Hybrid cell line (options), drug antibodies (options), a method for diagnosing multiple sclerosis in humans (options), a method of treating human polypeptide

 

(57) Abstract:

The invention relates to medicine, more specifically to the Neuroimmunology, namely to test for antigens associated with multiple sclerosis. Also the subject of the present invention is the generation of hybridomas that produce monoclonal antibodies that are specific against antigens associated with multiple sclerosis, as well as to the polypeptide. Possible applications of the present invention lies in the diagnosis of multiple sclerosis and in the current dispensary observation of patients with multiple sclerosis from the point of view of the development of the disease or response to therapy. The technical result of the invention is the possibility of diagnosis and treatment of multiple sclerosis and other demyelinating diseases. 12 C. and 10 C.p. f-crystals, 5 Il., 3 table.

The present invention relates to the detection of diseases associated with demyelinization, such as multiple sclerosis. More specifically, an object of this invention is the test for detection of antigen(s) associated with multiple sclerosis and related diseases. The object of the present invention is the generation of hybridomas that produce monoclonal to altoviti in the diagnosis of multiple sclerosis and in the current dispensary observation of patients with multiple sclerosis from the point of view of the development of the disease or response to therapy.

Multiple sclerosis is a slowly progressive disease of the Central nervous system (CNS), the hallmark of which from the point of view of pathology are scattered plaques of demyelination in the spinal cord and brain, and clinically - multiple objective and subjective symptoms with periods of remissions and exacerbations. Demyelination is the disappearance of myelin, the protective lipid material that surrounds the axon of the nerve fibers. Diseases related to multiple sclerosis, include a condition in which there is demyelination of the nerves.

The beginning of multiple sclerosis usually occurs without symptoms. Usually minor visible abnormalities, superficial ocular palsy, a temporary weakness, some stiffness or unusual fatigue extremities, minor violations of the gait, difficulty with bladder control, occurring occasionally dizziness or light emotional disorder - the symptoms of multiple CNS lesions - see for months or years before the disease is diagnosed.

Among the various symptoms of multiple sclerosis distinguish the following: parasthesia, ohvatyvaya, such as partial blindness in one eye, diplopia, dullness of sight or visual scotomas; urination disorders; depression. (Paresthesia is an abnormal sensation, such as burning, tingling and formicaria).

Spontaneous remission complicate the assessment of treatment effectiveness. Despite the fact that the average life expectancy after the onset of the disease ranges from 10 to 20 years, many patients are living longer. Some patients have frequent attacks, and they quickly lose their ability to work, while others are remission, lasting up to 25 years.

Diagnosis is difficult because of the imposition of the above symptoms of multiple sclerosis and similar symptoms of other disorders of the Central nervous system. Diagnosis of multiple sclerosis is most often carried out taking into account the history of remissions and exacerbations of symptoms for several years in combination with the systematic exclusion of other possible diseases that have similar symptoms. In order to eliminate these diseases, which have similar symptoms: intracranial injury of vascular lesions of the brain, acoustic neuromas, cerebellar tumors, gliomas, brain stem, op is the same disk, plastibase and hereditary ataxia conduct extensive and expensive examination, often physically uncomfortable for the patient.

Significant efforts were made to develop diagnostic methods and substances that can be used for early diagnosis of multiple sclerosis. The positive results of the analyses of the cerebral spinal fluid using colloidal gold is considered confirmatory, but not definitively establish a positive diagnosis. The same applies to testing for elevated levels of gamma-globulin in the cerebral spinal fluid test confirms the diagnosis at later stages, but it does not give results in the diagnosis in the early stages. Similarly, an active demyelinization associated with the disease often manifests itself in the increase of the analytical results in the identification of a core protein when testing the cerebrospinal fluid, but test levels decrease very rapidly immediately after the exacerbation. Identified indirect correlation between the existence of painful conditions and elevated levels of measles antibodies in serum and cerebral spinal fluids. And finally, some researchers suggested menini can serve as a serious basis for the diagnosis of multiple sclerosis.

Known [1] possible immunological marker for multiple sclerosis. This marker was not purified or characterized. In addition, nowhere is stated that all or most of the animals reacted by production of the appropriate isotype and the number of antibodies, and has not been shown that all or most animals of different species should respond in a typical way. Meet some of these requirements are necessary for commercial use, for example when using antisera in the diagnostic test.

The development of diagnostic methods and materials for fast, easy and accurate detection of multiple sclerosis remains a significant need. Such methods must be highly specific in relation to multiple sclerosis and related diseases, in order to avoid obtaining false positive results due to other diseases of the Central nervous system, other immune disorders or taking drugs. Further, such methods should provide the ability to diagnose multiple sclerosis in its early stages, should not include a painful or dangerous seizures of drugs to tissues of the patient and should predmore.

