SU1541254A1 - Strain of hybrid cultivable mus musculus cells used for producing monoclonal antibodies to thermally stable o-antigen of cholera germ - Google Patents

Abstract

The invention relates to hybridoma technology and can be used for the serological identification of the cholera pathogen serovar Ogawa. The purpose of the invention is to obtain a strain of hybrid cultured animal cells MUS MUSCULUS producing monoclonal antibodies (monAT) to the thermostable O-antigen of cholera vibrio serovar Ogawa. The strain is obtained by hybridizing the cells of the mouse myeloma P3-X63 / Ad8.653 with the spleen cells of mice immunized with the bacterial suspension VIBRIO CHOLERAE strain 3119 of the Ogawa serovar. Cells are cultured in RPMI 1640 medium with 10% emb. tel. Syv. pass 1 time in 3 - 4 days, sowing ratio 1: 4-1: 5. MonAT belongs to the class Idd, their titer in the culture fluid is 1: 2000 (ELISA), 1:60 (NIF), in ascetic fluid - 1: 80000 (ELISA), 1: 6000 (NIF). MonAT are specific to the thermostable O-antigen of Ogawa serovar strains and can be used for diagnostic purposes. The strain deposited under the number VSKK (P) N 237 D.

Description

The invention relates to biotechnology and can be used for the serological identification of the cholera pathogen serovar Ogawa.

The purpose of the invention is to obtain a hybrid strain of cultured animal cells Mus. musculus producing monoclonal antibodies (monAT) to the thermostable 0-antigen of cholera vibrio serovar Ogawa.

Strain was prepared as follows.

White mice are immunized intraperitoneally three times with an interval

10 days of bacterial suspension of Vibrio cholerae serovar Ogawa, heated at a temperature of 100 ° C at a dose of | 10 m0kl.

A booster injection of the antigen is carried out 4 days before hybridization. P3-X63 / Ag 8.653 mouse myeloma cells were fused to mouse spleen cells using 50% polyethylene glycol with a molecular weight of 1500 D. Under sterile conditions, the spleen is extracted and crushed using a homogenizer. Cell suspension

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rugot through 2 layers of gauze, and red blood cells are destroyed with a 0.83% solution buffered with Tris buffer. Lymphocytes are washed twice with Kgla-MEM medium and mixed with twice washed myeloma cells in a 5: 1 ratio (spleen cells: myeloma cells). The cell mixture was washed once in a 50 ml centrifuge tube, the supernatant was decanted, and 1 ml of 50% polyethylene glycol solution was added dropwise to the dry precipitate at 42 ° C for 1 min (in a water bath). After that, the serum-free Eagle-MEM medium is introduced into the centrifuge tube slowly. Cell suspension is incubated at 37CC for 10 min, then the supernatant is decanted and the mixture of cells is carefully resuspended in 40 ml of growth medium (RPMI 1640 or DMEM medium with 20% calf fetal serum, 4 g / l glucose,. 0.1 M pyruvate sodium) containing GAT components (0.0131 mg / ml of hypoxanthine, 0.000191 mg / ml of aminopterin, 0.00385 mg / ml of thymidine). Cell suspension in HAT medium was poured over 0 ml of B four 96-well flat-bottomed culture plates onto the feeder layer of pre-harvested (per day) peritoneal macrophages of white mice at a dose of 10,000 per well. Visible colonies of mice of hybrid cells appear on days 7-10. Hybridomas are cultured for 14 days on HAT medium, then 7-10 days on HT medium, after which they are transferred to germinating medium without selective components. Hybridoma is cloned twice by limiting dilution method on a feeder from peritoneal macrophages of white mice. As a result of cloning, a productive strain of hybrid cultured mouse cells stably producing MA of a given specificity is obtained.

The strain is designated GC-B7 / Og, it is stored in the Specialized Collection of Transplanted Somatic Cells of the Vertebrates of the Institute of Cytology of the Academy of Sciences of the USSR under the number VSKK (P) No. 237 and is characterized by the following features.

