Strain of hybrid cultivated animal cells mus musculus a 6g2-producer of monoclonal antibodies specific to heat shock protein 70

FIELD: medicine.

SUBSTANCE: invention can be used for production of monoclonal antibodies (MCAs) to heat shock protein 70 (HSP 70). A hybridoma strain is made by immunisation of BALB/c mice with bovine HSP 70 within 78 days. For the third days, splenocytes of immune mice (108 cells) are hybridised with murine myeloma cells P3-X63 Ag/8-653 (107 cells). A fusion agent is polyethylene glycol of molecular weight 4000 (Merk, Germany). The hybridisation is followed with selection, screening, cloning and cryopreservation of hybridoma. Hybridoma 6G2 is deposited in the microorganism collections of "ГНТТ ПМБ" under No. H-2. MCA.

EFFECT: produced hybridoma under the invention is more evident to be detected as HSP 70 on the cell surfaces, and change of endocellular HSP 70 level when exposed to the stress factors.

4 dwg, 1 tbl, 6 ex

 

The invention relates to biotechnology and can be used to produce monoclonal antibodies (MAB) to heat shock protein 70 (HSP 70).

Heat shock proteins play an important role to ensure the homeostasis of the organism at all stages of its development and in extreme conditions, such as exposure to stress, toxic and narcotic substances, radiation, sublethal temperature and others.

Currently sufficiently studied in detail the basic functions of intracellular HSP associated with the participation of these molecules in the processes of synthesis, folding, transport, reparatii intracellular protein (chaperone function [1] and the important role of HSP in protecting cells from the damaging effects (protective functions) [2]. Registered surface localization of HSP in a number of lines of tumor cells, virus - living lymphocytes, activated macrophages and T-lymphocytes [3], and also discovered the existence of free HSP in the intercellular space [4]. Based immunoadjuvant properties HSP intensively conducted research to develop vaccines for immunotherapy of malignant tumors [5, 6].

One of the important approaches for the determination of HSP 70 in biological organisms is the use of MCA produced by hybridomas.

You know, any protein structure bears on its surface the displacement certain number of epitopes, including structural. As a rule, the higher the molecular weight of the protein, the more epitopes. They have different immunogenicity and define the uniqueness of varying protein molecule in the implementation of its functional properties. Therefore, the larger the panel hybrid antibodies each of them only work with one of the epitopes of the protein, the more tools for the study of these properties. Therefore, obtaining new hybrid is always true. Monoclonal antibodies produced by hybridomas, are used as an important tool to identify the main structural and functional properties of proteins, including HSP 70.

Abroad some firms, for example, Sigma, Cayman chemical, Biocompare (USA), StressGen, Canada), HyTest (Finland) sell monoclonal antibodies to HSP 70 [7].

Known hybrid strain of cultured animal cells Mus musculus BRM 22 (firm Sigma, USA), producing µa to HSP 70. Commercial MCA produced by hybridomas BRM 22, do not reveal the indirect immunofluorescence analysis (e.g. cell lymphoma EL-4) different pools of living cells, which limits their use to determine HSP 70 under the influence of stress factors on subpopulations of living cells [8].

The objective of the invention is the obtaining of a hybrid strain of cultured cells that produce monoclonal antibodies specific for HSP 70 and fit on what I determine HSP 70 when exposed to stress factors.

The problem is solved in that a new hybrid strain of cultured animal cells Mus musculus 6G2 producing monoclonal antibodies to HSP 70.

The strain is deposited in the collection of microorganisms of the State scientific center of applied Microbiology and biotechnology (SSC BCH) collection number is H2.

Strain hybridoma obtained by immunization of mice of BALB/c N-terminal fragment of HSP 70 for 78 days. On the third day after the last booster injection spend hybridization of immune splenocytes of mice (108cells) with cells of the mouse myeloma P3-H Ag/8-653 (107cells). As agent for the merge to apply the polyethylene glycol with a molecular weight of 4000 (MEGC, Germany). After hybridization carry out the selection, screening, cloning and cryopreservation of hybridoma.

Description hybridoma 6G2.

Morphological characteristics. Culture of hybrid cells consists of subprinciples to the substrate rounded cells with the size of the original myeloma cell.

