WO1992015884A1

WO1992015884A1 - Novel protein, method and products for detection of oligodendrocytes

Info

Publication number: WO1992015884A1
Application number: PCT/US1992/001907
Authority: WO
Inventors: Eldon E. Geisert, Jr.; Charissa A. Dyer
Original assignee: UAB Research Foundation
Current assignee: UAB Research Foundation
Priority date: 1991-03-08
Filing date: 1992-03-09
Publication date: 1992-09-17
Anticipated expiration: 1993-09-08
Also published as: AU1765692A

Abstract

A novel myelin/oligodendrocyte specific protein found in central nervous system (CNS) white matter that is expressed exclusively in CNS myelin and the surface of oligodendrocytes is provided. A ligand that reacts with an antigenic epitope of the myelin/oligodendrocyte specific protein (MOSP) is also provided. Hybridomas producing antibodies reactive with the novel protein as well as nucleic acids encoding the protein are additionally provided. Finally, methods of detecting demyelination and multiple sclerosis are also provided.

Description

NOVEL PROTEIN, METHOD AND PRODUCTS FOR DETECTION OF OLIGODENDROCYTES

TECHNICAL FIELD

The myelin sheath is a lipoproteinaceous envelope in vertebrates surrounding most axons of greater than 0.5 μH diameter and consists of plasma membrane of oligodendrocytes or Schwann cells wound tightly around the axon in a variable number of turns. This myelin membrane is a bylayer consisting of about 80% lipid and 20% protein. In the central nervous system (CNS) , oligodendrocytes produce multiple wraps of myelin membrane that ensheath axons and alter the passive electrical properties of the axons, increasing the conduction velocity and efficiency of action potentials. Oligodendrocytes are one of five types of glial cells (astrocytes, microglia, ependymal cells and Schwann cells) which together make up about 90% of all neural tissue; neurons make up the other 10% of neural tissue. Glial cells surround neurons, both their cell bodies and their processes, providing structural and functional support to the neurons. The myelin of the peripheral nervous system (PNS) is produced by a different cell type, the Schwann cell.

BACKGROUND ART

Both CNS and PNS myelin share a variety of unique lipids and proteins; until now only myelin/oligodendrocyte glycoprotein (MOG) has been shown to be selectively expressed in CNS myelin. A number of proteins, such as proteolipid protein (PLP) , MOG, cerebellar soluble lectin (CSL) , and myelin basic protein (MBP) are found in myelin-producing cells. Although these proteins are highly enriched in myelin, PLP and MBP and CSL are not specific to CNS myelin, and MOG is present in only small or undetectable levels in mature oligodendrocytes, thus making them unsuitable for identification of oligodendrocyte cell bodies in adult tissue. Other markers, such as 2 ' , 3' cyclic nucleophosphohydrolase (CNPase) , carbonic anhydrase, galactocerebroside and sulfatide are present in oligodendrocytes but are not unique to the CNS.

Antibodies to the above markers have been used to detect the presence of the markers in CNS and PNS.

The destruction of myelin or the cells that produce it often results in a debilitating loss of sensory and motor function. Some demyelinating diseases appear to be CNS specific, such as multiple sclerosis. A clear understanding of the pathogenesis of multiple sclerosis has been hindered by the lack of a specific marker for mature oligodendrocytes. Defining the differences between CNS and PNS myelin may provide dramatic insights into the process of demyelination and the pathogenesis of certain idiopathic diseases involving myelin such as multiple sclerosis, certain leukodystrophies and other myelinopathies. We describe herein a novel myelin/oligodendrocyte specific protein (MOSP) that is not present in Schwann cells or in PNS myelin. It is present on the cell surface of oligodendrocytes and continues to be expressed in the cell body in adulthood. In addition, the protein appears to be highly conserved since it is present in higher vertebrate species including human, monkey, cat, ferret, rat, mouse and chick. In vitro studies demonstrate that MOSP has biological properties distinct from previously described myelin proteins.

