SU1446156A1 - Strain of hybrid cultivated cells of mus musculus for producing monoclonal antibodies to hemagglutinin of influenca virus - Google Patents

Abstract

This invention relates to biotechology and can be used to diagnose influenza. The aim of the invention is to obtain a hybrid strain of cultured animal cells MUS musculus, producing Monoclonal antibodies (MAB) against hemagglutinin of influenza virus type A. The strain is obtained by hybridization of splenocytes of the BALB / c lineus immunized with the influenza A virus (Krasnodar) T01 / 59 ( H2N2), with NSO myeloma cells. ICAs belong to class (K). They specifically interact with the hemagglutinin of influenza A viruses in the reactions of direct immuno-fluorescence, inhibition of hemagglutination, and neutralization in chickens. Stable production of ICA is maintained for 70 in vitro passages and 5 passages in BALB / C mice. 3 tab. i (L

Description

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This invention relates to biotechnology and can be used to diagnose influenza.

The aim of the invention is to obtain a strain of hybrid cultured animal cells Mus Musclus, producing monoclonal antibodies (mAbs) to hemagglutinin of influenza A virus,

Strain was prepared as follows.

Six-week-old female BALB / c mice are used for immunization. The immunization scheme of the animal includes intraperitoneal B1 (concentration-15). Selection of hybrid cells is carried out

0,83% solution in 5-fold volume. After lysis of erythrocytes, cells are indifferently precipitated and washed twice in serum-free RPMI-1640 or DMEM. For the fusion, a 50% solution of polyethylene glycol with mol. weighing 1000.

Immune splenocytes are fused to NSO cells in a 5: 1 ratio. Immune splenocytes from two mice are fused with NSO cells and distributed into 20 96-well plates with a layer of a coryphus of non-immune splenocytes.

and purified influenza A virus (Krasnodar) 101/59 (H2N2). The dose of the virus is 100 10 GAI per 0.5 ml in full Freund's adjuvant. Two weeks later, an antigen was introduced in the same ddze in Freund's complete adjuvant. Three days before the anti-. About 1 ml of antigen in saline buffer solution was administered intravenously at a dose of 50.003 GAU per mouse.

A murine myeloma line NSO was used as a malignant partner. The NSO cells are incubated under steady-state conditions in 800 ml plastic matrices. A week before they are merged, they are supported in the logarithmic growth phase by daily subculture in new matrices with the index .1: 2-1: 3. Myeloma cells grown in HAT medium composed of growth medium with m hypoxanthine, 1.6-10 M thymidine and 4 aminopterin.

20 Starting from 3 days from the moment of cultivation and on the following days, 2 drops of fresh selective medium are added to the wells of the culture plates every 3–4 days

25 O is removed, 1 ml of the culture medium and replaced with fresh. Starting from 20 days from the moment of cultivation, the cells from the wells, in which they observe the growth of hybrid cells, are subcultured

30 to 24-well plates with a layer - feeder of non-immune splenocytes. Non-immune splenocytes are resuspended in NT medium (selective medium without aminopterin) and O, 1 ml distributed in the growth medium of the next group is sprinkled into 24-well plates (splenocia: RPMI-1640 medium or DMEM with 0.13% you, obtained from one mouse, are distributed by NaHCOj, glucose Up to 4 g / l, 5% serum of cow embryos and 5% serum of dried sheep, purified with polyethylene glycol, pyruvate sodium 0.05 40 mg / ml, oxaloacetate 0.15 mg / ml, insulin 0.2 u / ml, 2-mercaptoethanol

5-10 M, HEPES 5-10 shM, gentamicin

) - 50 µg / ml.

Immune splenocytes are obtained on the third day after the 45th booster intravenous injection of antigen to mice from sterile-extracted spleens. Splenocytes are removed by repeatedly injecting spleen O into the pulp with 0.1-0.2 ml of cold growth medium RPVI-1640 or DMEM using a thin needle with a syringe.

Extracted splenocytes are collected in plastic cooled | 1e tubes, j precipitated by centrifugation at 1000 rpm for 10 minutes, the cell sediment with erythrocytes is treated for 5 minutes in the cold

divided into two 24-well plates). Hybrid-cell clones growing in 24-well plates with a layer - feeder are grown until a continuous layer is formed, and then cells are subcultured, t into 24-well plates without a layer - feeder. After 10-14 days, the hybridoma medium with a high serum content is replaced with medium with a reduced concentration of sigorotki (2-4%).

