Animal mus musculus hybrid cell clone l-producer of monoclonal cholera toxin antibodies

FIELD: medicine.

SUBSTANCE: there is produced a new CT-F5/H3 hybrid cell clone producing monoclonal cholera toxin antibody (MCAB) in the environment of cell culture and abdominal cavity of syngeneic animals. The clone is produced by the fusion of mouse myeloma SP-2/0 cells with popliteal lymph node cells of BALB/c mice immunised with a commercial preparation of cholera toxin (SIGMA) in posterior pads. A fusing agent is polyethylene glycol of molecular weight 4000. Hybridoma selection has been performed on Dulbecco modified Eagle's medium with bovine foetal serum and hypoxantine-aminopterin-thymidine added. Hybridoma synthesises MCAB specifically interacting with cholera toxin and not interacting with thermolabile E.coli toxin. The antibody titre reaches 1:20000 1:40000 in the cultural fluid, 1:4000000 in ascitic.

EFFECT: antibodies can be used for designing immunobiological systems of cholera toxin detection exceeding available analogues in sensitivity.

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The invention relates to the field of Microbiology and biotechnology, specifically to hybridoma technology, and is a clone of the hybrid cells CT-F5/H3 animals Mus Musculus L producing cell cultures and peritoneal cavity of syngeneic animals monoclonal antibodies (hereinafter MCAT) to the V. cholerae toxin (XT). Hybridoma can be used in diagnostic test systems for the specific indication of the cholera toxin in the food industry, environmental protection, medicine.

Vibrio cholerae (V. cholerae) and the toxin produced by them are a cause of acute intestinal diseases of humans, often ending in death. The V. cholerae toxin completely determines the symptoms of cholera and is one of the Central objects of monitoring of bacterial toxins in the environment and food. Currently, the analysis of toxins of great importance in connection with the threat of bioterrorism, as many natural toxins, including cholera toxin, can be used as components of biological weapons. The dose of the toxin that causes the disease, as a rule, extremely small [Scarlatos, A., Welt, C., C. Cooper et al // J. Food. Sci. 2005. V.70(8). P.121-124.], therefore the objective of the laboratory diagnosis of toxins is to develop tests with the highest possible sensitivity is almostly. XT should be tested at a concentration not exceeding 1 ng/ml [Ramamurthy T., Bhattacharya S.K., Uesaka y, Horigome, K., Paul M., Sen D., Pal, S.C., T. Takeda, Takeda y, Nair G.B. // J. Clin. Environ. 1992. V.30(7). P.1783-1786].

Now for fast and sensitive diagnosis of toxins is the most used and reliable systems are based on enzyme-linked immunosorbent assay (ELISA) using antibodies against toxins [The EFSA Journal. 2007, V.130. P.4-352].

The sensitivity and specificity of ELISA is determined largely by the quality of the antibodies. Monoclonal antibodies have advantages over polyclonal, because based on them receive standardized preparations. Antigenic similarity between representatives cholerophobia group of toxins, primarily this refers to the similarity between XT and thermo-labile toxin of E. coli (LT), dictates the feasibility of using MCAT with strict specificity to XT.

To determine toxins MCAT standard used in the competitive and sandwich options ELISA. In the competitive version of MCAT adsorb on a solid substrate, then add a solution of purified toxin conjugated to peroxidase, Biotin or fluorescent label, and the sample containing the toxin. The toxin contained in the sample compete for binding sites on MCAT with highly purified, and conjugated with accurate toxin. Concentration is the Oia test toxin is determined by the calibration curve, obtained in a competitive ELISA with a toxin known concentration. The sensitivity of the determination of XT in the competitive ELISA is 26 ng/ml [Rucker V.C., Havenstrite K.L., A.E. Herr Antibody microarray for native toxin detection // Anal. Biochem. 2005. V.339. P.262-270]. In the test used MCAT ("Biodesign International).

In the sandwich version of the toxin from solution is MCAT, adsorbed on a solid substrate, then (the second layer) with toxin other contact detecting MCAT conjugated with a label (for example, peroxidase, Biotin or fluorescein). The concentration of the test toxin is determined by a calibration curve obtained in the sandwich variant of the ELISA toxin known concentration. The sensitivity of the determination of XT in the sandwich variant of ELISA reaches 1 ng/ml [N. Koike engineering Germany, K. Okada, Y. Yabushita, D.Y. Zhang, K. Yamamoto, T. Miwatani, T. Honda. Rapid and differential detection of two analogous enterotoxins of Vibrio cholerae and enterotoxigenic Escherichia coli by a modified enzyme-linked immunosorbent assay // FEMS Immunol Med Environ. 1997 17(1):21-25].

When using MCAT ("Biodesign International) in sandwich version immobilized on the liposomes by ganglioside GM1 - cell receptor XT and LT the detection sensitivity up to 10 FG/ml [Ann-S. Yoon, DeCory T.R., A.J. Baeumner, Durst R./Anal Chem. Ganglioside-liposome immunoassay for the ultrasensitive detection of cholera toxin. 2003.75. 2256-2261]. The use of ganglioside GM1 limits the specificity of the assay, since GM1 binds both XT and LT.