DISCLOSURE OF THE INVENTION

Previously only one protein, MOG, was known to be exclusively expressed in central nervous system (CNS) myelin. This invention provides a novel surface membrane protein expressed only in CNS myelin and oligodendrocytes of higher vertebrates which has been identified by a monoclonal antibody (CE1) . This CNS myelin/oligodendrocyte specific protein, MOSP, has a molecular weight of about 48 kDa and a pi of approximately 6.7. In the presence of the monoclonal antibody, MOSP remains on the surface of cultured oligodendrocytes but becomes associated with cytoplasmic microtubules, indicating that MOSP is capable of forming a stable link with the oligodendrocyte cytoskeleton, unlike any other known myelin protein.

It is an object of this invention to provide a method for detecting acute multiple sclerosis events. It is a further object of this invention to provide a method for studying membrane cytoskeletal interactions during the formation and maintenance of central nervous system myelin. It is a further object of this invention to provide a means for detecting the occurrence of demyelination in the central nervous system. It is a further object of this invention to provide a method of studying the pathogenesis of disease of central nervous system myelin. It is a still further object of this invention to provide a method for diagnosing multiple sclerosis in humans. Objects are encompassed herein as described below.

BEST MODE OF CARRYING OUT THE INVENTION

The present invention provides the discovery of a novel protein found in central nervous system myelin and oligodendrocytes. The protein has a molecular weight of about 48,000 and an isoelectric focusing point of about 6.7. In addition, the protein is specific for central nervous system myelin and oligodendrocytes. Standard methods can be applied to determine the amino acid sequence and the nucleic acids which encode the protein. Once the sequence is determined, nucleotide probes can be made and standard methods, such as polymerase chain reaction, can be used to detect the nucleic acids.

Since the invention provides a novel protein antigen, one skilled in the art will readily recognize that modifications in the protein, such as substitutions and deletions, which do not affect the essential nature of the protein, can be made. Thus, the claims defining the protein are intended to cover these modifications. In addition, post-translational modifications or pre- translational modification variants are intended to be within the scope of the claims. Functional equivalents, such as smaller or larger proteins or polypeptides, are also within the definition of the protein as long as they retain an identifying number of the a ino acids of the protein.

The invention also provides ligands which are specifically reactive with the protein. While only antibodies are exemplified below, the skilled artisan will recognize that modifications of the antibodies or other ligands would be effective in the methods described in the example. Such modifications include isotype switching.

The following specific examples are intended to illustrate more fully the nature of the invention without acting as a limitation upon its scope.

EXAMPLE

A. INITIAL CHARACTERIZATION OF MOSP

1. Production And Purification Of Monoclonal Antibody

The monoclonal antibody CE1 is a ligand which reacts with the antigenic epitope of MOSP. It was produced from mice first immunized with rat glial membrane proteins and then with a final boost of rat CNS white matter. Five days following the final boost, the animals were anesthetized with KETALAR (Parke-Davis) (10 mgs) and the inguinal lymph nodes were dissected from the mice. Lymphocytes, obtained from the lymph nodes, were disassociated and added to an equal number of AG8 myeloma cells (American Type Culture) or other myeloma cell lines such as SP20; fusion was performed in PEG 1000 (Boehringer Manneheim) . CE1 was detected in an ELISA (enzyme linked immunosorbent assay) using live cultures of mixed glial cells.

CE1 was purified and concentrated from tissue culture supernatents with ammonium sulfate precipitation followed by dialysis in distilled water. The water insoluble IgM was resuspended in Delbeco's Modified Eagles Medium (DMEM) (Gibco) . Other antibodies were used in the characterization of CE1 and its antigen MOSP. A rabbit polyclonal antibody directed against sulfatide (007) was a gift from Dr. Robert Lisak. The murine monoclonal antibody Tu27 was a gift from Dr. Lester Binder. The rabbit polyclonal antibody directed against galactocerebroside was produced in the laboratoy of Dr. Joyce Benjamins. A variety of second antibodies were used to detect antibody:antigen complexes. Second antibodies, goat anti-mouse IgM conjugated to rhodamine (GAM-TRITC) and goat anti-rabbit IgG conjugated to fluorescein (GAR- FITC) , were purchased from Organon Technika (Malvern, PA) . GAM-TRITC crossreacts well with rat IgM and to a lesser degree with rabbit IgG. Therefore, to remove the population of antibodies in GAM-TRITC that reacted with rabbit IgG, GAM-TRITC was passed through a rabbit affinity column.