Cultural properties. Cultivation of strain hybridoma 6G2 carried out at 37°C in atmosphere containing 5% carbon dioxide. The cultural medium is DMEM or RPMI - 1640 containing 10% fetal calf serum, 2 mm L-glutamine, 50 μg/ml gentamicin. Cells cultivated in the form of stationary suspen the AI in plastic mattresses "Costar". The passage of cells hybridoma carried out 2 times a week with multiplicity dilution 1:2 to 1:3. The seeding cell concentration is 105in 1 ml of medium. The maximum concentration of hybrid cells during culturing is 1051 ml of medium. Hybrid clone does not lose the ability of antibody synthesis in 10 passages in vitro (observation period).

The cultivation of hybridoma in the body of the animal. Species of animal is inbred mouse strain BALB/c. The dose of cells at the inoculation is 106one mouse during the initial introduction and 105one mouse in the secondary introduction of immunoassay fluid. At least 14 days prior to the introduction of cells hybridoma, mice intraperitoneally injected with 0.5 ml of 2, 6, 10, 14-Tetraethylenepentamine (piers). Immunodeciency liquid is formed on a 14-20 day in a volume of 3-5 ml Synthesis of immunoglobulin produced by hybridomas, determined by indirect enzyme-linked immunosorbent assay (ELISA).

The characteristic of a useful product. Hybridoma 6G2 producing specific monoclonal antibodies to HSP 70, the title of which is in the culture liquid (QL) 1:1000, immunoscience fluid (IA) 1:100000. Monoclonal antibodies of QOL and IAG allocate using ammonium sulfate precipitation followed by affinity chromatography on protein a or G sepharose. The homogeneity of the selected antibodies is tested using ele is triarese in polyacrylamide gel in the presence of sodium dodecyl sulfate.

The productivity of the strain. The products of the ICA in the cultivation environment is 20-50 μg/ml, in the ascitic fluid is 2-4 mg/ml Products µa in culture liquid is stored for 10 passages (observation period) and 5 passages under cultivation in the form of ascitic tumors in mice (observation period).

Contamination of the strain. Contaminants hybrid lines, including bacteria, yeast, fungi, is not revealed. Microplasma was not determined.

Cryopreservation. Wednesday freeze contains fetal calf serum - 90%DMSO - 10%. The mode of cryopreservation and thawing. Hybrid cells contribute to cryoprobes, placed in a Styrofoam container with a wall thickness not less than 1 cm and leave for 16-24 hours in kelvinator at -70°C and then immersed in liquid nitrogen.

The defrosting is carried out quickly in a water bath at 37°C. the Vials containing 1.0 ml createsite environment with a concentration of hybrid cells 106. The viability of the restored hybrid after cryopreservation is 65-75%.

Properties useful product. Hybridoma produces monoclonal antibodies specific to lgGl subclass of mouse IgG. The MAB specific for HSP 70 bull and man (with other species of animals and microorganisms have not been tested).

Example 1. Getting hybridoma.

Immunizatory

M is our line of BALB/c mice subjected to immunization drug N-terminal fragment of HSP 70 bull scheme:

- subcutaneous injection of mice with 100 μg of the N-terminal fragment of HSP 70 in a few spots in complete Freund's adjuvant;

- after 28 days repeated administration to mice with 100 μg of the N-terminal fragment of HSP 70 in a few spots in incomplete Freund's adjuvant;

- after 28 days intravenous injection to mice 10 μg of the N-terminal fragment of HSP 70 in saline;

within 14 days of intravenous injection to mice 10 μg of the N-terminal fragment of HSP 70 in saline;

after 7 days of intravenous injection to mice 10 μg of the N-terminal fragment of HSP 70 in saline;

after 7 days of intravenous injection to mice 10 μg of the N-terminal fragment of HSP 70 in saline.

Blood from immune mice selected on the third day after the injection of the N-terminal fragment of HSP 70 according to the Animal protocol No. P 02-19, p.7. The titers of specific antibodies in the sera of animals is determined using an indirect solid-phase ELISA.

Hybridization

Through 3 days after the last intravenous injection remove the spleen of the mouse and hold the hybridization of 108the splenocytes of the mouse with 107cell myeloma line RZ-H Ag/8-653 in the presence of 1 ml of 50% solution of polyethylene glycol with a molecular weight of 4000 (Merk, Germany) for 1 minute.