The present invention relates to a method for detecting multiple sclerosis patients. The present invention relates also to either polyclonal or monoclonal antibodies specific against antigens associated with the presence of multiple sclerosis. Such antibodies used in immunological assays to detect antigens associated with multiple sclerosis, making thus possible diagnosis of multiple sclerosis.

The present invention relates to the characterization of cell lines producing monoclonal antibody, designated HB 11152 in the American type culture collection ("ATCC"), which shows specificity in relation to the epitope on the antigen associated with multiple sclerosis, patients suffering from multiple sclerosis. The present invention also relates to the characterization of cell lines producing monoclonal antibody, designated HB 11153 in the ATCC, which shows specificity in relation to the epitope on the antigen associated with multiple sclerosis, patients suffering from multiple sclerosis.

The invention also includes any drug antibodies comprising a diagnostically effective is social antibody produced by hybridoma cell line ATCC N HB 11152 or HB 11153, antigens associated with multiple sclerosis, as determined by enzyme immunoassay or other immunological analysis on competitive inhibition. It includes any antibody or its binding fragment that inhibits the binding of HB 11152 or HB 11153 antibodies with their respective epitopes on antigens associated with multiple sclerosis. Epitopes are the structural component of the antigen molecule responsible for its specific interaction with antibody molecules.

The present invention also provides a method and composition for alleviating symptoms of painful condition associated with demyelination that occurs in multiple sclerosis. The present invention involves the introduction of a person or an animal with multiple sclerosis effective dose of antigens associated with multiple sclerosis or their associated nucleotide sequences. Effective dose is a level that does not cause side effects such as anaphylactic reactions.

Prior to the present invention were unknown to specific antigenic the Tiki multiple sclerosis.

Accordingly, the objective of the invention is the development of a sensitive immunoassay for the detection of multiple sclerosis in humans.

Another objective of this invention to provide an immunological assay, which is highly specific in the detection of multiple sclerosis and does not give false positive results in patients with other diseases of the Central nervous system.

Another objective of this invention is the development of a sensitive immunoassay for the early diagnosis of multiple sclerosis in humans.

Another objective of the present invention is the development of a sensitive immunoassay for the current follow-up of patients with multiple sclerosis to assess disease progression and response to therapy.

Also another objective of the present invention is to develop a cost-effective way of detecting multiple sclerosis: as an alternative to the existing method, which consists of a series of tests designed to elimination of other similar, related nervous disorders.

Another objective of this infusion is practical analysis.

Another objective of the present invention is to develop a simple defining test for multiple sclerosis.

Another objective of the present invention is to provide as rapid as possible, the early treatment of patients with multiple sclerosis, because only one test is included in the diagnosis of multiple sclerosis.

Another objective of the present invention is to develop a method and composition for alleviating symptoms of painful condition associated with demyelination diseases such as multiple sclerosis.

Another objective of the present invention to provide a purified protein that is associated with the presence of multiple sclerosis or other related demyelinating disease in man or animal.

These and other objectives, features and advantages of this invention will become apparent after reviewing the following detailed description of the proposed implementation options and the accompanying claims.

Fig. 1 illustrates the results of immunological analysis of antisera obtained from several rabbits immunized with LPK extract multiple sclerosis and fathers-in-law who presented the results of blotting the Western forestry extracts from LPK from patients with multiple sclerosis and normal patients.

In Fig. 3 presents the results of analyses of sera from mice immunized with antigens associated with multiple sclerosis.

Fig. 4 is a graph showing the results of enzyme-linked immunosorbent assay (ELISA) of sera from normal patients and patients with polyclonal antibodies against antigens associated with multiple sclerosis, diagnosed with multiple sclerosis.

Fig. 5 is a graph showing the results of enzyme-linked immunosorbent assay (ELISA) of sera from normal patients and patients with 6D4.2 monoclonal antibody against an antigen associated with multiple sclerosis, diagnosed with multiple sclerosis.

The present invention provides a method of detecting multiple sclerosis patients. The present invention also provides polyclonal or monoclonal antibodies specific antigens associated with the presence of multiple sclerosis. Such antibodies can be used in immunological assays to detect antigens associated with multiple sclerosis, providing thus the possibility Diagnostika ciruelo cell line, marked HB 11152 in the ATCC, which has specificity against epitopes on the antigens associated with multiple sclerosis, which is observed in patients suffering from multiple sclerosis. The present invention also provides a characterization of monoclonal antibodies produced by the cell line, designated HB 11153 in the ATCC, which has specificity against epitopes on the antigens associated with multiple sclerosis in patients suffering from multiple sclerosis.