There is a method to quantify what about the detection of biological toxins-based ELISA using a biological microarray. Biological microchip is an ordered array of individual micro-cells on a solid substrate, in which immobilized MCAT to various biotoxins bacterial and plant origin. The use of microarrays allows you to simultaneously analyze the sample for the presence of several biotoxins with sensitivity, not inferior to the sensitivity of standard immunological tests. In the microchip using a competitive option the sandwich option ELISA. The detection sensitivity XT in biological chip reaches 1.6 ng/ml [F.S. Ligler, C. Rowe Taitt, L.C. Shriver-Lake, K.E. Sapsford, Ya Shubin and J.P. Golden. Array biosensor for detection of toxins. 2003. Anal Bioanal Chem 377(3):469-477]. Such sensitivity can be achieved using chip biosensor. Simultaneously analyze XT and four other toxin, namely the ricin, staphylococcal toxin SeB, botulinum toxin a and toxin of Bacillus globigii. The chip used sandwich option ELISA, in which binding of MCAT to XT and four other toxins (ricin, staphylococcal toxin SeB, botulinum toxin a and toxin of Bacillus globigii) locally immobilized on the glass surface by formation of a complex of avidin-biotinylated antibodies. For detection XT and other toxins using a fluorescein-labeled, MCAT. Fluorescent signals recorded by a confocal microscope. For detection XT use the form of MCAT clone of hybridoma 3D11 (firm "Biodesign International"), which do not have a strict specificity, and are associated with XT and LT.

Known hydrogel microchip, which is an ordered array of three-dimensional hydrogel cells on a solid substrate obtained by the method of photo - or chemically modified polymerization and containing immobilized MCAT to various biotoxins bacterial and plant origin [RF Patent N 2216547, C07K 17/08, publ. 2003]. Hydrogel microchip is designed for screening of a wide range of dangerous toxins which can be used as components of biological weapons. Currently, however, the chip detects a limited number of toxins and, in particular, will not detect XT, which poses a serious threat to the health and lives of people.

Known closest to the claimed hybrid clone of cultured animal cells Mus musculus L SCC (P) N 469D - producer of monoclonal antibodies to cholerea enterotoxin [RF Patent N1835848, MKI C12N 5/00, publ. 1995]producing monoclonal antibodies (N 469) class lgM obtained against chromatographically purified cholera toxin, when hybridization of splenocytes with myeloma cells X63.Ag8.653. MCAT N 469 contact b-subunit of cholera toxin. The main disadvantage of the clone producing MCAT N 469, is that with his participation in the sandwich variant of ELISA you the keys cholera toxin with high enough sensitivity 1 ng/ml In addition, clone N 469 characterized by a rather low productivity of the antibody titer in the culture fluid is 1:250-1:500, and in ascitic - 1:1000000. Clone N 469 produces antibodies of class lgM, as a result is difficult to obtain pure antibody preparations, as to obtain pure drug lgM antibodies unsuitable standard method - chromatography on protein a-sepharose.

The objective of the invention is to obtain clones of hybrid cells producing high-affinity, MCAT against the cholera toxin, non-binding thermo-labile toxin of E. coli and other bacterial toxins, with a limit of detection of toxin in IFA below 1 ng/ml.

The problem is solved by hybrid clone of cultured animal cells Mus musculus L CT-F5/H3 producer of monoclonal antibodies to cholerea toxin.

The specified clone hybrid of cultured animal cells Mus musculus L CT-F5/H3 obtained by fusion of myeloma cells lines SP-2/0 cells and lymph nodes of BALB/c mice immunized by cholera toxin (Sigma), when used as a melting agent, polyethylene glycol with molecular weight of 4000 and subsequent selection of hybrid cells on selective medium: medium Needle in the modification of Dulbecco with 20% serum fetal cow, 4 mm L-glutamine, 50 μm-mercaptoethanol and GAT (0,1 mm gipoksantin, 4×10-7M amino is terina, of 1.6×10-5M thymidine). The method of limiting dilutions selected stable clones producing antibodies in indirect ELISA specific binding XT and not connecting the LT. In the next phase of the clones producing MCAT specific to XT, select the clones that sandwich variant of ELISA define XT concentration below 1 ng/ml Then select the clones that the microchip is not cross-interact with other bacterial toxins, namely with ricin, a lethal factor toxin of Bacillus anthracis, the protective antigen toxin of Bacillus anthracis, diphtheria toxin, staphylococcal toxins SeA, SeB, Sel, SeG. The selected clone CT-F5/H3 produces antibodies, which upon detection XT in the sandwich variant of ELISA to identify XT in a concentration not exceeding 1 ng/ml In particular, the sensitivity of the sandwich variant of ELISA, where the binding of antibodies are used MCAT CT-F5/H3, as well as developing antibodies are used MCAT, recognizing different antigenic determinant XT, is 0.2 ng/ml the sensitivity persists when determining the cholera toxin in water from open water, broth and milk.