Breeding

After washing the poly (ethylene glycol) cells were seeded on 96-well plates at layer peritoneal what's macrophages, taken from mice of BALB/c. Selection of hybrid cells is performed on selective medium containing gipoksantin-aminopterin-thymidine (HAT). After 21 days from the environment clean, aminopterin. In subsequent cultivation is carried out at the DMEM Sigma with 15% fetal calf serum, 2 mm L-glutamine, 50 μg/ml gentamicin.

Screening of hybrid clones

For positive selection of hybrid clones producing µa, use indirect solid-phase ELISA.

For carrying out the indirect variant of ELISA in wells of 96-well polystyrene tablet for enzyme immunoassay make 100 ng of the N-terminal fragment of HSP 70 and HSP 70, prepared in 0.01 M carbonate buffer pH 9.6 volume of 100 μl, and then aged at a temperature of 6°C for 16 hours in the Wells washed buffered saline containing 0.05% Tween-20 (SVR-TV). Contribute to the wells, 100 μl of 0.5% solution of bovine serum albumin (BSA), tested in the absence of peroxidase activity and incubated at 37°C for 45 minutes Three times washed SFR-TV and contribute to the wells of the culture supernatant fluid volume of 100 μl. Panel incubated at 37°C for 1 hour. After that, the wells washed three times with a solution SFR-TV and add to the wells of the antibody against mouse IgG labeled with horseradish peroxidase in R is an operational breeding. Tablet incubated at 37°C for 40 minutes, then washed 5 times the wells with a solution SFR-TV. A positive reaction is judged by the appearance of a yellow-brown coloration of the product of the Chromogen - orthophenylene (4 μl of 30% H2O2and 4 mg of orthophenylphenol 100 ml of 0.1 M sodium citrate buffer pH 5.0). The reaction is stopped by adding to the wells, 50 μl of 8N sulfuric acid. Tablets scanned on a tablet reader Picon (Russia), measuring the absorbance at 492 nm.

Cloning

Cloning of hybrid cells is carried out by the method of limiting dilutions in 96-well tablets on the layer of peritoneal macrophages at the rate of 104cells per well. Screening of clones conduct indirect solid phase ELISA as described above.

Cultivation

The cultivation of clones of hybridoma carried out at 37°C in atmosphere containing 5% carbon dioxide. The cultural medium is the medium RPMI-1640 containing 15% fetal calf serum, 2 mm L-glutamine, 50 μg/ml gentamicin.

Cryopreservation

Clones of hybridoma are cryopreserved in freezing medium of consisting of fetal calf serum - 90%DMSO - 10%. Clones of hybridoma stored in the vessels of the Dewar with liquid nitrogen.

Check the specificity of the MCA.

Example 2. Indirect enzyme-linked immunosorbent assay.

For PR is doing a solid-phase ELISA in wells of 96-well polystyrene tablet for enzyme immunoassay make a N-terminal fragment of HSP 70 and HSP 70 in dilutions from 100 ng to 1 ng, prepared in 0.01 M carbonate buffer (pH 9,6) in a volume of 100 μl and incubated at 6°C for 16 hours. The next stage of the analysis carried out similarly to the stages described for screening of hybrid clones.

Monoclonal antibodies contribute to the wells at a concentration of 100 ng/ml.

Negative control serum intact mouse in a dilution of 1/1000.

In indirect solid-phase ELISA monoclonal antibodies of hybridoma 6G2 specifically interact with HSP 70 and define up to 3 ng of protein in the analyzed sample (figure 1).

Example 3. The immunoblot.

To verify the specificity of the MCA of hybridoma 6G2 to HSP 70 spend SDS-electrophoresis in 10% polyacrylamide gel (PAG) for 2 hours at 220 V, 20 mA. Proteins from SDS page transferred to nitrocellulose membrane (Hybond-P) for 1 hour at a current of 380 mA. After transfer the membrane block, immersing in 1% solution of BSA in 20 mm Tris buffer, pH 7.4 for 1 hour at room temperature. For washing of membranes and cultivation of proteins used 20 mm Tris buffer, pH 7.4, containing 150 mm NaCl and 0.5% tween-20. Blocked and washed membrane is cut into strips and incubated for every band in solutions µa of hybridoma 6G2 and MCA BRM 22 concentrations of 1 μg/ml for 1 hour at 37°C. after washing with buffer, the strips are treated with a solution artemisinin antibodies conjugated with p is roximity horseradish (Sigma) at a working dilution under the same conditions, washed with buffer and stained with diaminobenzidine using 0.5 mg/ml 3.3'-diaminobenzidine with 0.1 mg/ml NiCl2, 0.1 mg/ml ol2, 0.03% N2O2in SFR pH of 7.2. The reaction is stopped by washing with distilled water.

