Mus musculus cultivated hybrid cell strain producer of monoclonal antibodies specific to human protein c (versions)

FIELD: medicine.

SUBSTANCE: invention presents cultivated hybrid cell strains of Mus. musculus animals Sp2/0-BC/rhPC-4F10, Sp2/0-BC/rhPC-1C6, Sp2/0-BC/rhPC-3H6, producers of monoclonal antibodies specific to human protein C. The strains are deposited in the Russian National Collection of Industrial Microorganisms of Federal Unitary Enterprise State Research Institute 'Genetics', No. (VKPM H-111), (VKPM H-112), respectively. The antibodies belong to hPROC-specific murine immunoglobulin G possessing cross-responsiveness, are selectively bound with human protein C and form a stable complex.

EFFECT: antibodies under the invention may be used for purposes of pharmaceutical and biomedical analytical studies, particularly for quantitative detection of the human recombinant factor C.

1 dwg, 4 tbl, 4 ex

 

The technical field.

The invention relates to the field of biotechnology and biopharmacology, namely the technology of monoclonal antibodies (ICA) methods cell hybridization, more specifically to methods of obtaining MCA, specific protein From human (hPROC), with the use of hybrid strains of cultured cells of Mus musculus and creation on the basis of these µa immunoaffinity reagents for the quantitative determination of protein in conditioned culture fluid lines producing recombinant protein (rhPROC) and other fluids, including blood plasma.

The prior art.

Protein C is a vitamin K-dependent protein of blood plasma. It is included in the hemostatic system and plays a critical role in the regulation of blood coagulation. The protein is synthesized as an inactive molecule precursor undergoes a multi-stage post-translational processing in the cellular compartments and is secreted as intact (imagenow) form. Activation of protein C occurs in the bloodstream with the participation of thrombomodulin-thrombin complex. Activated protein C together with the cofactor protein S performs anticoagulant function. Activated protein C can prevent intravascular thrombosis and inhibit the growth of existing clots. The mechanism of action of Akti the new protein and the mechanism of activation of zymogen in the active protease described in the paper by T. Esmon Charles ("Protein-C: biochemistry, discrimination, and clinical implications" Blood, 1984, Vol.62(6) - P.1155-8).

Activation of protein C directly is thrombin, the latest the serine proteinase cascade of blood coagulation and indirectly associated with the membrane of endothelial cells glycoprotein of thrombomodulin. Thrombomodulin forms a strong non-covalent complex of structure 1:1 with thrombin and completely changes the functional properties of thrombin. Free thrombin in normal conditions, activates platelets, converts fibrinogen to fibrin and blood coagulation factors V and VIII to the active form Va and VIIIa. At the same time, thrombin in complex with thrombomodulin loses all of the above properties, but effectively activates the intact protein at physiological concentrations of calcium ions.

Activated protein C, in turn, selectively inactivates the activated forms of factors of blood coagulation V and VIII, but not their original form.

It should be noted that the main events at the start of the cascade of blood clotting occur on the surface of cells and require the involvement of membrane-associated proteins and phospholipids, while factors V and VIII freely circulate in the bloodstream and is involved in the positive feedback loop when the coagulation system. When they are activated by thrombin they accelerate the activation of factor X and prothrombin 5 orders of magnitude and dramatically accelerate the generation of thrombin at the site of the start of the clotting cascade.

Thus, the physiological role of activated protein C can be described as limiting the scope of the start of the blood coagulation system.

The main cofactor of activated protein C is a protein S, another vitamin K - dependent protein of blood plasma. Protein S more than 10 times increases caused by activated protein C the degradation of factors Va and VIIIa.

Protein C is an important therapeutic agent (EP 215549, EP 0191606) as a specific anticoagulant that has potentially broader scope than traditional anticoagulants heparin, warfarin (oksikumarin) or derivatives of the latter. Unlike traditional anticoagulants activated or intact protein With no effect on the blood clotting system, until the thrombin and does not cause a fall in the concentration zymogenic proteins of the coagulation system. As a result of anticoagulant therapy with protein C is potentially more secure, because it does not cause permanent risk of bleeding. Exogenous intact protein can also be used for replacement therapy in hereditary protein C deficiency that causes death in infancy when homozygous deficiency and frequent embolic when the heterozygous form of the deficit.

the Main area of clinical application of activated protein C are life-threatening thrombotic state, primarily disseminated intravascular coagulation (DIC), resulting in including in the development of acute sepsis.

Activated protein C is a marker of sepsis and its level and deficits correlate with the severity of the disease. So, protein C deficiency is observed in more than 85% of patients with severe sepsis; and protein C deficiency correlates with adverse clinical outcomes, such as septic shock (severe sepsis, accompanied by the development of arterial hypotension), DIC-syndrome and Polignano failure.

Since the concentration of protein C in plasma is very small, and its separation from other vitamin K-dependent proteins of the hemostatic system is very difficult, the only way to produce activated protein C for medical use is the biosynthesis, activation and purification of the recombinant protein. Creation of analytical systems for the quantitative determination of protein C is an important element as an industrial solution to this problem for biotechnological production and clinical application of the obtained pharmaceutical protein C and concomitant diagnosis of coagulopathy. One of the important methods for the determination of natural or recombinant protein C in biomedical applications t is aetsa using ICA, produced by hybridomas selected in such a way that secreted antibodies do not cross-reactive.

Known application of monoclonal antibodies to factor protein C, described in U.S. patent 5549893 (Johann Eibl et al; "Use of protein in the treatment of purpura fulminans"; 1995), while a patent protects therapeutic use of activated and intact protein From natural origin.