MCAT can be used for the quantitative detection of the cholera toxin in a simultaneous and parallel analysis with a number of other biological toxins in the format of a biological microchip. The sensitivity of ODA the division in the format of the microchip reaches of 0.44 ng/ml

Hybridoma CT-F5/H3 characterized by the following properties.

1. Karyological characteristics

The karyotype of the cells of the clone CT-F5/H3 by species mice, the modal number of chromosomes 88.

2. Morphological characteristics

Clone hybridoma CT-F5/H3 represented by large rounded cells, similar in morphology to cells of the initial myeloma line SP-2/0.

3. Standard growing conditions in vitro

The seeding concentration when grown in vitro (1.0 to 2.0)×105CL/cm3. The environment of the cultivation medium Needle in the modification of Dulbecco with 10% serum fetal cows. The cultivation temperature (37°C). The CO2in the atmosphere of cultivation - 5%.

4. Cultural properties of clone

Hybridoma CT-F5/H3 is the suspension-monolaurin, about 60% of the cells are in suspension, being attached to the surface of the culture vessel, the frequency of passage when sowing dose (1.0 to 2.0)×105CL/cm3- 3-4 days. Hybrid clone not lost the ability to synthesize antibodies when passirovannye in vitro for 6 months (time of observation). Productivity clone antibodies in vitro is in terms of titer supernatants in ELISA 1:20000 1:40000.

5. The cultivation of hybridoma in the animal organism

Species of animal is inbred mouse strain BALB/c. Cell dose of 1-2×106one mouse. 10-14 days before the introduction of hibri the ohms mice in the abdominal cavity injected with 0.5 ml of 2,6,10,14 - tetramethylpentane (piers). The tumor grows mixed ascitic is a solid way. Ascites is formed on 7-12 day in the amount of from 3 to 10 ml per mouse. Prevelement 100%. Hybrid clone retains the ability to produce antibodies at a constant level (control antibody titer) when perejivanii animal cells for 6 passages (observation period). The titer of MCAT in ascitic fluids in ELISA is 1:2×106-1:4×106.

6. Contamination clone

Contamination CT-F5/H3 bacteria, yeasts and fungi were not identified.

7. Biotechnology characteristic of a useful product

Antibodies produced by cells of the clone CT-F5/H3, belong to the class of immunoglobulins lgGl according to ELISA with teruyuki the monospecific antisera against certain classes of immunoglobulins mouse. Light chain MCAT CT-F5/H3 belong to "K" type.

By immunoblotting shows that MCAT CT-F5/H3 associated with b-subunit of cholera toxin.

The affinity constant calculated from Beatty [J.D. Beatty, Beatty B.G., and Vlahos G. W.: Measurement of monoclonal antibody affinity by non-competitive enzyme immunoassay. J Immnol Methods 1987; 100:173-179], is for MCAT CT-F5/H3 - 2,6×l09M-1.

8. Method of cryopreservation

Environment cryopreservation contains 90% serum fetal cows and 10% cryoprotectant is dimethyl sulfoxide (DMSO).

The mode of freezing and thawing.

Freezing: OS the iPod cells, obtained after low-speed centrifugation, suspended in the medium for the cryopreservation of calculation of 1-3×106CL in ml of medium. Cell suspension is stirred and poured into 1 ml of cryoprobes (Nunc). Labeled test tubes are placed in a low temperature refrigerator (-70°C) at night, and then in the Dewar vessel with liquid nitrogen for long term storage). Recovery of cells after thawing: rapid thawing in a water bath at 37°C until complete thawing. Then the cell suspension is transferred into a sterile centrifuge tube with 10 ml of culture medium without serum. Cells precipitated by centrifugation (10 min at 150g), then the sediment cells suspended in the medium Needle in the modification of Dulbecco containing 10% serum fetal cow and transfer to a container for culturing. Cell viability after cryopreservation is (80±10)%.

The invention is illustrated graphics.

Figure 1. Sandwich ELISA analysis of cholera toxin in PBS, milk, meat broth and water on the tablet using MCAT CT-F5/H3 as binding antibodies. The analysis of the obtained data the optical absorption (A 492 nm) calculated as the median of the optical absorption of three identical cells of the microplate. The analytical sensitivity of the assay, i.e., the lowest tested concentration CT, define the AK concentration, corresponding to the value of optical absorption, greater than, not less than two standard deviations, the optical absorption repeatedly measured zero point.

The value of the sensitivity is determined experimentally, according to the obtained calibration curves and calculated as follows:

IFo+2*SD,

where IFo- the value of the fluorescence intensity for each dimension zero samples; SD - standard deviation from the mean value IFo.

SD is calculated by the following formula:

where- average fluorescence intensity in the measurement of the zero samples; n is the number of dimensions.

For curve presents the sensitivity is 0.20 ng/ml.

