SU1631072A1 - Strain of hybrid cultured animal cells mus musculus l employed for producing monoclonal antibodies for glycoproteid structural protein of rabies virus - Google Patents

Description

 Eo petenene is a hybrid technology and can be used to diagnose rabies virus.

Usj. This invention is derived from a hybrid, cultured, sip strain that generates monoclonal antigens to the glycoprotein structural base i;: y of the rabies virus (VB).

 receive as follows.

ituuHHfcie myelomic ibredine cells with spleen cells

Balb / c mice immunized with rabies Vnu virus “- ovo-32, Hybrid clones are cultured on GAT selective medium. Using enzyme immunoassay and indirect immunolluorescence, clones that produce monoclonal antibodies to the glycoprotein protein of the rabies virus, Vnukovo-32 strain, are detected. By serial pass;, the cells of one of the most productive cichones are brought to a mass culture. Shtahm

s

X5

3

produces a monoclinal antibody to the glycoprotein protein of the rabies virus, the Vnukovo-32 strain for 17 passages (observation period).

The strain was named Kriv-1 C5 and deposited in the specialized collection of transplanted somatic vertebrate cells of the All-Union Cell Culture Collection at the Institute of Cytology, USSR Academy of Sciences, Leningrad under the number VSKK (N) No. 320D. Cultural features. The standard culture conditions are Dulbecco's medium with 20% fetal calf serum, 2 mM glutamine and 50 µg / ml gentamicin. Growth pattern — stationary suspension. Shooting method - shaking. Passing frequency - after 2-3 days. Seeding dose - 150,000 cells in 1 ml. The multiplicity of sieving - (1: 2) - (1: 3).

Cultivation in animals.

In the abdominal cavity of Balb / c mice, previously sensitized for 1-2 weeks with intraperitoneal injection of 0.3-0.5 ml of mineral oil 2, 4, 105 14 with tetramethyl pentadecane, hybrid cells are injected. The time of tumor formation is 9-12 days; over ten passages, 100% ascites transplantability is observed.

Cell morphology.

The culture consists of rounded cells of varying size with large cores that are weakly attached to the substrate. Cytoplasm has the appearance of a thin rim.

Cariologists.

The karyotype corresponds to the form (mouse). By staining with the C-method, three marker chromosomes introduced into this line from the parent myeloma line.

The productivity of the strain. The Kriv-1-C5 hybridoma strain produces monoclonal antibodies to the glycoprotein of the rabies virus when cultured for seventeen passages in cell culture and ten passages in animals (observation period). The antibody titer in the NIF for culture liquids is 1: 810 and 1: 65610 for ascent fluids. In the neutralization reaction in mice, the antibody titer is. 1: 500. Con

n .

fi

pc - 10

15

20

25

1072

the concentration of monoclonal in ascites fluid is 4-6 mg / ml.

Characteristics of the product obtained.

Mouse monoclonal antibodies to the glycoprotein protein of the rabies virus specifically react with the glycoprotein of the rabies virus in the reaction of indirect immunofluorescence (HMFA) 5 of the neutralization reaction in mice and the indirect reaction of immunofermental analysis (NIF).

The class of immunoglobulins.

The strain of hybrid cells Kriv-1-C5 secretes antibodies of the IgG class isotype 2a,

Cryoconservation Kriv-1-C5 cells are frozen at the tenth passage. The total number of ampoules is 20 by 2 to 3 million cells. Cryoprotective medium with 50% fetal bovine serum and 10% DMSO-dimethyl sulfoxide. Survival rate when defrosting 60-70%.

Contamination,

Bacteria, fungi and yeast were not found.

Using strain

Hybrid cells Curve-1-C5 are sown in a concentration of 100–150 thousand / ml in Talbecco’s medium with 20% fetal calf serum, 2 mM glutamine, 4 g / l glucose and 50 μg / culture culture mattresses with a capacity of 250–500 ml. ml of gentamicin. The cultures were incubated in an air atmosphere with 6.2-7.0% CO at 37 ° C in the steady state for 3-4 days and used as samples of the culture fluid (QOL). The cell pellet is resuspended in 1-1.5 ml of medium without serum and 3-5mln of cells are introduced into the abdominal cavity of Balb / c mice. After 8–12 days, ascites fluid is formed, which is used as antibody samples in ascites fluid (AM) after the cells are deposited.

The activity of monoclonal antibodies (monat),

MonAT activity to the glycoproid structural protein of the rabies virus is detected by an indirect immunoassay method (ELISA) with purified rabies virus and purified glycoprotein, in the reaction of indirect immunofluorescence (HMFA), in the neutralization reaction in mice, in protection mice from &: 3200 LD50 of rabies virus introduced into the brain, as well as in a pronounced

thirty

35

40

45

50

55

516310

therapeutic effect of pre-infected animals. In ELISA antibody titers vary 5-15 million

The obtained data show that MONAT Curve-1-C5 specifically reacts with the glycoprotein of various strains of the rabies virus and does not interact with the intracellular nucleoprotein.

It has been established that only antibodies to the glycoprotein have a virus-neutralizing and protective effect against the rabies virus.

Thus, the resulting hybrid secretes monAT to the main functionally significant determinant of the glycoprotein of the rabies virus.

Claims (1)

Invention Formula Strain hybrid cultured; Animal cells Hus musculus L. BCKK (II) No. 320D, used to produce monoclonal antibodies to the glycoprotein structural protein of the rabies virus.