METHOD OF DETECTING OR ESTIMATING BIOLOGICAL MATERIEL

Abstract: Monoclonal antibodies raised against cell walls of Group A Streptococci are specific to biological materials, e.g. immunoglobulins, having terminal N-acetyl glucosamine residues and can be used in their detection, e.g. in the diagnosis of diseases characterized by their presence, e.g. rheumatoid arthritis and Crohn's disease.

Description: This invention relates to the use of monoclonal antibodies as a method of detecting or estimating biological material. It has been reported that some patients with rheumatoid arthritis have raised levels of antibody to the polysaccharide/peptidoglycan complex of the cell walls of Group A Streptococci (Johnson et al , Clin. exp. Immunol., 55 , 115 and 61 , 373). It has also been reported that mice immunised with purified rheumatoid factor, i.e. immunoglobulin, from rheumatoid arthritis patients, develop antibodies to Group A Streptococci (Johnson et al , Clin. exp. Immunol., 61 , 373). It has recently been shown that the sugar residues on immunoglobulins taken from rheumatoid arthritis patients terminate with N-acetylglucosamine (GlcNAc) significantly more frequently than do normal immunoglobulins (Parekh et al , Nature 316 , 452). It is known that N-acetylglucosamine linked to polyrhamnose is a major determinant of Group A Streptococci. WO 84/04169 describes a method of assaying for Streptococcus Group A using anti-Strep. A antibodies which bind to the N-acetyl-D-glucosamine of Strep. A., in which assay the background resulting from cross-reaction with the N-acetyl-D-glucosamine moiety on the Strep. A. antibody or reaction with Staphylococcus A is reduced or eliminated. We have discovered that the N-acetylglucosamine present in the cell walls of Group A Streptococci is in essentially the same configuration as the N-acetylglucosamine present in the abnormal immunoglobulins of patients with rheumatoid arthritis. Consequently, antibodies raised against the cell walls of Group A Streptococci are capable of binding to N-acetylglucosamine residues in biological materials, e.g. the abnormal immunoglobulins of rheumatoid arthritis patients and may be used in assay methods for the detection and estimation of such biological materials. Using this discovery, we have now produced monoclonal antibodies to the cell walls of Group A Streptococci, or more specifically to the glycoconjugates having terminal N-acetylglucosamine residues present therein. Such antibodies are specific to biological materials, e.g. immunoglobulins, containing terminal N-acetylglucosamine residues. N-acetylglucosamine residues and the monoclonal antibodies can be used in assay methods for the purposes described below. It is particularly advantageous to be able to produce antibodies to abnormal immunoglobulins using an antigen which is not the immunoglobulin itself, since the monoclonal antibodies of the present invention can be made highly specific for abnormal immunoglobulins containing terminal N-acetylglucosamine residues and without the ability to bind other parts of immunoglobulins. The monoclonal antibodies used in the method of the invention may be produced in accordance with generally known techniques, see for example, "Monoclonal antibodies: production and maintenance" by U. Lovborg (1982), William Heinemann Medical Books, London. For example, mice are immunised with respect to Group A Streptococcal cell walls. The cell wall material is injected into the mice. Spleen cells from the mice are then fused with myeloma cells, e.g. of a mouse myeloma cell line. The hybridomas thus produced are screened for those producing antibodies possessing the correct specificity. This may be achieved by screening the antibodies by enzyme-linked immunoabsorbent assay (ELISA) on bovine serum albumin conjugated to N-acetylglucosamine (BSA-N-AG), enzyme treated fetuin (ETF) and fetuin. Fetuin is a glycoprotein found in serum which contains N-acetylglucosamine normally hidden in its structure. Treatment with sialidase and galactosidase cleaves the molecules to expose the N-acetylglucosamine and give ETF. Hybridomas producing antibodies positive both to BSA-N-AG and ETF but negative to fetuin are kept. The novel monoclonal antibodies to N-acetylglucosamine used in the present invention have a variety of significant utilities. In this instance they are used more particularly to detect and assay biological materials, i.e. materials of cellular origin, usually proteins, having exposed N-acetylglucosamine residues, e.g. abnormally glycosylated immunoglobulins in body fluids taken from patients suffering from, or suspected of suffering from, rheumatoid arthritis, tuberculosis, leprosy, Crohn's disease and similar diseases in experimental animals such as mice. Such monoclonal antibodies provide an important way of detecting and estimating the presence of the abnormal immunoglobulins believed to be associated with such conditions. The new monoclonal antibodies may also be used in the differentiation of certain types of tumour. There is evidence that tumours which bear terminal N-acetylglucosamine