Figure 2. The calibration curves to determine the concentration of CT in sandwich ELISA analysis on the microarray using MCAT CT-F5/H3 as binding antibodies. The calculation of the intensity of the fluorescent signal from each cell of the biochip and the determination of concentrations of toxins is carried out with the help of special software ImagelAssay (MPI, Moscow). The analysis of the obtained data, the fluorescence intensity is calculated as the median signal from four identical gel cell.

The value of the sensitivity define exp is rimentale, on the obtained calibration curves and calculated, as indicated in the legend to figure 1. For curve presents the sensitivity is of 0.44 ng/ml

The invention is illustrated by examples

Example 1. Obtaining a clone CT-F5/H3.

Hybridoma get when you merge a mouse myeloma SP 2/0 cells of lymph nodes mouse BALB/c conventional categories immunized with whole by cholera toxin (Sigma). On the first day of immunization toxin injected into the pads of the hind paws of mice at 2.5 µg/mouse cholera toxin diluted in 0.1 ml of physiological solution, with the addition of 0.1 ml of incomplete adjuvant's adjuvant. At 14 and 28 days injected cholera toxin with incomplete adjuvant's adjuvant at a dose of 5 and 20 μg/mouse, respectively. 6 days after the last immunization remove the popliteal lymph nodes and perform the hybridization of the 5×106lymphocytes of mice with 1×106cell myeloma line SP-2/0 1 ml of polyethylene glycol 4000 for 1 min joint incubation.

After removal of the polyethylene glycol by centrifugation, the cells were seeded on 96-well plates at a layer of feeder cells - macrophages isolated from the peritoneal cavity of a mouse inbred lines. Selection of hybrid cells spend in the environment of a needle in a modification of Dulbecco, with 20% serum fetal cow, 4 mm L-glutamine, 50 μm-mercaptoethanol, and GAT. After 21 days - time total loss of myeloma who's cells, cell hybridoma cultured on medium without aminopterin. Further cultivation of hybridomas is performed on the environment, containing no selective components GAT. 3-fold cloning of hybridomas is performed by the method of limiting dilutions in 96-well tablets on a layer of feeder cells. In the last two klonirovania the percentage of positive clones is 100%.

For screening of antibody-producing clones producing MCAT, specifically linking XT, using indirect ELISA with XT. In the wells of ELISA-tablet absorb cholera toxin at a concentration of 1 μg protein/ml in 0.1 M bicarbonate buffer pH 9.0 in. In each well contribute 100 μl of antigen solution and incubated overnight at 4°C. After sorption of free binding sites blocked with 1% solution of bovine serum albumin at 37°C for 1 h, the Wells washed FSB with 0.1% tween-20, then make 100 μl/well analyzed supernatants selected from the wells, which grow and produce antibodies of hybridoma. Incubated for 1 h at 37°C. Washed, as described above, contribute 100 μl/well peroxidase conjugate rabbit antibodies against mouse IgG, incubated for 40 min at 37°C, washed, and contribute 100 μl/well of peroxidase substrate: solution orthophenylene at a concentration of 1 mg/ml in 50 mm citrate buffer, pH 4.5, containing 0.015% hydrogen peroxide in Dorada. After development of the color reaction is stopped by adding 100 μl/well of 10% sulfuric acid. The colour intensity is recorded spectrophotometrically determining the optical absorption at a wavelength of 492 nm. Selected clones producing MCAT, specifically linking XT.

For screening of antibody-producing clones producing MCAT, not interacting with thermo-labile toxin of E. coli, using an indirect ELISA with thermo-labile toxin of E. coli. The analysis is performed as with the XT, but in the wells of ELISA-tablet absorb thermo-labile toxin of E. coli at a concentration of 1 μg protein/ml in 0.1 M bicarbonate buffer pH 9.0 in. In each well contribute 100 μl of antigen solution and incubated overnight at 4°C. Selected clones producing MCAT, not interacting with thermo-labile toxin of E. coli.

Example 2. The use of MCAT CT-F5/H3 in the sandwich variant of ELISA for cholera toxin.

In the sandwich variant of ELISA using MCAT CT-F5/H3 as binding antibodies, and detecting MCAT directed against epitopes of the molecule cholera toxin, which does not interact MCAT CT-F5/H3. MCAT CT-F5/H3 absorb the first layer at the bottom of the ELISA microplate, 100 μl/well at a concentration of 10 μg/ml of Free binding sites of the plastic block with 1%solution of dry milk for 1 hour at 37°C. Washed FSB with 0.1% tween-20. Then bring analyzed brazzi in the amount of 100 μl/well, containing cholera toxin in various dilutions, incubated for 45 min at 37°C, washed FSB with 0.1% tween-20. Make conjugate detecting MCAT with Biotin in the amount of 100 μl/well at the antibody concentration of 10 μg/ml is Incubated for 45 min at 37°C, washed, make conjugate with streptavidin horseradish peroxidase, incubated (45 min at 37°C) and washed. Then make the peroxidase substrate: solution orthophenylene at a concentration of 1 mg/ml in 50 mm citrate buffer, pH 4.5, containing 0.015% hydrogen peroxide. After 15 min the reaction is stopped by adding 10% sulfuric acid. The colour intensity is recorded spectrophotometrically at a wavelength of 492 nm. Analysis of solutions with known concentrations of cholera toxin shows that the detection sensitivity XT reaches 0.2 ng/ml During incubation the cholera toxin with MCAT CT-F5/H3 not in the FSB, and 10% milk, broth, water from an open reservoir detection sensitivity XT unchanged at - 0.2 ng/ml (figure 1).

