RU2425874C1 - CULTIVATED HYBRID CELL STRAIN OF Mus musculus L ANIMALS - PRODUCER OF MONOCLONAL ANTIBODIES SPECIFIC TO O-ANTIGENE OF CHOLERA VIBRIO SEROGROUP O1 - Google Patents

Abstract

FIELD: medicine. ^ SUBSTANCE: invention can be used in creating diagnostic test systems of monoclonal antibodies (MCA-01) for accelerated identification of cholera vibrios serogroup O1 of serovars Ogawa and Inaba. The cultivated cell strain of mouse hybridoma is produced by conventional methods used in the monoclonal antibody technology. A cloning procedure results in preparing a productive strain stably producing monoclonal antibodies isotype JgG specific to O-antigen of cholera vibrios O1 serogroup. The strain is deposited in Specialised Collection of continuous somatic cells of the Vertebrata Institute of Cytology (St.-Petersburg), No. RKKK (P) 386D. ^ EFFECT: hybridoma produced MCA-01 actively reacts with O-antigen determinants of the strains of serovars Ogawa and Inaba and do not react with related microorganisms. ^ 1 tbl, 3 ex

Description

The invention relates to biotechnology, in particular hybridoma biotechnology, and can be used in healthcare when creating diagnostic test systems based on monoclonal antibodies for the accelerated identification of cholera vibrios O1 of the serogov group Ogawa and Inaba.

Currently, the urgent problem of creating diagnostic products for serological and immunological methods for identifying the causative agent of cholera using highly specific monoclonal antibodies (MCAs) produced by hybridomas to increase the sensitivity and specificity of laboratory tests.

There is a method of enzyme immunoassay (see A.V.Sokolenko, L.P. Alekseeva et al., "The study of the interaction of uncultivated forms of cholera vibrios with monoclonal antibodies to lipopolysaccharide O1 and O139 serogroups", journal "Biotechnology", 2004, No. 3, p. 87-92), which consists in using a panel of monoclonal antibodies obtained against the O-antigen of serogroup O1 cholera vibrios, and then assessing the preservation of lipopolysaccharide (LPS) epitopes in O1 cholera vibrios during their transition to an uncultured state. Using the MCA kit, various LPS epitopes were identified, including epitopes that are often found in O1 cholera vibrios in an uncultured state.

However, the known method for producing MCAs was used to detect uncultivated forms of vibrios in microcosms of different compositions, while MCAs were not considered as diagnostic reagents for creating new generation preparations that could be used to analyze a wide range of strains of typical cholera vibrios O1 of the serogroup.

For the prototype, we chose a method for producing hybridomas producing monoclonal antibodies to V. cholerae O1 (see E.A. Mikheeva, N.E. Tereshkina et al., "Features of obtaining hybridomas producing monoclonal antibodies to" Vibrio cholerae O1 ", Problem materials Commission “Cholera and Vibrio pathogens for humans”, Rostov-on-Don, 2009, pp. 70-71), by immunizing mice disinfected with Na Merthiolate with whole cells of V. cholerae 569B (classic biovar, Inaba serotype), and then, after the fusion of immune splenocytes and myeloma, three types of hybridomas are produced that produce MCA, for example claimed to epitopes of the surface structures of the bacterial cell V. cholerae 569 B, mAb to the O-antigen mAb to surface structures and the O-antigen.

The disadvantage of this method is that the MCA of these hybridomas were tested only with cells of strain 569 B and with the O-antigen preparation, data on interaction with cholera vibrios of Serovar Ogawa and with cultures of closely related microorganisms are not presented, therefore it is not known whether hybridomas are species-specific or serovar-specific.

The technical result of the invention is the search for a strain of hybrid cultured animal cells of animals Mus. musculus producing diagnostically significant monoclonal antibodies to epitopes of the species-specific antigen of cholera vibrios O1 of the serogroup.

The specified technical result is achieved by obtaining a strain of cultured hybrid animal cells Mus. musculus L-producer of monoclonal antibodies specific for the O-antigen of cholera vibrios O1 of the serogroup deposited under the number RACC (P) 386D in the “Specialized Collection of Transplantable Somatic Vertebrate Cells” of the Institute of Cytology (St. Petersburg).