Application conformative-dependent antibodies for purification and activation of protein C is an object of protection in U.S. patent 4981952 (Betty Yan, S.; "Method for the purification of vitamin K-dependent proteins", 1989), however, the technological solution is based on a series of consecutive rounds chromatographic purification of the protein, where the basis immunoaffinity sorbent are MCA, the affinity of which is blocked by calcium ions, which leaves considerable potential for optimization of the technological process. Known application of conformation-dependent antibodies for analysis of biological activity and separation of modified forms of protein C. For such purposes can be used antibodies the chelator-dependent affinity (U.S. patent 5549893; Ohsawa K. et al. Purification of sufficiently gamma-carboxylated recombinant protein With and its derivatives. Calcium-dependent affinity shift in immunoaffinity and ion-exchange chromatography. // J Chromatography - 1992 - Vol.597(l-2) P.285-91) and calcinations affinity (Stearns D.J. et al. The interaction of a Ca2+-dependent monoclonal antiboy with the protein With the activation peptide region. Evidence for obligatory Ca2+binding to both antigen and antibody. // Biol Chem - 1988 - Vol.263(2) P.826-32; Nakamura, S., Sakata Y. Immunoaffinity purification of protein With by using conformation-specific monoclonal antibodies to protein C-calcium ion complex. // Biochim Biophys Acta. Biochim Biophys Acta - 1987 - Vol.925(2) P.85-93; Wakabayashi K. Conformation-specific monoclonal antibodies to the calcium-induced structure of protein C. // J Biol Chem - 1986 - Vol.261(24) P.11097-105; Tharakan J.P. Effect of feed flow rate, antigen concentration and antibody density on immunoaffinity purification of coagulation factor IX. // J Chromatography - 1990 - Vol.522 - P.153-62).

Monoclonal antibodies produced by hybridomas, are also used as a tool to obtain depleted by protein From human plasma used as a reagent for coagulopathies diagnosis. The shortage of appropriate natural plasma due to the extremely rare congenital genetic coagulopathy type PROC is found in 1 case out of a million.

There is a wide range of opportunities to further improve the known solutions for the quantitative determination of protein C human and recombinant variants. The possibility of such improvement is due to the fact that the selection of clones of hybridomas producing different MCA to protein C, it is possible to consider the influence of various factors on the course of determination of protein C and to select clones for sensitivity or resistance to the action of these factors. Examples of such factors may be the presence analiziruemoi other vitamin K-dependent plasma proteins, protein C cattle, different ratio of intact and activated form of protein C, protein C in complex with protein S, the concentration of products inactivation of activated protein C, etc.

Detailed description of the present invention

The technical problem solved by the authors, was to obtain high-affinity not cross-reactive antibodies to protein C human, applicable for pharmaceutical and biomedical analytical research, particularly quantitative detection of recombinant factor C person (rhPROC).

The technical result is achieved by creating new hybrid strains of cultured cells Sp2/0-BC/rhPC-4F10, Sp2/0-BC/rhPC-lC6, Sp2/0-BC/rhPC-3H6, secreting monoclonal antibodies to the protein With man and creation on their basis of immunoaffinity reagents for the quantitative analysis of protein per person (hPROC) and recombinant variants (rhAPC). The obtained strains deposited in Russian national Collection of Industrial Microorganisms (VKPM), FSUE gosniigenetika, September 17, 2010 under the registration room H-110, H-111 and H-112, respectively.

The main methodological approach to the development of strains of cell lines producing monoclonal antibodies is cloning cultivated hybrid cell hybrid. Hybridoma according to the present invention produces the t merge " non-secretory myeloma cells mouse (Mus musculus) and antireproductive b-lymphocytes in the presence of polyethylene glycol. Myeloma cells deficient in gipoksantin-guanine phosphoribosyltransferase (HGPRT) or timedancing (TC). Thus, they cannot survive when cultivated in a medium containing HAT (gipoksantin, aminopterin and thymidine). At the same time, any merged b-lymphocytes of the spleen and of themselves are not stable in culture for more than 3 days. Part of the hybrid cells B-lymphocytes and myeloma who received the corresponding chromosomes of the parent cell is stable in selective medium containing HAT. Such cells may be cultured for a long time and produce a potentially unlimited number of antibodies.

Supernatant culture hybridomas are analyzed for the presence of the target antibody by the method of enzyme-linked immunosorbent assay (ELISA). Then selected hybridoma cloned in order to obtain the subclones producing antibodies that do not have cross-reactivity in analytical tests. This applies the indirect ELISA in such a way that each of the selected antibodies are pairwise compared other as adsorbed on the surface of immunoplate and communicating from the solution with the antigen already contacting other adsorbed antibodies. Selected producers of monoclonal antibodies can be scaled in vivo as ascites mouse or in vitro in suspension cultures is (S.D. Pepper Selection antibodies for immunoaffinity chromatography // Methods in molecular biology ed. by Kenney A. and S. Fowell - 1992 - Vol.11 - P.136-171).

The term "monoclonal antibody" means an antibody artificially derived from cell clone and therefore contains only one type of immunoglobulin.

Antibody specific to the protein of the protein C person"means a molecule that selectively selectively binds to the protein C human rights and forms a stable complex. A stable complex is a complex in which the binding between partners takes place for a period of time sufficient to produce the specified detection of the complex. The term "selectively binds to the protein C human" means the ability of the specified molecule, it is preferable to contact the protein C human in contrast to binding to proteins that are unrelated to protein C person, or associate with non-protein components present in the sample. An antibody that preferentially binds to protein C is an antibody that binds to protein C human, but not associated to a significant extent with other molecules or components that may be present in the sample. Significant binding involves, for example, the binding of antibody binding to protein C man, molecule, non-protein C human,with affinato or power, sufficient to interfere with the ability of antibodies to bind to the protein C human.

