PURIFICATION OF A NOVEL GLYCOPHOSPHOLIPID

Abstract: The present invention relates to the discovery and purification of a novel glycophospholipid that contains an antigenic determinant for human thyroid stimulating immunoglobulins (TSI's). Such glycophospholipid has utility as a reagent for immunological tests for Graves' disease as well as for further biological and chemical studies of Graves' disease.

Description: PURIFICATION OF A NOVEL GLYCOPHOSPHOLIPID Background of the Invention The present invention relates to the discovery and purification of a glycophospholipid that is an antigenic determinant for human thyroid stimulating immunoglobulin. The glycophospholipid provides important information concerning the autoimmune nature of Graves' disease, reagents for immunological assays for Graves' disease and means for immunotherapy of Graves' disease. Graves ' disease is an autoimmune thyroid disease that involves the overproduction of thyroid hormone. The disease effects approximately 0.4% of the population in the U.S. With an incidence rate of 1-2 cases per 1000 population per year, it is the most common form of hyperthyroidism in people under age 40 (Tunbridge, W.M.G., Evered, D.E., Hall, R. et al . , "The Spectrum of Thyroid Disease in a Community: The Wickham Survey, " Clinical Endocrinology, 2:481-493 (1977)) . At present Graves' disease is treated in one of three ways : by destroying the thyroid gland with radioactive I 131 , by using antithyroid medication or by surgical removal of the thyroid gland. Each of these therapies has its related risks and side effects. In the case of I 131 ablation of the thyroid gland, the treatment with I 131 aims to destroy enough thyroid tissue to make the patient euthyroid or permanently hypothyroid. The therapy is contraindicated in pregnancy, and optimal doses are hard to achieve, so that most patients are rendered permanently hypothyroid or are under-treated and have recurrent hyperthyroidism. Antithyroid medications, such as thionamides (propylthiouracil and methimazole) work primarily by blocking organic binding of thyroidal iodine. The thionamides (especially methimazole) cross the placenta and can induce hypothyroidism in utero. Such treatment causes permanent remission in only about 40% of patients and has side effects such as granulocytopenia or agranulocytosis (Greer, M.A. , Antithyroid Drugs in the Treatment of Thyrotoxicosis Thyroid Today, 2:1 (1990) ) . Surgical removal of the thyroid gland is the least desirable treatment alternative for Graves' disease due to the risk of general anaesthesia and surgical complications. An ideal therapy for Graves' disease would reduce or prevent autoantibody-induced overproduction of thyroid hormone in a disease-specific manner and would lack toxicity. Oral immune tolerization to induce antigen-specific tolerance has shown promise in some autoimmune states (see, e.g., Trentham et al. , "Effects of Oral Administration of Type II Collagen on Rheumatoid Arthritis, " Science, 2£_-L:1727 (Sept. 24, 1993)) , and the development of oral immune tolerization for the prevention or treatment of Graves' disease would present an important advance. At present, the diagnosis of Graves' disease is made by a combination of history, physical exam, thyroid function testing and radioactive iodine uptake scanning. Measurement of thyroid stimulating antibody (immunoglobulin) ("TSI") aids in the diagnosis of Graves' disease in some instances (Morris J.C. et al . , "Clinical Utility of Thyrotropin-Receptor Antibody Assays: Comparison of Radioreceptor and Bioassay Methods," Mayo Clin. Proc, £1:707-717 (1988)) . There are two widely used indirect assays to measure TSI from patients with suspected or diagnosed Graves' disease. The first, a radioreceptor assay, involves inhibition of I 125 labelled bovine TSH binding to human or rat thyroid membranes by IgG from patients with Graves' disease (Pekonen, F. et al. , "Thyrotropin Receptors on Bovine Thyroid Membranes: Two Types with Different Affinities and Specificities," Endocrinology, 105 :352-359 (1979)) . In this assay, commonly called the thyroid binding inhibitory immunoglobulin assay (TBII) , approximately 90% of Graves' patients demonstrate the presence of inhibitory antibodies. However, the assay does not correlate with the presence of TSI as measured in the other frequently utilized cyclic AMP assay. Presumably, numerous IgG antibodies against thyroid antigens are present in patients with Graves' disease, but only certain antibodies are capable