residues may be more susceptible to recognition by cells of the immune response, and may trigger release of cytotoxic molecules from such cells (Dennis et al ., Eur. J. Biochem. (1986) 161 :359-373). Receptors able to recognise GlcNAc appear to exist on myeloid cells (Ross et al ., (1985), J. Immunol., 134 :3307-3315; Haltiwanger & Hill, 1986, J. Biol. Chem., 261 :7440-7444). A consequence of recognition of agalactosyl tumours by such cells may be a markedly reduced tendency of such tumours to metastasise. This has been demonstrated in a murine model by Dennis et al . The monoclonal antibodies used in the method of the invention can therefore be used on cancerous cells,e.g. from biopsy material, or tumours removed at surgery (e.g. in cancer of the breast), to predict the likely prognosis (i.e. their tendency to metastasize). The monoclonal antibodies used in the method of the invention do not bind to cell membranes on normal tissues, but they bind strongly to cell membranes of tumour cells having terminal N-acetylglucosamine residues on the surface, and when revealed by a suitable colour-forming label they give strong membrane staining on such cells. This has been demonstrated on a murine tumour (L929) which triggers its own destruction via cytotoxin release from macrophages which it contacts. In clinical use, the monoclonal antibodies also make it possible to assay variations in levels of abnormally glycosylated immunoglobulins. This assists in predicting the nature of acute abdominal crises in Crohn's disease; assessing the efficacy of treatment in rheumatoid arthritis; monitoring the effects of immunotherapy on bladder cancer using Mycobacterium tuberculosis (var BCG); monitoring correction by immunotherapy of the immunopathological mechanisms seen in tuberculosis. The new monoclonal antibodies may also be useful in veterinary practice, for example for screening badgers for tuberculosis. As it is well known, wild badgers act as a reservoir of bovine tuberculosis from which domestic cattle can be infected. To prevent indiscriminate slaughter of badgers suspected of carrying bovine tuberculosis, a simple assay method is desirable, and the monoclonal antibodies of the present invention make this possible. For these purposes, the novel monoclonal antibodies may be used in, for example, enzyme-linked immunoassay in the same way as other monoclonal antibodies, but relying on the specific ability of the novel antibodies to bind with N-acetylglucosamine residues generally, and especially such residues present in immunoglobulin (IgG). Enzyme-linked immunoassay is a well known technique: reference may be made, for example, to Rook and Cameron, (1981), J. Immunological Methods, 40 , 109-114. By making monoclonal antibodies from the cell walls of Group A Streptococci rather than from the abnormal immunoglobulin of rheumatoid arthritis patients, it is possible to achieve a much higher specificity of action for N-acetylglucosamine without interference from other affinities. In general, the present invention provides a method of detecting and/or estimating biological materials having terminal N-acetylglucosamine residues, said biological material being an immunoglobin or a mammalian cell membrane, which comprises causing the said biological material to bind (a) to a monoclonal antibody specific to terminal N-acetylglucosamine residues, which has been raised against the glycoconjugate of the cell walls of Group A Streptococci having terminal N-acetylglucosamine residues, and (b) to a label, under conditions such that the bound or unbound label provides a measure of the said biological material, and detecting or estimating the said bound or unbound label. The label may be any of the labels currently used in immuno-assay techniques including radioactive, fluorescent and enzyme labels. Preferably an enzyme label is used along with a colour forming reagent which undergoes a chromogenic reaction catalysed by the said enzyme. Heterogeneous assay methods are usually preferred. The immunoglobulin or other biological material to be assayed is bound to a solid support, e.g. by adsorption to an appropriate surface, the monoclonal antibody is allowed to bind to the biological material on the solid support, a label is attached directly or indirectly to said monoclonal antibody, and the said label is then assayed. Where the biological material is a mammalian cell, e.g. from a tumour under investigation for the reasons explained above, a separate solid support is not required and the monoclonal antibody is allowed to bind to the cell itself, i.e. to any terminal N-acetylglucosamine residues present on the surface of the said cell, the label is attached directly or indirectly to any bound monoclonal antibody and the label is then observed or assayed. Single cells can be examined under the microscope in this way. Suitable enzyme labels include peroxidase, β-galactosidase, alkaline phosphatase, and glucose oxidase. In each case, a means for revealing or determining the bound enzyme label is provided in the form of a suitable substrate for the enzyme, e.g. a