RU1773938C - Strain of hybrid cultivated cells from mus musculus l-producent of monoclonal antibodies to k99-adhesive antigen of eschericia coli - Google Patents

Abstract

Usage: biotechnology, immunologists. SUMMARY OF THE INVENTION; get the strain by hybridization of myeloma cells of the line X-63-Ad-8.653 with splenocytes of mice immunized with K99. The strain is designated 3F6D and is stored under HSCC (P) No. 519 D. The strain produces monoclonal antibodies that specifically interact with the K99 E. coli antigen with a mole. weighing 18.5 KDa. The antibody titer in ascites fluid reaches 1: 200000. (L s

Description

The invention relates to hybrid technology and can be used in veterinary medicine for the diagnosis of gastrointestinal diseases in animals caused by Escherichia coli.

A known strain of xenohybridoma 94A1 producing monoclonal antibodies to the K99 antigen of E. coli. Murine myeloma NSO (subline NS / 1 Ad 4.1) and calf lymphocytes not secreting immunoglobulins were used to prepare it. At the same time, xenogibridomas 53B3 and 53B4, not secreting immunoglobulins, were obtained. Then xenogibridoma 53B3 merged with calf lymphocytes secreting immunoglobulins to the E.99H antigen K99. As a result of these experiments, xenohybridoma 94A1 was produced producing monoclonal antibodies to the K99 antigen of E. coli (DVAnderson et al., Bovine monoclonal antibodies to the F5 (K99) pilus antigen of E. cololl produset by nurine / bovine Nybridomas .. Vet, Immunology and Immunopatology, 1987, v. 15, p. 233-237).

In addition, a known strain of hybrid cells 2VD4E4 producing monoclonal antibodies to the K99 antigen of E. coli strain B44 and B41. The antibodies produced are of class G1, and the titer of antibodies purified from ascites fluid was

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1: 12000. The concentration of immunoglobulins was 45-50% ascites fluid protein. The hybridoma was obtained by fusion of splenocytes of immunized mice and myeloma cell line P3-NS 1-Ag 4/1. Monoclonal antibodies produced by 2VD4E1 hybridoma were used to treat calves experimentally infected with K99 positive E.coH strains. The results of the experiment showed that the incidence in the control group was 82%, dehydration of the animal organism 82% and mortality 82%. while in the experimental group of animals the incidence was 75%, dehydration 29% and mortality 29%. Thus, the possibility of using monoclonal antibodies for treating calves of patients with colibacteriosis has been shown. This strain is taken as a prototype (Sadowski P. L. et al., Protection of Calvel Against Fatal Enteric Colibaclllosls by Oralil Administered Escherichla coli K99-speciflc monoclonal Antibody. - Infection and Immunity, 1983, v. 42, No. 2.

Thus, an analysis of the literature shows that two strains of hybrid cells producing monoclonal antibodies to the K99 antigen are currently known. The above monoclonal antibodies have been used for therapeutic purposes.

Due to the fact that antibodies from ascites fluid produced by 28D4E4 hybridoma have a relatively low activity of 1: 12000 and a productivity of 45-50%, they are less suitable for diagnostic purposes.

In view of the foregoing, a 3F6D hybridoma was obtained that produces monoclonal antibodies to the K99 antigen of E. coii with a sufficiently high affinity. This makes it possible to use these monoclonal antibodies for diagnostic purposes.

It is an object of the invention to provide a strain of hybrid cells producing monoclonal antibodies to the K99 adhesive antigen of E.coll.

