The hybrid strain of cultured animal cells mus musculus l. used to obtain monoclonal antibodies to protein 105 kda excretory-secretory antigen of liver flukes person opisthorchis felineus

 

(57) Abstract:

The inventive strain hybrid transplantable cells MPI 649 was obtained by cell fusion of mouse myeloma P3/NS1/1-Ag4.1 with cells of syngeneic BALB/c mice immunized with purified preparation of total antigen liver flukes Opisthorchis felineus. Strain hybrid transplantable cells MPI 649 secretes monoclonal antibodies lgG2a, specifically interacting with the protein 105 kDa excretory-secretory antigen Opisthorchis felineus in immunoblotting. Monoclonal antibodies of this invention are used as reagents for new test systems for hepatic trematode person Opisthorchis felineus. 1 Il., table 1.

The invention relates to biotechnology and is concerned with the obtaining of monoclonal antibodies for the hepatic trematode Opisthorchis felineus, which can find application in medicine, immunology and biotechnology to improve methods for the identification and diagnosis, as well as for the standardization of antigen in immunodiagnostics.

Famous collection of mouse monoclonal antibodies (MAB) to the hepatic trematode Opisthorchis viverrini obtained abroad [1, 3]. Information about the analogs of such monoclonal antibodies to liver treena is getting hybridoma mouse origin secreting μa to the immunogen 105 kDa excretory-secretory antigen (e-C) liver flukes Opisthorchis felineus that can be effectively used for the identification of antigen liver flukes Opisthorchis felineus in physiological fluids or specific immunoglobulins in the serum of patients with opisthorchiasis caused by hepatic trematode Opisthorchis felineus, making preparations of antibodies promising reagents for the improvement of existing and the design of innovative diagnostic test kits on the hepatic trematode person Opisthorchis felineus.

The problem is solved by cell fusion of mouse P3/NS1/1-Ag4.l (NSI) myeloma cells in the spleen of female mice BALB/c mice weighing 15-20 g (SRC VB Vector) immunized with purified total drug antigens trematode Opisthorchis felineus, which is obtained during the cultivation of Opisthorchis felineus within 2 weeks at the minimum support environment the Needle.

Use the immunization scheme, presented in the table.

To merge use 150 million splenic cells and 50 million cells NSI. The mixture of cells centrifuged, the supernatant carefully removed and the cells draught add 0.4 ml of 50% solution of polyethylene glycol (PEG) with a molecular is Verena, then the precipitate resuspended and centrifuged. Cells are distributed in five culture 96-hole blade 100 ál to well. Selection of hybrid cells is carried out in a NAT environment, consisting of a nutrient medium, DMEM(M), to which was added 15% fetal cow serum, 0.1 mm gipoksantina, 0.04 mm thymidine and 0.01 mm aminopterin.

The selection of specific hybrids performed enzyme-linked immunosorbent assay (ELISA). In the wells of polystyrene blade ("Dynatech", Switzerland) as antigen contribute 10 ng of purified drug trematode Opisthorchis felineus and after drying in air, the antigen is fixed for 10 min with cold ethanol. Place of nonspecific binding is saturated with 0.5% solution of casein. Then in the hole transfer 50 ál of culture medium analyzed hybrid and incubated for one hour at 37oC. After incubation the wells are washed 3-5 times with distilled water. Later in the tablets make 50 ál individualo conjugate (immunoglobulin-rabbit peroxidase labeled against mouse lgG) and Sorb 45 min at 37oC. the Tablets are washed and carry out the enzymatic reaction. The results of the analysis determined on the spectrophotometer at a wavelength of 492 nm. Positive consider clones, antibodies to the limit dilutions, translated in popular culture and frozen in liquid nitrogen.

The strain is characterized by the following features:

Morphological features. Culture consists of large round cells with similar morphology and size to the original parent myeloma NSI. Oval nucleus is eccentric and a large part of the cytoplasm.

Cultural properties. The medium for culturing medium Needle MEM modification of Dulbecco containing increased amounts of arginine, 200 mg/l of folic acid to 12 mg/l, asparagine up to 36 mg/l, and 0.05 mm 2-mercaptoethanol and 20 mm HEPES, 4 mm L-glutamine. The content of fetal serum in the growth medium is 10%. Wednesday also add 80 μg/ml gentamicin sulfate. Hybridoma 1A1/B2 grows in the form monocline-suspension cultures. Sowing dose of 100-200 thousand cells per milliliter, the multiplicity of sieving - 1:5-1:6 every 3-4 days. Cultivation was performed at 37oC in an incubator with 5% carbon dioxide, in the atmosphere of 95% humidity or in airtight vials without feeding CO2.

