The hybrid strain of cultured animal cells rattus norvegicus used to obtain monoclonal antibodies to hepatitis a virus human

 

(57) Abstract:

Strain hybrid transplantable cells MPI 612 was obtained by fusion of cells of rat myeloma 210RC.Y3-Agl.2.3 with spleen cells of rats LOU immunized with purified preparation of hepatitis A. Strain hybrid transplantable cells MPI 612 secretes a monoclonal antibody IgG2Binteracting specifically with hepatitis A. Monoclonal antibodies of this invention are used as reagents in diagnostic test systems for the detection of antigen of hepatitis a virus and antibodies to it. table 1.

The invention relates to biotechnology, more specifically to medical Virology, and for obtaining strain rat hybrid cells producing monoclonal antibodies (MAB) to hepatitis A virus (HAV), which can find application in medicine, immunology and biotechnology to improve methods for the identification and diagnosis of this dangerous infectious diseases of humans, can be used to develop and manufacture a new generation of diagnostic test systems, as well as to standardize the production process and the development of vaccines against HAV.

Famous collection of mouse hybridisation strain is a hybrid strain of cultured animal cells Mus Musculus L producing monoclonal antibodies to unknown for us strain of hepatitis A virus [4]. To get mouse monoclonal antibodies used hybridoma murine origin, making it difficult to obtain significant quantities of these antibodies, so that the output ascitic fluid from one mouse relatively small 3-5 ml.

An object of the invention is to obtain hybridoma rat origin, providing a high level synthesis µa to hepatitis A, which can be used effectively to determine viral antigen or specific immunoglobulins in the serum of patients with hepatitis A, which makes the products of these rat monoclonal antibodies promising substitutes mouse monoclonal and polyclonal antisera of different species origin in diagnostic test systems for the detection of antigen of hepatitis A virus and antibodies to it.

The problem is solved by merging cells of rat myeloma 210RC.Y3-Agl.2.3. with spleen cells of rats LOU immunized with purified hepatitis A. this strain HAS-15/4647 HAV cultivated for 3 weeks in culture of kidney cells of the embryo green monkey 4647, at a minimum support environment Needle with 2% fetal serum and purified in chloride density gradient C is manisali use female rats LOU (SRC VB "Vector"), weighing 180-200 g (table).

To merge use 150 million splenic cells of rats LOU and 50 million cells Y3. The mixture of cells centrifuged, the supernatant carefully removed and the cells draught add 0.4 ml of 50% solution of polyethylene glycol (PEG) (Merck, Germany) with a molecular weight of 2000. The mixture was centrifuged 15 min at 600 rpm After 3-5 min pause layer PEG slowly diluted with a solution of versene, after which the precipitate resuspended and centrifuged. Cells are distributed in five 96-well culture 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 hepatitis A virus and after drying in air, the antigen is fixed for 10 min with cold ethanol. Place of nonspecific binding is saturated with a 1% solution of bovine serum albumin (BSA). Then in the hole transfer 50 ál of culture medium analyzed hybrid and incubated for cha is individualo conjugate (rabbit immunoglobulins, peroxidase labeled IgG against rats) and Sorb 45 min at +37oC. Board washed and carry out the enzymatic reaction. The results of the analysis are recorded using the spectrophotometer at a wavelength of 492 nm. Hybrid strain 9B12 twice clone method of limiting dilutions and transferred into the mass culture. Cryopreservation is performed by freezing 107hybrid cells in liquid nitrogen in DMEM(M) with 10% dimethyl sulfoxide and 40% fetal serum.

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 Y-3. Oval nucleus is eccentric and a large part of the cytoplasm.

Cultural properties. The strain is monocline-suspension culture, in which up to 30% of the cells are in suspension, being attached to the surface of the culture dishes. Sowing dose of 100-200 thousand cells per milliliter, the multiplicity of sieving - 1:5-1:6 every 3-4 days. The index of proliferation - 10. Cells from the surface of the culture dishes are removed by vigorous shaking or after processing layer of cells with a solution of versene and trypsin in a ratio of 3:1.

The cultivation of hybridoma in the body of the animal. Female rats LOU (SRC VB "Vector") sensibiliser intraperitoneal introduction 5 ml of paraffin oil. After 2-4 weeks animals injected 10 million hybrid cells. 10-14 days is formed ascitic tumor. From one animal you can get 20-50 ml of ascitic fluid. Hybridoma inoculated in 100% of cases.

