RU2092554C1 - Strain of the hybrid cultured animal cell mus musculus l - a producer of monoclonal antibody raised to aujeszky's disease virus, strain uniiev-18b - Google Patents

Abstract

FIELD: veterinary biotechnology, virology. SUBSTANCE: invention relates to preparing the strain of the hybrid cultured cells Mus musculus L. producing antibodies raised to Aujeszky's disease virus, strain UNIIEV-18B. Strain is obtained by hybridization of murine myeloma cell (strain PAI) with splenocytes of murine strain Balb/c immunized with the strain UNIIEV-18B. Strain GVBAV-94/1 produces monoclonal antibodies of isotype IgG and their concentration in cultural and ascetic fluids - 10-20 mcg/ml and 20-30 mg/ml, respectively. Strain can be used for development of preparations used for diagnosis and prophylaxis of Aujeszky's disease. EFFECT: strain indicated above, enhanced effectiveness of antibodies. 1 tbl

Description

 The invention relates to virology, immunology and biotechnology, in particular to hybridoma technology and is a new strain of hybrid cultured animal cells of Mus musculus L animals that produce monoclonal antibodies (Mabs) to Aujeszky's disease virus (WBA) in cell cultures and ascitic fluids, which can be used for scientific research and preparation of diagnostics, prevention and treatment of Aujeszky’s disease (BA).

 AD is acute in the form of epizootics and sporadic cases of viral disease of farm animals of all kinds, fur animals and rodents. It is characterized by signs of damage to the nervous system and lungs, severe itching and scratching (in all species of animals, except pigs, minks and dogs). AD is a typical herpes virus infection, which has many forms of the course, including with a long, almost lifetime latency / 1 /.

 AD is one of the most common diseases of pigs, causing serious material damage to countries with developed livestock. In connection with the intensification of pig breeding, there is a tendency toward a wide enzootic distribution of AD throughout Europe, North and South America. This disease is classified as a viral infection especially dangerous for pig farming. All this forces to intensify the work on creating more effective means of diagnosis, prevention and treatment of AD. The problem of creating effective protective drugs remains very relevant. This is due to the characteristics of the pathology, epizootology and immunology of herpes virus infections.

 One of the areas of research to create a new generation of AD vaccines is the search for new field vaccines and the production of vaccine strains of IBA from them, suitable for the manufacture of effective vaccines.

 The first VBA vaccine strain widely used in Europe was the BUK strain obtained by a series of passages on chicken fibroblasts.

 Currently, there is information about the use of live and attenuated, virulent and avirulent strains of BWA: Barta K61, MK 25 and 35, Nia 3.4 and 14, Alfort 26, Arsky, VGNKI, K and others / 2, 3, 4, 5 /.

 Enzyme-linked immunosorbent assay using monoclonal antibodies, which are widely used by foreign researchers / 6, 7, 8, 9, 10, 11, 12, 13 /, is the most promising for the identification and identification of BVA and antiviral antibodies. Monoclonal antibodies are the most effective tool for studying the patterns of interaction between the virus and the cell, revealing the pathological, functional characteristics and biological properties of the pathogen. Monoclonal antibodies can be used to study the antigenic relationship of VBA strains, which makes it possible to predict the protective properties of vaccines prepared from field isolates. Mabs have several advantages over traditionally used polyclonal antisera. They are characterized by chemical homogeneity, strictly defined specificity for antigenic determinants of the virus. Sources of their production (hybridomas) are characterized by genetic stability and can be stored for a long time in liquid nitrogen. Mabs can be produced in any quantities in the form of ascitic fluid in mice or supernatants of cell culture. Immunofluorescence and immunoperoxidase methods using monoclonal antibodies / 7, 8, 9, 10, 11, 12, 14 / are widely used for the diagnosis and identification of IBA.

 At present, strains of hybrid cultured animal cells of Mus musculus L animals that produce monocdonal antibodies to BWA or to its main immunizing component of the Gp 50 virion are already known. Foreign researchers report data on the preparation of monoclonal antibodies for both whole BVA virion and individual virus-specific Virion proteins GI, G II, G III, Gp 50/6, 8, 9, 10, 11, 13 /.