Negative control serum intact mouse in a dilution of 1/1000.

Monoclonal antibodies produced by hybridomas 6G2, interact with HSP 70 in immunoblot and identify the same protein bands that commercial µa BRM 22 (figure 2).

Example 4. The dot-blot.

The dot-blot performed on nitrocellulose membrane (Hybond-P). Boarding dose for HSP 70 and its fragments is 50 ng to a point with a diameter of 2 mm Nitrocellulose membrane, blocked in 1% solution of BSA in SFR for 1 hour at room temperature, immersed in a solution UA of hybridoma 6G2 at a concentration of 1 μg/ml in SFR-TV, incubated for 1 hour at 37°C and washed SFR-TV. Then the membrane is placed in a solution of peroxidase conjugate (Sigma) in SFR-TV in working dilution for 1 hour at 37°C and washed SFR-TV. As a substrate for horseradish peroxidase using the solution of diaminobenzidine (0.5 mg/ml 3.3'-diaminobenzidine with 0.1 mg/ml NiCl2, 0.1 mg/ml l2, 0.03% N2About2in SFR pH of 7.2. The reaction is stopped by washing with distilled water.

Negative control membranes treated with serum in the akt mouse in a dilution of 1/1000. Positive control membrane treated with immune serum of the mouse in a dilution of 1/1000.

Monoclonal antibodies produced by hybridomas 6G2, specifically interact in the dot-band solid HSP 70 and its N-terminal fragment (aal-437) and does not interact with its C-terminal fragment (AA-641) (figure 3).

Example 5. Cytometrical testing UA.

Measuring the level of expression of surface HSP 70 is produced by flow cytometry after indirect immunofluorescence staining of cells using the tested monoclonal antibodies to HSP 70 (E, BRM 22 - "first" antibody titer of 1:500) and "second" antibody - conjugated antibodies against Fab fragments of mouse IgG with fluoresceinisothiocyanate (FITZ) (Sigma, USA) at a working dilution. For this purpose cells after centrifugation at 200g for 10 minutes, transferred to SFR containing 0.1% NaN3and 0.5% BSA, and incubated for 30 min at 4°c with the first antibody. The cells are then washed twice SFR using centrifugation at 200g for 10 min and incubated for 30 min at 4°C with the second antibody. After incubation, the cells are again washed twice in SFR by centrifugation in the same mode. Painted samples will be analyzed by a flow cytometer (EPICS XL, Coulter Corporation, USA). Each sample is carried out measuring the level of fluorescence is not me is it 10000 cells.

Figure 4 presents the results of a comparative cytometrical testing the obtained monoclonal antibody hybridoma 6G2 and commercial antibodies BRM 22 in the model of their interaction with HSP 70 present on the surface of the cell lymphoma EL-4. As controls use the typical distribution of cells, EL-4 on the options front (FS) and side (SS) light scattering, revealing pools of living and apoptotic cells (figure 4, A); control of autofluorescence (figure 4, B) and the control dyeing second, FITZ-labeled antibodies (figure 4, B).

Commercial µa BRM 22 are associated mainly with a pool of apoptotic cells and only a slight vzaimodeistvovat with the surface of living cells (figure 4, D). Monoclonal antibodies 6G2 paint pools apoptotic and living cells. However, in living cells, they discover three subpopulations, characterized by different levels of surface expression of HSP70 (figure 4, D).

Example 6. The definition of HSP 70 when exposed to heat shock.

A suspension of living cells lymphoma EL-4 cells were then incubated for 10 min at a temperature of 43°c subsequent indirect immunofluorescent staining of intracellular HSP 70 using ICA BRM 22 and MCA of hybridoma 6G2. As a control using cells, EL-4 without exposure to heat shock.

Indirect immunofluorescent staining of cells, EL-4 to evaluate the wew is kletochnogo of contents HSP 70 carried out as described in example 5 Protocol. However, before staining are prefixal cells and perforation their plasma membranes to ensure penetration of the antibodies into the intracellular space. For fixation, cells add 2% solution of paraformaldehyde in SFR and incubated for 30 min at room temperature and washed twice SFR. For punching plasma membranes of cells treated with 0.5% solution of Triton-X100 for 30 min at room temperature. After that, the cells washed twice SFR, transferred to a solution SFR and conduct the procedure for indirect immunofluorescent staining. The samples will be analyzed by a flow cytometer (EPICS XL, Coulter Corporation, USA). Each sample is carried out measuring the level of fluorescence of not less than 10,000 cells.