Morphological signs. Hybrid cells GC-V7 / OG are large round cells with narrow

rim of basophilic cytoplasm. The nucleus is hyperchromic, irregular-oval in shape, large, with a rough structure. nuclear chromatin swarm In a significant part of the cells, the cytoplasm has outgrowths,

Cultural characteristics are typical of mouse mouse cells.

plasma cell GC-B7 / Og cells are cultivated as a stationary suspension at 37 ° C in plastic or glass containers with a seed dose of 100–1000 thousand / ml for 3–4 days to%

 density of saturation 800-900 thous. Cells are passaged once every 3-4 days, with the same dose. Sowing ratio 1: 4-1: 50 Cultivation is carried out on a nutrient medium RPMI 1640 or DMEM,

0 containing J 0% fetal bovine serum, 2 mM glutamine, 8-10 mM HEPES buffer, 50 µg / ml gentamicin Cultivation of the strain in the body of experimental animals Knduk5 Day of ascites tumors in inbred mice of Ba1b / s line, intraperitoneally primed 14 days before injection prystanom hybridomas in a dose of 0.5 ml / mouse, carried out by inoculation

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1010 hybrid cells per animal. Immune ascitic fluid is collected as ascites tumors form in 12-14 days in a volume of 2-6 ml. Cell culture is maintained for two passages of an ascites tumor in mice (observation period).

Cariological signs, Modal chromosome number 87, C-markers of the parent myeloma line P3-X63 / Ag 8.653 o

Characteristics of monoclonal antibodies

MonAT belongs to the IgG class. They only revealed the Ogawa serovar 0-antigen of Vibrio cholerae, not giving cross-reactivity with the Inaba serovar vibrios and vibrios of non-01 groups and representatives of closely related families. Antibodies are tested in direct immunofluorescence, enzyme immunoassay, expanded agglutination, co-agglutination, and precipitation using the unstable 0-antigen of the cholera vibrio seros Ogaw and Inaba as the antigen preparation. The class of immunoglobulins is determined in an Ouchterloni precipitation reaction.

The titer of monAT in the culture fluid is 1: 2000 (ELISA), 1:60 (NIF), in ascites - 1: 80,000 (ELISA), 1: 6000 (NIF).

Contamination Bacteria and fungi are not detected in cell culture. Mycoplasma infection is not revealed.

Cryopreservation0 Strain cells are resuspended in RPMI 1640 medium containing 50% fetal calf serum and 10% glycerol or 10% dimethyl sulfoxide. Freezing is carried out in a programmatic freezer with a decrease in temperature of 1 ° C (down to -4 ° C), then 1 0 ° С (up to -1 00 ° С). The cells are then placed in liquid nitrogen. The defrosting is carried out quickly in a water bath at 37 ° C. The cell viability of trypan blue incorporation is 78-92%.

The study of the specificity of monat.,

A set of cloned cholera vibrio strains of Ogawa and Inaba serovars and representatives of the families of Vibrionaceae, Pseudomonadaceae, Enterobacteriac eae is used to determine the specificity of monat produced by hybridomas GC-Br / Og.

Bacterial cells of cholera vibrios of Ogawa and Inaba serovars and closely related microorganisms are used as antigenic preparations in the serological reactions studied.

When studying the specificity of monAT in the reaction of direct immunofluorescence and enzyme immunoassay, an active interaction of immunoglobulins with determinants of the 0-antigen of Ogaw's serovar strains was established.

In the antibody precipitation reaction, the hybridoma GC-B7 / Og, in afape, forms one clear precipitation line with the Vibrio cholera cells of the Ogawa serovar and does not interact with the Inaba serovar cells and closely related microorganisms.

When formulating the reaction of the unfolded agglutination of monat, produced by hybridomas GC-B7 / Og, have

5 the ability to agglutinate cells of Vibrio cholerae serovar Ogawa in titers up to 1: 8000.

EXAMPLE From the studied culture, isolated from objects of the environment or from humans, 1 billion suspensions are prepared. A drop of cell suspension is pipetted onto the glass and mixed with a drop of the monAt drug, gently shaking the glass „Education

5 large agglutinates within 30 s - 5 min indicates the presence in the sample of Vibrio cholerae serovar Ogawa.

The use of monats produced by hybridomas allows for early and rapid diagnosis of the cholera pathogen.

Claims (1)

Invention Formula The strain of hybrid cultured animal cells Mus musculus VASK (P) No. 237D used to obtain monoclocal antibodies to the thermostable Ogawa Vibrio 0-antigen of the cholera vibrio.