The hybrid cell strain was obtained by fusion of X-63-Ad-8-653 myeloma cell with splenocytes from mice immunized 4 times with a two-week interval purified by differential ultracentrifugation of K99 antigen of E.coH strain 09. The first immunization was carried out with 10 μg K99 antigen with full Freund's adjuvant, 2 and 3 immunization with the same dose of antigen with incomplete Freund's adjuvant. The last immunization was carried out with 10 μg of antigen in 0.15 M phosphate-buffered saline

and after 2 days, spleen cells were used for hybridization. Hybridization was carried out by adding to the mixture of 4x106 myeloma cells and 20x10 splenocytes 1 ml of solution,

containing 45% PEG-4000, 10% dimethyl sulfoxide and 45% RPM1-1640 medium. Then allowed to incubate for 10 minutes. After this, the cells were besieged by centrifugation at 1000 rpm for 10 minutes

Cell pellet was resuspended in RPM1-1640 medium supplemented with 10% by volume fetal calf serum and plated in 96-well plates at 100 μl per well. The next day, medium containing hypoxanthine, aminopterin, and thymidine (GAT) was added to these wells. Every 3-4 days, cells were fed. 10-14 days after fusion in wells having 2-3 mm cell colonies,

culture medium was selected to determine the productivity of the hybrid cells. Testing was carried out by enzyme-linked immunosorbent assay using purified K99 antigen by differential ultracentrifugation and rabbit immunoglobulins against peroxidase-labeled mouse antibodies. For 21 days after fusion, the cells were in GAT medium. Then gradually translated

on the GT environment and the complete RPM1-1640 environment. Monoclonal antibody producing cell clones are double cloned by limiting dilution. After the second cloning of 100% subclones

produced antibodies to the K99 adhesive antigen of Escherichla coll. The hybrid cell strain produces antibodies in the culture medium and in ascites fluid.

The 3F6D hybrid cell strain is stored in a specialized collection of transplantable somatic vertebrate cells of the Institute of Cytology of the USSR Academy of Sciences under the number VSCK (P) 519 D. The strain is characterized by the following properties.

Morphological properties. Hybrid cells are rounded cells weakly attached to the carrier the size of the original myeloma cell. The nucleus occupies most of the cell. The cytoplasm has the appearance of a thin rim.

Cultural properties. Hybrid cells were grown in RPM1-1640 medium containing heat-inactivated serum of fetal cows (10-20%), Lglutamine 200 mM (20 ml / l), HEPES- (3.375 g / l), sodium pyruvate; 2-mercaptoethanol (5x1 OM), penicillin (100 u / ml), streptomycin (100 μg / ml), sodium bicarbonate 37 g / l). Cells were cultured at 37 ° C in an atmosphere of 5%. The nature of growth is the stationary suspension. Sow cell concentration of 2x10 cells in 1 ml. The frequency of passage of cells after 2-3 days. When administered intraperitoneally to syngeneic mice, the hybridoma strain forms a tumor in ascites form on days 14-18. The dose of cells inoculated with 2x105 cells. 14 days before the introduction of the hybridoma, mice were injected with 2,6,10,14-tetramethylpentadecane at a dose of 0.5 ml per head. The strain retained the ability to produce specific monoclonal antibodies for 4 passages on syngeneic mice (observation time).

Strain productivity. On day 8 of culturing the hybridoma, the antibody concentration reaches 5 µg / ml in culture medium. In purified ascites fluid, the concentration of immunoglobulins is 8 mg / ml, and their titer is 1: 200000.

The characteristic of a useful product. Monoclonal antibodies are immunoglobulins of subclass G1. Monoclonal antibodies specifically interact with K99 antigen of E. coli with a molecular weight of 18.5 KDa. The specificity of monoclonal antibodies was determined by immunoblotting.

Contamination. No contaminants in the cells, including bacteria, fungi, yeast and myoplasm, were detected.

Cryopreservation. Cow embryonic serum was added to the hybrid cell pellet and then dimethyl sulfoxide to a final concentration of 10%. The suspension was poured into sterile plastic ampoules with screw caps on 2x10 cells in a volume of 1 ml. Ampoules were placed in a foam box and left for a day at -70 ° C, and then transferred to liquid nitrogen,

Defrosting conditions. The frozen ampoule was quickly placed in a water bath at 37 ° С. Once the last pieces of ice have melted, the cell suspension is transferred sterile into a test tube containing 10 ml of cold serum-free medium, washed once and seeded in the wells of a 24-well plate with a feeding layer of pertonial macrophages. The number of viable cells after thawing is not less than 60%.