The ability of antibodies to bind to the antigen can be determined by the person skilled in the art using methods including, but not limited to ELISA methods and equilibrium dialysis. Methods for determining the affinity and the strength of binding is well-known to the specialist in the art and are described, for example, Janeway and others (Immunobiology: The Immune System in Health and Disease (Garland Publishing Company, 1996)).

The method of immunization. Strains of hybridoma obtained by immunization of mice of BALB/c recombinant protein C human within 49 days. On the third day after the last booster injection spend hybridization of splenocytes immunoserologic mice (1.2×108cells) with cells of the mouse myeloma Sp2/0-Agl4 (3×107cells). As agent for the merge to apply the polyethylene glycol with a molecular weight of 1450 (Sigma, Germany). After hybridization carry out the selection, screening, cloning and cryopreservation of hybridoma.

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

Cultural properties. Cultivation of strains of hybrid Sp2/0-BC/rhPC-4F10, Sp2/0-BC/rhPC-3H6 and Sp2/0-BC/rhPC-lC6 lead separation is about, preventing cross-contamination, at 37°C in atmosphere containing 5% carbon dioxide. The cultural medium is the medium RPMI-1640 containing 10% fetal calf serum, 2 mm L-glutamine, 8 μg/ml tylosin. Cells grown in stationary suspension in a ventilated plastic vials with adhesive surface area of 25 cm2and displacement of environment 5-10 ml. Passage cells hybridoma spend 3 times per week with multiplicity dilution of 1:4-1:6. The seeding cell concentration is 3×105in 1 ml of medium. The maximum concentration of hybrid cells during culturing is 1.2×1061 ml of medium. Hybrid clone does not lose the ability of synthesis of antibodies at 12 serial passages in vitro.

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 2×106cells in 0.5 ml of phosphate-saline buffer solution per mouse during the initial introduction. 1 day before injection of cells hybridoma mice intraperitoneally injected with 1 ml of incomplete adjuvant-blockers.

Immunodeciency liquid is formed on the 12-16 day in a volume of 3-5 ml Quantitative determination of immunoglobulin produced by hybridomas, produced by the method of indirect enzyme-linked immunosorbent assay (ELISA).

Feature useful about the ukta. Hybridoma Sp2/0-BC/rhPC-4F10, Sp2/0-BC/rhPC-3H6 and Sp2/0-BC/rhPC-lC6 produce specific monoclonal antibodies to hPROC, the title of which is in the culture liquid (QL) of about 1:90000, immunoscience fluid (IAG) not less than 1:200000. Monoclonal antibodies of QOL and IAG allocate using fractional ammonium sulfate precipitation followed by affinity chromatography on protein a - or protein G-sepharose. The homogeneity of the selected antibodies are checked by electrophoresis in polyacrylamide gel in the presence of sodium dodecyl sulfate. Belonging to the class of immunoglobulin G is determined by ELISA analysis of the class-specific antivirovym antibodies.

The productivity of the strains. The products of the ICA in the cultivation environment is 20-50 mcg/ml at time of 72 h cultivation and seeding concentration hybridoma 0.3 million cells/ml; in ascitic fluid is 2-4 mg/ml Products µa in culture liquid is retained for 12 consecutive passages and plays in instilling in the form of ascitic tumors in mice.

Contamination of the strain. Contaminants hybrid lines, including bacteria and fungi (determination method of direct seeding on nutrient medium LB and Saburo-Emmons), not detected; Mycoplasma by PCR and fluorescent analysis is not detected; viruses - no data.

Cryopreservation is aliquo the AMI resuspending cells at 3×10 6cells/ml Vials containing 1.0 ml createsite medium containing fetal calf serum - 90%DMSO - 10%.

The mode of cryopreservation and thawing. Hybrid cells contribute to ciampoli, placed in a special container with cooled isopentane and leave in the low-temperature freezer for 3-4 hours. Upon reaching a temperature of minus 70°C ciampoli transferred into liquid nitrogen. The defrosting is carried out quickly in a water bath at a temperature of +37°C. the Viability of the restored hybrid after cryopreservation is 80-90%.

Useful product are hybrid strains and produced by these monoclonal antibodies of class IgG mouse IgG specific to hPROC and low cross-reactivity. The properties of the obtained monoclonal antibodies (ICA) and the results of their practical application of the invention is shown in the following figures.

Brief description of drawings

Figure 1 shows the definition of rhAPC solid-phase ELISA using pairs µa 3H6 + IC6 and 4B12 + 4F10.

The practical applicability of the claimed invention is illustrated by the following examples and not limiting in any way the scope of the present invention.

Example 1. Getting hybridoma.

The immunization.

Mice of BALB/c mice aged 8-10 weeks of 8 pieces subjected to immunization rAPC scheme:

- intraperitoneal administration to mice of 200 µg rhAPC in complete Freund's adjuvant;

- after 21 days of repeated administration to mice of 500 µg rhAPC in incomplete Freund's adjuvant;

- after 35 days intravenous injection to mice 500 µg rhAPC in incomplete Freund's adjuvant;

through 42 days of sampling blood from the orbital sinus, identifying animals with the best titer of antibodies to protein C in the serum by ELISA;

- selected animals after 7 days is intravenous booster injection of 200 µg of rhAPC in saline;

after 7 days of content bystrovany mice repeated sampling of blood from the orbital sinus on ELISA. Is the final botirovna animal with a maximum titer of antibodies to rhAPC: intravenous injection of 200 μg rhAPC in physiological solution.

3 days after re-busterboy a comparison of titers of polyclonal antibodies in serum bystrovany animals. Titles, i.e. dilution of serum at which OD450=l amounted to bostonbingo animal No. 1 1/82500, No. 2 1/19000, No. 3 1/615000. To merge primary cultures of splenocytes with myeloma cells Sp2/0-Agl4 chose an animal No. 3.

The hybridization.