combination of hydrogen peroxide and a compound, e.g. o -phenylene diamine or 4-chloro-naphthol which produces a colour when oxidized by the hydrogen peroxide under the influence of the enzyme. The label may be attached to the monoclonal antibody by an anti-mouse immunoglobulin covalently linked to the said label. Alternatively, and preferably, the monoclonal antibody of the invention is biotinylated in known manner and the label is bound thereto using avidin covalently linked to the said label. The following Example illustrates the invention. Example Balb/C mice were immunised by an intraperitoneal injection of 50 µg of Group A Streptococcal cell walls (Johnson et al., (1984) Clinical & Experimental Immunology, 55 ,115) emulsified in Freund's incomplete adjuvant. The injection was repeated three weeks later, and a further booster injection was given intravenously 4 days before the mice were killed. (The intravenous dose was in an oil/water emulsion containing 10 µg squalene/ml of phosphate buffered saline + Tween ® 80). Spleen cells from the immunised mice were then fused with cells of the JK non-secreting mouse myeloma cell line (P3-X63-Ag 8, see Kearny et al (1979), J. Immunol. 123 ,1548). The hybridised cells were screened for production of the desired antibodies by enzyme-linked immunosorbent assay using the following antigens: (1) Bovine serum albumin to which N-acetylglucosamine residues had been bound by coupling the bovine serum albumin with diazotised amino-phenyl N-acetylglucosamine (BSA-N-AG) (Zopf et al (1978) Archives of Biochemistry and Biophysics, 185 ,61-71); and (2) Fetuin which had been treated with Sialidase and Galactosidase so that N-acetylglucosamine became the terminal sugar (ETF). Fetuin was purified by passage through a 5 micron TSK-250 (21.5 × 600 mm) gel filtration chromatography column equilibrated in 0.1M potassium phosphate at pH 7.4. A fraction of HPLC purified fetuin (10mg/ml) was subsequently incubated for 24 hours at 37°C under toluene with 10 units/ml of neuraminidase (sialidase) from Arthrobacter ureafaciens (from Boehringer Mannheim), and jack bean β-galactosidase (purified from jack-bean meal as described by R. Parekh, D. Phil, Thesis, Oxford, 1987 based on Lee & Lee, "Methods in Enzymology", (1972) 28 , 702-713) in 0.1M sodium citrate at pH 4.0. The asialo-, agalacto-fetuin was finally purified by gel filtration chromatography as described above. The most active cell lines identified by these methods were then further screened using immunoglobulin known to show raised levels of N-acetylglucosamine. For this purpose the abnormal immunoglubulin was denatured in 12 molar urea solution with 0.5 molar 2-mercaptoethanol at 40°C for 16 hours followed by dialysis against iodoacetamide to block the liberated mercapto groups. This treatment results in complete unwinding of the immunoglobulin chains and enhances exposure of the sugar residues. The denatured immunoglobulin obtained in this way was coated onto the polystyrene walls of a microtitre plate at a concentration of 10 µg/ml in a carbonate/bicarbonate buffer. For control purposes other wells in the microtitre plate were coated with the same amount of known normal immunoglobulin. Monoclonal antibody from a selected cell line, diluted in phosphate-buffered saline containing 0.05% of Tween® 20, was incubated in the immunoglobulin-coated wells of the microtitre plate. The quantity of bound monoclonal antibody was then assayed by adding peroxidase-conjugated rabbit anti-mouse IgG followed by the enzyme substrate and chromogen. To check that the test and control cells contained equal quantities of immunoglobulin, anti-human IgG was added to some of the immunoglobulin-coated wells so that total bound immunoglobulin could be estimated. In this way, six hybridomas producing monoclonal antibody positive both to BSA-N-AG and to ETF but negative to fetuin were located. Of these, two were chosen which had the greatest ratio of binding to ETF compared with the background binding to fetuin. This method for screening the mouse cell lines may be adapted to use the monoclonal antibodies of the invention in immunoassay. The immunoglobulin to be determined, e.g. in a serum sample taken from a patient, is coated in an appropriate dilution onto the walls of a microtitre plate made of polystyrene or other polymer capable of binding proteins. The monoclonal antibody is incubated in the immunoglobulin-coated wells of the microtitre plate. Peroxidase-conjugated rabbit anti-mouse IgG is then added to each well followed by the enzyme substrate (hydrogen peroxide) and chromogen (e.g. ophenylenediamine). The colour produced depends on the amount of bound enzyme which itself depends on the amount of immunoglobulin in the initial sample having free N-acetylglucosamine residues and therefore capable of binding the monoclonal antibodies used in the invention. Preferably, however, any assay using the monoclonal antibodies is carried out as follows: All the operations are performed at room temperature. The biological material which may have terminal N-acetylglucosamine residues