of causing thyroid stimulation. The antibody detected by the TBII likely is not a stimulatory antibody. The second assay for measuring TSI is based on the ability of IgG from patients with Graves' disease to stimulate cyclic AMP generation in the rat FRTL-5 thyroid cell line. This assay is more relevant for Graves' disease than the TBII assay, as it measures the amount of cellular activation caused by TSI. TSI, as measured in this assay, is found in approximately 90-95% of patients with active Graves' disease. One commercial assay used by Nichols Institute involves the following: "TSI activity is measured in an in vitro bioassay using a thyroid cell line (FRTL-5) . The gamma globulin fraction is purified from the serum sample using DEAE sephadex. The fractionated gamma globulin is incubated with the thyroid cells and the production of cyclic AMP is determined. The concentration of cyclic AMP is then measured by a sensitive radioimmunoassay. The effect of stimulation is expressed as a percentage of basal activity. Basal production of cyclic AMP by FRTL-5 cells is estimated at 100%. The range in normal serum is 70-130%. Thus, any result exceeding 130% is considered abnormal." See Nichols Institute Reference Laboratories Assay Summary For Thyroid Stimulating Immunoglobulin (Revised 12/88) . More recently, the cloning of the TSH receptor (see Misrahi, M. et al. , "Cloning, Sequencing and Expression of Human TSH Receptor, " Biochem. and Biophys. Res. Comm. , 166 :394-403 (1990) ; Nagayama Y., et al . , "Molecular Cloning, Sequencing and Functional Expression of the cDNA for the Human Thyrotropin Receptor," Biochem. and Biophys. Res. Comm., 165 : 1184- 1190 (1989) ) has allowed researchers to develop assays that detect anti-TSH receptor antibodies in patients with Graves' disease. The antibodies detected are directed at conformational peptide epitopes of the TSH receptor (Kosugi, S. et al . , "Identification of Thyroid-Stimulating Antibody-Specific Interaction Sites in the N-Terminal Region of the Thyrotropin Receptor, " Mol. Endocrin. , 7:114-130 (1993)) . In pregnant women with active Graves' disease, the TSI bioassay is essential because its presence correlates with development of neonatal Graves' disease (Zakarita, M. et al. , Thyroid Stimulating Antibodies (TsAb) as a Prognostic Index for Neonatal Graves' Disease (Abstr. 121) , 11th Annual Meeting of the European Thyroid Association) . The general utility of TSI measurement in the diagnosis of Graves' disease may increase, however, if a better assay were available. The research and development of diagnostics and therapies for Graves' disease patients has been hampered by the lack of a good animal model for the disease. Several animal models (for example obese strain (OS) chicken, BUF rat and experimental autoimmune thyroiditis (EAT) mouse) (Weetman, A.P. et al . , "Autoimmune Thyroid Disease: Developments in Our Understanding," Endocrine Rev., 5_:309-355 (1984)) develop transient hyperthyroidism or chronic thyroiditis, but none mimic the humoral and cellular immune dysfunction seen in the human Graves' disease. Interestingly, immunization of mice with a highly immunogenic extracellular region of the human TSH receptor was unable to produce Graves' disease even though a proliferative T-cell response to thyroid antigens was present and high titres of anti-TSH receptor peptide antibodies were documented. (Carayanniotis, G., Huang, G.C., Nicholson, L.B. et al . , "Unaltered Thyroid Function in Mice Responding to a Highly Immunogenic Thyrotropin Receptor: Implications for the Establishment of a Mouse Model for Graves' Disease," Clin. Exp. Immulol . , 99 :294-302 (1995)) . Accordingly, there has remained a need for improvements in both the diagnosis and treatment of Graves' disease. A need also remains for a good animal model exhibiting the immune dysfunction seen in human Graves' disease. Summary of the Invention The present invention provides, in purified form, glycophospholipid (GPL) that is associated with a target antigen for autoantibodies (TSI) in Graves' disease. In one aspect, the present invention relates to such GPL per se . Other aspects of the invention relate to immunoassay methods and reagents employing GPL for the detection of TSI. The invention further provides anti-GPL antibodies, which also are useful in such immunoassay methods. The invention provides a method of diagnosing Graves' disease in a susceptible host, comprising obtaining a physiological specimen from the host and determining the