Strain 8c12 of permanent inter-species hybrid line of cells of mouse mus. musculus and sheep ovis aries - producent of monoclonal antibodies to glycoproteidal antigen of virus of cattle leucosis

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

 

The invention relates to the field of veterinary biotechnology, namely, to obtain cell hybrids used in immunochemical studies.

Methods of hybridization of somatic cells currently widely used for solving many problems in biology, medicine, veterinary medicine. Hybrid cell line animals use to solve the problems of Virology, pathology farm animals, production of biologically active substances and monoclonal antibodies that can be used for the preparation of highly specific diagnostic reagents to detect the virus or antibodies in biological fluids.

The leukosis virus in cattle causes malignant lymphoproliferative disease of cattle - leucosis, which ranks first in the structure of infectious diseases of cattle in the Russian Federation. The infectious process is characterized by the absence of viremia with simultaneous production of antibodies to proteins of the virus, which are dominated by antibodies to glycoprotein viral envelope (gp51).

For diagnosis of the disease are widely used serological methods, namely the reaction diffusion precipitation in agar gel (RDP) and enzyme-linked immunosorbent assay (ELISA). The sensitivity of serology is of ecogov high in cases when they are aimed at the detection of antibodies to glycoprotein gp51.

The need for this work due to the fact that the greatest sensitivity and specificity in detecting antibodies to the virus leukemia have a test system based on the principle of enzyme immunoassay using monoclonal antibodies for the implementation of the fixation of the antigen at the stage of sensitization of solid-phase basis. Such test systems are far superior in sensitivity and specificity of traditional, in which sensitization of the solid phase is carried out with the direct adsorption of purified leukosis virus of cattle.

Known strains of hybridoma culture of cells producing antibodies to glikoproteinom antigen leukosis virus in cattle. Strains of cells producing monoclonal antibodies against gp51 leukosis virus of cattle, received in the merger lymphocytes of mice immunized with the surface antigen of the virus leukemia cattle gp51 with cells of the mouse myeloma. After selection and cloning of the resulting hybrid cells were injected vnutriarterialno mice. Ascitic fluid was used as a source of monoclonal antibodies to gp51 leukosis virus (1).

Immunization of mice with leukemia virus bovine insufficient efficiency is positive, because the mouse is not sensitive to this virus species of animals and is responsible for immunization is not enough intensive education of cells producing antibodies; in addition, the contents glikoproteinov antigen in preparations of purified virions very low. Immunization of mice with purified glikoproteinami antigen more effective, however, the cleaning procedure is quite time-consuming if you have a low product yield. In turn, themselves monoclonal antibodies must possess a number of properties that determine the possibility of their use in a diagnostic test system enzyme-linked immunosorbent assay. Monoclonal antibodies that can be used for fixation of the antigen on the solid phase carrier, should be firmly bind the antigen and to ensure the accessibility of its epitopes analyzed for antibodies.

A known strain of interspecific hybridoma culture of cells producing antibodies to glikoproteinom antigen leukosis virus of cattle b/SS-1, obtained by fusion of peripheral blood lymphocytes calf Holstein breed, hyperimmunizing inaktivirovannye antigen VLCRC with cells of the mouse myeloma SP2/0-Agl4. The strain has a high production of monoclonal antibodies in cattle subclass IgG1specific to glikoproteinom antigen VL is RS in ELISA. When studying in a Western blot analysis of monoclonal antibodies revealed immunoreactivity of monoclonal antibodies against a viral antigen, which is a glycoprotein (mol. weight of 69 KD. Ascitic antibodies resulting from intraperitoneally inoculation of 1×107cells hybridoma mice BALB/c-nu/nu, four breeding, had a 50% inhibitory effect on early polykaryocytes (2).

The objective of our research was to obtain a strain of hybrid cells with in vitro high production of monoclonal antibodies to glikoproteinom antigen leukosis virus of cattle with high avidity and affinity for optimal spatial orientation of the antigen fixed with the help of these antibodies on the solid phase carrier, when used in a diagnostic test system.