Through 3 days after the last intravenous injection of animals are killed by cervical dislocation, aseptically remove the spleen and spend a hybrid of the tion of immune splenocytes of mice (1.2×10 8cells) with cells of the mouse myeloma Sp2/0-Agl4 (3×107cells) in the presence of 1 ml of 50% solution of polyethylene glycol (Sigma) with a molecular weight of 1450 in for 1 minute. Then within 15 minutes serial double dilutions volume was adjusted to 16 ml by adding dropwise serum-free medium RPMI-1640. Diluted hybridization, the suspension is incubated for 1 hour at 37°C in 150 ml of complete medium RPMI-1640 containing 10% fetal calf serum.

Selection.

After incubation, the cells were seeded on 96-well plates at a layer of cultured peritoneal macrophages taken from mice of BALB/c. Selection of hybrid cells is carried out on the selective environment system HAT (10 mm gipoksantina, 40 μm of aminopterin, 1.6 mm thymidine). After 14 days from the environment clean, aminopterin. In subsequent cultivation is carried out at the environment A-DMEM/F-12 with 10% fetal calf serum, 2 mm L-glutamine, 8 μg/ml tylosin.

Assessing the productivity of primary pools.

For selection of positive primary pools, producing µa, use the direct ELISA. For carrying out ELISA in wells of 96-well polystyrene tablet for ELISA make 100 ng rhAPC in phosphate-saline buffer solution (FBI; pH=7.4; KN2RHO41.7 mm, Na2HPO45.2 mm, NaCl 150 mm) in a volume of 100 ml and then incubated at 6°C for 16 hours in the Wells three times smyvayut phosphate-saline buffer solution, containing 0.05% tween-20 (FBI-TV). Make holes in 300 µl of 1% solution of bovine serum albumin in the FBI (1% BSA) and incubated at 37°C for 60 minutes Three times washed wells FBI-TV and contribute to the wells of the culture supernatant fluid volume of 100 ál, if necessary, making dilution in 1% BSA. Tablet incubated at 37°C for 1 hour. After that, the wells washed three times with a solution of the FBI-TV and add to the wells of the antibody against mouse IgG labeled with horseradish peroxidase in a working dilution of 1/3500. Tablet incubated at 37°C for 60 minutes, then washed 5 times the wells with a solution of the FBI-TV. A positive reaction is judged by the development of yellow-brown staining adding a chromogenic substrate TMB (4 μl of 30% H2O2, 4 mg 3,3',5,5-tetramethylbenzidine in 100 ml of 0.1 M sodium citrate buffer pH 5.0). The reaction is stopped by adding to the wells, 100 μl of a 5% solution of N3RHO4. Measure the optical density at a wavelength of λ=450 nm (OD450). Construct a standard curve based OD450from the concentration of pre-characterized antibodies to hPROC in the linear range of sensitivity of the method. Determine the titer of antibodies and analyzed the affinity of samples.

According to the results of direct ELISA primary pools hybrid maximum signal polyclonal colon and the ELISA was OD 450=3,304, 6 samples had signal OD450>2.0, while the top 5% percentile covered the range of activity OD450=3,304-0,150. For scaling, we selected 48 colonies (i.e. holes) with the signal in ELISA OD450>0,100.

Selected for scaling 48 polyclonal pools within the next 10 days were scaled to cultivation volume of 5 ml. Scaled to cultivation volume of 5 ml colonies were tested for the production of polyclonal antibodies to protein C in two consecutive tests direct ELISA with an interval of 48 hours. The results are presented in Table 1.

For further cloning were selected 4 primary clone with a maximum signal in ELISA (OD450from 0,257 to 2,265).

Example 2. Cloning and selection of monoclonal hybridomas secreting the protein C-specific antibodies.

Cloning of hybrid cells was performed by serial dilution method in 96-well tablets on the layer of peritoneal macrophages at the rate of 104cells on the first hole and the subsequent two-fold dilutions in a vertical row, and then in horizontal rows. Detection of the ICA in the wells was performed by direct ELISA as described above.

The cultivation of clones of hybridoma conducted at 37°C in atmosphere containing 5% carbon dioxide. The cultural medium is the medium RPMI-1640 containing 10% e is brinley calf serum, 2 mm L-glutamine, 8 μg/ml tylosin.

Clones of hybridoma has kryokonservierung in the freezing environment, consisting of 90% fetal calf serum and 10% dimethyl sulfoxide 1-3 million cells/ml of the freezing Procedure described above.

Selected according to the method described in example 1, the cell pools with the highest level of production of antibodies were cloned in limiting dilution was used on five 96-well plates in 1 pool. 8 days was found a total of 23 wells with single colonies. Colonies were suspended by pipetting and transplanted into the wells of a 24-hole tablet in cultivation volume of 1 ml/well.

7 days after reseeding was registered on 7 holes, for which the signal in the direct ELISA exceeded OD450=1 and persisted during the subsequent cultivation of the colonies in medium containing HAT. The results of ELISA of primary clones are shown in table 2.

Selected primary clones were scaled up to a volume of 5 ml, the fraction of the cells are frozen, the remains used to re-cloning, screening conducted on 5 96-well plates for each clone.

8 days after re-cloning was discovered a total of 12 wells with single colonies. In the next 14 days was conducted stepwise scaling up cultivation volume of 10 ml and defined 7 viable clones.

After 24 days after the second clone was conducted by direct ELISA in the presence of calcium ions and parallel ELISA in the presence of EDTA for supernatants clones with time of cultivation for 48 hours. Were recorded 5 clones of the second generation, producing μa to the protein With a person with a high titer and a constant signal in the presence and absence of calcium ions (signal for ELISA with 2 mm CA2+OD450=2.165-1.571, the signal with 2 mm EDTA-Na >88%). The ELISA results are presented in table 3.

The scaled hybrid clones of the second generation were cryopreservative, survival after defrost amounted to not less than 60% on coloring Trifanova blue.