Screening of supernatants from growing hybrid cultures is carried out with the help of a solid phase enzyme immunoassay (ELISA), influenza A virus antigens for the ELISA are prepared by infecting kidney cells of dogs (MDSK line). As a control antigen, similarly prepared noninfi schr, analogous cells of MDSC, serve. Antibody-producing clones of hybrid cells are grown in plastic bottles

in a HAT medium composed of a growth medium with m hypoxanthin, 1.6-10 M thymidine and 4 aminopterin.

Starting from 3 days from the moment of cultivation and on the following days, 2 drops of fresh selective medium are added to the wells of the culture plates every 3–4 days

0 ml of the culture medium is removed and replaced with fresh. Starting from 20 days from the moment of cultivation, the cells from the wells, in which they observe the growth of hybrid cells, are subcultured

in 24-luyichny plates with a layer - a feeder from not immune splenocytes. Non-immune splenocytes are resuspended in NT medium (selective medium without aminopterin) and O, 1 ml distributed in 24-well plates (splenocytes obtained from a single mouse, distributed

divided into two 24-well plates). Hybrid-cell clones growing in 24-well plates with a layer - feeder are grown until a continuous layer is formed, and then cells are subcultured, t into 24-well plates without a layer - feeder. After 10-14 days, the hybridoma medium with a high serum content is replaced with medium with a reduced concentration of sigorotki (2-4%).

Screening of supernatants from growing hybrid cultures is carried out with the help of a solid phase enzyme immunoassay (ELISA), influenza A virus antigens for the ELISA are prepared by infecting kidney cells of dogs (MDSK line). As a control antigen, similarly prepared noninfi schr, analogous cells of MDSC, serve. Antibody-producing clones of hybrid cells are grown in plastic bottles

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25 cm long with a layer of a feeder from splice cells and after accumulating cells in sufficient quantity (continuous coating of the growth surface of the vial), the assessment of the MCA in culture fluids (KJ) for activity and specificity is carried out. According to the study, clones were selected for their accumulation, in mass culture and cryopreservation

As a result, a collection of hybrids with different reactivity to the influenza A virus (Krasnodar) 101/59 (H2N2) .15 is obtained. 15

The most productive strain is brought into mass culture and is designated as Cr 3-30. Strain Kp 3-30 is stored in the Speed alized collection of transplanted somatic cells of 20 vertebrates. The Institute of Cytology, USSR Academy of Sciences under the number 88D and is characterized by the following features.

Morphological signs. The cell morphology of hybridoma Kp 3-30 does not differ from the parental line. The cells are round, proliferate in suspension composition and have poor adhesion to the surface of the glass or glass.30

Cultural features. Cultivation medium - RPMI-1640 wm DMEM with 0.13%, glucose up to 4 g / l, 5-10% of embryo serum; cows or dry sheep (purified with polyethylene glycol), sodium pyruvate (0.05 mg / ml), oxaloacetate (Oj15 mg / ml), insulin (0.2 u / mp), 2-mercaptoethanol (5--10 M), HEPES-buffer (5-10 tM). Cultivation temperature of 36.5 C.

Cells grow in suspension, have a weak adhesive ability to the surface of plastic or glass. A seeding dose of 200-300 10 cells / ml. The duration of sowing is 1: 4-1: 6, passage twice a week.

Cultivation of hybridoma in the organism of the animal. Female BALB / c mice; at the age of 2-2.5 months, sensitization of the Y-C, 5 ml of pristane intraperitoneally 7-10 days prior to the introduction of 2-4-10 hybrid cells, after 10-14 days an ascites tumor is formed. Having transferred the GUEST in 100% of cases. Crocus strain activity. The titer of ICA, you are in the - via immunoassay -. available, 1-4: 10 in culture I

liquid fluid, 1:10 in ac7 1; iti

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comical fluid (as much). Stable antibody production is maintained for 70 passages in vitro and 5 passages on BALB / c arms.

Characteristics of a useful product. ICAs are classified in class (K). They specifically interact with the hemaglutinin of influenza A virus (Krasnodar) 101/59 (H2N2),

The activity of the MCA in the reaction of direct immunofluorescence (NIF) is 1: 102 (kj) and 1:10 (already), in the reaction the inhibition of geneglutination (rtga) - 1: 103 (kj) and 1: 5,140 (as much) in the neutralization reaction (PH) in chickens of embryos (with 100 EIA of the virus) - 1: 3.2–103 (already).