Obtained interspecific hybrid strain of murine myeloma cells P3-X63-Ag-8. 653 with splenocytes sheep, infected with a virus leukemia in cattle. Strain interspecific hybrid cells S is a permanent cell line with an unlimited lifespan, suitable for biotechnology for producing drugs.

The purpose of virus infection leukemia cattle sheep subcutaneously introduced 2 ml of culture liquid culture of kidney cells of sheep embryo, x is onicescu infected leukosis virus of cattle. 2 weeks after inoculation vaccinated material in the blood of sheep by the method of the RDP were identified antibody glikoproteinom antigen gp51 VLCRC. After 1 month introduced intravenous drug, antigen WERS obtained by precipitation with polyethylene glycol (7%) from the culture fluid virusprobleme culture.

3 days after the booster injection, the sheep were killed, removed the spleen and perform the fusion of spleen cells infected VLCRC sheep with cells of the mouse myeloma line P3-X63-Ag-8. 653.

The cell fusion was performed by standard methods using PEG with 1500 mm. The ratio of myeloma cells and lymphocytes was 1:4-1:10. Selection of hybridomas was carried out on a medium containing gipoksantin, aminopterin and thymidine. Hybridoma were cultured on 96-well panels manufactured by "Nunc" in the medium RPMI-1640 with the addition of 10% serum of cow embryos in an atmosphere containing 5% CO2.

The screening of hybridomas for the production of specific monoclonal antibodies was performed by indirect solid-phase ELISA using purified preparation VLCRC at a concentration of 5 μg/ml for sensitization of the solid phase and species-specific immunoperoxidase conjugate for detection of bound peroxidase antibodies. Cloning and reklamiranje cell cultures were performed on 96-well panels method of limiting time is of edeni using macrophage feeder layer.

The result was selected and propagated clone of cells producing monoclonal antibodies to glycoprotein leukosis virus in cattle. This clone of cells was serially propagated in 96-, 24-hole panels, and then in the culture mattresses.

Was identified species and specificity of monoclonal antibodies produced by the selected clone methods ELISA, Western blot turns and immunocytochemistry and quantify the production of antibodies.

The strain has the following properties.

Morphological features: the Cells are uniform, round shape with a large oval nucleus containing from 1 to 3, usually 1-2 nucleoli. When seeding the cells are evenly distributed on the surface of the substrate and in the culture fluid. The proliferation index is 5-6.

Cultural properties. Interspecific hybrid strain IS cultured in medium RPMI-1640 with the addition of 10% serum of cow embryos. Cells grow in suspension and partially attached to the surface of the culture vessel. The seeding concentration of 1×104cells in 1 ml of medium. 5-7 days after sowing 80% of the culture fluid is removed along with her cells. The remaining cells add the same volume of fresh medium. pH of 7.2 to 7.4. The frequency of subculturing - 1 every 3-5 days. The multiplicity of races the eve of 1:5.

Immunological properties. Monoclonal antibodies recognize a common antigenic determinant outer glikoproteid gp51 and transmembrane glikoproteid gp 30 leukosis virus of cattle.

The contamination. Contamination protozoa, fungi, bacteria, mycoplasmas, viruses are not detected.

Storage of cultures. Environment for freezing: 70% RPMI-1640 medium, 20% whey of cow embryos, 10% dimethyl sulfoxide. The concentration of cells in plastic cryovials 1,C6cells/ml. Mode freeze: up to -70°C -1°C/min, followed by liquid nitrogen (-196°C) Recovery after freezing: rapid thawing at 37°C, centrifuged at 1200 rpm for 10 min, re-suspension in the growth medium. Viability after thawing 70-80%.

The proposed hybrid strain of mouse cells Mus. Musculus S producer of monoclonal antibodies sheep to shell glycoprotein gp51 VLCRC is stored in a Specialized Collection of transplantable somatic cell cultures of agricultural and game animals (she RAAS) under No. 72.