The table presents the results obtained when using different antibodies for flow-cytometrical analysis of changes in the content of HSP 70 in the cells, EL-4, subjected to heat shock.

Table
The results of flow-cytometrical analysis of changes in the content of HSP 70 in the cells, EL-4, subjected to heat shock, using ICA
ΜaThe relative level of fluorescence of the cells, EL-4
ControlHeat shock% change
BRM 2248,573,2+50,9%
6G251,692,8+79,8%

The table shows that the MCA hybridoma 6G2 give a higher level of fluorescence compared to ICA BRM 22 as in the control cells and in cells subjected to heat shock. In addition, registered tested µa effects of hyperthermia vary by size. Heat shock leads not only to increased synthesis of HSP 70, but also to significant changes in its condition and conformation. In particular, this effect can cause the formation of solid complexes of HSP 70 with other intracellular proteins and their aggregation, which is reflected in detektiruya µa level of HSP 70. Monoclonal antibodies of hybridoma 6G2 reveal a higher percentage increase in the synthesis of HSP 70 in the cell than MCA BRM 22, which allows to obtain a more complete picture of the quantitative changes in the synthesis of this protein in response to exposure to heat shock.

The advantage of hybridoma 6G2 compared with hybridomas BRM 22 is that its ICA more clearly identify how exp is essay HSP 70 on the surface of cells, and the change in the level of intracellular HSP 70 when exposed to heat shock (stress factor). In addition, the use of ICA opens the perspective of analysis of subpopulations of living cells with different levels of expression of surface HSP 70.

Monoclonal antibodies of hybridoma 6G2 specific for HSP 70.

These properties hybridoma 6G2, as well as the interaction of its ICA with a number of HSP 70 and with its N-terminal fragment suggests the possibility of using MCA as the component test systems for determination of HSP 70.

References

1. Craig E.A., J.S. Weissman, Horwich A.L. Heat shock proteins and molecular chaperones: mediators of protein conformation and turnover in the cell // Cell - 1994. - Vol.78. - P.365-372.

2. Parsell D.A., Lindquist S. The function of heat-shock proteins in stress tolerance: degradation and reactivation of damaged proteins // Annu. Rev. Genet. - 1993. - Vol.27. - P.437-496.

3. Multhoff G. and Hightower LE Cell surface expression of heat shock proteins and the immune response. // Cell Stress &Chaperones - 1996. - Vol.1. - P.167-176.

4. Hightower LE, Guidon P.T. Selective release from cultured mammalian cells of heat-shock (stress) proteins that resemble glia-axon transfer proteins. //J. Cell. Physiol. - 1989. - Vol.138, - P.257-266.

5. Udono, H. and Srivatstava P.K. Heat shok protein 70-associated peptides elicit specific cancer immunity // J. Exp.Med. - 1993. - Vol.178. - P.1391-1396.

6. Li Y., Yang G., Repasky, E., Wang, X.-Y. Generation of anti-tumor immunity using mammalian heat shok protein 70 DNA vaccines for cancer immunotherapy // Vaccine - 2006. - Vol.24. - P.5360-5370.

7. /www.caymanchem.com, www.biocompare.com, www.stressgen.ru, www.abcam.com. /

8. Sapozhnikov A.M., Ponomarev E.D., Tarasenko T.N. and Telford G.W. Spontaneous apoptosis and expression of cell surface heat shock proteins in cultured EL-4 lymphoma cells // Cellproliferation - 1999. - Vol.32. - P.363-378.

The hybrid strain of cultured animal cells Mus musculus 6G2 producing monoclonal antibodies specific to heat shock protein 70, deposited in the collection of microorganisms of the State scientific center of applied Microbiology and biotechnology (GNT MBP), collection number N-2.



 

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1 tbl, 4 ex

FIELD: medicine.