The present invention also includes any drug antibodies containing diagnostically effective amount of the antibody or its binding fragment, which competitively inhibits the binding of monoclonal antibodies produced by hybridoma cell line ATCC N HB 11152 or HB 11153, antigens associated with multiple sclerosis, as determined by enzyme immunoassay or any other immunological analysis on competitive inhibition. This includes any antibody or its binding fragment that inhibits the binding of HB 11152 or HB 11153 antibodies with their respective epitopes on the antigens associated with the multiple scle deistvie with antibody molecules.

According to the present invention are antibodies, which are produced by the cell line HB 11152 and/or HB 11153, or antibodies that inhibit competitive binding of antibodies produced HB 11152 or HB 11153 cell lines can be applied in any analysis for the detection of antigens using antibodies. These include immunological assays binding enzymes, radioimmunological assays, fluorescent immunological assays, bioluminescent and chemiluminescent immunological assays, competitive immunological assays, dot-lettenhove methods and analyses using blotting for Western.

The present invention also includes a purified protein or proteins or their associated RNA-type or DNA-type nucleotide sequence, which has been shown to be associated with the presence of multiple sclerosis and related diseases associated with demyelinization. Analysis of DNA-type sequences are well known in the art and includes a blot on the Southern and hybridization on Northern [2]. The antigen(s) associated with multiple sclerosis, is a protein that can be isolated from white blood cells. Protein is molecule is. It was shown that the protein is present in patients suffering from multiple sclerosis.

The present invention also includes a method of obtaining monoclonal antibodies produced by a cloned cell line(s) hybridoma In against antigen(s) associated with multiple sclerosis. Getting hybridoma and production of monoclonal antibodies can be made in a variety of ways well known in the prior art. Apply standard methods of immunization, test, merge, cultivation and cloning [3], which is included here in a footnote. The purpose of this work is to have specific antibodies that can be purified and used successfully in a variety of methods, including immunological tests.

According to the present invention, these antibodies are produced by hybrid cell lines, obtained by merging and cloning producing antibodies cells, such as, for example, immune cells of the spleen, and partner to merge, such as, for example, cell line SP2/o Immune spleen lymphocytes obtained from a suspension of individual cells, prepared from spleens of BALB/c mice, which is known that they produce antibodies to ant is the API (ELISA) in serum samples from immunized mice. These antibodies may react against the epitope(s) on the surface of antigen(s) and get them from a cloned cell line(s).

Antibodies, which are produced as described herein are specific against the antigen(s) associated with multiple sclerosis, and characterize them according to the isotype: HB 11152 produces 6D4, which is lgG subclass 3; and HB 11153 produces 3D3, which is lgM antibody. When hybridoma grow continuously, they are a constant source of specific antibodies. On Deposit with ATCC patent store in Rockville, MD., included two hybridoma designated as HB 11152 and HB 11153.

In addition to the use of purified monoclonal antibodies for sensitization as exciting reagent, there are other areas where the analysis could be strengthened in terms of specificity, sensitivity and analysis, including, but not limited to, application: 1) right conjugatesand with the enzyme (including horseradish peroxidase, alkaline phosphatase, glucose oxidase, urease and others ) antibodies or anti-bilinovich antibodies, 2) Biotin-conjugatesand monoclonal antibodies, 3) bio - or chemiluminescent labeled antibodies, 4) fluoresc is against antigens associated with multiple sclerosis, can be applied in a number of different diagnostic tests. Such assays include enzyme-linked immunosorbent assay (ELISA), blotting the Western, radioimmunoassay analysis, bioluminescent analysis and chemiluminescent analysis, but are not limited to. Such immunological assays well known in the art; their description can be found easily [4].

The methodology and results of a sandwich enzyme-linked immunosorbent assay (sandwich ELISA) using antibodies according to the invention described in examples 11 and 12.

One example of analysis using antibodies specific against antigens of multiple sclerosis according to the present invention is a method of enzyme-linked immunosorbent assay (ELISA). According to the present invention the person taking the blood sample. Preferably, the peripheral blood lymphocytes ("LPK") was isolated from the blood sample, and then carried out the extraction of mitogens associated with multiple sclerosis, from the General pool of LPA, if they are present. The present invention encompasses the use of nucleotide sequences or their fragments associated with bellerose diseases with the use of such methods, as hybridization on Northern and hybridization on Southern or nucleotide sequences.

Then LPK extract is injected into a specially designed container which contains antibodies specific against the antigen of multiple sclerosis that are already sewn to the container. "Exciting" antibodies reactive against antigens associated with multiple sclerosis. Antigens associated with multiple sclerosis present in the TIC of the extract, then contact with the antibodies attached to the container.

Then add to the mixture a second series of antibodies, primary antibodies that are specific against antigens associated with multiple sclerosis. These primary antibodies will contact only those antigens associated with multiple sclerosis, who are connected with breathtaking antibodies are attached to the container. Primary antibodies differ also in that they have been modified by adding a label such as an enzyme. Then the containers are washed to remove all unbound antibodies. The label therefore will be present only to the extent that present antigens associated with multiple sclerosis.