The cultivation of hybridoma in the body of the animal. Female BALB/c mice (SRC VB "Vector") sensibiliser intraperitoneal introduction 5 ml of paraffin oil. After 2-4 weeks animals injected 10 is staticheskoi fluid. Hybridoma inoculated in 100% of cases.

The characteristic of a useful product. Hybrid clone 1A1/B2 provides obtaining of mouse immunoglobulin lgG at a dose of 10-20 mg of antibodies from ml ascitic fluid and 1-5 μg of milliliters of culture medium. They specifically interact with the protein 105 and 160 kDa replica of the preparation of excretory-secretory antigen Opisthorchis felineus in response immunobloting. Stable products µa persists for at least 30 passages in vitro.

Cryopreservation. Environment for freezing - environment DMEM(M) - 50%, fetal serum - 40%, dimethylsulfoxide - 10%. 0.5 ml of cell suspension is transferred into a plastic cryoprobes and placed in a Styrofoam container with a wall thickness of 1.5 see Container bring in a pair of liquid nitrogen. The next day the tubes are transferred into liquid nitrogen. Defrosting is carried out, omitting the test tubes in the water with the temperature of the 41oC. cells of the plant with the environment DMEM(M) and centrifuged at 600 g. Sediment resuspended in the growth medium at a concentration of 200-300 thousand cells per milliliter and transferred to culture flasks. Viability after thawing is 60-80% (the color of 0.25% Trifanova blue).

The invention Illus is Oh 1A1/B2, in immunoblotting with excretory-secretory antigen Opisthorchis felineus.

Example 1. Cultivation of strain 1A1/B2, secreting monoclonal antibodies to the trematode Opisthorchis felineus in animals, BALB/c mice.

Option 1. Cultured cells of strain 1A1/B2, located in the logarithmic growth phase, sterile centrifuged for 5-10 minutes at 1000 rpm in a centrifuge ARF-3. Adosados removed, and the residue suspension in a sterile solution of Earl or Hanks. Female BALB/c mice (SRC VB "Vector") weighing 15-20 g intraperitoneally injected every 3-5 ml of cell suspension, containing 15-20 million hybrid cells. After 7-10 days the animals are euthanized and the peritoneal cavity extract 10-12 ml of ascitic fluid. Cells from ascitic fluid is separated by centrifugation and used for further perelivania hybridoma, and the supernatant determine the titer of antibodies using enzyme immunoassay, as described above.

Option 2. Pre-mice BALB/c (SRC VB "Vector") weighing 15-20 g not less than 10 days before inoculation of hybridoma cells injected intraperitoneally in 5 ml of paraffin oil. Suspension cultured cells of strain 1A1/B2, prepared as described above, vaccinated animals intraperitoneally. It is vivki hybridoma cells formed 15-30 ml of ascitic fluid, which is used to obtain preparations of monoclonal antibodies, as described below.

Example 2. The selection of the purified monoclonal antibodies produced by the hybrid strain of cultured cells 1A1/B2 from ascitic fluid and culture medium.

Option 1. To the selected volume of ascitic fluid or culture medium containing MCA and pre-centrifuged at 5000 g for 20 min, add an equal volume of a saturated solution of ammonium sulfate. The resulting suspension after incubation for 30 min at +4oC again centrifuged 20 min at 5000 g. The precipitate, containing enriched fraction of immunoglobulins, dissolved in 0.01 M phosphate buffer solution, at a pH of 7.2. The remainder of the ammonium sulfate is removed by gelfiltration. Used for this purpose is the column h cm, filled with Sephadex G-25 and balanced 0.01 M phosphate buffer, pH 7,2.

A further selection of ICA is carried out by chromatography on DEAE-Sephadex A-50. The column size 2.5x20 cm, filled with DEAE-Sephadex A-50, suspended in 0.02 M phosphate buffer (FB), pH 6.3. apply the selected antibodies (1 mg gel 1 mg protein). Elution of immunoglobulins spend FB, pH 6.3. A fraction of specifices the KA, using PEG-20000.