Alternatively, other detectable agents such as horseradish peroxidase, alkaline phosphatase and β- galactosidase may be conjugated to other second antibodies directed against mouse IgM and mouse IgG and anti-rabbit IgG. Also, biotin coupled to a primary antibody or a biotin-streptavidin sandwich assay may be used to detect the presence of complexes.

The term "complex" refers to a "ligand complex," an "immune complex" and an "antigen:antibody complex," which are treated herein as synonymous and are defined as the reaction of an antigenic epitope with a ligand specific for that epitope. The term "immunoreactivity" as used herein denotes immune complex formation. A ligand is a substance capable of reacting with, binding to, or •forming a stable interaction with another substance. "Indirect immunohistochemistry," as used herein, is a means for detecting the formation of complexes between a first ligand and its antigen using a second more readily detectable ligand which reacts with the complex formed between the first ligand and its antigen. Indirect immunohistochemical means include immunostaining, enzyme- linked immunosorbent assay (ELISA) , immunoaffinity chrαmatography, immunoprecipitation, immunoblotting (Western blot and dot blot) and radioimmunoassay.

2. Localization Of MOSP By Immunostaining

For initial characterization of the protein antigen, MOSP, a number of rat tissues were screened using indirect immunohistochemical methods. The biological samples were sectioned on a freezing microtome at 40 μm or on a cryostat at 20 μm. The sections were placed in a blocking solution of borate-buffered saline (BBS) with 4% bovine serum albumin and 2% normal goat serum. The primary antibody was then added to the sections ovenight at 4'C to allow formation of antigen:antibody complexes. The next day the sections were rinsed with BBS and placed in second antibodies, goat anti-mouse IgG and IgM fluorescein conjugated (Pierce) or other second antibodies directed against mouse IgM, at a dilution of 1:500 for 2 hours at room temperature and allowed to form immune complexes. The sections were again rinsed in BBS and glass coverslips were applied.

In the immunostained sections of the CNS, only myelin and oligodendrocytes were labeled. No specific staining is observed in sections from any of the other tissues tested, including peripheral nerve, retina, liver, skin and kidney. Immune complexes that form between the antigenic epitope on MOSP and CE1 are detected in tissue sections of CNS myelin from chick, mouse, rat, ferret, cat monkey and man. No immunoreactivity is detected in sections of frog or goldfish brain. Thus, the epitope recognized by CE1 is CNS myelin and oligodendrocyte specific and appears to be conserved in higher vertebrate species.

The cellular distribution of MOSP was further characterized by immunostaining a variety of culture systems: enriched mouse oligodendrocytes, mixed rat glial cells, meningeal fibroblasts, rat neurons, and Schwann cells. In these cultures, only oligodendrocytes were labeled and all other cell types including type 1 astrocytes, type 2 astrocytes, fibroblasts, neurons, macrophages, microglia and Schwann cells were negative. Indirect immunofluorescent staining for the CE1 antigen was performed on live cultures by treating them at 37°C with CE1 for 15 minutes and then with second antibody rhodamine-conjugated goat anti-mouse IgM for 15 minutes. This permitted the formation of immune complexes between MOSP and CE1. Cells were then fixed with 4% paraformaldehyde for 10 minutes. For positive identification of oligodendrocytes and Schwann cells, cultures were double stained for galactocerebroside by addition of rabbit anti-galactocerebroside IgG for 15 minutes followed by second antibody fluorescein-conjugated goat anti-rabbit or other second antibodies directed against rabbit IgG.