Regardless of the use of primary conjugatophyceae antibody or a two-stage, primary plus secondary, conjugatophyceae antibody conjugatophyceae antibody can be observed compound belonging to the following group: a radioactive isotope, enzyme, fluorogenic material or a fluorescent marker. Determining the presence of antigens associated with multiple sclerosis, can be performed in the following relevant methods: radiometrically, enzymatic, fluorometrically and through luminescence.

For diagnostic purposes can be skompanovany antibodies and other necessary reagents and appropriate devices in the form of a kit for performing a convenient, accessible analyses.

Nadobny diseases. The present invention provides for the introduction of human or animal suffering associated with demyelination disease such as multiple sclerosis, effective doses of antigens associated with multiple sclerosis, or their fractions or their associated nucleotide sequences or their fractions. Effective dose is a level that does not cause anaphylactic reactions.

In the present invention under the factors associated with multiple sclerosis, understand one or more proteins associated with multiple sclerosis, fractions or derivatives of proteins associated with multiple sclerosis or RNA-type or DNA-type sequence or their fractions that are associated with the protein component associated with multiple sclerosis. Practically, the present invention provides for the introduction of less than approximately 10-2mg single dose a person or an animal with multiple sclerosis. The preferred dose factors associated with multiple sclerosis, or their associated nucleotide sequences or their fractions or their active derivatives, lies between approximately 10-2mg to 10-10mg. More pre mg and 10-8mg. the Most preferred dose factors associated with multiple sclerosis, is approximately 10-7mg it is Preferable that the total periodic daily dosage does not exceed about 10-2mg per patient, and even more preferably it does not exceed values of approximately 5 to 10-3up to 10-4mg.

The present invention provides for the introduction of a number not exceeding approximately 10-2mg, although in certain cases the total number of factors associated with multiple sclerosis entered in a single day, may exceed the preferred limit. Factors associated with multiple sclerosis, may be introduced in liquid form, or they may be introduced in solid form in the case when the factors associated with multiple sclerosis, introduced or mixed with Foundation, subject to biological degradation or recycling. This framework can release factors gradually. Such bases are well known in the art and are not fundamental to the present invention. Factors associated with multiple sclerosis, can be introduced preferably by subcutaneous injection of whether the military or intradermal.

In one implementation, the filler is used an aqueous solution, which is contained within an inert container. In another embodiment, the composition is in the form of a suppository. The liquid form of the composition is preferably administered subcutaneously, although valid, other types of injections as part of the present invention. In addition, the composition may be introduced through the membrane mucous membranes such as nasal membrane. The liquid carrier includes 0.1% phenol in saline solution (0.9% NaCl), but is not limited to them. For the introduction of factors associated with multiple sclerosis, can be applied to other pharmaceutically acceptable carriers.

Factors associated with multiple sclerosis, can be introduced by standard methods, which include intramuscular and subcutaneous routes of administration, but are not limited to. Factors associated with multiple sclerosis, can also be entered sublingual and intranasal routes. Since the effective number of factors associated with multiple sclerosis, the dose is very small, the composition according to the present invention may also be injected intradermally, anal or oral. A single dose can be either liquid or solid. Ka is ü introduced a greater number of doses.

Further, the present invention is illustrated by the following examples, which should not be construed in any way as limiting its capabilities. On the contrary, it should be absolutely clear that can be accessed in various other implementations, modifications and equivalents, which after reading the descriptions given here may arise from the experts, without deviation from the spirit of the present invention and/or boundaries of the attached claims.

Example 1.

Collection, separation and homogenization antigenic LPA is produced as follows.

Blood samples from patients diagnosed with multiple sclerosis, or other neurological condition, were provided by Dr. Gary Duncan of Neurological Association, Nashville, Tennessee. In addition, also provided samples of blood group members contribute to the fight against multiple sclerosis, Greenville, South Carolina.

Antigenic peripheral blood lymphocytes ("LPK") receive from people suffering from multiple sclerosis or other disorders, as follows: blood is collected by venipuncture from the veins of the hands, unless shown separately. From each patient take pribi tube is of 8.5 ml of blood and each contains 1.5 ml citrate dextrose (Acid Citrate Dextrose Solution), which acts as an anticoagulant. Filled hermetically sealed tubes, stored at room temperature (15-30oC), quickly transported from various places in laboratory Cell Med, Inc., Columbia, MD.