SUBSTANCE: strain A-4A7 is prepared by fusion of mouse myeloma cells of line SP2/0.Agl4 with mouse lymphocytes of line Balb/c immunised by introduction in pads of a purified preparation AMGF (alpha2-microglobulin of fertility) separated from amniotic fluid, and deposited in the transplantable mammal cell culture collection of the Research Institute of Human morphology of the Russian Academy of Medical Science numbered 131/2002. Strain A-4A7 synthesises monoclonal antibodies (MCA) of IgGI class specifically reacting in solid-phase immune-enzyme analysis (IEA) with AMGF isoforms of endometrial, follicular and sperm nature. Activity of the strain: cultural supernatant contains MCA 3-5 mkg/ml, while ascetic fluid contains MCA 2-5 mg/ml. The antibody titre in cultural fluid is 1:500-1:1000, in ascetic fluid up to 1:1×107. A-4A7 bonds various AMGF protein glycoforms produced in male and female reproductive organs. Application of MCA A-4A7 as an immunodiagnosis test systems allows for high-specific and high-sensitive (1 ng/ml) quantitative analysis of various AMGF/glykodeline isoforms in biological liquids.

EFFECT: new compounds are characterised with valuable biological properties.

2 ex

FIELD: biology.

SUBSTANCE: present invention relates to biotechnology. An A-5D1 strain is obtained from fusing mouse myeloma cell line SP2/0.Ag14 with mouse lymphocyte line Balb/c, immunised by introduction into pouches of a purified preparation of alpha 2-fertility microglobulin, extracted from amniotic fluid, and deposited in the collection of passaged culture of mammal cells of the state research institute of human morphology (Russian Academy of Medical Sciences) under the number 144/2002. The strain of hybredome A-5D1 synthesises the IgG1 class monoclonal antibody, which specifically interacts in enzyme-linked immunosorbent assay and immunohistochemical assay with isoforms of alpha 2-fertility microglobulin of endometrial or special origin. Monoclonal antibodies do not have cross reaction with placental α 1-microglobulin, trophoblastic β1-globulin, human chorionic gonadotropin, α-fetoprotein, bovine serum albumin, and C-reactive protein. Monoclonal antibodies detect alpha 2-fertility microglobulin/glycodelin in cells and tissue of female and male reproductive organs.

EFFECT: use of monoclonal antibody A-5D1 as an immunodiagnostic system allows for quantitative analysis of isoforms of alpha 2-fertility microglobulin/glycodelin in body fluids with high sensitivity (1 ng/ml) and specificity.

3 ex

FIELD: biochemistry.

SUBSTANCE: invention refers to antibodies that link with CTGF. Antibodies, in particular, are directed to the areas of CTFG participating in different types of biological activity related to fibrosis. The invention also refers to the method of antibodies application in pharmaceutical compositions for the treatment of CTGF-related diseases including localised and systemic fibrotic diseases such as diseases of lungs, liver, heart, skin and kidneys, for neutralisation of biological activity of CTGF and method of treatment and prophylaxis of CTGF-related diseases. The invention covers polynucleotide sequence and its variants coding the specified antibody as well as the host cell and cell line № PTA-6006 (ATCC) producing the specified antibody.

EFFECT: use of the invention provides new specific means - antibodies which effectively neutralise certain types of CTGF activity in pathology and provide specificity and pharmacokinetic profile suitable for a therapeutic agent.

82 cl, 33 dwg, 4 tbl, 12 ex

FIELD: biotechnology, immunology.

SUBSTANCE: invention describes a monoclonal anti-IFNα antibody that binds with the following subtypes of IFNα: IFNα1, IFNα2, IFNα4, IFNα5, IFNα8, IFNα10 and IFNα21 and comprises three CDR-sites of heavy chain. Amino acid is given in the invention description. Invention discloses heavy chain of anti-IFNα antibody or its fragment that comprise indicated CDR-sites also. Invention describes anti-IFNα antibody that comprises at least one light chain and one heavy chain. Invention discloses variants of nucleic acids encoding indicated antibodies and variants of vectors used for expression of nucleic acids, and variants of transformed host-cells. Among expression vectors invention describes also vectors deposited at № 2881 and № 2882 carrying heavy and light chain of antibody, respectively. Invention describes a method for preparing antibody from indicated cells. Invention discloses the murine hybridoma cell line deposited in ATCC at number № РТА-2917, and antibody produced by indicated cell line. Also, invention describes variants of the antibody-base pharmaceutical composition and a method used for diagnosis of autoimmune disease. Also, invention discloses using antibodies in treatment of disease or state associated with enhanced level of IFNα in a patient. Using the invention provides inhibiting biological activity of at least seven human IFNα subtypes simultaneously, namely: IFNα1, IFNα2, IFNα4, IFNα5, IFNα8, IFNα10 and IFNα12 that can be used in diagnosis and therapy of different human diseases mediated by IFNα, such as insulin-dependent diabetes mellitus or erythematosus lupus.

