RU2583306C1 - Hybrid strain of cultured cells of animals mus musculus -t 2e5 - producer of monoclonal antibodies isotype g 1 to b subunit of cholera toxin - Google Patents

Abstract

FIELD: biochemistry.

SUBSTANCE: invention relates to the field of biochemistry. Disclosed is a strain of cultivated hybrid animal cells Mus musculus XT 2E5-producer of monoclonal antibodies isotype G 1 k-Cholera toxin b subunit. Strain is deposited in the state collection of pathogenic microorganisms and cell cultures "GKPM-Obolensk, no. n-44.

EFFECT: invention enables obtaining of highly specific monoclonal antibodies which can be used for constructing the enzyme immunoassay diagnostic test systems for determination of production of cholera toxin in V.cholerae strains.

1 cl, 3 tbl, 5 ex

Description

The invention relates to the field of microbiology and hybridoma biotechnology and can be used to create immunodiagnostic test systems based on monoclonal antibodies (MCAs) to determine toxigenic strains of Vibrio cholerae from cholera toxin (XT) production.

Cholera is a severe acute infectious disease caused by toxigenic strains of cholera vibrio. In the context of the ongoing seventh cholera pandemic, active migration processes and the existence of modern high-speed vehicles pose a constant threat of infection from entering anywhere in the world as soon as possible.

Currently, immunological methods for the diagnosis of cholera are used both in the framework of bacteriological research (accelerated methods for the detection of pathogens and identification of cultures), and independently - to indicate cholera vibrio in the material infected with this pathogen [Laboratory diagnosis of cholera: MUK 4.2.2218-07. -M .: Federal Center for Hygiene and Epidemiology of Rospotrebnadzor; 2007.87 p .; Kaper J.B., Morris J.G., Levine M.M. Cholera Clin. Microbiol. Rev. 1995; P.48-86].

The most promising approach in this direction is to determine the production of V. cholerae cholera toxin using enzyme-linked immunosorbent assay using MCA. Since the concentration of enterotoxin causing the disease is small [Scarlatos A., Welt B., Cooper B. et al. // J. Food. Sci. 2005.V. 70 (8). P. 121-124], it is important to develop an enzyme-linked immunosorbent assay system with high sensitivity.

A known strain of hybrid cultured animal cells of mus musculus L is a producer of class M monoclonal antibodies to cholera enterotoxin, which can be used to construct immunobiological preparations that detect cholera toxin in an amount of 1.0 ng / ml (patent RU 1835848).

Clones of hybrid cells CT-B1 / F8, CT-E6 / E10, CT-F5 / H3 of animals Mus Musculus L are also known - producers of monoclonal antibodies to cholera toxin, the use of which in the enzyme immunoassay allows to determine 0.2 ng / ml of cholera toxin ( patent RU 2401299, RU 2401300, RU 2401301).

The objective of the invention is to obtain a strain of a hybridoma producer of highly specific MCAs intended for use as immunoreagents in the design of enzyme-linked immunosorbent diagnostic test systems in order to determine the production of cholera toxin in V. cholerae strains that are superior in sensitivity to existing analogues.

The problem is solved by obtaining a strain of hybrid animal cells Mus musculus XT 2E5, a producer of monoclonal antibodies of isotype G 1 to the B subunit of cholera toxin.

The inventive strain of hybrid animal cells of Mus musculus XT 2E5 animals is obtained by hybridization of BALB / c immune mouse splenocytes with murine Sp 2/0 myeloma cells in the presence of a polyethylene glycol (PEG-4000) merging agent, followed by screening of the cell suspension in wells of 96-well culture plates cultivation in selective media with a gradual transition to a complete hybrid cell culture medium, selection of the primary clone in terms of the production of specific antibodies, its cloning and reclamation by the method of limiting dilutions.

The strain of hybrid cells of animals Mus musculus XT 2E5 is characterized by the following features.

Marker signs and methods for their assessment. The strain secrets murine immunoglobulins that specifically interact with cholera toxin. Analysis of murine immunoglobulins is carried out by enzyme-linked immunosorbent assay using XT as antigen and antibodies against mouse immunoglobulins labeled with horseradish peroxidase.

Contamination by bacteria and fungi was not detected.

Morphological signs. The culture consists of large rounded cells, similar in morphology and size to the original parental myeloma Sp 2/0.