presence of antibodies to a glycophospholipid of the present invention in the physiological specimen. Additional aspects of the invention relate to immunotherapy methods for the prevention or treatment of autoimmune hyperthyroidism (Graves' disease) . Thus, the invention provides a basis for inducing a state of immunotolerance in a patient in need thereof, by administering a therapeutically-effective amount of a GPL (or a functional analog thereof) , resulting in immune tolerance and normalization of thyroid hormone production. The invention also provides methods for treating a patient with receptor-blocking anti-GPL antibody. Yet another aspect of the invention is the development of an animal model of Graves' disease. Suitable animals inoculated with an immunostimulant, such as GPL of the present invention either alone or in combination with purified TSH receptor, will exhibit a Graves' disease like state. Thus, the administration of the GPL of the present invention to non-human animals will be useful for medical research. Brief Description of the Drawings Figure 1 shows TLC results after staining purified glycophospholipid with resorcinol and ammonium molybdate-perchloric acid. Figure 2 shows TLC results after incubating purified glycophospholipid with hydrofluoric acid (HF) . Figure 3 shows results of fluorophore assisted carbohydrate electrophoresis (FACE) of purified glycophospholipid. Figure 4 shows FACE enzyme analyses of purified glycophospholipid. Detailed Description of the Preferred Embodiments Novel glycophospholipid (GPL) has been isolated and purified from animal tissues. GPL is isolatable from human thyroid tissues and, in view of demonstrated in vitro immuno-reaction with TSI from Graves' disease patients, is considered to be a thyroid target for TSI. GPL of the present invention has been isolated from porcine pancreatic tissues. The fact that GPL is isolatable from porcine pancreas is consistent with the autoimmune nature of Graves' disease in humans and the fact that patients with Type I diabetes are predisposed to Graves' disease, as are their family members. Others have shown that both the thyroid and pancreas are endodermally derived and that autoimmune thyroid disease and autoimmune diabetes should share common antigenic targets (Tadmor, B. et al . , "Embryonal Germ-Layer Antigens: Target for Autoimmunity, " Lancet, 2-22.: 975-978 (1992)) . Thus, the theory of common shared antigens in autoimmune diseases further supports the role of such GPL as a target of human autoantibodies in Graves' disease. The following procedure describes and enables the isolation of GPL, which, according to the present invention, is immuno-reactive with human TSI. l Tissue Extraction: 2.5 kilograms of porcine pancreas were obtained upon slaughter and snap frozen in liquid nitrogen. Pancreatic tissue was homogenized in distilled water (3 ml per gram of tissue) and combined with methanol (8 ml per gram of tissue) and chloroform (4 ml per gram of tissue) . The mixture was stirred at room temperature for 20 minutes and centrifuged at 2000 x g for 15 minutes. The supernatant was saved and the pellet re-homogenized in the same solvents as above. After the second centrifugation the supematants were pooled and poured off into a large separatory funnel. Distilled water was then added to create an upper and lower phase (chloroform:methanol :water (C:M:W) ratio 1:2:1.4) . The phase separation was allowed to take place overnight and the upper phase was collected and saved. The lower phase was then re-extracted in methanol (half of the lower phase volume added) and 0.01 M KCl (one-fifth the total phase volume plus added methanol) , thus causing another phase separation (Svennerholm, L. et al., "A Procedure for the Quantitative Isolation of Brain Gangliosides, " Biochem. Biophys. Acta., 617:97-109 (1980)) . The upper phases were then combined, evaporated to dryness and taken up in distilled water. The reconstituted upper phase was dialyzed for 3 days against distilled water with Spectrophor #7 MWCO 1000 dialysis tubing to desalt the lipid preparation. 2 ^ _ Ion Exchange Chromatography: The lipid preparation was again rotary evaporated to dryness and taken up in C:M:W (30:60:8) . The preparation was then passed over a DEAE-Sephadex A-25 anion exchange column and monosialogangliosides were eluted off from the column in 0.02 molar sodium acetate. The monosialogangliosides were dried down via rotary evaporation, reconstituted in distilled water and dialyzed against distilled water as before. The dialyzed 0.02 M glycolipids were evaporated to dryness and taken up in C:M:W (65:25:3) . 