Live oligodendrocytes were intensely immunostained following brief (ten minute) exposures to CE1 and second antibody directed against mouse IgM. Immunofluorescent staining of live cultured rat oligodendrocytes was detected after a 5 minute application of CE1 followed by fixation and addition of goat anti- mouse IgM or other anti-mouse IgM conjugated to fluorescein. These results indicate that the antigen detected by CE1 is constitutively expressed on the external surface of oligodendrocyte cell membranes. The results also show that immunohistochemical staining can detect the presence or absence of oligodendrocytes in both live and fixed biological samples. Oligodendrocytomas, tumors of oligodendrocyte origin, may also be detected by immunostaining as described above.

The distribution of MOSP was further characterized in normal and pathological human tissues. Surgically obtained and autopsy human CNS tissue were fixed with 4% paraformaldehyde, sectioned at 20μm on a cryostat and immunostained with CE1. In sections of normal spinal cord, the tissue distribution of MOSP is similar to that of myelin basic protein (MBP) , a cytoplas ic protein found specifically in myelin and myelin-producing cells in both the CNS and the PNS. In the spinal cords of patients with multiple sclerosis, both MBP and MOSP are absent from the sclerotic plaques. This demonstrates that CE1 stains normal CNS myelin and that when demyelination occurs, immunoreactivity for MOSP, like MBP, is no longer detected. Thus CE1 may be used to detect abnormal distributions of MOSP, and thereby provides a means for diagnosing multiple sclerosis in humans. B. ELISA CHARACTERIZATION OF MOSP

To define the antigen recognized by CE1, the reactivity of the antibody with myelin lipids and proteins was examined. A variety of glycolipids, including the two highly antigenic myelin-enriched glycolipids, galactocerebroside and sulfatide, were screened in a liposome enzyme linked immunosorbent assay (ELISA) . The reactivity of CE1 with mixed gangliosides, ceramide, sulfatide, galactocerebroside, glucocerebroside, ganglioside, asialo ganglioside, and phosphatidyl choline:cholesterol vesicles (negative control) was determined in the ELISA. Polyvinyl chloride 96-well microtiter plates were coated with liposomes consisting of lipid antigen:phosphatidyl choline:cholesterol. CE1 and anti-sulfatide (positive control) were incubated with the lipids followed by addition of second antibody directed against mouse IgM conjugated to alkaline phosphatase. Color was developed by adding the substrate, p-nitrophenyl phosphate (Sigma, St. Louis, MO) . No CE1 immunoreactivity was detected with any of the lipids tested, indicating that the antigenic epitope of CE1 is not a lipid.

C. IMMUNOBLOTTING OF MOSP

Electrophoresis and immunoblotting with a peroxidase- conjugated second antibody were performed. This sequence of protein detection steps is designated "Western blotting." Samples of CNS proteins were separated by 4 to 16% SDS polyacrylamide gel electrophoresis (SDS-PAGE) . The samples were transferred to nitrocellulose, blocked with 5% non-fat dry milk in borate buffer (pH 8.4) and stained with the monoclonal antibody CE1. After rinsing in borate buffer 3 times, the blots were placed in second antibody (peroxidase- conjugated goat anti-mouse IgG and IgM, Pierce) for two hours at room temperature. After further rinsing in two changes of borate buffer and two changes of Tris buffer (0.1M, pH 7.4), the blot was developed in 0.5mg DAB/ l of Tris Buffer (filtered) with 3 μl of 3% H202/ml of tris buffer. A protein band of approximately 48 kDa was detected on immunoblots of rat CNS myelin probed with CEl.

Alternatively, other second antibodies directed against mouse IgM and IgG conjugated to a variety of other detectable agents such as fluorescein, rhodamine, alkaline phosphatase or β-galactosidase, or a biotin-streptavidin sandwich assay, may be used. Alternatively, dot blot hybridization alone may be used to detect the presence of immune complexes between MOSP and CEl.

D. IMMUNOAFFINITY PURIFICATION OF MOSP

To confirm the detection of MOSP by immunoblot, immunoaffinity column chromatography and immunoprecipitation with CEl were performed and the bound protein was analyzed by SDS-PAGE.