Upon receipt of the samples resuspended by tilting the tubes several times immediately before centrifugation. The tubes centrifuged for 10 minutes at 2000 rpm in a centrifuge IC. Using a disposable plastic Pasteur pipettes collect leukocyte film located on the boundary plasma/cells, and bring the total volume up to 6.5 ml with phosphate buffered saline ("SFR"). SFR contains 9.0 g/l NaCl, 0.8 g/l Na2HPO4and 0.15 g/l KH2PO4pH brought to 7.4. Equal amounts of material leikocitarnuu film layer on the surface 3 ml industrial environment division (Histopaque H-1077, Sigma Chemical Company, St. Louis, MO) in two centrifuge tubes with a capacity of 15 ml of the material is Then centrifuged for 40 minutes at 2000 rpm forestry can also be separated from the raw blood, layered on the surface of ficoll.

After stage centrifugation Pasteur pipette to collect the TIC-enriched boundary of the section and bring it up to 15 m is more back and forth across the eyedropper capacity of 5 ml, at least 5 times and transferred to a Dounce glass homogenizer. Cells are homogenized by 20 passages of the pestle up and down. In the alternative case, the cells are destroyed and then homogenized by passing through a needle 27-caliber 20 times. These extracted materials are stored and then frozen at -70oC.

When, for rapid processing of collected too many samples, the TIC can be collected in SFR stage after Histopaque and instead of resuspendable in SFR for homogenizing the cells can be resuspendable in 1 ml Cryomedia (RMPI 1640, 10% fetal bovine serum and 7.5% DMSO) and frozen at -70oC. the cells are Then thawed at 37oC, bred 13 ml SFR, harvested by centrifugation for 10 minutes at 2000 rpm, resuspended in SFR and homogenized as described above.

Example 2.

Preparation of LPA extracts for immunization of rabbits and mice do the following

LPK extracts obtained from patients suffering from multiple sclerosis, unite in order to have enough material for immunization of several animals from an exponential number of patients with multiple sclerosis. When immunogenic pool of trained, all material mix several times by inversion, Sobir the holding of protein, determined by the method of Pierce ICA (Pierce Rockford, IL.) ranges of 0.8-1.1 mg/ml for several different representative groups. This material is then stored frozen at approximately -70oC until use

For the purposes of the immunization antigen(s) described above, thawed and mixed with an equal amount of Freund adjuvant (Sigma Chemical Company, St. Louis, MO). Adjuvant is a full primary vaccination or incomplete for subsequent immunizations. The mixture emuleret by mixing in a tightly closed vessel containing glass beads (4-6 mm diameter). Emulsified composition is left for deposition alone for 10 minutes in order to ensure that the emulsion is stable and not divided before being transferred to the syringe and the introduction of animals.

Example 3.

Immunization of rabbits and mice to obtain antisera against factors associated with multiple sclerosis.

Each rabbit (new Zealand white, 4-6 pounds each) are vaccinated at least in two places, one on each side. Each vaccination consists of 0.5 ml of the emulsified composition described above. For mice (Balb/C, Harlan Sprague Dawley, Indianapolis, IN.) General immunization dose of 0.1 ml, which jut from the ear vein by venipuncture, leave to collapse, and then the serum is separated from the cellular elements in the same or next day. In mice blood analyze only once or twice by a fence from orbital or tail vein. If you carry out other treatments, such as vaccinations antigen, all analyzed blood samples collected before producing operation immunization.

Example 4.

Analysis of the immune response is produced as follows.

Sera from immunized mice or rabbits then test for specific reactivity against associated with multiple sclerosis antigen(s) using a variety of methods, most often, enzyme-linked immunosorbent assay (ELISA) and leukocyte (WB) analysis.

First, microanalytical tablet (High binding, Nunc, Helsinki, Finland) sensibiliser due to physical absorption immunizing antigen(s) in the optimal dilution was estimated at approximately 1/100 to approximately 1/1000, in bicarbonate buffer (1,602 g/l Na2CO3, of 2.93 g/l NaHCO3and 0.2 g/l NaN3within one to seven days at approximately the 4oC. Before use, the Sensi is SFR). Can be applied to other blocking agents, such as gelatin and gidrolizirovanny skim milk. After blocking for 15 minutes tablets washed SFR with 0.05% tween-20 (as BSA and twin production Sigma Chemical Company). All incubation is conducted at approximately 37oC for 1-2 hours.

Use the volume of the specimen, conjugate and substrate, 100 μl/well, and five volumes of 200 ál, for washing, as the buffer used for washing SFR-twin. Serum samples containing antibody(a), bred approximately 1/100 or stronger in SFR. Conjugates (horseradish peroxidase - antimachine or horseradish peroxidase - anti-rabbit was obtained from Kirkegaard and Perry Laboratories ("KPL"), Gaithersburg, MD.) diluted 1/1000 in 5% BSA in SFR. Color changes due to the transformation of the substrate, O-phenylenediamine ("OPD")(Sigma Chemical Company), yellow product and transformations carried out at the end of the reaction by adding 100 µl per well of 4N sulfuric acid, which gives an orange product. The orange product register at 490 nm on a Bio Tech ELISA analyzer tablets (the reader).