A method of obtaining a strain includes several stages:

I - receiving immune splenocytes. For this, white mice are immunized intraperitoneally three times with an interval of 10 days by bacterial suspension of cholera vibrios O1 of the serogroup, heated for 15 min at a temperature of 100 ° C in a dose of 10 ⁸ -10 ⁹ mcl A booster injection of antigen is carried out 4 days before hybridization. In sterile conditions, the spleen is removed and crushed using a homogenizer. A suspension of cells is filtered through 2 layers of gauze, and red blood cells are destroyed by 0.83% NH ₄ Cl solution, buffered Tris buffer (pH 7.4). Lymphocytes are washed twice with Igla-MEM medium.

II - hybridization. For this, mouse myeloma P3-X63 / Ag 8.653 cells are fused to spleen cells of immune mice using 50% polyethylene glycol with a molecular weight of 1500 D (Serva). For this, twice washed myeloma cells and lymphocytes are combined in a 5: 1 ratio (spleen cells: myeloma cells). The mixture of cells is washed once in a 50 ml centrifuge tube, the supernatant is decanted and 1 ml of a 50% solution of polyethylene glycol is added dropwise to the dry precipitate for 1 min. After this, the serum-free Igla-MEM medium is slowly added to the centrifuge tube. The cell suspension is centrifuged at 37 ° C for 10 min, after which the supernatant is decanted and the cell mixture is carefully resuspended in 40 ml of growth medium (RPMI 1640 or DMEM supplemented with 20% fetal calf serum, 4 mM / ml glutamine, 4 g / l glucose , 0.1 M sodium pyruvate) containing GAT components (0.0131 mg / ml hypoxanthine, 0.000191 mg / ml aminopterin, 0.00385 mg / ml thymidine).

III - cultivation of hybridomas. A suspension of fused cells in a GAT medium is poured into 0.1 ml into four 96-well flat-bottomed culture plates onto the feeder layer of pre-seeded (per day) peritoneal macrophages of white mice at a dose of 10,000 per well. Visible colony of mouse hybrid cells appear on day 7-10. The hybridoma is cultivated for 14 days on GAT medium, then 7-10 days on GT medium. Then transferred to the growth medium without selective components.

IV - hybridoma screening. Hybridoma culture fluids were carefully selected for ELISA testing.

V - cloning. The hybridoma is twice cloned by the method of limiting dilutions on a feeder from peritoneal macrophages of white mice. Cloning yields a productive strain of hybrid cultured mouse cells that stably produce MABs of a given specificity.

The strain was deposited in the Specialized collection of transplantable somatic cells of vertebrates of the Institute of Cytology (St. Petersburg) under the number RKKK (P) 386 D.

The inventive strain of hybridomas has the following characteristics:

Morphological signs. Hybrid cells are large rounded cells with a narrow rim of the basophilic cytoplasm. The nuclei are hyperchromic, irregularly oval, large, with a rough structure of nuclear chromatin. In a significant part of the cells, the cytoplasm has outgrowths.

Cultural traits are typical of transplanted mouse cells with plasmacytomas. Cells are cultured in the form of a stationary suspension at 37 ° C in a plastic or glass dish at a sowing dose of 100-150 thousand cells / ml for 3-4 days to a saturation density of 800-900 thousand cells / ml cells are passaged once every 3- 4 days with the same dose. Sifting ratio 1: 4-1: 5. Cultivation is carried out on RPMI 1640 or DMEM nutrient medium containing 10% fetal calf serum, 2 mM glutamine, 8-10 mM HEPES buffer, 50 μg / ml gentamicin.

The cultivation of the strain in the body of experimental animals. The induction of ascites tumors in white mice, intraperitoneally primed with marinas at a dose of 0.5 ml / mouse 14 days before the introduction of the hybridoma, is carried out by inoculation of 3-10 million hybrid cells per animal. Immune ascitic fluid is collected as ascites tumors form after 12-14 days in a volume of 2-6 ml. The cell culture was maintained for two passages of an ascites tumor in mice (observation period).

Karyological signs. The modal number of chromosomes is 87, C-markers of the parent myeloma line P3-X63 / Ag 8.653.