1. Antibody Affinity Column

Purified CEl was conjugated to CNBr-activated

SEPHAROSE CL-4B beads (Pharmacia) according to the protocol suggested by the supplier. Oligodendrocyte enriched shake off cultures were labeled 18 hr with 150 uCi/ l (³H) leucine (111 Ci/mM) . A TRITON X-100 (Sigma, St. Louis) (0.5% TRITON X-100 in Tris-buffered saline (TBS) plus 2mM phenylmethylsulfonylfluoride (PMSF)) soluble lysate was prepared from the cells and the lysate was incubated with the CEl affinity beads overnight with constant mixing at 4^eC. Nonadherent proteins were washed from the column with TBS. Adherent proteins were eluted with 0.2M glycine, pH 2.3 plus 0.02% TRITON X-100 (Sigma); column fractions were immediately neutralized with 0.5 Tris. pH 8.5. The column fractions containing radioactivity were separated on a 5-20% SDS polyacrylamide gel to identify the molecular weight of the isolated protein (standard molecular weight markers were also electrophoresed) . Gels were sliced, treated with PROTOSOL (New England Nuclear) overnight, scintillation cocktail was added and samples were counted in a Beckman counter for 10 min per sample. A 48 kDa protein corresponding to MOSP was detected.

2. Immunoprecipitation

Immunoprecipitations were performed on TRITON X- 100 (Sigma) soluble lysates of cultured oligodendrocytes radiolabeled with (³⁵S)methionine (New England Nuclear) . Cultures were labeled 18 hrs with 250 μCi/ml (³⁵S) methionine (1160 Ci/mM) . The cultures were solubilized with TRITON X-100 (Sigma) and the lysate was treated sequentially with the following: 1) 50 μg of mouse monoclonal anti-sulfatide IgM plus 50 μl of protein

A-SEPHAROSE (Pharmacia) bound to goat anti-mouse IgM, 2) another 50 μg of anti-sulfatide IgM and protein A- SEPHAROSE (Pharmacia) bound to goat anti-mouse IgM, and 3) 50 μl of protein A-SEPHAROSE (Pharmacia) bound to goat anti-mouse IgM or other anti-mouse IgM. After overnight incubation with constant mixing at 4"C the beads were sedimented by low speed centrifugation and the lysate removed and used for the next immunoprecipitation step. Steps 1-3 were used to clear proteins from the lysate that non-specifically adhere to either antibodies or to protein A-SEPHAROSE (Pharmacia) . The cleared lysate was then treated with CEl followed by protein A-SEPHAROSE (Pharmacia) to precipitate the antigen recognized specifically by CEl. After immune complex formation and removal from the lysate, protein A-SEPHAROSE beads (Pharmacia) were washed six times in TBS and Laemmli sample buffer was added to dissociate the immune complexes. The samples were boiled 3 min and loaded on a 5-20% SDS polyacrylamide gel for analysis. Gels were sliced and radioactivity detected as described for antibody affinity columns above. This procedure resulted in the detection of a major radioactive peak at 48 kDa (MOSP) precipitated by CEl that was not precipitated by the anti-sulfatide control.

D. ISOELECTRIC FOCUSING

Equilibrium isoelectric focusing (EIF) of precipitates from (³⁵S) methionine labeled oligodendroglial culture TRITON X-100 (Sigma) lysates was performed. The lysate was first treated with anti-sulfatide IgM as described for immunoprecipitation above and then treated with CEl and protein A-SEPHAROSE (Pharmacia) bound to goat anti-mouse IgM or other anti-mouse IgM. The immunoprecipitates were washed extensively and loaded onto separate IEF tube gels. The pH gradient in the focused gels was determined by slicing duplicate gels into 0.5 cm sections, placing each section into 0.5 ml dH₂0 and measuring the pH after 30 min. The pH ranged from 3.2 to 9.3. The pi of the CEl protein spots detected on the two- dimensional gel autoradiograph was determined by measuring the distance the proteins migrated through IEF gel and comparing that to the pH gradient obtained from the sliced gels. After focusing, each gel was placed on top of a SDS 5-20% acrylamide gradient slab gel and run on the second dimension. The gels were stained, destained and impregnated with the scintillant PPO (New England

