RU1798372C - Strain of hybridous cultured mammalian cells - mus musculus l - a producer of protective monoclonal antibodies to equine east encephalitis virus - Google Patents

Abstract

. Usage: biotechnology, immunologists. SUMMARY OF THE INVENTION: A strain of hybrid transplantable cells was obtained by fusion of mouse P3NS1 / 1-Ag4.1 mouse myeloma cells with BALB / c syngenic cells immunized with a purified virus preparation, Southern variant. The hybridoma secretes lgG2 monoclonal antibodies that specifically interact with the E2 glycoprotein of the VCEL virus, neutralizing the infectivity of the virus on 1: 200 Vero cells and protecting sensitive animals from lethal doses of the virus upon passive administration. The strain is designated 1B2 and is stored under the number (I) 479D. The antibody titer reaches 1: 5000 in the culture fluid and 1: 300000 in ascites. 1 tablet, 1 ill. ate with

Description

The invention relates to biotechnology, and more specifically to medical virology, and for the production of monoclonal antibodies, to the Echoes encephalomyelitis virus Los El Edea (VESEL), which can be used for treatment and prevention in medicine and veterinary medicine.

The aim of the invention is to obtain a strain of hybrid cells secreting MCAs that can effectively protect sensitive animals with lethal doses of the VCEL virus, which makes preparations of these antibodies promising as agents for the prevention and treatment of severe viral diseases.

This is achieved by fusion of mouse P3NS1 / 1-Ag4.1 mouse myeloma cells with BALB / c mouse selenium cells immunized with Purified BEL virus.

The strain is obtained as follows.

Female BALB / c mice were used for immunization. The immunization schedule includes intraperitoneal administration of the VESEL virus preparation three times at a dose of 50 μg per mouse with an interval of 2 weeks with complete Freund's adjuvant. 3 days prior to fusion, the animals were given 50 µg of the virus intravenously in saline. Mice are sacrificed with chloroform and spleens are removed under sterile conditions from which a cell suspension is prepared.

400 million spleen cells and 80 ml NS1 cells are used as fusion partner. The mixture of cells is centrifuged, the supernatant is thoroughly removed, and 0.5 ml of a 2000% polyethylene glycol (PEG) solution with a molecular weight of 2000 is added to the cell pellet.

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The mixture was centrifuged for 4 minutes at 600 d. After 8 minutes, the PEG layer was slowly diluted with Versen's solution, after which the precipitate was resuspended and centrifuged. Cells are distributed in 10 culture microplates. Selection of hybrid cells was carried out in HAT medium consisting of DMEM (M) growth medium, to which 20% fetal bovine serum, 0.1 mM hypoxanthine, 0.04 mM thymidine and 0.01 mM aminopterin were added. .

Specific hybrids were selected by enzyme-linked immunosorbent assay using 200 ng of purified VSEL virus as antigen. Hybridoma 1B2 was cloned twice by limiting dilution, transferred to mass culture and frozen in liquid nitrogen.

The strain of hybrid cells was deposited in the collection of the Institute of Cytology of the Academy of Sciences of the USSR.

The strain is characterized by the following features.

Morphological signs. The culture consists of large rounded cells that are similar in morphology and size to the original parent NS1 myeloma. The oval core is located eccentrically and occupies a significant part of the cytoplasm.

Cultural properties. The cultivation medium is DMEM medium (manufactured by VNII MB) containing increased amounts of arginine up to 200 mg / l, folic acid up to 12 mg / l, asparagine up to 36 mg / l, as well as 0.05 mm 2-mercaptoethanol and 20 mM HEPES (DMEM (M)}. The content of fetal serum in the growth medium is 15%. 1 μg / ml gentamycin sulfate is also added to the medium. Hybridoma 1B2 grows in a visually-layer-suspension culture. Seeding dose is 100-200 thousand cells per milliliter, sieving ratio -1: 3-1: 4.

The cultivation of hybridomas in the body of the animal. Female BALB / c mice are sensitized by intraperitoneal administration of 0.5 ml pristan. After 2-4 weeks, 5 million hybrid cells are introduced into the animals. After 10-12 days, an ascites tumor forms. 3-5 ml of ascitic fluid can be obtained from one animal. Hybridoma is vaccinated in 100% of cases.

The characteristic of a useful product. MCAs belong to the subclass fgG2a. They specifically interact with aphidoprotein E2 of the VSE virus in the immunoblotting reaction. The activity of MCA in the neutralization of the VES virus in Vero cell culture is 1: 200. MCA 1B2 effectively protects mice from administering a lethal dose of VES virus. The solubility index is 6.5 Ig. ICA titer in

ELISA is 1: 5000 for culture fluid and 1: 300000 for ascites. Stable MCA production is maintained for at least 30 passages in vitro ..