Biotechnological characteristics of a useful product. MonAT belong to the IgG isotype. They detect the O-antigen of the cholera vibrios of the Ogawa and Inaba serovars, without cross-activity with non-O1 group, O139 serogroup vibrios and representatives of the genera Plesiomonas, Aeromonas, strains of Vibrio parahaemolyticus, and Comamonas. Antibodies are tested in slide agglutination reactions, indirect immunofluorescence, enzyme immunoassay, extensive agglutination, and precipitation reactions. The class of immunoglobulins is determined in the Ouchterloni precipitation reaction. Using the immunoblot method, it was found that FATG12 MonAT hybridomas are directed to species-specific epitopes of the polysaccharide part of the O antigen with Mr 20-25 kDa, common for Ogawa and Inab serovar. The monAT titer in the culture fluid is 1: 2000 (ELISA), 1:64 (NIF), in ascites - 1: 100000 (ELISA), 1: 6000 (NIF).

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

Cryopreservation. The cells of the strain are resuspended in RPMI 1640 medium containing 50% fetal calf serum and 10% glycerol or 10% dimethyl sulfoxide. Freezing is carried out in a program freezer with a temperature decrease of 1 ° C (to -40 ° C). Further at 10 ° C (up to -100 ° C). Then the cells are placed in liquid nitrogen. Defrosting is carried out quickly in a water bath at 37 ° C. Cell viability for trypan blue inclusion is 78-92%.

MonAT specificity study. To determine the specificity of monAT produced by the hybridoma, a set of strains of cholera vibrios of the Ogawa and Inaba serovars and representatives of the genera Plesiomonas, Aeromonas, strains of Vibrio parahaemolyticus, Comamonas is used. The bacterial cells of cholera vibrios of the Ogawa and Inaba serovars and closely related microorganisms are used as antigenic drugs in the studied serological reactions.

When studying the specificity of monAT in the reaction of indirect immunofluorescence and enzyme-linked immunosorbent assay, an active interaction of immunoglobulins with the determinants of the O-antigen of the Ogawa and Inaba serovar strains was established. In the agglutination reaction on glass with strains of cholera vibrios O1 of the serogroup, agglutinate flakes are formed with enlightenment. In the precipitation reaction, antibodies of the hybridoma form in agar one clear precipitation line with the cholera vibrio cells of the Ogawa and Inaba serovars and do not interact with closely related microorganisms. When setting the reaction of expanded agglutination, monAT produced by a hybridoma have the ability to agglutinate cells of the cholera vibrios of the Ogawa and Inaba serovars in titers up to 1: 8000.

Example 1. The use of strain RKKK (P) 386 D.

To obtain monoclonal antibodies produced by the MKA-O1 hybridoma, the RKKK (P) 386 D strain is grown in vitro to a final concentration of 1 · 10 ⁶ -1.5 · 10 ⁶ cells. in 1 ml of medium. Cells are pelleted by centrifugation at 1000-1500 rpm for 10 min and injected intraperitoneally to Balb / c mice pretreated with piers at (5-10) · 10 ⁶ cells in a volume of 1.5-2 ml. After the formation of an ascites tumor, immuno-ascitic fluid is sampled. Cells are precipitated from the IAH by centrifugation at 1000-1500 rpm for 10 min and used for subsequent passage in vitro and in vivo.

The supernatant containing MAB is used in ELISA, slide agglutination, indirect immunofluorescence to identify Vibrio cholerae O1 strains of the serogroup, or is used as a semifinished product for isolation of monoclonal antibodies by double precipitation with 50% saturation with ammonium sulfate, more suitable for long-term storage at minus 20 ° C, without losing specific activity for 2 years (observation period), which can be used in the development of immunoglobulin preparations and test systems for laboratory diagnosis of cholera Ry.

Example 2. The use of the product of the strain.

