Strain of hybrid culturing rattus norvegicus 122h9 cells which is producer of cross-reactive neutralizing monoclonal antibodies against pathogenic for human orthopoxviruses

FIELD: biotechnology and virology.

SUBSTANCE: strain of hybrid culturing Rattus norvegicus 122H9 cells is disclosed. Said strain is obtained by fusion of rat myeloma cells 210RC.Y3-Ag1.2.3 with rat spleen cells LOU, immunized with purified ectromelia virus of K-1 strain. Said strain is producer of cross-reactive neutralizing monoclonal antibodies against pathogenic for human orthopoxviruses.

EFFECT: vaccines for prophylaxis and therapy of diseases associated with pathogenic for human orthopoxviruses.

2 tbl, 5 ex

 

The invention relates to biotechnology and Virology and the receipt of a new strain of hybrid cells of Rattus norvegicus - the product of cross-reactive neutralizing monoclonal antibodies (MAB) against orthopoxviruses. MCA can find application in medicine, immunology and biotechnology for the development and improvement of the means of prevention and therapy of diseases caused by orthopoxviruses pathogenic for humans, is the variola virus (UPE), Monkeypox (SBI), ospowiki (WWII) and smallpox cows (wok).

Well-known foreign collection murine monoclonal antibodies (MAB) to orthopoxviruses [1-5], produced by strains of hybrid animal cells Mus musculus.

However, analogues of hybrid strains of animal cells Rattus norvegicus - producers of monoclonal antibodies to orthopoxviruses unknown.

The technical result of the claimed invention to provide a strain of hybrid cells of Rattus norvegicus 122H9, secreting MCA, which have cross-reactivity with VE, BOB, wok and UPE and neutralizing activity against SAI and variola virus. This technical result is achieved by fusion of cells of rat myeloma 210RC.Y3-Ag1.2-3. with spleen cells of the rat LOU immunized with purified virus ectromelia (strain K-1), which was obtained from kultivirovanii purification of the virus.

The inventive strain of hybrid cells of Rattus norvegicus 122H9 received at the State research center of Virology and biotechnology Vector (SRC VB "Vector") of the Ministry of health of the Russian Federation and deposited in the Institute of cell cultures SRC VB "VECTOR" under no NIMB-281. Author name hybridoma cell line - N.

The strain of Rattus norvegicus 122H9 characterized by the following features:

Pedigree strain. The hybrid strain of cultured cells obtained by fusion of cells of rat myeloma 210RC.Y3-Ag1.2.3. (Y-3) with spleen cells of rats LOU immunized with purified preparation of virus ectromelia. As a melting agent used a 50% solution of polyethylene glycol firm Sigma with a molecular weight of 2000. The strain was grown on selective medium GAT, then thrice cloned by the method of limiting dilutions, using as cells kormeluk peritoneal macrophages of rats LOU. The output of positive clones in the last clone was 100%.

The number of passages by the time escrow is -12 passages.

Marker signs and methods of their evaluation. Strain secretes a rat immunoglobulins that specifically interact with the VE. Analysis of rat immunoglobulins spend enzyme-linked immunosorbent assay, using as antigen 200 ng purified VE. The interaction of MCA with VE detected using conjugate antibodies against rat IgG labeled with horseradish peroxidase.

Contamination by bacteria and fungi were not found.

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 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. The content of fetal serum in the growth medium is 10%. Wednesday also add 80 μg/ml gentamicin sulfate. Hybridoma 122H9 grows in the form monocline-suspension cultures. Sowing dose of 100-200 testlets in milliliter, the multiplicity of sieving - 1:5-1:6 every 3-4 days.

The cultivation of hybridoma in the body of the animal. Female rats Lou (SRC VB "Vector") sensibiliser intraperitoneal introduction 5 ml of paraffin oil or Wharf. After 2-4 weeks animals injected 10 million hybrid cells. Ascitic tumor is formed in 10-12 days. From one animal you can get 20-50 ml of ascitic fluid. Hybridoma inoculated in 100% of cases.