3. HPLC: The 0.02 M fraction was further purified by HPLC using a 10 μm Hypersil semi¬ preparative silica column (250mm x 10mm ID) on a Gilson HPLC system. Ion exchange-purified 0.02 M glycolipids were eluted with a linear gradient of C:M:W (65:25:3) to (60:35:8) for 180 minutes. The flow rate was maintained at 2 ml/min and 4 ml fractions were collected. Each fraction was dried down under nitrogen and reconstituted in 200 μl of C:M:W (60:30:4.5) . Fractions were run on TLC in order to analyze the HPLC separation. (See Gottfries, J. et al., "High Performance Liquid Chromatographic Separation of Gangliosides, Combined with Immunological Detection and Fast Atom Bombardment Mass Spectrometric Characterization, " Journal of Chromatography, 490 :263-274 (1989)) . In particular, ten to twenty μl of each fraction was spotted on E. Merck Silica Gel 60 HPTLC plates and thin layer chromatography was performed with a solvent system of C:M:0.25% KCl (50:40:10) . Each fraction was run twice, once on a TLC plate stained with resorcinol (for detecting the presence of gangliosides) and again on a TLC plate stained with perchlorate-molybdate (for detecting the presence of phospholipids) . One fraction [29] was subjected to further analysis on analytical HPLC using a 5 μm Lichrosorb- NH2 column (250mm x 4 mm ID) . GPL was eluted from the column using a gradient of acetonitrile :phosphate buffer according to the method of Gazzotti (See Gazzotti, G. et al. , "Normal-Phase High-Performance Liquid Chromatographic Separation of Non-Derivatized Ganglioside Mixtures," J. Chromatography, 348 :371-378 (1985) ) . The elution was monitored by uv-detection at 206 nm. Two other fractions [35 and 38] of the fractions subjected to TLC, as described above, were combined and further purified on a Lichrosorb-NH2 semi¬ preparative HPLC column (250mm x 10mm - E.M. Sciences) using a phosphate buffer gradient, also in accordance with the above-cited method of Gazzotti. The flow rate was set at 6 ml/min and fractions of u.v. absorbing peaks (206 nm) were collected. The two fractions were selected because they both contained a phospholipid, and were observed migrating between ganglioside standards GM3 and GM2 in the TLC system described above. (See Gottfries, J. et al . , "High Performance Liquid Chromatographic Separation of Gangliosides, Combined with Immunological Detection and Fast Atom Bombardment Mass Spectrometric Characterization," Journal of Chromatography, 490:263- 274 (1989)) . Since the lipid entity detected with Graves' TSI migrates between GM3 and GM2 gangliosides in the TLC system employed, we further characterized this lipid. Each of the major peaks collected was subjected to gas chromatography-mass spectroscopy (GC- MS) and carbohydrate analysis, as described below. L. Mass Spectroscopy and GC-MS: Mass spectroscopy was performed in either of two ways - with a mass detector having a mass range up to about 3000 MW or GC-MS on samples using a Hewlett-Packard model 5890A gas chromatography system linked to a Hewlett-Packard model 5970 mass selective detector having a MW range up to 800. Mass spectroscopy was performed on fraction 29 using a time-of-flight mass spectrometer. (Kompact Maldi III instrument (Kratos Analytical, Manchester, U.K.) ) . As with fractions 35 and 38, fraction 29 was selected because it is a lipid that migrated between ganglioside standards GM3 and GM2 in the above- described TLC system. The results showed a lipid having a molecular weight of 917, accompanied by major fragments having molecular weights of 693, 671, 599, 571 and 447. Carbohydrate analyses also were performed on lipid fraction 29 by using the protocol of Supelco, Carbohydrate Analyses by GC and HPLC, copyright 1985, Supelco Inc. Beliefonte, PA 16823-0048. In particular, fatty acid methyl esters (FAME) of the lipid fraction were produced by hydrolyzing the lipid material in methanolic-HCl according to the procedure of Supelco. Specifically, 10 ml of 0.5 N methanolic- HCl and 10 mg of the lipid material were heated at 80°C for 18 hours in a reaction vessel sealed with a Teflon-lined cap. The resulting mixture was extracted four times with equal volumes of hexane and FAME was recovered in the lower phase. The FAME were then injected onto a Hewlett Packard GC-MS. A total ion chromatogram (TIC) of the FAME was produced along with a single ion scan. The single ion scan showed a peak at 11.241 minutes