To study the specificity of monoclonal antibodies produced by RKKK (P) 386 D hybridoma, we studied the agglutination reaction on a glass of a culture of cholera vibrio O1 serogroups, as well as cultures of closely related microorganisms (see table). When setting up the reaction, a drop of MKA-O1 preparation in working dilution and a drop of 0.9% sodium chloride solution are applied to the glass, and then the test culture is added to them, thoroughly rubbing with a loop or a glass rod, while shaking the glass. A positive reaction is characterized by the formation of a clear coarse or fine-grained agglutinate within 30 seconds, which indicates the presence of serogroup cholera vibrios O1 in the test sample. In the control with a 0.9% sodium chloride solution, the microbial suspension remained homogeneous. The absence of visible agglutinates is regarded as a negative reaction.

Thus, from the results presented in the table, it is clear that of 62 strains of cholera vibrios eltor serovar Ogawa, all were agglutinated with MKA-O1, and of 35 strains of Inaba, only one strain was not agglutinated with MKA-O1. All studied strains of the classical biovar were agglutinated with MKA-O1. From this it follows that the specificity of the studied reaction with cholera vibrios O1 of the serogroup presented in the table is 99%. The absence of cross-reactions with closely related microorganisms presented in the table is also an indicator of the high specificity of MCA hybridoma RKKK (P) 386 D.

Example 3. Confirmation of the specificity of MKA-O1 in the formulation of an indirect version of the method of luminescent antibodies (NIFM).

To conduct a reaction to a fixed smear of the test culture, a drop of MKA-O1 preparation is applied in working dilution, incubated in a moist chamber at 37 ° C for 30 minutes. The smear is washed, anti-mouse immunoglobulins labeled with FITC are applied, incubated in a humid chamber at 37 ° C for 30 minutes. Then the glass is washed, dried and the smear is examined under a microscope in luminescence mode. The characteristic luminescence of +++ and ++++ indicates the presence of cholera vibrios O1 of the serogroup. The absence of a specific glow or dim glow less than +++ is regarded as a negative reaction. From the results of the table it follows that the specificity of MKA-O1 in the indirect immunofluorescence reaction is 97%.

Thus, summarizing the results of our studies (see the table), we can conclude that the monoclonal antibodies produced by the hybridoma are directed to such an epitope in the structure of the O antigen, which is widely represented both in cholera vibrios of Serovar Ogawa and in strains of Serovar Inaba . This was confirmed with respect to the studied strains of V. cholerae O1 in the reactions of slide agglutination and indirect immunofluorescence, which in this case belong to accelerated testing methods.

In addition, when studying strains of closely related microorganisms, namely, 7 RO strains, 100 strains of cholera vibrios of not O1 and not O139 serogroups showed a negative reaction, which together confirms the high specificity of the MCA of the claimed hybridoma. High specific activity is explained by the presence in the structure of the O antigen of a repeating epitope recognized by the MCA hybridoma, which determines the ability of O1 cholera vibrios to precipitate and agglutinate.

Strain RKKK (P) 386D produces class G monoclonal immunoglobulins specific for the O antigen of serogroup O1 cholera vibrios, allowing the identification of Vibrio cholerae O1 strains of Serovar Ogawa and Inaba.

In addition, MKA-O1 can be used in healthcare in the form of immunochromatographic test systems (bands), which can be effectively used in a regulated scheme of laboratory analysis for cholera at the stage of selection of suspicious colonies and obtaining pure cultures to establish their belonging to Vibrio cholerae O1.

Table No. p / p Strains The number of studied strains The number of strains agglutinating MCA-O1 The number of positive strains in NPIF with MCA O1 one V.cholerae eltor Ogawa 62 62 61 2 V.cholerae eltor Inaba 35 34 33 3 V. cholerae cholerae ogawa 7 7 7 four V. cholerae cholerae Inaba 3 3 3 5 V.cholerae O139 17 6 V.cholerae RO 7 7 V.cholerae not O1 / not O139 (2002-2008) one hundred Negative agglutination results NPIF negative results 8 Plesiomonas 7 9 Aeromonas 108 10 Comamonas thirty eleven Vibrio parahaemolyticus 12 12 E. coli 2 13 Brucella sp. four fourteen Salmonella sp. four

Claims (1)

The strain of cultured animal hybrid cells Mus. musculus L is a producer of monoclonal antibodies specific for the serogroup O1 cholera vibrio O-antigen, deposited under the number RKKK (P) 386D in the Specialized Collection of Transplantable Somatic Vertebrate Cells of the Institute of Cytology (St. Petersburg).