To highlight the ICA with each end of the clone of the second generation was obtained in 30 ml air-conditioned environment.

Example 3. Isolation, purification and biotinylation µa.

For the studied 5 µa held office globulin fraction, affinity chromatographic purification using protein G-sepharose and desalting by dialysis. To do this, the culture fluid (48 h cultivation) was mixed with a saturated solution of ammonium sulfate to 45% saturation. The suspension was mixed and stirred for 2 h at 4°C, and then centrifuged at 14500 m-1within 20 minutes after removing the supernatant, the precipitate was dissolved in the FBI, 1/8 of the initial volume, separating the precipitate of denaturirovannym the proteins by repeated centrifugation. The obtained supernatant is re-precipitated with ammonium sulfate at saturation of 50%, the precipitation-dissolution was repeated twice.

Chromatographic purification was performed using column with protein G-separate (GE Life Sciences, USA) according to the manufacturer's Protocol. Removal of aminecontaining compounds neutralized eluate from the column was concentrated using ultrafiltration centrifuge tubes Vivaspin 500 3 kDa (Sartorius Stedim, Germany) to a volume of 75 μl, concentrates were dialyzed against the FBI on the membrane with cut-off at the sedimentation mass 7000 Yes within 24 h at 4°C. Was prepared 5 specimens treated μa, the concentration of protein in solution was for MCA from Sp2/0-BC/rhPC-4F10 370 µg/ml, Sp2/0-BC/rhPC-lC6 250 μg/ml, Sp2/0-BC/rhPC-2F8 77 µg/ml, Sp2/0-BC/rhPC-3H6 93 µg/ml, Sp2/0-BC/rhPC-4B 12 239 µg/ml, sample size of 75 µl. The degree of concentration relative to the original environment was 400x.

The resulting solution treated MCA were divided into 2 aliquots volume of 20 ál 55 ál. A smaller aliquot was used for modification of sulfo-N-hydroxysuccinimide (Sulfo NHS-LC-biotin, pierce, USA) in a molar ratio of 1:100 in the FBI at pH 7.0. The reaction was conducted for 1 hour at room temperature, and then was terminali the addition of a solution of Tris-HCl pH 7.5 50 mm.

To determine the preservation of the affinity of the MAB in the allocation and conjugation against the Wali samples of the original air-conditioned environments in a dilution of 1/400, the obtained preparations of purified µa in 1/16000 dilution (i.e. presumably in the same concentration) and biotinylated µa breeding 1/160000. Testing was performed by direct ELISA according to the method described in example 1 using conjugate antivitamin antibodies to horseradish peroxidase (antimist - HRP) conjugate neutravidin and horseradish peroxidase (Neu-HRP, Pierce, USA). The results are presented in table 4. According to the data obtained it was concluded that biotinylation µa applicable as a component of an analytical system for hybrid clones 4F10 and C, the adsorption of the antibodies can be used µa hybrid S, 3H6 and 4B12. The full name of the corresponding hybrid clones: Sp2/0-BC/rhPC-4F10 etc.

Thus, a possible set of analytical reagents on the basis of the MCA to the protein C includes biotinylated MCA as detectiong reagent and purified unmodified antibody adsorption of the antigen in indirect typhoid analysis.

Example 4. Determining the sensitivity of a detection system protein C on the basis of pairs of the MCA.

To determine the sensitivity of system prototypes detection of protein C, that is, pairs of the ICA, was performed using an indirect ELISA on a "sandwich". For this purpose, the holes 96-well polystyrene tablet for enzyme immunoassay act and "adsorption" antibodies to the FBI at a concentration of 4 μg/ml, 100 μl per well and kept at 4°C for 16 hours. Then, after blocking nonspecific adsorption of a 1% solution of BSA in the FBI for 1 h, wells were made in 100 μl of a solution of recombinant activated protein C in 1% BSA at a concentration of from 78 ng/ml to 10 μg/ml, twofold dilutions (normal concentration of protein C in the plasma of human blood - 4 µg/ml). The next stage of the analysis was similar to the stages described for screening clones of hybridomas. Biotinylated µa was used in a dilution of 1/1000, Neu-HRP at a dilution of 1/8000. As a negative control instead of biotinylated MCA used the serum intact mouse in a dilution of 1/1000.

It was shown that in this formulation the reaction biotinylated µa clone 4F10 specifically interact with activated protein C, the limit of detection of antigen a lot of below 0.15 ág/ml when used as adsorption of antibodies µa hybrid S and 3H6 (figure 1).

These properties of monoclonal strains of Sp2/0-BC/rhPC-4F10, Sp2/0-BC/rhPC-lC6 and Sp2/0-BC/rhPC-3H6, as well as the interaction produced their µa with protein C human suggests the possibility of using data µa to get immunoaffinity reagents for the quantitative analysis of protein C.

Table 2.
Ranked activity of antibodies to rhAPC for primary hybrid clones.
Pool No.AddressActivity, TH
Selected for re-cloning
22/263,690
32/133,615
44/G73,219
11/D22,918
35/D72,834
310/C82,411
22/H71,707
Rejected
22NS0,583
22/F10,410
2 11/N20,117
424/N80,176
124/D40,155
125/E70,149
323/C90,139
225/e0,129
422/C11to 0.127
224/A30,124
125/H50,115
22/120,114
421/D100,112
321/A70,101
124/H120,100
325/A60,085

Table 3.
The activity of antibodies to rhAPC end-scaled hybrid clones by ELISA
End No.Address primary cloneThe address of the target cloneActivity
Wash buffer containsThe residual activity
2 mm CA2+, OD4502 mm EDTA, OD450
12/131/C62,1652,14899,2%
22/264/F102,2161,96888,8%
32/132/F82,0091,94496,8%
45/D73/N61,64995.6%of
52/134/B121.5711.40289.2%

Table 4.
Changes in the activity obtained μa at the selection and modification of Biotin.
SampleThe culture fluidAfinno-purified IgGBiotinylated IgGBiotinylated IgG
Address cloneActivity, TH
Antimist - PHNeu - HP
4F10to 2.570,900,85the 1.44
S2,392,351,061,71
3H62,851,300,92 0,51
2F82,750,380,450,06
4B122,861,540,890,10

1. The hybrid strain of cultured animal cells Mus musculus Sp2/0-BC/rhPC-4F10 producing monoclonal antibodies specific to the protein With human, deposited in Russian national Collection of Industrial Microorganisms FSUE gosniigenetika, registration number H-110.