Contamination. The hybridoma is free of bacterial contaminants and mycoplasmas.

Cryoconservation Freezing medium - bovine fetal serum 90%, dimethyl sulfoxide 10%. 1 ml of cell suspension with a density not lower than 2-10 viable cells resuspended in a cold cryo-preservative are transferred to plastic 1.8 ml ampoules, placed in a foam box with a wall thickness of at least 1.5 cm and immediately placed on the cold ( -70) - (-80) C. On the following day, the ampoules are transferred to liquid nitrogen. Frozen ampoules are thawed in water at 37-39 ° C. Cells were diluted 10 times embryonic and shortened and centrifuged at 500 rpm for 10 minutes. The recovered cells resuspend in a growth medium concentration of 2.5. U - 3.0.-10; viable cells in 1 ml; vials transferred to plastic cultures-alyoshev. The viability after thawing is 40-60%, as determined by the differential coloration of the cells using a 0.25% trypan blue solution.

Example. ICA was extracted from as much as 20% polyethylene glycol, mol. mass 6000 and prepared on the basis of their immunoferment conjugate (the ratio of protein and re-oxidase 2: 1), immunofluorescent conjugate (the ratio of dye FITC and protein 1:50) and erythrocyte antibody diagnostic for the reaction of reverse i passive hemagglutination (RPHA).

The results of the study obtained diagnostic products with. Antiviral diagnostics.

P:. Dp mule invention

in table 1-3.

The results are shown in Table. 1-3, testify to the high actuality and specificity of the obtained diagnostic preparations “In this case, the ICA recognize a common epitope on the hemagglutinin of influenza viruses of the type.) And can be used for the ideology of the virus, influenza A (Krasnodar), the identification of viruses type in Clay- 101/59 (H2N2),

The hybrid strain of cultured animal cells Mus musculus VS M 88 Dj used to obtain monoclonal antibodies to hemagg

Control A / PR / 8 (34 / HINI)

Measles virus (L-15)

Noticeable

Control: A / PR / 8/34 (H1N1)

Measles virus (L-16) VDSK cells

EXAMPLE D)} I and e.

+4 - luminescence of .100% of cells in the visual field; 3 - glow. 75% of cells in the visual field; - - Otsugskie glow.

virological diagnosis.

P:. Dp mule invention

virus virus, influenza A (Krasnodar) 101/59 (H2N2),

virus virus, influenza A (Krasnodar) 101/59 (H2N2),

Strain of hybrid cultured animal cells Mus musculus VSCC (PM 88 Dj used to produce monoclonal antibodies to hemagglutis) Table 1

activity MKAj uelo ed,

absence of

table 2

Influenza A virus

(Krasnodar) 101 512 32

Measles virus Oh About

Table

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512 o

Claims (1)

Claim The strain of hybrid cultured animal cells Mus muscuius BCKK (Π) Μ 88 D, used to obtain monoclonal antibodies to hemaggluti Influenza A virus (H2K.) Indicators for peroxidase ata based on monoclonal antitags (inverse values) for conjun: G 1000 .2000 '· ·' < 40.00 8000 10,000 Krasnodar (101) 59 y +4 +4 +3 4- Leningrad (134) 57 v4 :-4 +4 + 3 4- Leningrad (549) 80 ^; • M h-4 +4 +3 4- Control A / PR / 8 (34 / HINI) - - - - Measles virus (L-1 S) t - Note. +4, +3, + - activity of MCA, conv. units - ι reactions. lack of table 2 Leningrad (134) 57 +4 +4 +4 +4 +3 Leningrad (549) 30 +4 +4 +4 +4 +3 Control: A / PR / 8/34 (H1 N1) Measles virus (L-16) MDS cells Note: +4 - luminescence of .100% of cells in the field of view; +3 - glow. 75% of the cells in sight; - - lack of a luminescence. Table 3 Test material ROPGA antigen titer (inverse) Extinguishing ROPHA with antisera (antigen titers, inverse values) pluripotent homologous monoclonal homologous heterotropic Influenza A virus (Krasnodar) 101 512 32 0 512 Measles virus 0 0 0 0