The characteristic of a useful product. Typing monoclonal immunoglobulins conducted by the immunodiffusion method according Ouchterlony. MCA belong to the IgG class. They specification is Eski interact with the protein of 12.8 kDa (gene 214L) virus ectromelia in the reaction of the Western blot turns. The title of the ICA in the ascites is not less than 1:2200000 ELISA with VE. Stable products µa persists for at least 10 passages in vitro. One ml of ascitic fluid can be obtained 5-7 mg of purified monoclonal antibodies. Strain stably produces µa during one month of continuous perelivania in culture.

Cryopreservation. Environment for freezing: Wednesday, DMEM(M) - 50%, fetal serum - 40%, dimethylsulfoxide - 10%. Cell suspension in a volume of 0.5-1 ml is transferred into a plastic cryoprobes and placed in a Styrofoam container with a wall thickness of 1-1,5 cm Container bring in 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 37-41°C. Cells diluted in 5 ml of medium, DMEM(M) and centrifuged at 1000 rpm Sediment resuspended in the growth medium and transferred into culture flasks at a concentration of 200-300 thousand cells per milliliter. Viability after thawing is 60-80% (the color of 0.25% Trifanova blue).

The method of obtaining the claimed strain

The strain of hybrid cells of Rattus norvegicus 122H9 obtained as follows.

Female rats LOU, weighing 180-200 g (vivarium GMCVB "Vector") are subjected to immunization according to the scheme below in table 1.

To merge use 150 million splenic cells and 50 million cells 3. 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 molecular weight of 2000. The mixture was centrifuged 15 min at 600g. After 3-5 min pause layer PEG slowly diluted with a solution of versene, after which the precipitate resuspended and the suspension 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 of aminopterin.

Table 1

Scheme immunization
DaysThe diluent antigenAntigen dose (μg/rat)Area injections
0Full adjuvant's adjuvant7,5Intraperitoneally
7Incomplete adjuvant's adjuvant7,5Intraperitoneally
10Saline75Intravenous
11Saline75Intravenous
14Hybridization (The fence spleen)

The selection of specific hybrids performed enzyme-linked immunosorbent assay (ELISA), using as antigen 200 ng of purified virus ectromelia. Place of nonspecific binding is saturated with 0.5% solution of casein (ICN). Then transferred into the wells, 100 μl of culture medium analyzed hybrid and incubated for 45 min at 37°). After incubation the wells are washed 3-5 times with physiological saline containing 0.05% tween-20 (Sigma). In the tablets make 100 ál individualo conjugate (rabbit immunoglobulins against rat immunoglobulins labeled with horseradish peroxidase and incubated for 45 min at 37°C. Then the tablets are washed and carry out the enzymatic reaction staining. The results of the analysis to determine the MULTISCAN spectrophotometer at a wavelength of 492 nm.

Hybrid strain N twice clone method of limiting dilutions, translated in popular culture and frozen in liquid nitrogen.

The following examples detail reveal the essence of the invention.

Example 1. Cultivation of a strain of hybrid cells of Rattus norvegicus 122H9, secreting µa against orthopoxviruses, in the body of rats LOU.

Cultured cells 122H9 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 is a sterile solution of Earl or Hanks. Pre rats LOU (GMCVB "Vector"), weighing 180-200 g, not less than 10 days before inoculation of hybridoma cells injected intraperitoneally in 5 ml of paraffin oil or Wharf. Suspension cultured cells of strain-producer 122H9, prepared as described above, vaccinated animals intraperitoneally, 10 million cells in 1-3 ml of medium without serum 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.

Example 2. The selection of the purified monoclonal antibody produced by a strain of hybrid cells of Rattus norvegicus 122H9 from ascitic fluid.