Strain 3F6D is used as follows.

EXAMPLE 1. Hybrid cells are placed in plastic mattresses with an area of 25 cm by 5x10 cells in 5 ml of culture medium and incubated for 3 days at 37 ° C in an atmosphere of 5% CO2. The culture medium is collected and centrifuged for 10 min at 800 d in order to

separating the cells from the supernatant containing antibodies. When hybridomas were cultured in mice, immunoglobulins from ascites fluid were obtained by salting out with ammonium sulfate to 50% saturation. For this, an equal volume of 0.15 M phosphate buffered saline (PBS) is added to the ascites fluid. pH 7.2. An equal volume of a saturated solution of ammonium sulfate was then added and left stirring overnight at 4 ° C. Immunoglobulins are separated by centrifugation at 5000 rpm for 30 minutes at 4 ° C. Antibody sediment was resuspended in a minimal volume of PBS. PRI me R 2. On nitrocellulose membrane, previously cut into

8 lanes, 1 µl of the propagated K99 antigen is applied. The culture is carried out from 10 µg / ml to 1 ng / ml. Membrane strips were incubated for 1 hour at 37 ° C in 1.5% BSA to block free membrane surfaces. After incubation, the strips are washed 3 times for 10 minutes in 0.15 M PBS with 0.1% Tween-20 (PBS-Tw). The washed strips are incubated for 1 hour at 37 ° C in 8 dilutions of monoclonal antibodies from 1: 1000 to 1: 12000. After incubation, the membrane strips are washed as described above and incubated in a solution with rabbit antibodies against immunoglobulins from horseradish peroxidase labeled mice. After 1 hour of incubation at 37 ° C, the washing procedure is repeated to remove unbound reaction components. The reaction was manifested by the addition of a substrate. The substrate is prepared by dissolving 8 mg of 4-chloronaphthol in 1 ml of ethanol and adding

9 ml FBI and 5 μl of a 30% hydrogen peroxide solution. The working dilution of monoclonal antibodies is 1: 1000, i.e. such dilutions of the antibody reveal 1 ng / ml K99 antigen.

Example 3. To determine the molecular weight of the protein to which monoclonal antibodies are obtained, electrophoresis and transfer of the protein to a nitrocellulose membrane is carried out, followed by immunochemical manifestation (immunoblotting). Protein electrophoresis was carried out on an 11% polyacrylamide gel in the presence of 0.1% sodium dodecyl sulfate. Protein transfer to the nitrocellulose membrane was carried out by semi-dry electroblotting using a Hoefer Scientific Instruments apparatus at a current strength of 0.8 A per 1 cm2 gel for 2 hours. The stained gel was examined on a densitometer with Hoefer Scientific computer software.

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Nitrocellulose membrane is used-Result. Immunoblotting showed that

for immunochemical manifestation, monoclonal antibodies are specifically

antibodies. To do this, nitrocellulose binds to a protein; the molecular masme membrane is incubated in 1.5% BSA for 18.5 KDa. According to the literature K99

overnight at room temperature, then5 the Eschertchla coll antigen has molecules that are incubated in a purified solution of 18.5 KDa. The resulting antibody monoclorate at a dilution of 1: 500 into current antibodies will be used in

1 hour. After this, the carrier is incubated in te-ELISA for the diagnosis of colibacteriosis.

Claims (1)

1 hour at 37 ° C with rabbit antibodies against mouse immunoglobulins, conjugi-10 Hybrid cultured strain cultivated with horseradish peroxidase. Reactions of animals Mus Musculus L- VSCC (P) Ms exhibit a substrate containing 4-519 D-producing monoclonal antibodies to chlorine-naphthol, hydrogen peroxide, FBI. K99 adhesive antigen Escherlchla coll.