The strain used in the manufacture of a diagnostic test system for the detection of antibodies to the virus leukemia cattle enzyme-linked immunosorbent assay.

Example 1. Cultivation of the strain in vitro and quantifying the production of antibodies. Cells of strain S cultivate the glass mattresses at a temperature 37,0°C in a nutrient medium, consisting of RPMI-1640 medium and 10% serum embryos blood of antibiotics (penicillin and streptomycin at 500000 IU/l). The optimum planting density of 2×104cells/ml of medium. After 3-5 days after the formation of the suspension culture fluid partially remove and add the required amount of fresh nutrient medium. The titer of antibodies to glikoproteinom antigen VLCRC in the culture fluid determine enzyme-linked immunosorbent assay with purified antigen VLCRC and species-specific rabbit antibodies against sheep globulin labeled with peroxidase. In native (non-concentrated) culture fluid it is 1:32.

Example 2. Immunological properties of monoclonal antibodies. Purified virus leukemia cattle were separated by the method of electrophorese 12.5% gel SDS-page (dodecyl-sodium sulphate-polyacrylamide) and after transfer to nitrocellulose filter were incubated with monoclonal antibodies produced by the culture S and then, after washing the filter with peroxidase labeled antibodies against sheep globulin. Bound peroxidase peroxidase showed a solution containing 3,3'-diaminobenzidine in 0.1 M Tris-model HC1, pH 7.4, and 1:5000 hydrogen peroxide H2O2. Were shown two coloured stripes in two zones corresponding in molecular mass to uznomu glycoprotein gp51 and the transmembrane glycoprotein gp 30 leukosis virus cattle (with a molecular mass of 67 and 31 KDa).

Example 3. Immunological properties of monoclonal antibodies. From the culture fluid culture S, obtained as described in example 1, was isolated immunoglobulins three-precipitation of a solution of ammonium sulfate to 50% saturation with subsequent dialysis. The antibodies were labelled with peroxidase controllable periodic destruction method (3). Primary cultures of chicken fibroblasts were infected with the recombinant strain of vaccinia virus WR 601 BLV with a built-in DNA genome env leukosis virus in cattle. The recombinant virus is characterized by high expression of the complete env gene product VLCRC (gp51+gp30). Multiplicity of infection was 2-3 COMBAT by 1 cell. After 48 hours of cultivation at 37°C was deleted culture liquid, the monolayers were fixed with a solution Buena, and incubated with monoclonal antibodies produced by the culture C peroxidase labeled. The reaction showed staining DUB-H2About2. Tricks of the JRS were painted in brown color.

Example 4. Conducting enzyme immunoassay using monoclonal antibodies. Monoclonal antibodies sheep to glikoproteinom antigen leukosis virus in cattle produced by strain S were used for fixation of the antigen VLCRC on the solid phase in indirect enzyme-linked immunosorbent assay. For immobilization of the antigen used three is tadinya method of fixation. In the first stage, the surface of the plastic panel for micrometrology was senzibilizirani due to nonspecific adsorption of the monoclonal antibody N to sheep immunoglobulins produced by strain N (deposited in she RAAS under No. 73) from a solution with a concentration of 5 µg/ml at pH of 7.2-2-7,4. Incubation was carried out for 16-20 hours at 4°C. In the second stage conducted immobilization due to specific immunological interaction of monoclonal antibodies to glikoproteinom antigen gp51 VLCRC from the culture fluid of strain S (deposited in she RAAS under No. 72) for 1.5 hours at 37°C. In the third stage, conducted immobilization also due to specific immunological interaction of antigen leukosis virus of cattle from the culture fluid virusprobleme culture. Excess reagents after each stage of sensitization of the solid phase and conducting immunoassay washed with a solution of detergent in phosphate-saline buffer solution. Made test and analyze samples of blood serum of cattle and incubated for 1.5 hours at 37°C. as the detecting antibody used peroxidase labeled monoclonal antibodies against globulin cattle, do not give cross-reactions with antibodies OVC is, produced by strain A (deposited in she RAAS under No. 71)Incubatio conducted for 1.5 hours at 37°C. the reaction Accounting conducted by the change in optical density of samples compared to control samples after addition of the substrate mixture containing tetramethylbenzidine (TMB) and hydrogen peroxide H2O2. Positive thought of the sample, the optical density in which two or more times greater than the optical density of the negative control