One volume of ascitic fluid containing MAB, diluted with 4 volumes of 0.6 m acetate buffer (0,04M citric acid, 0.2m sodium acetate), pH 4.0 and bring the pH to 4.5 with 0,1N 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 +4°C. Then centrifuged for 30 min at 8000g and the precipitate removed and adosados mixed with 10x phosphate-saline buffer (FSB) and set pH 7.4 with a solution of 1.0 N sodium hydroxide. Equal volume (V:V) saturated solution of ammonium sulfate is added to this solution, shaken and incubated overnight at 4°or 30 minutes at 0-25° C. Centrifuged 15 min at 5000 g.

Adosados drained and the sediment resuspended the FSB, pH 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 by ELISA cross-interaction MCA produced by a strain of Rattus norvegicus 122H9, with orthopoxviruses.

ELISA was carried out on polystyrene tablets; antigen (purified virus) barbirolli the FSB, pH 7.4, in the amount of 100 µl/well. Place nonspecific binding was saturated 45 minutes at 37°With 0.5% solution of casein in buffer TSB-twin (0.145 M sodium chloride, 20 mM Tris-HCl, 5 mM PMSF (Sigma), 0.1% Tween-20 (Serva), pH 7.4). The tablets were made µa (45 minutes at 37° (C) binding of the MAB to the antigen was revealed by peroxidase labeled antibodies against immunoglobulins of the rat. Next was added a Chromogen, a 0.1% O-phenylenediamine in citrate-phosphate buffer (0.2 M citric acid, 0.5 M Na2HPO3pH 5.0) with 0.03% hydrogen peroxide. Stopped the reaction by adding 100 µl per well of 1N HCl and measured the optical density of samples on a spectrophotometer "Multiscan" (Finland) using a filter with a maximum transmission 492 nm. As negative and positive control were used respectively homologous immune (normal) and hyperimmune serum.

Example 4. Identifying the immunoblot interaction MCA produced by strain-about what wantom Rattus norvegicus 122H9, protein of 12.8 kDa (gene 214L) virus ectromelia, strain K-1.

Viral proteins after 12% SDS page electrophoresis were transferred to nitrocellulose membrane (Millipore, USA). Place nonspecific binding was saturated with 0.5% solution of casein in buffer TSB-twin (0.145 M sodium chloride, 20 mM Tris-HCl, 5 mM PMSF (Sigma), 0.1% Tween-20 (Serva), pH 7.4) for 2 hours at 37°C. Then the individual strips of the membrane were incubated with monoclonal antibodies for 4 h at 20-22°C. Specific binding of antibodies that interact with viral proteins were detected using a conjugate of an antibody against rat IgG labeled with horseradish peroxidase. As a negative control was used Y3-ascitic fluid at a dilution of 1/5000, as a positive control serum to VE in a dilution of 1/5000 from rats used to obtain hybrid. The results are shown in the drawing, regarding the detection method immunoblot of proteins VE (K-1)that interact with antibodies in the immune serum against orthopoxviruses and MCA N. Where the digits represented by the following notation:

1 - anticavity rats to VE (K-1) (1/5000), nereguliruemyi conditions;

2 - anticavity rats to VE (K-1) (1/5000), reducing conditions;

3 - MCA N(1/5000), nereguliruemyi conditions;

4 - ICA N(1/5000), reducing conditions;

5 - ascitic fluid of rat myeloma Y3(1/5000);

6 - normal mouse serum 1/5000).

In parentheses are the cultivation of drugs. The right shows the location of the marker proteins 10, 20, 30, 40, 50, 60,70 kDa on the left labeled proteins of the virus ectromelia.