Example 3. Quantitative analysis of the cholera toxin using a biological microchip

On hydrogel chips immobilized MCAT CT-F5/H3, MCAT against 8 other toxins (ricin, a lethal factor toxin of Bacillus anthracis, protective antigen toxin of Bacillus anthracis, diphtheria toxin, staphylococcal toxins SeA, SeB, Sel, SeG). The concentration of each antibody in the polymerization mixture is 0.8±0.2 mg/ml, the volume of the gel element of 0.1 nl. Each gel element contains one of the nine above MCAT, in addition, the chip includes a control gel elements containing no antibody.

Add 30 ál of the test sample and 30 μl of a solution exhibiting a biotinylated monoclonal antibody specific for XT (as in example 2) and incubated on the chip for 17 h at 37°C. After washing (20 min, the FSB with 0.1% tween-20) add fluorescently labeled streptavidin, incubated 10 min at 37°C, washed and re-register the fluorescent signals (figure 2).

Hydrogel chips obtained as follows [RF Patent N 2216547, MKI SC 17/08, publ. 2003]. Glass slides for the manufacture of microchips process solutions 1 N. NaOH, H2SO4, washed with water, then immersed in a 1% solution of 3-methacryloxypropyltrimethoxysilane in ethanol, washed in ethanol, water and dried. The polymerization mixture containing the gel-forming monomers on the basis of methacrylamide and N-substituted amino sugars, and subject to immobilization MCAT, applied with the help of a robot QArray (Genetix, UK) in the form of droplets with a volume of 0.1 nl to the surface of the activated substrate. Polymerization of the gel cells are under the lamp ultraviolet light with a maximum emission at 350 nm (GTE Sylvania lamp, F15T8/350B1, UK) on the Sam is anii 8 cm from the lamp, during 50 min at 20°C in a stream of nitrogen. When applying PTS 150 μm get gel elements hemispherical shape with a diameter of 120 μm. Biochips after polymerization washed for 40 min FSB with 0.1% tween-20. To reduce nonspecific interaction of the reagent with the surface of the biochip surface treated with a blocking buffer for 1 h, then washed with distilled water.

The quality of the biochips check in transmitted light using a biochip analyzer (MPI RAS), equipped with special software TestChip and QualityControl (MPI RAS). In the quality assurance rejected biochips, for which the deviation of the values of the radii of gel elements exceed 5% within each biochip and 8% among all biochips of this party.

Example 4. Simultaneous analysis of several biotoxins, including the cholera toxin on a single microchip.

The analysis were carried out as in example 3, but biotinylation MCAT, detecting XT, contribute in a mixture with biotinylating antibodies to other toxins (ricin, a lethal factor toxin of Bacillus anthracis, the protective antigen toxin of Bacillus anthracis, diphtheria toxin, staphylococcal toxins SeA, SeB, Sel, SeG). The limits of detection of biotoxins for parallel analysis are the same as the definition of each toxin alone. For XT minimally detektiruya the dose of 0.44 ng/ml

The proposed analysis can be used to test for the presence of toxin in food, in water and biomaterials.

Clone hybrid of cultured animal cells Mus musculus L ST-F5/H3 producing monoclonal antibodies to cholerea toxin.



 

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EFFECT: produced antibodies exceeds the analogues available in sensitivity.

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EFFECT: use of the invention increases reliability of enzyme immunoassay.

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SUBSTANCE: invention relates to biotechnology and can be used in immunodiagnosis of Marburg haemorrhagic fever. A strain of hybrid animal cells Mus musculus L. 3F9 is formed, which is deposited in the collection of cell cultures of The State Research Center of Virology and Biotechnology VECTOR. The hybridoma strain produces monoclonal antibodies which are specific to the VP35 protein of the Marburg virus (Popp strain) (hereinafter MCA). MCA 3F9 produced by hybrid animal cells Mus musculus L. 3F9 relate to a subclass of immunoglobulins IgGI, having a heavy 55 kDa and a light 25 kDa chain and having a unique feature of detecting the VP35 protein of the Marburg virus (Popp strain) in a "sandwich" immunoenzymometric system format owing to antigen "capture" properties and simultaneously be an indicator, labeled biotin. The antigen epitope for MCA 3F9 produced by the 3F9 hybridoma is localised between 252 and 278 aminoacid residues.

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2 cl, 3 dwg, 1 tbl, 5 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to biotechnology and can be used in immunodiagnosis of human cytomegalovirus. The strain of hybrid animal cells Mus musculus L.5F10 is obtained by merging mouse myeloma cells p3-X63/Ag8.653 (NS/1) with mouse spleen cells BALBc, immunised by an affinity purified recombinant protein pp65. The hybridoma strain is deposited in the collection of cell cultures of The State Research Center of Virology and Biotechnology VECTOR and is used as a producer of monoclonal antibodies for detecting the pp65 protein of human cytomegalovirus.