Nuclear) . The gels were then dried onto filter paper and exposed to X-AR5 (Kodak) X-Ray film for 1 week. A 48 kDa protein with a pi of about 6.7 was identified on the CEl gel and not on the control anti-sulfatide gel. The above results show that MOSP is a 48 kDa protein with a pi of about 6.7. When a second sample, immunoprecipitated in an identical manner, was examined by two-dimensional gel electrophoresis as above, a pair of labeled proteins with isoelectric points of approximately 6.7 were observed at 48 kDa. A pH gradient ranging from 3.2 to 9.3 was reproducibly obtained. These two radiolabeled proteins probably represent a single protein, since they migrate as a double in two-dimensional gels and share the CEl epitope. The slight difference in isoelectric points suggests that MOSP can be post-translationally modified.

E. LECTIN AFFINITY CHARACTERIZATION OF MOSP

To determine if MOSP contains carbohydrate side chains containing mannose or sialic acid, the radiolabeled lysates were passed through two lectin affinity columns. 0.5% TRITON X-100 (Sigma) lysates from (³⁵S)methionine labeled oligodendrocyte shakeoff cultures were passed through concanavalin A-AGAROSE (Sigma) and wheat germ agglutinin-AGAROSE (Sigma) columns. The columns were washed extensively with 0.2% TRITON X-100 (Sigma) Tris buffered saline (TBS), pH 7.4, until background counts were obtained. Glycoproteins were eluted from the concanavalin A column with 0.2M D-mannose in 0.2% TRITON X-100 TBS and from the wheat germ column with 0.2% N- acetyl D-glucosamine in 0.2% Triton X-100 TBS. The adherent glycoprotein fractions were then immunoprecipitated and analyzed by gel electrophoresis. No proteins were immunoprecipitated. MOSP was not retained on either column, indicating that carbohydrates containing high mannose (concanavalin A) or polysialic acid (wheat germ agglutinin) are not covalently attached to MOSP.

F. BIOLOGICAL PROPERTIES OF MOSP In tissue culture, murine oligodendrocytes elaborate extensive membrane sheets that have uniform surface distributions of MOSP, and other selected myelin markers such as galactocerebroside and myelin/oligodendrocyte glycoprotein (MOG) . These membrane sheets contain an internal network of microtubular veins that surround cytoplasmic domains of myelin basic protein (MBP) . The normal distribution of surface antigens on cultured oligodendrocytes can be visualized by fixation and then immunocytochemical staining.

When antibodies specific for selected myelin markers bind to live oligodendrocytes, the antibody:antigen complexes reorganize to form characteristic patterns. For example, when antibodies reactive with either galactocerebroside or sulfatide bind to oligodendrocytes, the result is the redistribution of the antibody:glycolipid complexes into surface patches directly over the cytoplasmic domains of MBP. Data show that the binding of a monoclonal antibody to MOG on the surface of cultured oligodendrocytes also results in redistribution of the antibody:protein complexes over cytoplasmic domains of MBP. The surface expression of proteolipid protein (PLP) on cultured oligodendrocytes is diffuse and faint, unlike MOSP which is abundantly expressed on the membrane surface.

In contrast to the redistribution pattern of the above myelin markers, MOSP redistributes in a lacy surface pattern that directly overlies internal microtubular veins. Furthermore, this redistribution pattern is dependent upon the integrity of the microtubular network. Following pretreatment of oligodendrocytes with colchicine, a drug which depolymerizes microtubules, the redistribution of MOSP to produce the lacy surface pattern does not occur; the membrane sheets remain solidly stained for MOSP. These observations indicate that MOSP is capable of forming stable transmembrane associations which are dependent upon the presence of polymerized microtubules.

MOSP has physical and biological properties that are distinct from MOG and PLP, the only other known integral membrane proteins described to date that are specific to CNS myelin and oligodendrocytes. PLP is also present in the cytoplasm of Schwann cells, but is not inserted into the plasma membrane. The molecular weight of MOSP (48 kDa) is considerably higher than MOG (26 and 28 kDa) or PLP (27 kDa) . These data show that MOSP is a novel myelin/oligodendrocyte specific protein.