As a result, the sequence of reactions in the methodology is the following: sensitization, blocking, washing and antibody solution, the washing solution conjugate, n is the logical analysis on anticigarette, obtained from several rabbits immunized with LPK extract multiple sclerosis and tested with antigen(s) of multiple sclerosis, or extract from normal cells. Samples were collected on the days indicated on the x-axis. Arrows indicate the points at which the animals were immunized. Fig. 1 shows that the extract multiple sclerosis was present specific antigens that were not detected in the normal extract.

Example 5.

The answer is in murinova system.

In Fig. 3 presents the results of the tests described in example 4, for murinova immune response, but only for one point (time), since multiple charges are inappropriate. As in the case of rabbits it is obvious that in the extract multiple sclerosis was present specific antigens that were not detected in the normal extract. The blotting for Western confirmed this fact. In addition, certain antigens that reacted mice and rabbits showed the same mobility.

Example 6.

The blotting for Western antisera against the immunizing antigen(s).

Extracts LPK from healthy patients and multiple sclerosis patients, quenching the Bio-Rad Laboratories, Richmond, CA. ). After electrophoresis, the samples are subjected to translating according to the recommendations, then block with the same 0,2% BSA as described above.

Electrophoretic separation of different samples is performed using DDS and treatment with mercaptoethanol. This method allows to separate polypeptide chains of the basic subunits of molecular weights. These electrophoretic materials are then transferred from polyacrylamide gels ("PAG") on a substrate, such as nitrocellulose paper, which binds proteins.

Incubation of nitrocellulose paper, subjected to translating with rabbit or moremovie antibodies ("anti-MS) was performed at room temperature for approximately 2 hours, and for all leaching apply the same wash buffer (SFR-twin), as in example 4. Samples containing antibodies, diluted 1/100 in SFR. A conjugate of alkaline phosphatase anti-rabbit or anti-purinovy lgG (KPL), diluted in 5% BSA in SFR. Positive color reaction obtained by conversion of the substrate BCIP/nicrosini tetrazole (KPL) into an insoluble blue compound.

Summarize the sequence of reactions: antibodies, washing, alkaline phosphatase - anti-Crawley the CIP/nicrosini tetrazole, and finally, the water to stop reaction.

Selected serum samples analyzed, compared with the sample normal and multiple sclerosis TIC of the extract according to the recommendations of Bio-Rad. This sample gives the option of two major bands and several minor bands on a solid substrate, which, apparently, is unique to extract multiple sclerosis (Fig. 2). The application of molecular weight standards possible to determine the molecular weight for a minor band of 14000 to 16000 daltons, which is easily distinguishable by reaction with monoclonal antibodies. Other great bands may reflect the shape of the predecessor, as it often is, or modified form, such as deep-glycosylated form, or neuregulirovannaya form. Murinova monoclonal antibodies interact initially with the band with the lowest molecular weight.

Example 7.

Processing and conjugation rabbit lgG against antigens in multiple sclerosis.

Antisera from rabbit 5866, taken on the 42nd day, was treated with the standard by precipitation with 40% ammonium sulfate followed by dialysis reconstituted precipitated protein PRC ml, containing these antibodies, then extensive absorb normal forestry extract stitched to the Af-fi-Gel 10 (Bio-Rad Laboratories). And finally, spend conjugation rabbit lgG against antigens of multiple sclerosis with Biotin, NHS-Biotin, following the manufacturer's recommendations (Pierce Chemical, Rockford, IL.). Conjugate Biotin - rabbit lgG against antigens of multiple sclerosis in this case is determined using streptavidin-horseradish peroxidase enzyme (KPL).

Example 8.

Application murinova immunoglobulin in the enzyme-linked immunosorbent assay (ELISA).

Antigens of multiple sclerosis can be detected through the use of enzyme-linked immunosorbent assay, in which sensitization microanalytical holes used purified monoclonal antibody that is used to capture the antigen(s) derived from forestry extract multiple sclerosis. Immobilized antigen(s) are then detected, and the signal obtained by the second antibody.

This example used a polyclonal immunoglobulin obtained from rabbits. For the initial stage of use murinova antibodies from mouse 2. Murinova immunoglobulins obtained by precipitation with 40% ammonium salt, easibility microanalytical tablets in sensitizing buffer, but when the protein concentration 5 mg/ml Tablets block and washed in the same way as described in example 4.

This analysis is similar to analysis for the detection of antibodies described in example 4, except that the tablets sensibiliser antibodies, blocked, washed, incubated with LPA antigenic extract, washed as before, incubated with conjugate antibodies, washed, incubated with the second conjugatesand view (if necessary) and incubated with enzyme substrate. The enzyme binds only when one or both antibodies when using a double antibody system, revealed the antigen(s). The volume also cut in half, to 50 μl. Incubation continued for approximately 1-2 hours at approximately 37oC. Antibodies may be directly konjugierte with horseradish peroxidase ("HRP) or alkaline peroxidase ("PS").