EFFECT: invention enables to widening of range of strains of hybrid cells Mus musculus L - producers of MCA for detecting the pp65 protein of human cytomegalovirus and production of domestic diagnostic test-systems for detecting cytomegaly.

2 dwg, 1 tbl, 5 ex

FIELD: chemistry.

SUBSTANCE: invention relates to humanised anti-TGF-beta-antibody which is linked to TGF-beta. The humanised antibody has a variable domain VH which contains residues of the hypervariable region (non-human), which are contained in the human domain VH which includes a modified framework region (FR) (amino acid and nucleotide sequences are given in the list of sequences). The humanised antibody can contain residues of the complementarity determining region (CDR) of the variable domain of the light strand VL. The invention also relates to a composition for treating TGF-beta mediated disorders, e.g. malignant tumours, nucleic acid, coding monoclonal antibody, and a method of obtaining the latter using host cells. The invention provides a method of treating and detecting TGF-beta in a sample from the body using the disclosed antibody, as well as to a product which contains the humanised antibody and directions for use for treating TGF-beta mediated disorders.

EFFECT: invention enables control of TGF-beta molecules, which can prevent possible changes in antibodies, enables preparation of high-affinity humanised antibodies which act as TGF-beta antagonists.

57 cl, 45 dwg, 4 tbl, 8 ex

FIELD: medicine.

SUBSTANCE: invention aims at preparation of new strain of hybrid cells Mus. Musculus 6F3 - a producer of monoclonal antibody (MCA) to hemagglutinin protein of high-pathogen avian influenza virus A/duck/Novosibirsk/56/05. Strain 6F3 is prepared by fusing murine myeloma cells Sp2/0 with murine spleen cells BALB/c, immunised with a purified and inactivated preparation of avian influenza virus A/H5N1 (strain A/duck/Novosibirsk/56/05). Hybridoma produced MCA belong to IgA class. Strain 6F3 is deposited in the Collection of cell culture of Ivanovsky State Research Institution of Virology of the Russian Academy of Medical Sciences, No. 8/2/3. Using hybridoma allows producing specific monoclonal antibodies to hemagglutinin protein of avian influenza virus A/H5N1.

EFFECT: possibility to use antibodies to studying the antigenic structure of hemagglutinin for differential diagnostics of avian influenza virus A/H5 serotype.

1 dwg, 6 ex

FIELD: medicine.

SUBSTANCE: there is produced a new CT-E6/E10 hybrid cell clone producing monoclonal cholera toxin antibody in the environment of cell culture and abdominal cavity of syngeneic animals. The clone is produced by the fusion of mouse myeloma SP-2/0 cells with popliteal lymph node cells of BALB/c mice immunised with a commercial preparation of cholera toxin (SIGMA) in posterior pads. A fusing agent is polyethylene glycol of molecular weight 4000. Hybridoma selection has been performed on Dulbecco modified Eagle's medium with bovine foetal serum and hypoxantine-aminopterin-thymidine added. Hybridoma synthesises monoclonal antibodies specifically interacting with cholera toxin and not interacting with thermolabile E.coli toxin. The antibody titre reaches 1:10000 in the cultural fluid, 1:1000000 in ascitic.

EFFECT: produced antibodies exceeds the analogues available in sensitivity.

2 dwg, 4 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: invention discloses a strain of hybrid animal cells Mus musculus L.4 A2, which is deposited in the Collection of cell cultures of the State Research Center of Virology and Biotechnology VECTOR, which is a producer of monoclonal antibodies which are specific to the matrix protein VP40 of the Ebola virus, Zaire subtype (Mainga strain), and a strain of hybrid animal cells Mus musculus L. 1C1 which is deposited in the Collection of cell cultures of the State Research Center of Virology and Biotechnology VECTOR, which is a producer of monoclonal antibodies which are specific to the matrix protein VP40 of the Ebola virus, Zaire subtype (Mainga strain). The invention is also aimed at obtaining monoclonal antibodies 4A2 which are produced by the 4A2 hybridome, (subclass of immunoglobulins IgGl which have a heavy 55 kDa and a light 25 kDa chain) and are used as binding antigens in the "sandwich" format immunoenzymometric system for exposing the matrix protein VP40 of the Ebola virus, Zaire subtype (Mainga strain), and monoclonal antibodies 1C1 produced by the 1C1 hybridome (subclass of immunoglobulins IgGl which have a heavy 55 kDa and a light 25 kDa chain), used as biotin labelled indicators in the "sandwich" format immunoenzymometric system for exposing the matrix protein VP40 of the Ebola virus, Zaire subtype (Mainga strain). The disclosed antibodies are used together in a "sandwich" format immunoenzymometric system for exposing the matrix protein VP40 of the Ebola virus, Zaire subtype (Mainga strain).