Cultural properties. Somatic cells are incubated at 37 ° C in an atmosphere with 5% CO2 in plastic 96-well plates. Cultivation of myeloma lines is carried out in a growth medium of the following composition: Hybridomia Express Plus hybridoma medium (with HEPES, without L - Glutamine) supplemented with antibiotics (streptomycin, gentamicin, penicillin, amphotericin), 3% horse serum ("RAA", Austria) or 10 % fetal cow fetal serum ("RAA", Austria) for hybridomas, respectively.

At all stages of the hybridization of immune splenocytes with murine plasmacytoma cells, serum-free Hybridomia Express Plus medium with the above ingredients is used. This medium was used to prepare a solution of a merging agent, polyethylene glycol 4000. For this, 1 g of PEG was autoclaved at 110 ° C for 30 min and dissolved in 1 ml of medium immediately before the fusion.

The selective medium consists of a hybridoma culturing medium supplemented with a 50-fold solution of HAT or NT. To dissolve HAT or NT, deionized water was used, which was sterilized using membrane filters with a pore diameter of 0.22 μm. After repeated screening to accumulate a preparative amount of hybrid cells synthesizing monoclonal antibodies, further cultivation was continued on hybridoma medium.

The strain is a monolayer-suspension culture in which up to 15% of the cells are in suspension, without attaching to the surface of the dishes for cultivation. Sowing dose - 10 ⁵ cells / ml. Passaging frequency is 2-3 days.

Cultivation of the strain of hybrid cells 2E5 in the animal.

8-12 week old mice weighing 20 g of the BALB / c inbred line, syngeneic to Pl.2 plasmacytoma lines Sp.2 / 0-Ag.8, previously primed with 0.5 ml pristan, were injected intraperitoneally with 2 × 10 ⁶ hybrid cells. The formation of ascites tumors is recorded in 100% of bio-producers at the same time (on day 9-10). On average, the volume of immuno-ascitic fluid from one mouse was 6.5–7.0 ml at a cell concentration of 7–8 × 10 ⁶ m. in 1 ml.

Antibody-producing activity, which was determined by indirect enzyme-linked immunosorbent assay, remained constant for 1 year and reproduced well in cell culture, which indicated the stability of the main properties of strain 2E5 both in vivo and in vitro.

The characteristic of a useful product. The strain of hybrid cells XT 2E5 produces specific monoclonal antibodies to cholera toxin, the titer of which is 1: 20,000 in the culture fluid, and 1: 2,000,000 in the immunostasic fluid. The immunoglobulin fraction was isolated by double precipitation with a saturated to 45-50% solution of ammonium sulfate. The concentration of immunoglobulins was determined electrophoretically at a wavelength of 280 nm.

Strain productivity. MCA production in the cultivation medium ranged from 15 μg / ml to 50 μg / ml, in ascitic fluid - 10-30 mg / ml. Stable MCA production is maintained for at least 10 passages in vitro and 10 passages in vivo (observation period).

Cryopreservation. Plasmocyte lines and positive clones of hybrid cells outside the period of experiments on the accumulation of preparative amounts of MCA 2E5 hybridoma cells were stored in cryopreservation tubes with fetal serum and added as cryoprotectant 10% dimethyl sulfoxide at minus 196 ° C in Dewar vessels with liquid nitrogen. Thawing was carried out at 37 ° C in a water bath for 2-3 minutes. Cell viability after thawing is at least 80%.

Properties of a useful product. Isotyping of the resulting MCA was performed using the Mouse Monoclonal Antibody Isotyping Reagents kit (Sigma, USA) according to the manufacturer's instructions. The XT 2E5 hybridoma produces MCAs belonging to class G immunoglobulins of subclass 1. The affinity constant was determined by the JD Beatty method. - 2.0 ± 0.10 10 ⁹ M ^-1 [Beatty JD, Beany BG, Vlahos WG Measurement of monoclonal antibody affinity by non-competitive enzyme immunoassay // J. Immunol. Methods - 1987. - 100. - P. 173-179].

The strain of hybrid cells XT 2E5, a producer of MCA of isotype G 1 to B, a subunit of cholera toxin, was obtained at the Russian Microbiological Research Institute of Anti-Plague Rospotrebnadzor and deposited at the State Collection of Pathogenic Microorganisms and Cell Cultures GKPM-Obolensk under number H-44.

The invention is illustrated by examples.

Example 1. Obtaining a strain of hybrid cells of animals Mus musculus XT 2E5.

The strain is obtained by hybridization of splenocytes of BALB / c hyperimmune mice with Sp.2 / 0-Ag.8 myeloma cells defective in the synthesis of the hypoxanthine-guanine-phosphoribosyltransferase enzyme. For immunization of BALB / c mice, the optimal schedule was for a 5-fold intraperitoneal administration of XT at a dose of 2 μg with a 3-day interval.