Finally, the linked enzyme is able to convert the substrate solution in the solution with a new product. This new product absorbs or in the case of fluorescent products emits at a wavelength different from the wavelength of the unreacted substrate.

Fig. 4 represents the analysis of normal patients and patients where b is the circle the higher end of the scale, and known negative around the lower end.

Example 9.

The creation of HB 11152 and HB 11153 hybrid deponirovannye in ATCC.

To generate monoclonal antibodies are subjected to immunization Balb/C mice as described in example 3, two pools multiple sclerosis-LPK antigens: one of the patients from South Carolina, and earlier - from a group of patients from Tennessee.

For a hybrid, described in the present invention, the animals are killed by cervical dislocation and prepare the standard preparation of individual lymphoid cells by destroying the shell of the spleen and release of lymphocytes from other components of the connective tissue by mechanical grinding back and forth between two frosted glass plates. Spleen cells taken twice washed with serum-free medium and conduct merger in polyethylene glycol. Cells incubated in medium with hypoxanthine-aminopterin-thymidine (GAT) at approximately 37oC, in conditions of 100% relative humidity in an atmosphere consisting of 95% air and 5% CO2. For cells see on a planned schedule on the subject of early growth on the 10th day.

Microwell culture, exposed to fusion, washed, and cells feed("FCC"), SPI (mailwoman acid, pyruvic acid, and insulin), Fungizone, penicillin and streptomycin. Starting from the 10th day, watching the holes on the growth potential hybrid, excessive growth of fibroblasts and fungal and bacterial contamination.

Example 10.

Analysis of monoclonal antibodies produced by hybridomas obtained in example 9.

Table 1 presents a summary of the characteristics of the two monoclonal antibodies raised against antigenic forestry extract multiple sclerosis.

Table 1 gives a summary of these cell lines and their products, the respective monoclonal antibodies. Table 2 presents a comparison of the signal distribution of absorption between the two monoclonal antibodies and third monoclonal antibody A.

The 6D4 antibody is an IgG3 monoclonal antibody reactive with blotting for Western.

Table 2 presents an analysis of the distribution of signals generated in a 1:4 dilution of serum (or plasma) from normal patients and patients diagnosed with multiple sclerosis. In the analysis using sensitization affination anti-marinovich and the sample of the patient and sequentially with rabbit antibody against multiple sclerosis, labeled with Biotin, and then streptavidin-HRP.

Example 11.

Getting sensitizing antibodies for use in the enzyme-linked immunosorbent assay (ELISA) is conducted as follows.

Antibody 3D3.2, clone monoclonal antibody 3D3, partially purified from ascitic materials. First, each ml of ascitic fluid diluted with equal volumes of cold distilled water. Then add an equal amount of 100% saturated ammonium sulfate, the sample is thoroughly mixed and left to settle for several hours at approximately 4oC. and Then precipitated immunoglobulins, including 3D3.2 IgM, harvested by centrifugation (10000 x G for 15 minutes). The supernatant was removed and the precipitate immunoglobulins resuspended in a minimum amount of water. Then the resulting material deleteroute against 2X STR for 3 days with three changes of buffer solution. After dialysis, the sample becomes transparent and the resulting material was stored at approximately -20oC.

For the purposes of sensitization sample frozen antibodies thawed and diluted approximately 1:1000 in the same buffer for sensitization, which was used in example 4. Sensitized the th bovine serum albumin and process, as described in example 4.

Example 12.

Obtaining a conjugate of a monoclonal antibody for use in the enzyme-linked immunosorbent assay (ELISA) is conducted as follows.

Another monoclonal antibody (6D4), from clone 6D4,2, conjugates with horseradish peroxidase according to the procedure described in [5]. First antibody purified from ascitic material using a set with a penetrating protein A for the purification of antibodies as follows: 1.5 ml sample of ascites diluted with a volume equal to 1.5 ml binding buffer, centrifuged for 15 minutes at 10000 x G and applied to a column with staph A capacity of 5 ml, washed thoroughly with binding buffer, and then elute the antibody buffer for elution. Because this antibody behaves as crioglobulin, especially when the value of the ionic strength equal to physiological or below, all steps performed at room temperature, except for stage dialysis and centrifugation, which are carried out at approximately +4oC. similarly, if possible, apply 2X SFR, especially in cases when the preferred high concentrations 6D4. Antibody kongugiruut with the enzyme (1:4) after the standard period of activation of HRP. Separation askanywhere and scanalizer against 2X STR and stored at approximately -20oC.

The analysis performed as described in example 8, except that since the conjugate is HRP-6D4, you need only a single stage rather than the two required in the case when using the Biotin-antibody and streptavidin-HRP sequence.