in a sample to be assayed is adsorbed onto nitrocellulose sheets. The high affinity of the biological material, which is proteinaceous, for this material (which does not bind sugars) results in enhanced exposure of any sugar residues that are present. In the first stage, 0.5 µg of each sample of biological material (previously purified, e.g. by adsorption on protein A in the case of IgG) in 5 µl of phosphate buffered saline is spotted onto nitrocellulose. Conditions are controlled so that the size and concentration of each spot are kept constant. After drying for one hour, the nitrocellulose is boiled for 5 minutes. This further enhances exposure of the sugar residues and eliminates non-specific effects caused by rheumatoid factor activity, or any other antibody activity in the sample. The nitrocellulose is then incubated in 1% bovine serum albumin in phosphate buffered saline at pH 7.0 containing 0.05% Tween® 20 (PBS/BSA/Tween®) to block any other protein-binding sites. The number of terminal GlcNAc,s on each sample spot is then revealed by determining quantitatively the binding of the monoclonal antibody. To do this the monoclonal is biotinylated in known manner, e.g. by incubation overnight in phosphate-buffered saline of pH 7.0 containing N-hydroxy-succinimidobiotin (Sigma, catalogue number 11-1759). Conjugation occurs spontaneously. The nitrocellulose is then incubated in biotinylated monoclonal (diluted 1:2000 in PBS/BSA/Tween®), and then an avidin-peroxidase complex (from Amersham Internation plc, GB; diluted 1:500 in PBS/BSA/Tween; see Yolken et al , J. Immunol Methods (1983), 56 , 319-327), and finally hydrogen peroxide and 4-chloronaphthol, are added. This results in blue spots the intensity of which is related to the amount of terminal GlcNAc's present in the sample. The blue spots are measured by sandwiching the nitrocellulose between a red light emitting diode and a photodiode, linked to a suitable amplifier. The apparatus (based on that described by Rook and Cameron (1981), J. Immunological Methods 40 , 109-114) can be calibrated using samples of IgG of known terminal N-acetylglucosamine content. In an alternative procedure, protein A or protein G is first adsorbed into the nitrocellulose, e.g. by incubation of the latter in 100 µg/ml protein A in phosphate-buffered saline (PBS) for two hours. Then remaining protein-binding sites are blocked by incubation overnight in PBS/BSA/Tween® 20. Then the nitrocellulose bearing the protein A is incubated in serum diluted 1:2 in PBS for two hours, in order to saturate the IgG-binding sites on the protein A with IgG. Then it is washed 4 times in PBS, and fixed in 0.5% glutaraldehyde (Electron microscopy grade) in PBS for 30 minutes at 4°C to ensure permanent binding of the IgG to the protein A. Then the nitrocellulose now bearing both protein A and IgG bound to the protein A, is incubated in 0.1 molar lysine in PBS to block remaining aldehyde groups. Then the nitrocellulose is dried, boiled, and treated exactly as for the nitrocellulose-based assay described above.

Claims: A method of detecting or estimating a biological material having terminal N-acetylglucosamine residues, said biological material being an immunoglobulin or a mammalian cell membrane, which comprises causing the said material to bind (a) to a monoclonal antibody specific to terminal N-acetylglucosamine residues, which has been raised against glycoconjugate of Group A Streptococci cell wall having terminal N-acetylglucosamine residues and (b) to a label, under conditions such that the bound or unbound label provides a measure of the said biological material, and detecting or estimating said bound or unbound label. A method according to claim 1 in which the biological material having terminal N-acetylglucosamine residues is bound to a solid support, the said monoclonal antibody is allowed to bind to the said material on the solid support, a label is attached directly or indirectly to said monoclonal antibody, and the said label is then assayed. A method according to claim 2 in which the biological material is an immunoglobulin which is bound to the solid support by reaction with Protein A or Protein G which is itself bound to the said support. A method according to claim 1 in which the biological material is a mammalian cell, the said monoclonal antibody is allowed to bind any terminal N-acetylglucosamine residues on the surface of the said cell, a label is attached directly or indirectly to any bound monoclonal antibody, and the said label is then observed or assayed. A method according to any one of claims 1 to 4 in which the said label is an enzyme, which is assayed by a chromogenic reaction catalysed by said enzyme. A method according to any one of claims 1 to 5 in which the label is attached to the monoclonal antibody by an anti-mouse immunoglobulin covalently linked to the said label.

Inventors: EDGE, JENNIFER JANE, ROOK, GRAHAM ARTHUR WILLIAM
Applicants: G.D. SEARLE & CO.
Application year: 1987
Main classification: G01N33
IPC codes: C07K14, C12P21, C12Q1, C12N5, G01N33, C07K16, C07K19, C12N15, C12R1