Strain as25 of constant hybridoma cell line of mouse mus musculus-producer of monoclonal antibodies to antigen h3 of equine influenza virus

FIELD: biotechnology.

SUBSTANCE: strain of permanent hybridoma cell line of mouse Mus.musculus is proposed. The present invention can find application as "capturing" and detecting antibodies in enzyme immunodetection during diagnosing equine influenza.

EFFECT: monoclonal antibodies produced by the strain are specific to the surface glycoprotein H3 of equine influenza virus H3N8 and have no cross-reactivity relating to antigens of equine influenza virus of 1 subtype of H7N7.

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The present invention relates to the field of veterinary biotechnology, in particular to hybrid technology, and relates to the obtaining of monoclonal antibodies to the H3 antigen of influenza virus of equine H3N8. The invention can be used to create the diagnostics and immunoassay test kits for the detection of surface glycoproteins (HA) of influenza virus horses in biological fluids.

Flu in horses - an acute highly contagious respiratory disease characterized by lesions of the mucous membranes of the upper respiratory tract, fever, symptoms of intoxication, disturbances of the cardiovascular and nervous systems. According to the classification of the International Committee on taxonomy of viruses influenza virus horses belongs to the family Orthomyxoviridae (from the Greek. orthos - straight and Greek. myxa - goo), the genus by influenzavirus A, referring to Influenza A virus. Virions of influenza virus are spherical particles with a diameter of 80-120 nm, in the center are RNA fragments enclosed in lipoproteina shell on the surface of which there are "spikes" consisting of hemagglutinin (H or) and neuraminidase (N or NA). Antigenic variants of surface H and N glycoproteins isolated serotypes, two of which H7N7 and H3N8 cause flu horses. Currently, however, the epidemic of influenza of horses caused by a virus of the 2nd under�IPA - H3N8, and the first subtype virus (H7N7) wholly eliminated in the population of horses, or this subclinical infection [1].

Influenza infection remains a serious problem for horse breeding, causing mass outbreaks of respiratory disease in horses worldwide. The disease causes significant harm to breeding due to the reduction of breeding and sport value animals recover from, treatment costs, quarantine measures, derailing the plan sporting events and other economically important reasons.

Diagnosis of influenza of horses is carried out by evaluating the presence of antibodies in the sera of animals, the most widely used serological methods of investigation, namely the reaction of hemagglutination-inhibition (hi) and single radial hemolysis (ORG). This determine increase the level of antibodies to influenza virus in horses examined paired trial of blood serum taken from animals in the first days of illness and after 2-3 weeks. Currently in serological diagnosis methods are not used, allowing to identify the causative agent of the disease in the initial period of infection before the appearance of antibodies to influenza virus horses that reduces the effectiveness of therapeutic and preventive measures.

Antigen detection of influenza virus horses most sensitive and specific�STU have a test system, based on the principle of labeled antibody - enzyme-linked immunosorbent assay (ELISA). The use of monoclonal antibodies for the preparation of conjugates of antibodies with the label allows to increase the specificity of the analysis, to ensure greater accuracy and information content analysis. In addition, the methodology of statement of ELISA designed for use in mass trials.

In the literature available to us information about hybrid strains of cultures of cells producing monoclonal antibodies to the H3 antigen of influenza virus horses, could not be found.

The objective of our research was to obtain a constant strain hybrid cell line having high in vitro production of monoclonal antibodies to the surface glycoprotein (H3) influenza virus horses that can be used as "exciting" and a detection antibody in ELISA.

Method for producing the strain is as follows.

The proposed strain of hybrid cells obtained by fusion of cells of a continuous cell culture of mouse myeloma P3-X63-Ag-8. 653 with spleen lymphocytes mice of line BALB/c immunized twice intraperitoneal and one intravenous injection of 100 μg of purified antigen of influenza virus of equine H3N8 strain "A/horse 2/Bitza/07".

At the height of the immune response (3 days after the last injection) conducted a merger of splenii�s immunized mice with cells of the mouse myeloma line P3-X63-Ag-8. 653.

Cell fusion was carried out according to standard methods using PEG with M. M. 1500. The ratio of myeloma cells and lymphocytes was 1:4-1:10. Selection of hybridomas was performed on a medium containing hypoxanthine, to produce remissions in childhood and thymidine. The hybridomas were cultured in 96-well panels manufactured by "Nunc" on RPMI-1640 medium with addition of 10% serum of cows embryos in an atmosphere containing 5% CO2.