Option 2. One volume of ascitic fluid or culture medium containing MCA, diluted with 4 volumes of 0.6 M acetate buffer (0.04 M citric acid, 0.2 M sodium acetate), pH 4.0 and pH adjusted to 4.5 with 0.1 N sodium hydroxide solution. To the diluted sample is added dropwise, with constant stirring, Caprylic acid at the rate of 25 µl per 1 ml and incubated for 30 min at +4oC. and Then centrifuged for 30 min at 8000 g, and the precipitate was removed and the supernatant is mixed with 10x phosphate-saline buffer (FSB) and establish a pH of 7.4 with a solution of 1.0 N sodium hydroxide (supernatant optionally filtered). Add an equal volume of a saturated solution of ammonium sulfate (V:V), shaken and incubated overnight at +4 C or 30 min at +20-25oC. Centrifuged 15 min at 5000 g. The supernatant is drained and the sediment resuspended the FSB, a pH of 7.4. The remains of ammonium sulfate is removed by dialysis against 50 to 100 volumes of the FSB, pH 7,4.

Example 3. Determination of the specific interaction between the MAB 1A1/B2 with excretory-secretory antigen of liver flukes Opisthorchis felineus by ELISA.

Option 1. ELISA performed on polystyrene tablets; the antigen is applied in a volume of 15 µl/well on tablets, and after pagsusuri CE-twin (0.145 M sodium chloride, 20 mM Tris-HCl, 5 mM PMSF (Sigma, USA), 0.1% Tween-20 (Serva), pH 7.4) at 37oC 45 minutes.

Then incubated with monoclonal antibody 1A1/B2 45 minutes at 37oC. Specific binding reveal antivirovym peroxidase labeled antibodies against lgG. The Chromogen is of 0.1% o-phenylenediamine in citrate-phosphate buffer (0.2 M citric acid, 0.5 M Na2HPO3, pH 5.0) with 0.03% hydrogen peroxide). Stop the reaction by adding 100 µl per well of I N HCl and measure the optical density of samples on a spectrophotometer scanreceiver (Finland) using a filter with a maximum transmission 492 nm. As a control using homologous normal and hyperimmune serum.

Option 2. ELISA is carried out with a replica of the preparation of excretory-secretory antigen, subjected to electrophoresis and transfer to nitrocellulose membrane in the band [2, 4, 5]. Place of nonspecific binding is saturated with 0.5% solution of casein in buffer TSB-twin (0.145 M sodium chloride, 20 mM Tris-HCI, 5 mM PMSF (Sigma, USA), 0.1% Tween-20 (Serva), pH 7.4) at 37oC 45 minutes.

Then incubated with monoclonal antibody 1A1/B2 45 minutes at 37oC. Specific binding reveal antivirovym labeled with horseradish peroxidase antibodies against lgG. The Chromogen - 0.1% 4-chloro-1-h is t parent myeloma NSI.

Cell line 1A1/B2 provides obtaining of mouse immunoglobulin lgG (subclass lgG2a) in an amount of 5-10 mg of purified antibodies from ml ascitic fluid and 0.5-2 μg of milliliters of culture medium. MCA 1A1/B2 have a high specificity to excretory-secretory antigen Opisthorchis felineus, while binding in ELISA with e-antigen by the method of limiting dilution titer of the culture fluid 1/64.000 and the title of ascites 1/1.280.000, the signal in ELISA when linking with 15 ng e-antigen per well OP492: 2.00. In immunoblotting monoclonal antibodies bind to a protein immunogen 105 kDa liver flukes person Opisthorchis felineus.

The above properties of the strain of hybrid cells 1A1/2 allows to conclude that for the first time on the basis of mouse myeloma received hybridoma-producer µa to protein 105 kDa excretory - secretory antigen of liver flukes Opisthorchis felineus, which allows you to get mouse monoclonal antibodies for use in research and production purposes.

The above properties of strain 1A1/B2 distinguish it from all previously described hybridomas producing µa to hepatic trematode of the genus Opisthorchis.

The list used is 7

2. Laemmli U. K. // Nature, - 1970, - vol. 227, p. 680-685.

3. Sirisinha , S., Chawengkirttikul R., Tayapiwatana C., Naiyanetr C., Waikagul J. , Radomyos, P. and Podoprigora G. I. Specific and cross - reactivemonoclonal antibodies to the 89-kDa antigen of Opisthorchis viverrini. Southeast Asean J. Trop. Med. Public Health, - 1992, - vol. 23, - N 3, - p. 489-490.

4. Towbin H., Gordon J. // J. Immunol. Methods, - 1984, - vol. 72, p. 313-340.

5. Towbin H. Staehelin T., Gordon. // Proc. Nat. Acad. Sci. USA, - 1979, - vol. 76, p. 4350-4354.

The hybrid strain of cultured animal cells Mus musculus L. MPI 649 used to obtain monoclonal antibodies to protein 105 kDa excretory-secretory antigen of liver flukes person Opisthorchis felineus.

 

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