Cryopreservation. The freezing medium is DMEM (M) medium - 50%, fetal serum - 40%. dimethyl sulfoxide - 10%, 0.5 ml of the cell suspension is transferred into plastic cryoprobes and placed in

A foam container with a wall thickness of 1.5 cm. The container is introduced into liquid nitrogen vapors. After one day, the tubes were transferred to liquid nitrogen. Defrosting is carried out by lowering the tube into water at a temperature of 41 °

5 C. Cells were diluted with DMEM (M) medium and centrifuged at 600 d. The pellet was resuspended in growth medium at a concentration of 200-300 thousand cells per milliliter and transferred to culture bottles. The viability after thawing is 60-80% (dye 0.25% trypan blue). The invention is illustrated in the drawing. .

Immunoblotting of sucrose-purified VSE cell sucrose density gradient. After the viral protein was electrically transferred, nitrocellulose strips were treated with a 0.5% casein solution. After saturation of the nonspecific binding sites, the strips were incubated in 2 ml of the culture medium of the corresponding hybridoma. MCA binding sites were determined using peroxidase-labeled antibodies against mouse immunoglobulins. 1 - cultural

5 medium hybridoma 1B2; 2, 3 - negative controls with normal mouse serum, mouse myelo culture medium. we are NS1, 4 - a mixture of three types of MCA to E1 proteins. E2iSvirusa VSEL (9F2, 10G8, 1B11).

0 PRI me R 1. Definition of viral

protein specifically reacting with MCA,

synthesized by 1B2 tibridoma,

. Viral target protein for MCA 1B2

determined by immunoblotting. Proteins of VSE virus separated by polyacrylamide gel electrophoresis were transferred to a nitrocellulose membrane (Mllltpore, United States) according to the method of Roehrig et a. Transfer is carried out for 1.5 hours on the device

0 Transphor (KLB, Sweden) at a voltage of 80 in 0.025 M Tr, is-HC buffer containing 0.192 M glycine (pH 8.3) and 20% ethanol. Control of protein transfer to nitrocell. The vine is carried out by staining the polo-5 membrane with amide black 10B. The remaining strips are placed in a 10 mM Tris-HCI pH 7.2 buffer containing 0.145 m NaCl, 0.05% Tween 20 (TBZ-Tween) and 0.5% Casein overnight at 4 ° C; The strips thus treated are incubated for one hour at 37 °

C with preparations of ascitic fluid MCA in a dilution of 1: 100. The excess antibodies are eliminated by washing the TVS-tween membranes three times. The membranes are then treated with rabbit anti-mouse IgG serum labeled with peroxidase at a dilution of 1: 200. Stand for 2 hours at room temperature. The strips are washed and developed in a chromogen solution containing 1 mg / ml 4-chloro-1-naphthol and 0.03% hydrogen peroxide in 50 mM Tris-HCI buffer pH 7.4 and 0.145 MNaCI. The interaction of MCAs with viral proteins appears as bright blue-violet bands. According to immunoblot data, MCA 1B2 interact with glycoprotein E2 (55 kD) of the VSEL virus (see drawing).

Example 2 Determination of the protective activity of MCA 1B2 from lethal VES infection with passive administration.

To determine the protective activity of MCA 1B2, two groups of non-primordial white mice weighing 8-10 g were used. One group of animals was injected intraperitoneally with 0.1 ml of ascitic fluid containing MCA 1B2, the other used as a control was administered ascitic fluid containing MCA hybrid We 7V6 (prototype), not having a protective effect. One day after the administration of MCA, the animals were subcutaneously infected with various doses of VES virus with a TO-fold dilution step. Observation of the animals was carried out for 10 days. The protective activity of MABs was expressed through a resistance index, which is the difference of logarithms of virus titers determined in animals passively immunized with the studied and control MABs. The virus titer was calculated by the method of Kerber. The results of the experiment are presented in the table.

Thus, the 1B2 hybrid cell strain derived from murine myeloma produces protective MABs that can be used as prophylactic and treatment agents. MCA data in a dose of 1-2 mg protect white mice from more than 3 million LD.so of the VESEL virus. MCA data belong to the lgG2a subclass.

The above properties of the strain of hybrid cells 1B2 allow us to conclude that for the first time a hybridoma was synthesized synthesizing protective MABs for the VES virus.

Claims (1)

The claims The strain of hybrid cultured animal cells Mus musculus L BCKK (II) No. 479 is a producer of protective monoclonal antibodies to equine eastern encephalomyelitis virus .. The protective activity of MCA 1 B2 against lethal infection of VES viruses during passive immunization of white mice is protective.  234