Example 5. The study of the neutralizing activity of the ICA N on the culture of Vero cells When setting the neutralization (PH) used viruses ospowiki (strain LIVP) and smallpox (strain India). Work with variola virus was carried out in conditions of maximum security (BSL-4) with the use of protective suits with a positive pressure in the who collaborating centre, Koltsovo. The Novosibirsk region. The nature of the conducted work with variola virus has previously been approved by who. Virus infectivity was determined by the method of plaques on the culture of Vero cells. Neutralizing activity of the MAB was determined in monolayer culture of Vero cells in 96-well tablets using ascitic fluid or purified µa. Serial two-fold dilution of the MCA in a supportive environment (50 μl) were introduced into the hole of the tablet containing 100 μl of a supportive environment, then made 1000 PFU of infectious virus in a volume of 50 μl. The initial dilution of the ICA in the hole was 1:40-1:50 for BOB and 1:400-1:500 for EIT. The plates were incubated for 3 days in CO2-incubator at 37°With BOB and 35°With variola virus. After that, cells progresiv is whether a 0.1% solution of crystal violet in 10% formalin and the number of plaques was considered visually. About the presence of neutralizing judged by the absence or reduction in the number of plaques. As a positive control was used monoclonal antibody J2D5, courtesy of Ichihashi Y [4], as a negative ascitic fluid NSO myeloma. The results are shown in table 2. As can be seen from the presented data, the ICA N, secreted by strain-producer Rattus norvegicus NIMB-281, inhibited reproduction WWII and UPE in the culture of Vero cells.

Table 2

Determination of neutralizing activity µa N
ΜaProtein target (kDa)The virus immunogenThe title of the ICA in PH (arr. value)*.
BOBVNO
100%≥50%≥50%
N12,8VE5012000500
* - in % indicated the suppression of belascoaran.

The above properties of the strain of hybrid cells of Rattus norvegicus N allow to conclude that for the first time on the basis of rat myeloma received hybridoma Rattus norvegicus 122H9 producing cross-reactive neutralizing µa to proteins of orthopoxviruses. The hybrid strain CL is current provides reception of rat immunoglobulins of the IgG class in the amount of 7.5 mg of purified antibodies from ml of ascitic fluid. The use for the generation of monoclonal antibodies of hybridoma rat origin has advantages over murine hybridomas in volume produced ascitic fluid. It is known that the yield of ascitic fluid from one mouse is 3 to 5 ml using a strain of hybrid cells of Rattus norvegicus 122H9 allows you to get 15-30 ml of ascitic fluid from one animal. Peeled µa 122H9 specific reacted in ELISA with virus ectromelia (titer MCA was 1:2200000) and was detected in the immunoblot protein of 12.8 kDa VE, strain K-1 (gene 214L). received µa showed cross-reactivity in ELISA with viruses ospowiki, smallpox cows and smallpox. In addition, as shown by the results of the neutralization, MCA 122H9 possessed neutralizing activity against BOB and UPE (strain India), the most pathogenic representative of orthopoxviruses. The above properties of a strain of Rattus norvegicus 122H9 distinguish it from all previously described hybridomas producing µa to proteins of orthopoxviruses.

Sources of information

1. Baker O.R., M. Bray, J.W. Huggins//Antiviral. Research - 2003 - Vol.53 - P.13-23.

2. Czemy C.P., Johann S., Holzle L., Meyer H. //Virology - 1994 - Vol.200, N2 - P.764-77.

3. Dallo S, Rodriguez JF, Esteban M.A //Virology - 1987. - Vol.159, No. 2 - P.423-32.

4. Y. Ichihashi, Oie m //Virology - 1988. - Vol.163, No.1 - P.133-44.

5. Hooper J.W., Schmaljohn, A.I., C.S. Schmaljohn//Prophylactic and therapeutic monoclonal antibodies. - Patent Number: 6, 451, 309 B2 (US)., Sep. 17, 2002.

6. Meyer H, Osterrieder N, Czemy CP. // Virology - 1994 - Vol.200, No. 2, P.778-83.

The strain of hybrid cells of Rattus norvegicus 122H9 deposited in the Institute of cell cultures SRC VB "Vector" under no NIMB-281 used to obtain cross-reactive neutralizing monoclonal antibodies against orthopoxviruses pathogenic for humans.



 

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