EFFECT: invention enables to obtain monoclonal antibodies which are specific and do not compete with each other for antigen epitopes and which, when used together in a "sandwich" format immunoenzymometric system, ensure high reliability of results for exposing the matrix protein VP40 of the Ebola virus.

5 cl, 3 dwg, 1 tbl, 6 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: invention discloses a strain of hybrid animal cells Mus musculus L. 1B2, which is deposited in the Collection of cell cultures of the State Research Center of Virology and Biotechnology VECTOR, which is a producer of monoclonal antibodies which are specific to the nucleoprotein of the Ebola virus, Zaire subtype (Mainga strain) and are used as binding antigens in a "sandwich" format immunoenzymometric system for exposing the neucleoprotein of the Ebola virus, Zaire subtype (Mainga strain), and a strain of hybrid animal cells Rattus Norvegicus 7B11 which is deposited in the Collection of cell cultures of the State Research Center of Virology and Biotechnology VECTOR and which is a producer of monoclonal antibodies which are specific to the nucleoprotein of Ebola virus, Zaire subtype (Mainga strain) and are used as biotin labelled indicators in the "sandwich" format immunoenzymometric system for exposing nucleoprotein of the Ebola virus, Zaire subtype (Mainga strain). The invention describes monoclonal antibodies 1B2 which are produced by the strain of hybrid animal cells Mus musculus L. 1B2, which relate to the subclass of immunoglobulins IgGl which have a heavy 55 kDa and a light 25 kDa chain, and monoclonal antibodies 7B11 which are produced by the strain of hybrid animal cells Rattus Norvegicus 7B 11 related to the subclass of immunoglobulins IgG. The antibodies are used together in the "sandwich" format immunoenzymometric system for exposing nucleoprotein of the Ebola virus, Zaire subtype (Mainga strain).

EFFECT: use of the invention enables to obtain results during "ВЭ" laboratory reseach and when designing a test system for highly reliable exposure of an antigen.

5 cl, 3 dwg, 2 tbl, 7 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: invention discloses a strain of hybrid animal cells Mus musculus L. 7D8, which is deposited in the Collection of cell cultures of the State Research Center of Virology and Biotechnology VECTOR, which is a producer of monoclonal antibodies which are specific to the VP40 matrix protein of the Marburg virus (Popp strain) and are used as binding antigens in a "sandwich" format immunoenzymometric system for exposing the VP40 matrix protein of the Marburg virus (Popp strain) and a strain of hybrid animal cells Mus musculus L. 7H10 which is deposited in the Collection of cell cultures of the State Research Center of Virology and Biotechnology VECTOR and which is a producer of monoclonal antibodies which are specific to the VP40 matrix protein of the Marburg virus (Popp strain) and are used as biotin labelled indicators in the "sandwich" format immunoenzymometric system for exposing the VP40 matrix protein of the Marburg virus (Popp strain). Monoclonal antibodies 7D8 which are produced by the strain of hybrid animal cells Mus musculus L. 7D8 relate to a subclass of immunoglobulins IgGl and have a heavy 55 kDa and a light 25 kDa chain. Monoclonal antibodies 7H10 which are produced by the strain of hybrid animal cells Mus musculus L. 7H10 relate to a subclass of immunoglobulins IgGl and have a heavy 55 kDa and a light 25 kDa chain. Monoclonal antibodies 7D8 and antibodies 7D8 and 7H10 are used together in the "sandwich" format immunoenzymometric system for exposing the VP40 matrix protein of the Marburg virus (Popp strain).

EFFECT: use of the invention increases reliability of enzyme immunoassay.

5 cl, 3 dwg,1 tbl, 6 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to biotechnology and can be used in immunodiagnosis of Marburg haemorrhagic fever. A strain of hybrid animal cells Mus musculus L. 3F9 is formed, which is deposited in the collection of cell cultures of The State Research Center of Virology and Biotechnology VECTOR. The hybridoma strain produces monoclonal antibodies which are specific to the VP35 protein of the Marburg virus (Popp strain) (hereinafter MCA). MCA 3F9 produced by hybrid animal cells Mus musculus L. 3F9 relate to a subclass of immunoglobulins IgGI, having a heavy 55 kDa and a light 25 kDa chain and having a unique feature of detecting the VP35 protein of the Marburg virus (Popp strain) in a "sandwich" immunoenzymometric system format owing to antigen "capture" properties and simultaneously be an indicator, labeled biotin. The antigen epitope for MCA 3F9 produced by the 3F9 hybridoma is localised between 252 and 278 aminoacid residues.

EFFECT: invention enables to obtain MCA with specificity to VP35 protein of the Marburg virus (Popp strain), suitable for immunodiagnosis of Marburg haemorrhagic fever.

2 cl, 3 dwg, 1 tbl, 5 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to biotechnology and can be used in immunodiagnosis of human cytomegalovirus. The strain of hybrid animal cells Mus musculus L.5F10 is obtained by merging mouse myeloma cells p3-X63/Ag8.653 (NS/1) with mouse spleen cells BALBc, immunised by an affinity purified recombinant protein pp65. The hybridoma strain is deposited in the collection of cell cultures of The State Research Center of Virology and Biotechnology VECTOR and is used as a producer of monoclonal antibodies for detecting the pp65 protein of human cytomegalovirus.