The study 662 blood sera from disadvantaged by the leukemia economy in parallel in the reaction diffusion precipitation (RDP) and enzyme-linked immunosorbent analysis results (RDP+/ELISA+; RDP-/ELISA) was noted in 560 or 84.6%of the mismatch (API+/ELISA; RDP-/ELISA+) - 15.4% of cases. Only 3 positive results RDP-samples reacted negative in ELISA (0.5 percent). The sensitivity of enzyme immunoassay surpassed RDP with glikoproteinami antigen and did not yield to her specificity. In ELISA were identified 190 (28,7%) positive samples, whereas in the RDP - 94 (14,2%), i.e. by 14.5% or twice.

Table
Comparative analysis of the results of RDP and ELISA.
ELISAELISA+ELISATotal
REID
REED+91 (13,7%)3 (0,5%)94 (14,2%)
RIED99 (15,0%)469 (70,8%)568 (85,8%)
Total190 (28,7%)472 (71,3%)662 (100%)

The proposed strain tested with positive results in the laboratory, all-Russian Institute of experimental veterinary medicine named. Arrowlink and Kursk bio from 2006 to 2007

The feasibility study. The resulting strain S hybrid cells producing monoclonal antibodies characterized by the following useful properties:

- is a permanent cell line with an unlimited lifespan, suitable for biotechnology in producing the products;

- has a high level of production of monoclonal antibodies. Antibody titers to native culture fluid was 1:32-1:64 in the enzyme-linked immunosorbent assay;

- monoclonal antibodies specific for common antigenic determinants picop is otaigbe leukosis virus - external gp 51 and transmembrane gp30.

- Monoclonal antibodies provide a strong binding glikoproteinov antigen leukosis virus of cattle with a solid-phase medium during the assay.

Spatial location glikoproteinov antigen associated with a solid-phase carrier by means of monoclonal antibodies when conducting immunoassay that detects antibodies in the serum of naturally infected with a virus leukemia cattle with high sensitivity and specificity.

Strain S producing monoclonal antibodies to glikoproteinom antigen leukosis virus of cattle - will find application in the manufacture of enzyme immunoassay system for the diagnosis of leukemia in cattle. Using this test system will improve the effectiveness of the Wellness interventions, to reduce the duration of recovery affected with leukemia livestock farms and eventually dramatically reduce the incidence of bovine leukemia.

Sources of information

1. Log. Virology. the 1982 - v.l22, - p.342-352.

2. Log. Jpn.J.Vet.Sci. - 1988, - v.50, - R-1138.

3. The book "Immunofluorescence and related staining techniques" Ed. W Knapp. Elsevier: North-Holland Biomedical Press. - 1978. - 215-221.

Strain IS constant interspecific g is Britney cell line mouse Mus. musculus and sheep Ovis aries producing monoclonal antibodies sheep to glikoproteinom antigen leukosis virus of cattle, deposited in a "special Collection transplantable somatic cell cultures of agricultural and game animals (she RAAS)" Moscow under No. 72.



 

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3 tbl, 1 ex

FIELD: biotechnology, virology.