The characteristic of a useful product. The immunodiffusion method according Ouchterlony it was found that MCA belong to the class IgG2B. They specifically interact with the antigen of the virus HAV in the enzyme-linked immunosorbent assay (ELISA). The title of the ICA in ELISA is 1:16000 to the culture fluid and 1:1024000 for ascitic fluids. From 1 ml of ascitic fluid and culture medium is allocated when cleaning 6-10 mg 100-300 mcg of specific immunoglobulins, respectively. Stable products µa stored for 2 months of continuous perelivania in cell cultures and 10 passages on animals.

Cryopreservation. Environment for tumor in plastic cryoprobes and placed in a Styrofoam container with a wall thickness of 1.5 cm The container make a pair of liquid nitrogen. The next day the tubes are transferred into liquid nitrogen. Defrosting is carried out, omitting the tubes in water with a temperature of 41oC. cells of the plant with the environment DMEM(M) and centrifuged at 600 rpm 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 following examples detail reveal the essence of the invention.

Example 1. Cultivation of strain 9B12, secreting monoclonal antibodies to hepatitis A virus in animals rats LOU.

Option 1. Cultured cells of strain 9B12, 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 is suspended in a sterile solution of Earl or Hanks. Female rats LOU (SRC VB "Vector"), weighing 160-180 g, intraperitoneally administered 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 the private analysis as explained above.

Option 2. Pre rats LOU (SRC VB "Vector"), weighing 180-200 g, not less than 10 days before inoculation of hybridoma cells injected intraperitoneally in 5 ml of paraffin oil. Suspension cultured cells of strain 9B12, prepared as described above, vaccinated animals intraperitoneally. Each injection contains 10 million cells in 1-3 ml of serum-free medium or solution Earl. 10-14 days after inoculation of hybridoma cells formed 15-30 ml of ascitic fluid, which is used to obtain preparations of monoclonal antibodies, as described previously.

Example 2. The selection of the purified monoclonal antibodies produced by the hybrid strain of cultured cells 9B12, from ascitic fluid and culture medium.

Option 1. 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 using a 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 rpm and remove sieges is atra. (Supernatant optionally filtered). Add an equal volume of a saturated solution of ammonium sulfate, shaken and incubated overnight at +4oC or 30 min at +20-25oC. Centrifuged 15 min at 5000 rpm, 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.

Option 2. The selected volume of ascitic fluid, culture medium or purified Caprylic acid µa, 1-fold in phosphate-buffered saline applied to a column Packed with Sephacryl-300 (Pharmacia, Sweden) in physiological solution containing Tris-HCl (100 ml gel 2 ml protein). The eluate is collected in portions of 5 ml, Stripping immunoglobulins saline pH 7.3. Faction specific IgG released in the first peak. After gelfiltration and elution can concentrate fraction containing MCA, using a saturated solution of ammonium sulfate or PEG-20000.

Example 3. The definition of competitive interaction µa 9B12 with faction specific IgG isolated from polyclonal antisera against hepatitis A, by ELISA.

Option 1. For competitive ELISA was used the test-system "Vektora A-IgM (SRC VB-Vetrovi person by ELISA, and can be used for early differential diagnosis of hepatitis A in clinical and epidemiological studies. In payment for immunological studies (strips) with immobilized antibodies to Ig-M person bring positive control human serum (K+) containing anti-HAV IgM and negative control serum (K) and incubated at +37oC within 90 minutes after the time of incubation of the serum removed and the Board is washed 3-5 times with distilled water. Next to all wells of the card in addition to A-1 contribute 50 μl of the antigen solution and placed in a thermostat at +37oC. after incubation, the antigen solution is collected and fees washed 3-5 times with distilled water. Then in the holes absorb 50 ál of conjugate solution (IgG-anti-HAV Guinea pigs, labeled with horseradish peroxidase) and 50 μl of drugs µa (culture medium, ascites fluid or purified monoclonal immunoglobulins), restaranah 2-10-fold increments in 1% solution of bovine serum albumin, and incubated for 1.5 hours at +37oC. Board washed and carry out the enzymatic reaction, the optical density measurement. The results of the analysis take into account spectrophotometrically at a wavelength of 492 nm is the consumption of staining in the holes in the competitive binding rat MCA 9B12 on the hav antigen. For a competitive title is accepted that breeding, where the fixed 50% reduction in binding of the conjugate to the antigen.