2. The hybrid strain of cultured animal cells Mus musculus Sp2/0-BC/rhPC-1C6 producing monoclonal antibodies specific to the protein With human, deposited in Russian national Collection of Industrial Microorganisms FSUE gosniigenetika, registration number N-111.

3. The hybrid strain of cultured animal cells Mus musculus Sp2/0-BC/rhPC-3H6 producing monoclonal antibodies specific to the protein With human, deposited in Russian national Collection of Industrial Microorganisms FSUE gosniigenetika, registration number N-112.



 

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2 cl, 9 dwg, 1 ex

FIELD: medicine.

SUBSTANCE: strain of hybridioma is obtained by immunization of mice of line BALB/c. Mice are immunised by conventional method by double with thirty day exposition subcutaneous introduction of inactivated spores of B. Anthracis strain "СТИ-1". On the third day after last buster-injection carried out is hybridisation of splenocytes of immune mice (1×108 cells) with cells of mouse myeloma P3-X63 Ag/8-653 (1×107 cells), using for fusion polyethylene glycol (Sigma, USA). After hybridisation carried out are selection, screening, cloning and cryopreservation of hybridoma. Obtained hybridoma is deposited in collection of microorganisms of FSSE SRC AMB under number H10. Invention relates to biotechnology and can be used for obtaining monoclonal antibodies (MCA) to spores Bacillus anthracis. Strain productivity is 20-50 mcg/ml of monoclonal antibodies (MCAla in ascitic liquid- 5-10 mg/ml. Hybridoma produces MCA, related to IgGl subclass of mouse immunoglobulins and spores specific to Bac.anthracis.

EFFECT: increase of strain productivity.

7 dwg, 2 tbl, 9 ex

Fused partner cell // 2431667

FIELD: medicine.

SUBSTANCE: invention describes fused partner cells making it possible to produce heterohybridomas of cells of biological species different from a mouse. Heterohybridomas are produced by fused myeloma cells produced from an animal of a first biological species with leukemia cells produced from an animal of a second biological species which have an additional S-phase in a cell cycle and exhibit a diploidisation property. The fusion partner cell SPYMEG is deposited, No. FERM BP-10761 and can be used for hybridoma production. What is described is hybridoma producing antibodies produced by fusion of fusion partner cell and a third cell with antibody-producing ability. The invention describes methods for producing a fusion partner cell, a hybridoma and an antibody-producing cell, and also methods for producing antibodies. Use of the invention provides stable and simple production of the substances with the use of hybridomas in a wide range of species of animals.

EFFECT: hybridomas stably keep a phenotype throughout the whole process of cloning.

20 cl, 10 dwg, 2 tbl, 10 ex

FIELD: medicine.

SUBSTANCE: cultivated cell strain of Mus. musculus L animals that is a producer of monoclonal antibodies (MCA) specific to LPS of cholera vibrios 0139 serogroup is produced. Hybridoma is deposited in Specialised Collection of continuous somatic cells of the Vertebrata Institute of Cytology (St.-Petersburg), No. RKKK (P) 674D. The specific activity of the MCA produced by hybridoma under the invention as a diagnostic reagent is proved in a slide agglutination reaction. The produced hybridoma can be applied in creating diagnostic test systems of the monoclonal antibodies for accelerated identification of cholera vibrios O139 serogroup.

EFFECT: invention use allows producing the diagnostically significant monoclonal antibodies to LPS epitopes of cholera vibrios 0139 serogroup that allows higher effectiveness of laboratory cholera researches.

3 tbl, 4 ex

FIELD: medicine.

SUBSTANCE: invention can be used in creating diagnostic test systems of monoclonal antibodies (MCA-01) for accelerated identification of cholera vibrios serogroup O1 of serovars Ogawa and Inaba. The cultivated cell strain of mouse hybridoma is produced by conventional methods used in the monoclonal antibody technology. A cloning procedure results in preparing a productive strain stably producing monoclonal antibodies isotype JgG specific to O-antigen of cholera vibrios O1 serogroup. The strain is deposited in Specialised Collection of continuous somatic cells of the Vertebrata Institute of Cytology (St.-Petersburg), No. RKKK (P) 386D.

EFFECT: hybridoma produced MCA-01 actively reacts with O-antigen determinants of the strains of serovars Ogawa and Inaba and do not react with related microorganisms.

1 tbl, 3 ex

FIELD: medicine.

SUBSTANCE: hybridoma techniques involving the fusion of myeloma of Sp-2/0 mice and lymphocytes and splenocytes of BALB/c mice immunised by a conjugate of benzo[a]pyrene and a bovine serum albumin, with using polyethylene glycol of molecular weight 1500 for cell fusion, followed by limit dilution cloning are used to produced a Mus. Musculus BpBal animal hybrid cultured cell strain deposited in RKKK(P) of the Institute of Cytology of the Russian Academy of Sciences, Registration No. 723 D producing the benzo[a]pyrene and benz[a]anthracene specific monoclonal antibodies. The hybridoma strain produces the monoclonal antibodies mAB showing high Bp and Ba specificity and low cross-responsiveness with non-cancerogenic polycyclic aromatic hydrocarbons (PAH) and with endogenous compounds (aromatic amino acids and steroid hormones).