After a complete immunization cycle, the titers of specific antibodies in animal sera are determined by indirect enzyme-linked immunosorbent assay.

Hybridization. Two days before the fusion, a booster vaccination is performed, and 96-well plates with a feeder layer of cells are prepared. Hybridization of mouse immunosplenocytes and myeloma cells is carried out in a ratio of 1: 5 in the presence of 1 ml of 4000 polyethylene glycol for 1 min.

Selection. After washing the polyethylene glycol, the cells are plated on 96-well plates with a feeder. In the first stage after hybridization, a selective NAT medium is used. After 5-7 days, clones of hybrid cells were observed in an inverted microscope. Starting from the 14th day after the fusion, hybrid cultivation was carried out on a medium with NT; after 5-7 days, they were transferred to a growth medium without selective components.

Screening of hybrid clones. To select hybridoma-producing MCAs for type-specific epitopes on the 14th day after hybridization, the culture fluid of the wells with proliferating clones is tested for the presence of antibody-producing hybridomas in an indirect enzyme-linked immunosorbent assay. Wells of polystyrene ELISA plates sensitize XT and its B subunit at a concentration of 20 μg / ml and 5 μg / ml, respectively. The plates are incubated at a temperature of 37 ° C for 2 hours or at a temperature of 4 ° C for 16-18 hours.

After the incubation time, the wells are blocked with a 0.5% solution of skimmed milk powder in phosphate-buffered saline (PBS). Incubated at 37 ° C for 30 minutes. After removing the blocking solution, the FSB wells are washed three times, then the culture fluid is added from those wells where the number of cells covers 1/3 of the bottom of the well. The plates are incubated at 37 ° C for 1 h. Then, the wells of the plates are washed three times with FSB and a solution of the white mouse labeled with horseradish peroxidase labeled with horseradish peroxidase labeled anti-species antibodies against Ig G (H + L) is added. As a diluting liquid, a 0.5% solution of Tween-20 in FSB and a 0.5% solution of lactalbumin in a ratio of 1: 9 are used. The plates are incubated at 37 ° C for 1 h. Shake up the liquid from the wells and wash 6 times. Then, 100 μl of substrate-indicator mixture is added to each well of the plates. The tablet is placed on a shaker and incubated for 30 minutes with gentle shaking. The results are taken into account spectrophotometrically, determining the optical absorption at a wavelength of 405 nm.

Cloning. Hybridomas with maximum and stably recorded optical density values were selected for cloning by limiting dilution in 96-well plates with a feeder layer of cells. Clone screening was performed by indirect enzyme-linked immunosorbent assay as described above.

Cryopreservation. Plasmocyte lines and positive clones of hybrid cells are stored in cryopreservation tubes with fetal serum and 10% dimethyl sulfoxide added as cryoprotectant at a temperature of minus 196 ° C in Dewar vessels with liquid nitrogen.

Example 2. Cultivation of a strain of hybrid cells XT 2E5, secreting MCA to cholera toxin, in vivo.

To mice weighing 18-20 g of the BALB / c inbred line, after implantation 14 days later, hybrid cells were injected intraperitoneally at a dose of 2 × 10 ⁶ per mouse. The formation of ascites tumors was observed in 100% of bio-producers at the same time (on days 9-10). On average, the volume of immuno-ascitic fluid from one mouse was 6.5-7.0 ml at a cell concentration of 7-8 · 10 ⁶ mk. in 1 ml. Both indicators were characterized by approximately the same values in all passages. Cells from ascitic fluid can be used for further transplantation or stored in a cryopreserved state, and the antibody titer is determined in the supernatant using an enzyme-linked immunosorbent assay as described above. The characteristics of the strain of hybrid cultured animal cells of animals Mus musculus XT 2E5 are presented in table 1.

Example 3. Determination of clone productivity in culture and immuno-ascitic fluids.

Wells of a polystyrene ELISA plate adsorb XT at a concentration of 20 μg / ml. The plate is incubated at a temperature of 37 ° C for 2 hours or at a temperature of 4 ° C for 16-18 hours.