representing an 18 carbon chain fatty acid C(18:0) with a molecular weight of 298 and no unsaturated bonds. Another single ion scan showed a peak at 10.167 minutes, which reflected a 16 carbon chain fatty acid C(16:0) with a molecular weight of 270. Thus, data showed that lipid fraction 29 possesses both a C(18:0) and a C(16:0) fatty acid chain. These data are significant because palmitic acid C(16:0) and stearic acid C(18:0) are common fatty acids found in many membrane phospholipids. Additional GC-MS was carried out on lipid fractions obtained from the semi-preparative HPLC of fractions 35 and 38, as described above. All of the samples showed C(18:0) , C(17:0) and C(16:0) . Some fractions showed C(18:0) and C(17:0) as major species, while other fractions showed primarily C(18:0) with lesser amounts of both C(17:0) and C(16:0) . ______ Immunoblotting: Aluminum backed Silica gel 60 HPTLC plates were washed in methanol and air dried to activate the silica. Purified GPL obtained from the treatment of lipid fractions according to the above purification schemes was applied to lanes of an HPTLC plate and run in a TLC tank equilibrated in C:M:0.25% KCl (50:40:10) . Known ganglioside and phospholipid standards were run in separate lanes along side the sample lanes. The HPTLC plate was developed and removed and dipped in polyisobutylmethacrylate for 50 seconds. The lanes were then cut out with scissors. Each individual lane was incubated for 6-8 hours with normal human serum from control patients or serum from patients having Graves' disease or multinodular goiter (MNG) . The lanes were washed and then incubated with goat antihuman IgG peroxidase conjugated antibody for 2-4 hours. The lanes were washed a second time and then incubated with 4-chloro-l-naphthol reagent to develop the color reaction. The purified GPL exhibited binding to IgG from 2/2 patients with Graves' disease, 0/1 patient with MNG and 0/4 control patients. ___. Thin Layer Chromatography: High performance Silica gel 60 thin layer chromatography plates were soaked in methanol and air dried. The chromatography tank was equilibrated with C:M:0.25% KCl (50:40:1) overnight before use. Lipid extracts or standards were applied to the bottom of the plate and allowed to dry. After running the TLC reaction the plate was then stained with the appropriate reagent for visualizing either gangliosides, sugars or phospholipids, as described above and below. The plates were then heated in an oven at 100° C for 10 minutes or until bands appeared. 7___ Fluorophore Assisted Carbohydrate Electrophoresis (FACE) : Purified GPL was treated with either ceramide glycanase (which cleaves carbohydrate from lipid in gangliosides) or hydrofluoric acid (HF) (which cleaves phosphodiester bonds) and conjugated with 7-amino-1, 3-naphthalene disulfonic acid according to the methods of GLYKO, Inc. Novato, CA. (See Basu, S.S., Hosseini, S.D., Hoover, G., Li, Z., and Basu, S., "Analysis of Glycosphingolipids by Fluorophore- Assisted Carbohydrate Electrophoresis Using Ceramide Glycanase from Mercenaria, Analytical Biochemistry, 222 : 270-274 (1994)) . In particular, fluorophore conjugated sugars were subjected to gel electrophoresis and bands were detected with a fluorescent spectrophotometer from GLYKO, Inc. Relevant carbohydrate bands were cut from the gel, followed by eluting out the carbohydrates from the gel and subjecting them to enzymatic digestion to determine their chain sequence. Figure 3 shows a FACE ® profile of ANDA labeled glycolipid samples on an N-linked gel. The samples contained in lanes 1-7 comprise the following: Lane 1 & 7 - Oligo Ladder Standard Lane 2 - Mixture of ceramide glycanase released globosides, Gb 4 and Gb 3 , and lactose, galactose, glucose Lane 3 - GPI Sample #31 treated with HF and HONO Lane 4 - GPI Sample #31 released with ceramide glycanase Lane 5 - Mixture of ceramide glycanase released gangliosides, ASGM lf GM α , GD la , GD lb , GT lb , GM 2 , GM 3 (co-migration of GD la and GD lb ) Lane 6 - Sample #43 released with ceramide glycanase The GPL [31] subjected to the above analysis is characterized in the following manner: (1) The GPL migrates between ganglioside standards GM3 and GM2 in the TLC system described above. The GPL is unstained by resorcinol indicating that it does not contain sialic acid. The GPL stains black with orcinol and bright blue with ammonium molybdate-perchloric acid, thus indicating that it contains a sugar sequence and a phosphate group, respectively. Specifically, in the photograph