Option 2. In the wells immunological boards absorb 50 ál of the solution of antibodies to HAV (IgG-anti-HAV Guinea pigs) 45-90 min at +37oC. Upon completion of the sorption wells washed 3-5 times with distilled water. Next to all wells cards make 50 μl of the antigen solution and put on 1.5 hours in a thermostat at +37oC. after incubation, the antigen solution adsorb and Board washed 3-5 times with distilled water. Then contribute to the wells, 50 μl of monoclonal antibody 9B12 and strength conjugate antibodies to HAV (IgG-anti-HAV Guinea pigs, labeled with horseradish peroxidase) and incubated for 1.5 hours at +37oC. Board washed 5-7 times with distilled water and carry out the enzymatic reaction. The density measurement and evaluation of results is carried out, as described above.

Competitive enzyme-linked immunosorbent assay between polyclonal human antisera system "Vektora A-IgM" and received the MCA showed that ICA is able to 25-70% to inhibit the interaction of human antisera with viral antigen. This indicates the identity of the antigenic determinate hybrid cells 9B12 allow to conclude, for the first time on the basis of rat myeloma received hybridoma 9B12-producer µa to hepatitis A, which allows to get a rat monoclonal antibodies for use in research and industrial purposes. Cell line provides reception of rat immunoglobulin class IgG2Bin the amount of 6-10 mg of purified antibodies from ml ascitic fluid and 100-300 mcg from milliliter of culture medium.

The above properties of strain 9B12 distinguish it from all previously described hybridomas producing µa to hepatitis A man.

The list of used literature

1. Hughes J. V., Stanton L. W., J. E. Tomassini, Long, W. J. and Scolnick, E. M.//J. of Virol. - 1984 - v. 52, N 2 - p. 465-473.

2. Hughes J. V., J. E. Tomassini, E. M. Scolnick//Hepatitis A-subunit antigen. U.S. patent N 4614793, MKI C 07 N 15/04.

3. Fridljanskaja I. I., Chernov So on, Antropov, O. Y., Kusov Y. Yu, Nastashenko T. A., Balayan, M. S., Tsarev, S. A., Markov, S. C., Sverdlov E. D. // hybrid Strain of cultured animal cells Mus Musculus L. producing monoclonal antibodies to the recombinant protein of hepatitis A man. - A. S. USSR N 1611930, MKI C 12 N 5/00.

4. Berkova N. P., Nastashenko T. A., Kusov Y. Yu, Shamborant O. G., Kojic A. T. , Balayan, M. S., Ivanov, C. T. //Piece of man. - A. S. USSR N 1657527, MKI C 12 N 5/00.

5. MacGregor A. et. al. //J. Clin. Micr. - 1984. - v. 18, N 5. - p. 1237-1243.

6. Provost P. J., Hilleman, M. R. (1979) Propagation of human hepatitis A virus in cell culture in vitro. Proc. Soc. Exp. Biol. Med. 160, 21-221.

7. Bishop N. E., Hugo D. L., Borovel S. V., Anderson D. A. (1994) Rapid and efficient purification of hepatitis A virus from cell culture. J. Virol. Meth. 47, 203-216.

The hybrid strain of cultured animal cells Rattus norvegicus MPI-612 that is used to obtain monoclonal antibodies to hepatitis a virus human.

 

Same patents:

The invention relates to medicine, in particular to Oncology and immunology, and for the treatment of b-cell lymphoma

The invention relates to the field of biochemistry, in particular to the new fused proteins, and can be used in the treatment of neoplastic diseases

The invention relates to the field of Virology, immunology and biotechnology, namely, hybridoma technology, and represents a new hybrid strain of cultured animal cells Mus musculus L. producing cell cultures and ascitic fluids monoclonal antibody (MAB) to the virus vesicular disease swine (WBS) strain T-75, which can be used for scientific research and preparation of diagnostics, prevention and treatment VBS

The invention relates to hybrid technology and can be used in veterinary medicine and medicine in the diagnosis of brucellosis

The invention relates to hybridoma technology and can be used in the diagnosis of hepatitis b

The invention relates to hybridoma technology and can be used in the diagnosis of hepatitis b

The invention relates to the field of veterinary biotechnology, in particular, to obtain strain of hybrid cells producing monoclonal antibodies that can be used for the preparation of highly specific diagnostic reagents for identification and quantitative determination of immunoglobulin G class in biological fluids pigs

The invention relates to the field of immunology, in particular the production and use of monoclonal antibodies
Up!