After the incubation time, the wells are blocked with a 0.5% solution of skimmed milk powder in the FSB. Incubated at 37 ° C for 30 minutes. After removing the blocking solution, the FSB wells are washed three times. The test culture and immuno-ascitic fluids are titrated, starting from a dilution of 1/5000 and 1/2000, respectively, up to the 12th well, sequentially transferring 100 μl of fluid from each previous well to the next. The plate is incubated at 37 ° C for 1 h. Then, the wells of the tablet are washed three times with FSB and a solution of the white mouse labeled with horseradish peroxidase labeled anti-IgG (H + L) conjugate is added in working dilution. As a diluting liquid, a 0.5% solution of Tween-20 in FSB and a 0.5% solution of lactalbulin in a ratio of 1: 9 are used. The plate is incubated at 37 ° C for 1 h. Shake up the liquid from the wells and wash 6 times. Then, 100 μl of substrate-indicator mixture is added to each well of the plate. The tablet is placed on a shaker and incubated for 30 minutes with gentle shaking.

In the wells of the plate where the reaction took place, staining from saturated green to light green appears. The staining intensity decreases as the concentration of specific antibodies introduced decreases. In wells that do not contain specific antibodies, the substrate is colorless or slightly stained. The results show that the productivity of the clone in the culture and immunoascytic fluids was 1: 20,000 and 1: 2,000,000, respectively.

Example 4. Isolation of purified monoclonal antibodies produced by the strain of hybrid cells XT 2E5.

Isolation of the immunoglobulin fraction is carried out by double precipitation with a saturated solution of ammonium sulfate to 45-50% saturation. To do this, the centrifuged ascitic fluid is diluted 2 times with distilled water and the first saturation with a saturated solution of ammonium sulfate is carried out to 45%, left overnight at 4 ° C. The day after centrifugation, a second saturation is carried out with a saturated solution of ammonium sulfate to 50%, also left overnight at 4 ° C. The release of the immunoglobulin fraction from ammonium sulfate is carried out using dialysis against saline.

Example 5. Determination of the specific interaction and sensitivity of the immunoglobulin fraction of the strain of hybrid cells XT 2E5 with cholera toxin in an enzyme-linked immunosorbent assay.

For non-specific sorption, MCA at a concentration of 10 μg / ml is added to the wells of the polystyrene tablet. The plates are incubated at 37 ° C for 2 hours or at 4 ° C for 16-18 hours. After the incubation time, the MCA solution is removed and blocked with a 0.5% lactalbumin solution, keeping the plate in an thermostat at 37 ° C in within 30 minutes 200 μl are added to the first well of the titration series, and 100 μl to the rest. Then, XT in an amount of 100 ng / ml is added to the first well and 100 μl of the well contents are transferred sequentially, with pre-mixing. The tablet is kept in a thermostat at a temperature of 37 ° C for 1 h. Next, the wells of the tablet are washed three times with FSB and a solution of peroxidase immunoglobulin monoclonal conjugate is added in working dilution. After incubation at 37 ° C for 1 hour, the wells of the plate are washed 6 times with PBS and the substrate mixture is introduced. The reaction is taken into account photometrically at a wavelength of 405 nm by a color change in the wells. To do this, calculate the arithmetic mean value in the wells with a negative control sample - Opsr (K-). The result of the analysis was considered positive if OPOb ≥ Opps (K-) × 2. The result of the analysis was considered negative if OPO <OPsr (K-) × 2, where OPOb is the optical density in the well with the analyzed sample).

According to the results of the analysis, the specific sensitivity of the interaction of monoclonal antibodies of the strain of hybrid cultured animal cells of Mus musculus XT 2E5 with cholera toxin is 0.05 ng / ml, which exceeds the known analogues. The results of specific sensitivity with optical density values are presented in table 2. Comparative characteristics of the strains of the analogues and the claimed, are presented in table 3.

Thus, the claimed strain of hybrid cultured animal cells of mus musculus XT 2E5 provides for the production of immunoglobulins specific for the B subunit of cholera toxin belonging to class G immunoglobulins of subclass 1. Purified monoclonal antibodies specifically interacted with the cholera toxin preparation in an enzyme immunoassay, the sensitivity of which is 0, 05 ng / ml.

Strain of hybrid cultured animal cells Mus musculus XT 2E5 - producer of monoclonal antibodies of isotype G 1 to B - subunit of cholera toxin

Strain of hybrid cultured animal cells Mus musculus XT 2E5 - producer of monoclonal antibodies of isotype G 1 to B - subunit of cholera toxin

Strain of hybrid cultured animal cells Mus musculus XT 2E5 - producer of monoclonal antibodies of isotype G 1 to B - subunit of cholera toxin

Claims (1)

The strain of hybrid cultured animal cells Mus musculus XT 2E5 is a producer of monoclonal antibodies of isotype G 1 to B, a subunit of cholera toxin, deposited in the State collection of pathogenic microorganisms and cell cultures "GKPM-Obolensk" under the number H-44.