol and 1% sodium azide in coating buffer), and maintained at room temperature for 30 minutes to block all the reactive sites on the bead surface.

After the foregoing procedure, the latex beads are ready to be examined for the presence of antibody on their surface and for the specific activity of these antibodies.

As stated earlier in the specification, the neat ascites may be purified prior to sensitizing the beads. This may be desirable because it

increases the stability of the antibody coated beads in relatively high temperature conditions (e.g. 37°C). Procedures such as ammonium sulfate precipitation (at 50% saturation) or Protein A chromatography have been used successfully to purify the neat ascites. Then, the procedure for coating the beads is carried out as outlined above with the following exceptions: 4 ml of 0.5% washed latex beads in coating buffer is sensitized with 0.6 ml of a mixture of 100 μl purified MAb (having 100K titer [approximately 5 mg antibody per ml]) and 500 μl 1% BSA. The mixture of MAb and BSA preferably are kept for 20 minutes at RT for stabilization before adding to latex beads.

Examination of the Presence of Antibody on Sensitized Latex Beads

Twenty μl of assay buffer (20m M Tris-HCl, 0.15M NaCl, ph 7.5), 40 μl of anti-mouse IgG (1:100) and 20 μl sensitized beads were delivered to the surface of a slide. The components were mixed with a mixing stick and the slide was left on a mechanical rotator for several minutes. A positive reaction is indicated by the appearance of large clumps of red beads, indicating the presence of the antibody on the surface of the beads. A negative control consisting of 60 μl assay buffer

and 20 μl sensitized red beads was delivered and tested using the same procedure. No agglutination was detectable

Selection of Concentration of 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.

To select the proper concentration of enhancer, 20 microliters of a defined concentration of enhancer, 40 microliters of assay buffer and 20 microliters of sensitized latex beads are delivered to a slide. These reagents are mixed on the surface of the slide, and the slide is rotated on a mechanical rotator for 5 minutes. Then, it is determined whether autoagglutation has occurred. Autoagglutation is considered to be present when the agglutination is visible to the naked eye. The test is repeated for various concentrations of enhancer. The results are shown in the table of Figure 2. A plus sign represents agglutination detectable with

the naked eye.

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%.

Examination of the Specific Activity of the Sensitized Latex Beads

An acetone precipitate of 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. 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, lipomannan (LM) and phosphoinositol mannoside (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 maximum yield during acetone precipitation.

Systems-Ultrasonic, Inc., Framingdale, NY) operating at optimal cavitation intensity. 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.)

To the precooled supernatant fluid, 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.

Twenty μl of enhancer (4% polyethylene glycol), 40 μl of an acetone precipitate of M. tuberculosis suspension (83 μg/ml) and 20 μl of sensitized red beads are delivered onto the surface of a slide and mixed with a mixing stick. The slide containing the mixture is placed on a mechanical rotator for five minutesN A negative control of 20 μl enhancer, 40 μl assay buffer [see Example II] and 20 μl sensitized red beads was subjected to the same procedure. The specific activity of the sensitized latex beads was determined by observing for agglutination of the red beads in the presence of the positive control (acetone precipitate of M. tuberculosis) and the absence of agglutination with the assay buffer.

Latex Agglutination Test for detection of Mycobacteria in Human Sputum

An equal volume of a solution containing 50 ml

of IN (4%) NaOH and 50 ml of 2.9% trisodium citrate, H₂0 and 0.5 g of 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 μl of PO^~ buffer (pH 6.8) is added to the pellet. Following sputum processing, the processed sputum is neutralized, if necessary, with 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.

In the agglutination assay, 20 μl enhancer, 40 μl processed sputum sample, and 20 μl 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 μl enhancer, 40 μl of the same processed sputum,

and 20 μl of sensitized negative latex beads (mixed with sputum and enhancer). 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.

EQUIVALENTS Those skilled in the art will be able to ascertain, using no more than routine experimentation, many equivalents of the specific embodiments of the invention described herein. These and all other equivalents are intended to be encompassed by the following claims.

Claims

1. A solution containing a monoclonal antibody reactive with a mycobacterial antigen, wherein the monoclonal antibody titer is at least 70K.

2. A solution as claimed in claim 1 wherein the monoclonal antibody titer is at least 100K.

3. A solution as claimed in claim 1 wherein the monoclonal antibody is capable of binding LAM.

4. A solution as claimed in claim 3 wherein the monoclonal antibody is ML9D3.

5. A monoclonal antibody capable of binding to the virulent Erdman strain of M. tuberculosis.

6. A monoclonal antibody as claimed in claim

5 wherein the antibody is capable of binding to all strains of M^ tuberculosis.

7. A monoclonal antibody as claimed in claim

6 wherein the antigen is LAM.

8. A monoclonal antibody designated ML9D3.

9. An immortalized stable cell line producing a monoclonal antibody as claimed in any one of claims 5, 7 or 8.

10. In an immunoassay for detecting the presence of a mycobacterial antigen in a biological sample, the improvement comprising, using the solution of any one of claims l, 2, 3 or 4.

11. In an immunoassay for detecting the presence of a mycobacterial antigen in a biological sample, the improvement comprising, contacting the biological sample with a monoclonal antibody as claimed in any one of claims 5, 6, 7 or 8.

12. A therapeutic composition comprising an effective amount of a monoclonal antibody as claimed in any one of claims 5, 6, 7 or 8 and a pharmaceutically acceptable carrier.

13. A kit useful in aiding in the diagnosis of mycobacterial disease comprising, a container including a monoclonal antibody as claimed in any one of claims 5, 6, 7 or 8.