in Figure 1 the TLC plate on the left is stained with resorcinol reagent and contains ganglioside standards (from top to bottom) GM3, GM2, GMl, GDla, and GDlb, as well as the purified glycophospholipid of the present invention. The glycophospholipid contains no sialic acid, as shown by the glycophospholipid' s inability to stain with resorcinol along with the sialic acid- containing ganglioside standards. The TLC plate on the right is stained with ammonium molybdate and contains the same quantity of standards and glycophospholipid as the TLC plate on the left . As observed in the TLC plate on the right, the glycophospholipid migrates between GM3 and GM2 and stains bright blue (as shown in Figure 1 by the additional band located adjacent to the arrow) indicating the presence of a phosphate group therein. (2) Incubation of the purified GPL with hydrofluoric acid (HF) , a reagent capable of cleaving phosphate bonds (see Shaw, N. and Stead, A., "The Reaction of Phosphoglycolipids and Other Lipids with Hydrofluoric Acid," Biochemical Journal, 143 :461-464 (1974)) , shows migration of the phospholipid, giving further evidence that phosphate is present. This is shown in the photograph of the TLC plate in Figure 2 containing ganglioside standards on the left and phosphotidylethanolamine (PE) and phosphotidylcholine (PC) standards on the right of the plate. The lane labelled HF- contains the purified glycophospholipid of the present invention which migrates between ganglioside standards GM3 and GM2. The lane labelled HF+ contains the glycophospholipid after treatment with hydrofluoric acid. As observed in the photograph, the band migrates near the top of the plate, thus indicating the loss of the polar sugar group. Staining was done with ammonium molybdate reagent . FACE analysis released a disaccharide that did not migrate with known oligo ladder standards (see Figure 3, lane 2) . The oligo ladder standards are glucose units (i.e., G_ = one glucose polymer, G 2 = two glucose polymers, etc.) . Carbohydrate sequencing is demonstrated in Figure 4, wherein the samples in lanes 1-4 comprise the following: Lane 1 Sample #31 treated with HF and nitrous acid in enzyme buffer Lane 2 Sample in Lane 1 plus β- galactosidase Lane 3 Sample in Lane 1 plus β-Hexase I Lane 4 Sample in Lane 1 plus - mannosidase Outside lanes Oligo Ladder Standard Lane 4 shows a major decrease in the intensity of band 2, which comigrates with G 2 after incubation with α-mannosidase. This decrease in band intensity reflects the presence of a terminal mannose residue on the purified GPL. Additional evidence, as reported in Figure 3, lane 4, shows that no carbohydrate release occurred upon treatment with ceramide glycanase, thus indicating that the purified GPL is not a ganglioside. Ceramide glycanase releases carbohydrates that are attached to ceramide. Because gangliosides contain ceramide as the core lipid, ceramide glycanase will release carbohydrates from all gangliosides. Thus, the GPL of the present invention contains phosphate, mannose, another saccharide moiety (perhaps glucosamine or glucose) , and lipid moieties. GPL described above is useful as a reagent in the further biological and chemical study of autoimmune diseases and Graves' disease in particular, as well as in clinical chemistry. For example (by way of illustration and not limitation) , GPL can be used per se. or in the preparation of immunoassay reagents for detecting TSI antibodies in blood or tissue samples from patients. GPL can be used in conventional direct and indirect immunoassay detection methods, for example, detection assays which use monoclonal or polyclonal antibodies directed against GPL, by well-known and conventional techniques. For example, polyclonal murine or rabbit antisera raised against GPL of the present invention can be employed for immunoaffinity purification of GPL and in an ELISA assay for GPL or TSI . The accuracy and precision of this new assay will be significantly better than the currently available diagnostic (bioassay) technology. Whereas currently available bioassays indirectly measure TSI based on cyclic AMP generation, an immunoassay according to the present invention provides direct quantitation of TSI in serum or other tissue samples. Animals can be immunized with GPL per se or an immunogenic GPL conjugate, and thereafter sera are screened for the presence of anti-GPL titer. Alternatively, spleen cells from immunized animals showing anti-GPL titer are fused to myeloma cells, or are treated in other