EFFECT: using the BpBal mAB allows producing sensitive and specific test systems for definition cancerogenic PAHs, the related antibodies in human and animal biological fluids, and searching immune mimetic peptides and producing anticancerogenic PAH vaccines.

1 dwg, 3 tbl, 3 ex

FIELD: medicine.

SUBSTANCE: invention concerns anti-PSMA antibodies which contain variable sites of heavy and light chains; the antibody does not contain fucosyl residues. Amino acid sequences of said chains are presented in the formula. Antibodies under the invention are a monoclonal antibody, a humanised or chimeric antibody, a human antibody. Also, there is described a method of inhibition of PSMA+ cell growth, such as tumour cells by contact of said cells with the anti-PSMA antibodies.

EFFECT: antibodies show higher, antibody-dependent prostate cancer cell cytotoxicity as compared with a fucosyl antibody form.

32 cl, 3 tbl, 6 ex

FIELD: medicine.

SUBSTANCE: there are described isolated mouse antibody selectively bound with antigen F1 of Yersinia pestis, and an antigen-binding fragment (Fab). There is presented yeast cell producing the described antigen-binding fragment (Fab). There is offered method for producing described antigen-binding fragment. Also, there is presented method for Yersinia pestis detection and a kit for detection of antigen F1 of Yersinia pestis.

EFFECT: invention presents a Yersinia pestis detection reagent.

9 cl, 12 dwg, 7 ex

FIELD: medicine.

SUBSTANCE: there are described humanized antibody selectively binding antigen F1 of Yersinia pestis and its antigen-binding fragment (Fab). There is presented yeast cell producing the described antigen-binding fragment. There is offered method for producing described antigen-binding fragment.

EFFECT: invention presents a Yersinia pestis detection and plague treatments reagent.

8 cl, 6 dwg, 3 ex

FIELD: medicine.

SUBSTANCE: there are presented versions of antibodies specific to claudin 18A2 produced by immunisation by a related amino acid sequence or a nucleic acid or a host cell expressing said peptide. The antibodies possess an ability to mediate elimination of cancer cells expressing claudin 18A2. There are disclosed versions of antibody-producing hybridomas. What is described is a conjugate or a pharmaceutical composition on the basis of antibodies or conjugates for elimination and/or inhibition of a cancer cell expressing claudin 18A2. There are disclosed versions of the method for growth inhibition and/or elimination of the cancer cell, as well as for treating or preventing a disease or a disorder involving cancer cells expressing claudin 18A2 with using the antibodies, conjugate and pharmaceutical composition under the invention.

EFFECT: use of the invention can find further application in medicine for treating cancer cells expressing claudin 18A2.

39 cl, 33 dwg, 5 tbl, 10 ex

FIELD: medicine.

SUBSTANCE: strain of hybridioma is obtained by immunization of mice of line BALB/c. Mice are immunised by conventional method by double with thirty day exposition subcutaneous introduction of inactivated spores of B. Anthracis strain "СТИ-1". On the third day after last buster-injection carried out is hybridisation of splenocytes of immune mice (1×108 cells) with cells of mouse myeloma P3-X63 Ag/8-653 (1×107 cells), using for fusion polyethylene glycol (Sigma, USA). After hybridisation carried out are selection, screening, cloning and cryopreservation of hybridoma. Obtained hybridoma is deposited in collection of microorganisms of FSSE SRC AMB under number H10. Invention relates to biotechnology and can be used for obtaining monoclonal antibodies (MCA) to spores Bacillus anthracis. Strain productivity is 20-50 mcg/ml of monoclonal antibodies (MCAla in ascitic liquid- 5-10 mg/ml. Hybridoma produces MCA, related to IgGl subclass of mouse immunoglobulins and spores specific to Bac.anthracis.

EFFECT: increase of strain productivity.

7 dwg, 2 tbl, 9 ex

FIELD: medicine.

SUBSTANCE: cultivated cell strain of Mus. musculus L animals that is a producer of monoclonal antibodies (MCA) specific to LPS of cholera vibrios 0139 serogroup is produced. Hybridoma is deposited in Specialised Collection of continuous somatic cells of the Vertebrata Institute of Cytology (St.-Petersburg), No. RKKK (P) 674D. The specific activity of the MCA produced by hybridoma under the invention as a diagnostic reagent is proved in a slide agglutination reaction. The produced hybridoma can be applied in creating diagnostic test systems of the monoclonal antibodies for accelerated identification of cholera vibrios O139 serogroup.

EFFECT: invention use allows producing the diagnostically significant monoclonal antibodies to LPS epitopes of cholera vibrios 0139 serogroup that allows higher effectiveness of laboratory cholera researches.

3 tbl, 4 ex

FIELD: medicine.

SUBSTANCE: invention can be used in creating diagnostic test systems of monoclonal antibodies (MCA-01) for accelerated identification of cholera vibrios serogroup O1 of serovars Ogawa and Inaba. The cultivated cell strain of mouse hybridoma is produced by conventional methods used in the monoclonal antibody technology. A cloning procedure results in preparing a productive strain stably producing monoclonal antibodies isotype JgG specific to O-antigen of cholera vibrios O1 serogroup. The strain is deposited in Specialised Collection of continuous somatic cells of the Vertebrata Institute of Cytology (St.-Petersburg), No. RKKK (P) 386D.

EFFECT: hybridoma produced MCA-01 actively reacts with O-antigen determinants of the strains of serovars Ogawa and Inaba and do not react with related microorganisms.

1 tbl, 3 ex

FIELD: medicine.