Cerebrospinal fluid (CSF) or other biological fluids such as urine or serum, from patients with neurological disorders such as multiple sclerosis can be screened for the presence of MOSP by, for example, an immunoblotting test using CEl and second antibody to detect MOSP. Similar screening has been performed using antisera raised against MBP in a radioimmunoassay to detect the presence of MBP in CSF of patients with acute demyelinating events (Cohen et al.. Annals of Neurology 25:107 (1989)). Similar methods can be applied to MOSP.

Alternatively, circulating CEl can be detected by contacting a sample of biological fluid with MOSP and detecting the presence of complexes between MOSP and CEl. This test can indicate an autoimmune response to MOSP before MOSP can be detected in the biological fluid. CSF and plasma from patients with serious neurological disorders has also been screened for the presence of autoantibodies to an endogenous mannose-binding protein, the cerebellar soluble lectin (CSL) , by means of an immunoblotting test with rat CSL as the antigen (Zanetta et al. Lancet 335:1482-4 (1990)). 47 of 51 patients with multiple sclerosis were positive for anti-CSL compared with 30 of 188 patients with other neurological disorders. The specificity of the CSL test for multiple sclerosis is 85% and the sensitivity 93.5% (Zanetta et al. (1990)). These methods can be applied to MOSP.

The effects caused by binding of the monoclonal antibody CEl to MOSP on cultured oligodendrocytes can also mimic the effects elicited by interaction of MOSP with an in vivo endogenous ligand present on other cell types or in myelin itself. The association of MOSP with cytoplasmic microtubules of cultured oligodendrocytes after antibody binding suggests an important role for MOSP in membrane/cytoskeletal interactions during assembly and maintenance of the multiple myelin sheaths elaborated by individual oligodendrocytes and in the interactions of oligodendrocytes with other cells of the CNS during development. Abnormalities in the metabolism of MOSP may be central to the pathogenesis of specific myelinopathies or leukodystrophies. Moreover, since MOSP is expressed on the extracellular surface of normal cultured oligodendrocytes and myelin, it is likely that MOSP serves as an antigenic target in certain idiopathic diseases such as multiple sclerosis that selectively affect CNS myelin.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.

Claims

WHAT WE CLAIM IS;

1. A ligand which reacts with an antigenic determinant of a myelin/oligodendrocyte specific protein of about 48,000 daltons determined by SDS-PAGE with an isoelectric focusing point of about 6.7.

2. A monoclonal IgM antibody capable of binding the antigenic epitope of a myelin/oligodendrocyte specific protein of about 48,000 daltons determined by SDS-PAGE with an isoelectric focusing point of about 6.7.

A protein reactive with the antibody of Claim 4.

4. A substantially pure central nervous system myelin and oligodendrocyte protein of about 48,000 daltons determined by SDS-PAGE with an isoelectric focusing point of about 6.7.

5. A method for detecting demyelination in an animal, comprising the steps of:

(a) exposing a ligand to a biological sample from the animal, wherein the ligand reacts specifically with an antigenic determinant of the myelin/oligodendrocyte specific protein of

Claim 4; and

(b) determining whether the antibody has failed to bind to the protein, the absence of binding indicating demyelination.

6. The method of Claim 5 wherein the biological sample comprises central nervous system white matter.

7. A method of Claim 5 wherein the biological sample is spinal chord tissue.

8. A method for determining when an acute multiple sclerosis event is occurring in a human, comprising the steps of: (a) exposing a ligand which reacts with the myelin/oligodendrocyte specific protein of Claim 4 to a biological fluid sample from the human; and (b) detecting the presence of immunoreactivity of the sample with the ligand, the presence of immunoreactivity indicating the presence of an acute multiple sclerosis event.

9. The method of Claim 8 wherein the detecting step is accomplished by means selected from the group consisting of immunoprecipitation, dot blot hybridization. Western blot hybridization, enzyme linked immunosorbent assay and radioimmunoassay.