conventional ways, to prepare GPL-specific monoclonal antibodies. GPL according to the present invention will be useful in the animal modelling of Grave's disease, the study of its causes and course, and in the evaluation of potential therapies. A variety of immunotherapy procedures are provided by the present invention, including the preparation and administration of a TSI-receptor blocking antibody, the preparation and administration of a cytotoxic antibody to destroy a B-lymphocyte clone that is secreting TSI, and immunizing patients with the purified GPL in order to induce tolerance and decrease or eliminate TSI production by clonally activated b-lymphocytes . Protocols for the establishment of such procedures will be known to the ordinarily-skilled clinician. GPL is administered to humans or other warm¬ blooded animals in order to ameliorate various disorders stemming from immune system dysfunction, including the disorders arising from Graves ' disease and the underlying overproduction of thyroid hormone . Treatment with GPL advantageously will be instituted early in the development of such disorders. Immunotherapy for the treatment of Graves ' disease uses GPL according to the present invention. Since thyroid hormone overproduction in Graves' disease is caused in part by the presence of TSI's, the GPL which is characteristic of TSI target thyroid antigen advantageously will induce a state of tolerance and hence ablate B-lymphocyte clones that produce TSI . While the exact mechanism by which tolerance induction takes place is unknown, many believe that autoreactive clones of B-lymphocytes are eliminated via a mechanism that involves apoptosis. Therapeutic GPL compositions will contain a therapeutically-effective dose of GPL in a pharmacologically acceptable carrier. The dose, carrier and route of administration selected will depend upon, among other factors, the condition of the patient, the desired route of administration and the activity of the selected GPL. This is readily determined and monitored by the physician during the course of therapy. One carrier for infusion or injection of GPL is a sterile isotonic aqueous solution, for example saline for injection or 5% dextrose. These preparations can be administered by oral, intranasal, subcutaneous, intravenous, intraperitoneal or other conventional routes. Oral tolerance to GPL may be dose-specific, and increased dosages may result in a loss of tolerance. Oral tolerance can be enhanced by the co¬ administration of immune adjuvants, such as lipopolysaccharides, which appear to stimulate additional populations of cells to down-regulate immune responses (See Weiner et al . , "Double-Blind Trial of Oral Tolerization with Myelin Antigens In Multiple Sclerosis," Science, 2J2__.:1321 (Feb. 26, 1993) ) . Although the present invention has been described in connection with certain preferred embodiments, it is not so limited. Modifications and variations will be apparent to those skilled in this field.

Claims: Claims; I claim: 1. Isolated and purified glycophospholipid that is an antigenic determinant for human thyroid stimulating immunoglobulin. 2. An isolated and purified glycophospholipid comprising an antigenic determinant for human thyroid stimulating immunoglobulin and migrating between GM3 and GM2 on a thin-layer chromatogram. 3. An isolated and purified glycophospholipid comprising an antigenic determinant for human thyroid stimulating immunoglobulin and migrating between GM3 and GM2 in the thin-layer chromatogram of Figure 1. 4. A method of identifying Graves' disease in a susceptible host, comprising obtaining a physiological specimen from said host and determining the presence of antibodies to a glycophospholipid of claim 1 in the physiological specimen. 5. A method of identifying the presence of thyroid stimulating immunoglobulin in a physiological sample, comprising reacting the physiological sample with a glycophospholipid of claim 1. 6. An immunoassay reagent comprising a solution comprising a glycophospholipid of claim 1. 7. A monoclonal antibody that specifically recognizes a glycophospholipid of claim 1. 8. A method of inhibiting the production of thyroid stimulating immunoglobulin in a host in need thereof, comprising administering to said host an effective amount of a glycophospholipid that is an antigenic determinant for said immunoglobulin.

Inventors: BLEICH DAVID, BLEICH, DAVID
Applicants: HOPE CITY, CITY OF HOPE
Application year: 1996
Main classification: C07H15
IPC codes: C07H3, G01N33