Screening of hybridomas for the production of specific monoclonal antibodies was performed by solid-phase ELISA using allantoic fluid containing influenza virus horses two subtypes: H7N7 and H3N8 ("1 Equine/Prague" and "/horse 2/Bitza/07"). Cloning and reklamiranje cell cultures were performed in 96-well panels by the method of limiting dilutions using a macrophage feeder layer.

The result was selected and propagated clone of cells producing monoclonal antibodies to the surface glycoprotein (H3) influenza virus horses. This clone of cells was serially propagated in 96-, 24-hole panels, and then in the culture mattresses.

Culture fluid was examined for the content of specific antibodies. The titer of specific antibodies in ELISA was 1:16-1:64. The monoclonal antibodies were obtained three times by precipitation with a solution of ammonium sulfate to 50% saturation with subsequent�brilliant dialysis. Was determined the species identity and the specificity of monoclonal antibodies by ELISA.

The proposed strain has the following properties.

MORPHOLOGICAL FEATURES. Cells are uniform, rounded shape with a large oval nucleus containing from 1 to 3, 1-2 nucleoli. When seeding, the cells are evenly distributed on the substrate surface and in the culture fluid. The proliferation index is equal to 5-6.

CULTURAL PROPERTIES. Cells are cultured in RPMI-1640 medium plus 10% serum embryo cows. Cells multiply in suspension, partly attached to the surface of the plastic culture vessel. The seeding concentration of 10,000 cells/cm3growth environment. In 2-3 days after the formation of the slurry, 80% of the culture fluid was removed together with her cells. The remaining cells are added the same volume of fresh nutrient medium, pH 7,2-7,4. The multiplicity of sieving 1:5. The frequency of sub-culturing - 1 time in 3-5 days. When the seed concentration of 20,000 cells/cm2the suspension is formed in 2-3 days. The proliferation index is equal to 5-6.

CULTIVATION IN ANIMALS. Cells cause the formation of ascites in linear mice BALB/c treated with pristane 7-10 days before intraperitoneal administration of 2-10×106cells/mouse. Activity in ELISA ascitic fluid is 1:1280-1:5120.

CONTAMINATION. Contamination with protozoa, fungi, bacteria, mycoplasmas, viruses are not detected.

STORAGE OF CULTURES. Freezing media: 70% of the medium RPMI-1640, 20% serum embryo cows, 10% of dimethyl sulfoxide. The concentration of cells in plastic cryovials 1.5 to 2.0×106cells/cm3environment. Freeze mode: up to minus 70°C to 1°/min, then liquid nitrogen (minus 196°C). Recovery after freezing: rapid thawing at 37°C, centrifugation at 1200 rpm for 10 minutes, resuspension in growth medium. Viability after thawing 70-80%.

The strain used in the manufacture of diagnostic test systems for the detection of influenza virus of equine H3N8 in biological fluids.

Strain of hybrid cells AC25 is a permanent line of cells with an unlimited lifespan, suitable for biotechnology in producing the drugs.

Properties of the strain's unstable past also produces and�creating them monoclonal antibodies are reflected in the specific examples.

Example 1. Immunological properties of monoclonal antibodies. The specificity of antibodies is determined by ELISA in plates sensitized with influenza horses two subtypes: H7N7 and H3N8 ("1 Equine/Prague" and "/horse 2/Bitza/07"). In tablet making culture fluid and incubated 1.5 hours at 37°C, introduce the conjugate antibodies against immunoglobulin of mouse IgG with horseradish peroxidase (manufactured by enterprise for the production of bacterial preparations IEM. N. F. Gamaleya RAMS), incubated in the same mode and make the substrate mixture containing hydrogen peroxide and TMB. After the development of staining for 10 min to stop the reaction by adding sulfuric acid solution and measure the optical density value. The values of optical density in the wells sensitized with influenza horses of the 2nd subtype A/horse 2/Bitza/07 and the 1st subtype "1 Equine/Prague", respectively 1,725 and 0.031 inch. The ratio of optical densities exceeding 55, suggests that monoclonal antibodies interact only with the antigen of the influenza virus horses 2nd subtypes (H3N8) and have no cross-reactivity against the antigen of the influenza virus horses 1 subtype H7N7.

Example 2. Cultivation of the strain in vitro and quantification of antibodies production. Cells of strain IS cultivated in glass mattresses, low temperature�re 37,0°C in a nutrient medium, consisting of medium RPMI-1640 and 10% serum embryo cows, antibiotics (penicillin and streptomycin 500,000 U/DM3). The optimum planting density of 2×104cells of 1 cm3environment. 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 the antigen of H3 influenza virus in horses in the culture fluid ELISA was 1:64.