10. The method of Claim 8 wherein the biological fluid sample is selected from the group consisting of cerebrospinal fluid, serum and urine.

11. An in vitro diagnostic method for the detection of an acute demyelinating event, comprising the steps of: (a) contacting a ligand specific for the myelin/oligodendrocyte specific protein of Claim 4, with a biological fluid for a period of time and under conditions sufficient for myelin/oligodendrocyte specific protein antigenic epitopes in the biological fluid and the ligand to form complexes; and (b) detecting the formation of the complexes, the presence of the complexes indicating a demyelinating event.

12. The method of Claim 11 wherein the detecting step is accomplished by indirect immunohistochemistry.

13. An in vitro diagnostic method for the detection of an autoimmune response to a myelin/oligodendrocyte specific protein of about 48,000 daltons determined by DSD-PAGE with an isoelectric focusing point of about 6.7 comprising:

(a) contacting a biological fluid sample from the human with the myelin/oligodendrocyte specific protein for a period of time and under conditions sufficient for antibodies in the biological sample which react with the myelin/oligodendrocyte specific protein to form complexes; and (b) detecting the formation of complexes, the presence of complexes indicating the existence of an autoimmune response to the myelin/oligodendrocyte specific protein.

14. A method for detecting oligodendrocytes in live cell cultures, comprising the steps of:

(a) contacting the cultures with an effective amount of a ligand specific for the myelin/oligodendrocyte specific protein of Claim

4 to form a complex; and

(b) detecting the presence of the complexes.

15. The method of Claim 14 wherein the step of detecting the complexes is accomplished by indirect immunohistochemistry.

16. Hybridomas that produce and secrete monoclonal antibodies specific for an antigenic epitope of a myelin/oligodendrocyte specific protein of about 48,000 daltons determined by SDS-PAGE with an isoelectric focusing point of about 6.7.

17. A composition comprising a ligand specific for the myelin/oligodendrocyte specific protein of Claim 4 and a conjugate effective to form a detectable immune complex in a diagnostic assay.

18. The composition of Claim 17 wherein the conjugate comprises a second ligand which reacts with the first ligand.

19. The composition of Claim 17 wherein the conjugate comprises a detectable agent selected from the group consisting of rhodamine, fluorescein, horseradish peroxidase, β-galactosidase, alkaline phosphatase and biotin.

20. A complex comprising a central nervous system myelin and oligodendrocyte protein of about 48,000 daltons determined by SDS-PAGE with an isoelectric focusing point of about 6.7 bound to a ligand.

21. A kit for the detection of demyelinating diseases, comprising:

(a) a ligand specific for myelin/oligodendrocyte specific protein of about 48,000 daltons determined by SDS-PAGE with an isoelectric focusing point of about 6.7;

(b) a conjugate effective to form an additional complex in a diagnostic assay; and

(c) means for detecting the immune complex.

22. The kit of Claim 21 wherein the conjugate is a second ligand conjugated to a detectable agent selected from the group consisting of fluorescein, rhodamine, horseradish peroxidase, β-galactosidase, alkaline phosphatase and biotin.

23. A method for detecting oligodendrocytomas in animals, comprising the step of: immunohistochemically staining animal tissue with a ligand specific for a myelin/oligodendrocyte specific protein of about 48,000 daltons by SDS-PAGE with an isoelectric focusing point of about 6.7. and detecting the staining.

24. A method of affecting the production of central nervous system myelin comprising regulating the amount of the protein of Claim 4 in a central nervous system myelin producing cell.

25. A nucleic acid encoding the protein of Claim 4.

26. A nucleotide probe capable of selectively hybridizing with the nucleic acid of Claim 25.

27. A host vector system comprising the nucleic acid of Claim 25.

28. A method of detecting the nucleic acid of Claim 25 comprising contacting a nucleic acid which encodes the protein of Claim 4 with a nucleotide probe capable of selectively hybridizing with the nucleic acid.

29. A method of detecting the nucleic acid of Claim 25 comprising amplifying the nucleic acid and determining the presence of the amplified nucleic acid.