SUBSTANCE: invention relates to preparing a new strain of hybrid cells of Mus musculus L., NIIMB-280 (9E2), as a producer of monoclonal antibodies to the West Nile virus (WNV) protein E. West Nile virus (strain WNV/LEIV-VIg99-27889) is isolated in Volgograd district in 1999 year from a patient. Producing monoclonal antibodies can be used effectively for detection of the strain WNV/LEIV-VIg99-27880 of WNV that causes human diseases in Russia territory. New hybrid strain of cells is obtained by fusion of murine myeloma cells p3-X63/Ag8.653 (NS0/1) with murine splenocytes BALB/c immunized with the purified and inactivated preparation WNV (strain WNV/LEIV-VIg99-27889). The strain of hybrid cells Mus musculus L., NIIMB-280 (9E2), is deposited in Collection of cellular cultures of NII cellular cultures GNTS VB "VEKTOR" at number № NIIMB-280. Author's name of hybridoma cellular strain is 9E2. Using hybridoma allows preparing specific monoclonal antibodies raised to the West Nile virus protein E that, in turn, gives a possibility for identification of WNV and to standardize the content of protein E in immunodiagnostics.

EFFECT: valuable properties of strain.

1 dwg, 3 ex

FIELD: biotechnology, hybridoma technology.

SUBSTANCE: hybridoma strain is prepared by fusion of murine plasmocytoma Sp2/0-Ag.8 and B-lymphocytes of murine spleen of the inbred strain BALB/c immunized with protein-polysaccharide complex from Y. enterocolitica. Hybridoma produces monoclonal antibodies of isotype IgG to Y. enterocolitica O3 and O9 serovars used as components of IFA-test-system for identification of indicated serovars that are isolated most often in European areas from sick humans, agricultural animals and from objects of environment. The usage of monoclonal antibodies producing by hybridoma allows carrying out the identification of Y. enterocolitica strains of indicated serovars representing the most epidemic danger among other intestine-persistent microorganisms. Invention can be used in the development of diagnostic test-systems for identification of Y. enterocolitica strains O3 and O9 serovars for aims laboratory diagnosis in the public health, veterinary science and in carrying out scientific investigations.

EFFECT: valuable properties of strain.

1 tbl, 2 ex

FIELD: biology, hybridoma technology.

SUBSTANCE: invention represents a new strain of mammalian hybrid cells C3/S-3E5 of Mus musculus L. producing monoclonal antibodies (MCAb) to Bernet's coxiellas (strain "Grita") in cell cultures and abdominal cavity of syngenic animals. Hybridoma C3/S-3E5 producing MCAb to this pathogen is obtained by fusion of murine myeloma of strain Sp-2/0 and murine splenocytes of strain BALB/c immunized with the concentrated and purified Bernet's coxiella preparation (strain "Grita) inactivated with formalin using polyethylene glycol of molecular mass 1000 Da as a fusing agent and the following cloning by method of maximal dilutions. Specificity of prepared MCAb: absence of cross-reactions in IFA with Provacheck's rickettsia antigen and with the non-infected accumulation substrate. Using prepared MCAb it is possible to carry out specific detection of Bernet's coxiellas by method IFA (direct and indirect variants). IFA sensitivity based on these MCAb is 2.0 x 103 ID50 x cm-3 for white rats. Applying the present invention allows detecting and identifying pathogens of rickettsial etiology.

EFFECT: improved method preparing, valuable properties of strain.

3 tbl, 1 dwg, 1 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: present invention pertains to biochemistry, specifically to obtaining a hybrid, and can be used for obtaining a strain-producer of monoclonal antibodies to the MUCI human antigen. Using monoclonal antibody technology, a strain of hybrid cultured animal cells "ВКПМ Н-105" - producer of clinical rat monoclonal antibodies, specific for hypoglycosylated and deglycosylated isoforms of tumours associated with the human MUCI antigen can be obtained.

EFFECT: possibility of identifying clinical isoforms of MUCI antigen using antibodies, produced by an obtained strain, the antibodies of which can be used determining concentration of MUCI in blood plasma of a person previously diagnosed with tumours.

1 dwg, 3 ex

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