EFFECT: invention enables to widening of range of strains of hybrid cells Mus musculus L - producers of MCA for detecting the pp65 protein of human cytomegalovirus and production of domestic diagnostic test-systems for detecting cytomegaly.

2 dwg, 1 tbl, 5 ex

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

FIELD: veterinary.

SUBSTANCE: strain 5A10 of hybridomal line of cells of mouse Mus. museums, producing monoclonal antibodies to immunoglobulin IgG of cattle (C) is permanent line of cells and is suitable for biotechnology in elaboration of preparations. Strain is deposited with Special Collection of re-inoculated somatic cell cultures of agricultural and commerciall sold animals by No 71. Antibody titers in native culture liquid constitute 1:32-1:64, in ascitic liquid 1:640-1:5120 in immuno-enzymatic analysys. Monoclonal antibodiesproduced by strain are specific to immunoglobulin IgG of cattle and do not react with immunoglobulins of sheep. Peroxydase-marked monoclonal antibodies ensure high sensitivity and specificity of IEA for detection of antibodies to C leucosis virus in biological material. Strain 5A10 - producent of monoclonal antibodies to immunoglobulin IgG of cattle can be used in production of immuno-enzymatic test-system for diagnostics of C leucosis.

EFFECT: application of said test-system will allow to increase efficiency of sanitation measures, reduce terms of enhancement of adverse in terms of leucosis cattle-breeding farms.

5 ex

FIELD: veterinary.

SUBSTANCE: obtained is strain 1H8 of hybridomal line of cells of mouse Mus. musculus - producent of monoclonal antibodies to IgG of sheep, suitable for biotechnology in elaboration of diagnostic preparations. Strain is deposited with Special Collection of re-inoculated somatic cell cultures of agricultural and commerciall sold animals by No 73. When determined by method of immuno-enzymatic analysys (IEA) antibodies titers in native culture liquid constituted in IEA 1:32-1:64, in ascitic liquid 1:640-1:5120. Monoclonal antibodies are specific to immunoglobulin IgG of sheep and do not react with immunoglobulin of cattle. When used for fixation on solid phase of glycoproteidal antigen of cattle (C) leucosis virus (in composition of complex glycoproteidal antigen- monoclonal antibodies of sheep to glycoproteidal antigen) in IEA, they ensure strength of fixation and optimal availability of antigen for antibodies in testes samples.

EFFECT: strain 1H8 can be used in production of immuno-enzymatic test-system for diagnostics of C leucosis, which will allow to increase efficiency of sanitation measures, reduce terms of enhancement of adverse in terms of leucosis cattle-breeding farms.

1 tbl, 4 ex

FIELD: veterinary.

SUBSTANCE: obtained is strain 8C12 of inter-species hybrid cells of mouse Mus musculus and sheep Ovis aries - producent of monoclonal antibodies of sheep to glycoproteidal antigen of virus of cattle (C) leucosis. Strain is deposited with Special Collection of re-inoculated somatic cell cultures of agricultural and commerciall sold animals by No 72. Strain is permanent hybrid line of cells and possesses high level of production of monoclonal antibodies of sheep. Antibody titers in native culture liquid constitute 1:32-1:64 in immuno-enzymatic analysys (IEA). Monoclonal antibodies are specific to general antigen determinant of glycoproteids of C leucosis - external gp51 and transmembranous gp30. When used in IEA for detection of antibodies in blood serum and milk of C infected with leucosis virus, antibodies provide strong selective binding with solid-phase carrier and optimal space orientation of glycoproteidal antigen.

EFFECT: strain 8C12 can be used in production of immuno-enzymatic test-system for diagnostics of cattle leucosis, which will allow to increase efficiency of sanitation measures, reduce terms of enhancement of adverse in terms of leucosis cattle-breeding farms and, as a result, reduce incidence of leucosis in cattle.

1 tbl, 4 ex

FIELD: medicine.

SUBSTANCE: there is produced a new CT-E6/E10 hybrid cell clone producing monoclonal cholera toxin antibody in the environment of cell culture and abdominal cavity of syngeneic animals. The clone is produced by the fusion of mouse myeloma SP-2/0 cells with popliteal lymph node cells of BALB/c mice immunised with a commercial preparation of cholera toxin (SIGMA) in posterior pads. A fusing agent is polyethylene glycol of molecular weight 4000. Hybridoma selection has been performed on Dulbecco modified Eagle's medium with bovine foetal serum and hypoxantine-aminopterin-thymidine added. Hybridoma synthesises monoclonal antibodies specifically interacting with cholera toxin and not interacting with thermolabile E.coli toxin. The antibody titre reaches 1:10000 in the cultural fluid, 1:1000000 in ascitic.

EFFECT: produced antibodies exceeds the analogues available in sensitivity.

2 dwg, 4 ex

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