SUBSTANCE: hybridoma techniques involving the fusion of myeloma of Sp-2/0 mice and lymphocytes and splenocytes of BALB/c mice immunised by a conjugate of benzo[a]pyrene and a bovine serum albumin, with using polyethylene glycol of molecular weight 1500 for cell fusion, followed by limit dilution cloning are used to produced a Mus. Musculus BpBal animal hybrid cultured cell strain deposited in RKKK(P) of the Institute of Cytology of the Russian Academy of Sciences, Registration No. 723 D producing the benzo[a]pyrene and benz[a]anthracene specific monoclonal antibodies. The hybridoma strain produces the monoclonal antibodies mAB showing high Bp and Ba specificity and low cross-responsiveness with non-cancerogenic polycyclic aromatic hydrocarbons (PAH) and with endogenous compounds (aromatic amino acids and steroid hormones).

EFFECT: using the BpBal mAB allows producing sensitive and specific test systems for definition cancerogenic PAHs, the related antibodies in human and animal biological fluids, and searching immune mimetic peptides and producing anticancerogenic PAH vaccines.

1 dwg, 3 tbl, 3 ex

FIELD: medicine.

SUBSTANCE: mouse hybridoma called S79 is produced by fusion of spleen lymphocytes of a mouse immunised by the recombinant protein SURF-6 fused with glutathione-S-transferase, expressed and recovered from E coli cells with P3X63-Ag8.653 mouse myeloma cells in the ratio 3:1 by 50 % polyethylene glycol of molecular weight 1450. The produced hybridoma secretes a monoclonal antibody S79 specific to human nucleolus protein SURF-6 with a known amino acid sequence regardless of tissue or linear origin of cells. The antibody S79 does not react with SURF-6 in mouse cells NIH/3T3 and rat cells C6. The monoclonal antibody S79 produced by clone S79 can be used for detecting the SURF-6 in proliferative normal and tumour human lymphocytes, and also for detecting a possible contamination of human cell cultures by the cells of other species, including a mouse and a rat.

EFFECT: produced monoclonal antibody is used for specific detection of the human nucleolus protein by an immune-enzyme method, in the immunofluorescence and immunoblot reactions in human cells of various linear and fabric origin.

11 dwg, 6 ex

FIELD: chemistry.

SUBSTANCE: clone of mouse hybridome cells called "S148" is obtained by merging splenic lymphocytes of a mouse which is immunised by human recombinant protein SURF-6, which is merged with glutathione S-transferase which is expressed and extracted from E. coli cells, with cells of mouse myeloma of the line P3X63-Ag8.653 in ratio of 3:1 using a 50% solution of polyethylene glycol with molecular weight 1450. The obtained hybridome secretes the monoclonal antibody S148, which is specific towards human, mouse or rat nucleolar protein SURF-6, having an established amino acid sequence.

EFFECT: disclosed monoclonal antibody S148 enables to detect nucleolar protein SURF-6 in mammal cells using immunocytochemistry, immunoblotting and immunoprecipitation techniques.

8 dwg, 7 ex

FIELD: medicine.

SUBSTANCE: offered are versions of antibodies each of which is specifically bound with IGF-IR, inhibits its activity and is its antagonist, not exhibiting substantially IGF-IR agonist activity. Each of the antibodies is characterised at least by the presence of a variable area of a heavy and easy chain. There are described: antibody conjugates with cytotoxic agents, and also versions of a pharmaceutical composition for cancer diagnosing and therapy, methods of cancer treatment and diagnosing, a cancer diagnosing reagent - on the basis of the antibodies. There are disclosed: a method of producing the antibodies; nucleic acid (NA) coding the antibody; an expression vector containing NA. Offered is a hybridoma EM 164 producing the antibody under the invention, deposited in ATCC, No. PTA-4457, and also the use of the antibody for IGF-IR linkage. The use of the invention presents the antibodies which allow inhibiting MCF cell growth approximately in 12 times that is higher approximately in 5 times than hen using the antibody IR3, and can be used for cancer diagnosing and treatment expressing higher levels of receptor IGF-I, such as breast cancer, colon cancer, lung cancer, prostate cancer, ovarian cancer, synovial carcinoma and pancreatic cancer.

EFFECT: more efficient diagnosing and treatment of said cancers.

58 cl, 28 dwg, 10 tbl, 1 ex

FIELD: medicine.

SUBSTANCE: strain 4C6 is produced received by cell fusion of line SP2/0 murine myeloma and splenocytes of Balb/c mice immunised by intraperitoneal introduction of the purified ivermectin-BsA conjugate, and is deposited in the Collection of Vertebrates Finite Body Cells of State Institution D.I.Ivanovsky Research Virology Institute of the Russian Academy of Medical Science, No. 05/09. The hybridoma strain 4C6 synthesizes class IgGl monoclonal antibodies (MCA) specifically reacting in a competitive solid-phase enzyme immunoassay (EIA) with a group of compounds referred to a group of avermectins: ivermectin, avermectin, abamectin, aversectin.

EFFECT: use of MCA 4C6 as an immune detector allows the high sensitive and specific quantitative evaluation of avermectins in biological fluids and tissues.

2 cl, 1 dwg, 2 tbl, 3 ex

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.

2 dwg, 4 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: group of inventions refers to medicine, namely to infectious diseases, and can be used for treating whooping cough and/or for Bordetella infection protection. Polypeptide under the invention represents a fragment of adenylate cyclase Bordetella containing CD11b/CD18 interaction domain from an amino acid sequence extended from position 1166 to position 1281 SEQ ID NO:1. Said invention also concerns the specific fragments of adenylate cyclase Bordetella containing CD11b/CD18 interaction domain, and to their application, particularly for targeting of molecules of interest to CD11b expressing cells.

EFFECT: use of the inventions allows extending the range of products for treatment and prevention of the Bordetella infection.

26 cl, 9 dwg

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