Example 3. Cultivation in animals and quantification of antibodies production. Cells of strain at a dose of 2-10×106cells/mouse in 0.5 cm3phosphate-buffered saline was injected into the abdominal cavity linear mice BALB/c, which previously 7 days were injected with 0.3 cm3of pristane. 10-14 days received ascitic fluid, the titer of specific monoclonal antibodies to the H3 antigen of influenza virus in horses in which was in ELISA 1:2560.

Example 4. Conducting enzyme-linked immunosorbent assay using monoclonal antibodies. The monoclonal antibodies were obtained from ascitic fluid three times by precipitation with a solution of ammonium sulfate to 50% saturation, followed by dialysis.

In the first stage, the surface of the plastic panel for micrometrology were sensibilized due to the nonspecific adsorption of monoclonal antibodies to the H3 antigen of the virus DOF�and horses produced by the strain AS, from a solution with a concentration of 10 μg/cm3at pH 7,2-7,4. Incubation was performed for 16-20 hours at 4°C.

Made control and analyze samples of swabs from the nasal cavity and incubated for 1 hour at 37°C.

The excess reagents after each stage of sensitization of the solid phase and enzyme-linked immunosorbent assay was washed with detergent solution in phosphate-saline buffer solution.

As detecting antibody used peroxidase-labeled globulin cock immunized with influenza virus horses 2nd subtype. Binding of antibodies with peroxidase from horseradish roots was performed by the method developed by Wilson and Nakane after activation of the enzyme by periodate sodium [2]. Incubation was performed for 1 hour at 37°C.

Accounting reactions were carried out by varying the optical density of the analyzed samples compared to negative control samples after addition of the substrate mixture containing tetramethylbenzidine (TMB) and hydrogen peroxide (H2O2. Positive thought of the sample, the optical density of which is two or more times greater than the optical density of the negative control.

The study 25 samples of swabs from the nasal cavity in animals during the period of infection management has determined that wee�us flu the 2nd subtype was detected in 15 animals.

The proposed strain was tested with a positive result in the State scientific institution "all-Russian research Institute of experimental veterinary medicine named Ya. R. Kovalenko RAAS" (GNU VIEV RAAS) in 2013.

Feasibility study. The resulting strain AS hybrid cells producing monoclonal antibodies to the surface glycoprotein (HA) of influenza virus of equine H3N8, characterized by the following useful properties:

- is a permanent line of cells 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 liquid was 1:16-1:64, ascitic fluid 1:1280-1:5120 in immune-enzyme analysis;

- monoclonal antibodies specific to surface glycoprotein (HA) of influenza virus horses H3;

- monoclonal antibodies do not react with the surface glycoprotein (HA) of influenza virus horses H7;

- monoclonal antibodies, fixed on a solid phase, provide a solid fixation of the antigen of the influenza virus horses and specificity of ELISA in the detection of influenza virus of equine H3N8 in biological material;

Strain AS deposited in a Specialized To�lectures transplantable somatic cell cultures of agricultural and game animals RCCC (she RAAS) of VIEV No. 86.

Strain AS-producer of monoclonal antibodies to the surface glycoprotein (HA) of influenza virus of equine H3N8 may find application in the manufacture of ELISA test systems for the diagnosis of influenza in horses. The use of such test systems will help to identify the causative agent of the disease in the first days of infection before the appearance of antibodies that will improve the effectiveness of anti-epizootic measures, to reduce the time of healing equine farms, disadvantaged on influenza of horses.

Sources of information

1. Manual of Diagnostic Tests and Vaccines for Terrestrial Animals. - 7thed. Volumes 1 and 2, 2012. - xxxv + 1404p.

2. The book "Immunofluorescence and related staining techniques" ed. by W. Knapp.- Elsevier: North-Holland Biomedical Press. - 1978. - P. 215-221.

Strain AC25 hybrid permanent line of cells of mouse Mus. musculus-producer mouse monoclonal antibodies to H3 influenza virus horses deposited in the Specialized Collection of transplantable somatic cell cultures of agricultural and game animals RCCC (she RAAS) of VIEV at the State scientific institution "all-Russian research Institute of experimental veterinary medicine named Ya. R. Kovalenko RAAS" (GNU VIEV RAAS) in Moscow under No. 86.



 

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