The results are presented in Fig. 5, where the first seven samples used were controls with known diagnosis; the last two groups of samples, 21 CU-CU 28 and 29 CU-CU-37 was an unknown tested samples. The first group of samples obtained under code was marked as CU 21-28; all of these samples gave negative results when tested. The second group of samples, denoted as CU 29-37, all gave positive results. Then the code was cleared and patients, from which he received the blood, have been identified, the first group consisted entirely of patients not suffering from multiple sclerosis, although one patient was the son of a patient who was diagnosed with multiple sclerosis, and the other suffered from epilepsy and perineal nerve palsy. The second group of samples was obtained from patients with multiple sclerosis or disorders, presumably prior to the release of multiple sclerosis.

As can be seen from table 3, the analysis using the method according to the present invention makes it possible to divide the normal patients and patients diagnosed with multiple sclerosis. It should be understood, of course, that all of the above relates only to preferred variants of realization of the present invention and that can be done numerous modifications or changes without going beyond the spirit and scope of the invention that is set forth in the accompanying claims.

Literature

1. U.S. patent N 4,294,818.

2. Current Protocols in Immunology, Vol. 2, edited by John E. Coligan et al., pgs 10.6.1 through 10.6.14 and 10.12.1 through 10.12.8, (1991).

3. Zimmerman et al., "Commercial Applications of Monoclonal Antibodies to Bacteria" (1985), Vol. II, pp. 283-320.

4. Current Protocols in Immunology, Coligan et a1., editor, Green Publishing Associates &Wiley Interscience, 1991, Chapter 2.

5. Nakane et al., "A Buffer Antibody: A New Method of Conjugation", Histochem Cytochem, 22: 1084, (1974).

1. Hybrid cell line for producing monoclonal antibodies exhibiting specificity in relation to the epitope on antigen 11152.

2. Hybrid cell line for producing monoclonal antibodies exhibiting specificity in relation to the epitope on the antigen associated with multiple sclerosis, deposited in the American type culture collection (ATSS) HB 11153.

3. The preparation of antibodies for the diagnosis of multiple sclerosis, characterized in that it contains antibody or binding fragment labeled D, secreted hybrid cell line under item 1.

4. The preparation of antibodies for the diagnosis of multiple sclerosis, characterized in that it contains antibody or binding fragment labeled D, secreted hybrid cell line under item 2.

5. The preparation of antibodies for the diagnosis of multiple sclerosis, characterized in that it contains a diagnostically effective amount of the antibody or its binding fragment, which competitively inhibits the binding of monoclonal antibodies produced by hybridoma cell line was ATSS HB 11152, with the antigen associated with multiple sclerosis, which is measured by enzyme immunoassay or other immune analysis on competitive inhibition.

6. The preparation of antibodies for diagnostici its binding fragment, which competitively inhibits the binding of monoclonal antibodies produced by hybridoma cell line was ATSS HB 11153, with the antigen associated with multiple sclerosis, which is measured by enzyme immunoassay or other immunological analysis on competitive inhibition.

7. A method for diagnosing multiple sclerosis in humans, characterized in that the sample from a person detect antigen associated with multiple sclerosis, using the antibody or binding fragment of the drug under item 3.

8. The method according to p. 7, characterized in that as the sample using the sample of blood.

9. The method according to p. 8, characterized in that as the sample use a serum specimen.

10. A method for diagnosing multiple sclerosis in humans, characterized in that the sample from a person detect antigen associated with multiple sclerosis, using the antibody or binding fragment of the drug under item 4.

11. The method according to p. 10, characterized in that as the sample using the sample of blood.

12. The method according to p. 11, characterized in that as the sample use a serum specimen.

13. Spacegun, associated with multiple sclerosis, using the antibody or binding fragment of the drug under item 5.

14. The method according to p. 13, characterized in that as the sample using the sample of blood.

15. The method according to p. 14, characterized in that as the sample use a serum specimen.

16. A method for diagnosing multiple sclerosis in humans, characterized in that the sample from a person detect antigen associated with multiple sclerosis, using the antibody or binding fragment of the drug under item 6.

17. The method according to p. 16, characterized in that as the sample using the sample of blood.

18. The method according to p. 17, characterized in that as the sample use a serum specimen.

19. Polypeptide associated with multiple sclerosis present in the fraction of cells of a patient suffering from multiple sclerosis or other demyelinizing disease, which has a mol.m. about 14000 - 16000 measured in DDS-polyacrylamide gel.

20. A method of treating multiple sclerosis or another demyelinating disease by injecting the patient preparation of the polypeptide, wherein the administered polypeptide under item 19 in the dose of 10-2

22. The method according to p. 20, wherein the polypeptide is administered sublingual.

Priority points:

18.12.92 - PP.1 - 18;

16.12.93 - PP. 19 - 22.

 

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