EFFECT: valuable biological and medicinal properties of antibodies.

53 cl, 4 tbl, 10 dwg, 2 ex

FIELD: biotechnology, immunology.

SUBSTANCE: invention proposes hybridomas producing monoclonal antibodies showing specificity to human epiregulin. Invention discloses monoclonal antibody recognizing specifically human epiregulin with the sensitivity limit 10 pg/ml. Also, invention describes methods for specific detection of human epiregulin in a sample in vitro and a method for detection of cells expressing human epiregulin in extracellular fluid in vitro by using monoclonal antibody to human epiregulin. Invention provides a simple and highly sensitive method for detection of human epiregulin that can be used in diagnosis of human epiregulin-expressing tumors.

EFFECT: valuable biological and medicinal properties of antibody and hybridoma.

8 cl, 7 dwg, 8 ex

FIELD: microbiological and immunological methods.

SUBSTANCE: invention is intended for use to study structural and functional arrangement of nucleolus and mechanisms of action of pharmacological preparations on human cells. Mouse hybridoma cell strain, called A3, is obtained through fusing mouse splenocytes immunized by coarse fraction of nuclei of human cell line RAMOS with cells of mouse myeloma line P30X63-Ag8.653. Resulting hybridoma secretes monoclonal antibodies against antigen, called A3 antigen, localized inhuman cell nucleoli irrespective of their tissue or line origin. In cases of different cell fixation ways. A3 antigen is revealed as incorporated in discrete (typically several tens) foci located exclusively in the zone of nucleoli. Unique property of A3 antigen is its high sensitivity to the action of various protein synthesis inhibitors, e.g. emetine, anisomicyne, cycloheximide, and puromicyne. During incubation of cells in presence of above-listed substances, A3 antigen migrates from nucleoli to numerous foci located in nuclear nucleoplasma. This A3 antigen migration precedes apoptotic death of cells. Monoclonal antibodies A3 produced by the strain A3 are recommended to reveal nucleoli and to estimate total level of protein synthesis in human cells by cell biology techniques. Antibodies can be used to reveal possible contamination of human cell cultures with another-species cells as well as to investigate biological activity of known and novel protein synthesis inhibitors, including pharmacological preparations in human in vitro cells.

EFFECT: expanded microbiological and immunological possibilities.

2 cl, 6 dwg

FIELD: biotechnology, hybridoma technology.

SUBSTANCE: hybridoma T2/S-6E11 is prepared by fusion of murine myeloma strain Sp-2/0 and murine BALB/c splenocytes immunized with the TOPC virus, strain CoD, purified preparation inactivated with concentrate formaldehyde solution and by using polyethylene glycol-1000 Da and the following cloning by method of limited dilution. Prepared hybridoma T2/S-6E11 produces monoclonal antibodies (MCAb) to epitopes of the abovementioned pathogen. Specificity of prepared MCAb is shown by absence of cross-reactions in IFA-test with Hantaan and Ebol viruses and with noninfected substrates accumulated by TOPC virus. IFA-test system based on MCAb provides carrying out the specific detection of TOPC virus in analyzed samples. The sensitivity of this test-system based on MCAb is estimated to be 2.5 x 103 plaque-forming units (virus) in cubic centimeter (PFU/cm3). Using the invention in immunological researches in creature of diagnosticum used for detection of TOPC virus in samples provides enhancing the specificity of analysis in detection of coronaviruses.

EFFECT: valuable properties of strain.

3 tbl, 1 ex

FIELD: pharmacology.

SUBSTANCE: present invention refers to immunology and biotechnology. There is offered recovered human antibody to RG1 polypeptide. There are described versions of antibodies, including one-chain antibody, and immunoconjugate based on said antibodies. There are disclosed methods of selective cell destruction, cell inhibition, treatment of disease state, detection of disease state, detection of RG1, monitoring of clinical course of prostate cancer, prediction in a person with using antibodies and immunoconjugate.

EFFECT: application of the invention provides new antibodies to RG1 polypeptide that can find application in treating tumours with RG1 overexpression.

16 cl, 4 dwg, 1 tbl, 13 ex

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