 Ben-Porat T. et al. Have studied the role of various BW glycoproteins in inducing the production of neutralizing antibodies in pigs. The authors analyzed the antigenic properties and immunogenic activity of BW glycoproteins identified in the immunoprecipitation reaction with Mab. Viral isolates isolated in different geographical areas of the United States, significantly differed in antigenic properties. Changes are noted in proteins G I and G III. The neutralizing activity of MAb is mainly associated with G III / 6 /.

 In Russia, the strain of UNIIEV 18V VBA is a production one and is used in the manufacture of vaccines and diagnostic preparations, however, we did not find information on the receipt of Mab for VBA strain UNIIEV 18V / 15, 16, 17, 18, 19, 20, which is available to us. 21 /.

 The objective of the invention was to obtain a strain of hybrid cultured cells (hybridomas) producing Mab to BWA strain UNIIEV 18B, highly active in enzyme immunoassay and neutralization reactions with a homologous virus.

 The problem is solved in that a new, previously unknown strain of hybrid cultured mouse cells (Mus musculus L) was obtained.

GV VAI-94/1 producer of MAb for BWA strain UNIIEV 18B. The culture is stored in the cell museum of the All-Russian Research Institute of Animal Welfare and on passage 17-20 in the culture of cells grown in plastic mattresses, as well as after grafting on mice of the BA LB / C line, was deposited in the All-Russian Collection of Cell Cultures of the Institute of Cytology RAS (St. Petersburg ) under the registration number VSCK (P) 647D
Characterization of the obtained strain
1. The pedigree of the strain. The hybrid strain N 1 GVBAU-94 (GVBAU-94/1) was obtained by fusion of murine PAI line myeloma cells with mouse spleen cells of the BAL B / line of the previously immunized WBA strain UNIIEV 18B.

 2. The number of passages at the time of certification and deposit. Cells were frozen at passage 17-20 in a culture of cells grown in plastic mattresses, as well as after transplantation on mice of the BA L B / C line.

3. Standard growing conditions. The cultivation temperature is +37 ^o C. DEMEM cultivation medium supplemented with 15 fetal bovine serum 50 μg / ml gentamicin, pH 7.0-7.2.

4. Cultural properties. The strain grows on mammalian cell culture media JMDM, DMEM, RPMJ-1640 supplemented with 10-20 fetal bovine serum with 50 μg / ml gentamicin. When hybrid cells are selected, hypoxanthine-aminopterinthymidine (GAT) is added to the culture medium in working dilutions. The optimal cell growth concentration is 150,000 cells / ml. The optimal pH of the medium is 7.0-7.2 cells are incubated in a humid atmosphere with 5 CO ₂ at 37 ^o C. When inoculated, single cells during the propagation process form round-shaped colonies with smooth edges, then the colonies merge and form a continuous monolayer. At a sowing dose of 150,000 cells / ml, a continuous monolayer is formed for 2-3 days. Cells have good adhesive ability. Easily removed from the walls of the dishes by shaking without the use of trypsin and versene. Cell viability is maintained by regular reseeding on a fresh nutrient medium. The frequency of reseeding 3 times a week. The sieving shortness is 1: 2 1: 5 Type of growth stationary suspension. When hybridomas are administered intraperitoneally, ascitic or solid tumors form in BA L B / C mice. Before the introduction of cells for 5-20 days, these animals are injected intraperitoneally with 0.3-0.5 ml of Angarsk oil (TU-38-101 84-81). The titer of antibodies in ascitic fluid in ELISA is 10 ³ -10 ⁵ . From one mouse receive 3-10 ml of ascites. Ascites is formed after 14-20 days.

 5. Characterization of the cultivation of hybridomas in the body of the animal. It forms ascitic tumors in mice of the BA B / s line. The dose of cells inoculated with 4-10 million / ml mice primed with 0.5 ml of hangar oil 5-20 days before the introduction of cells. The term for ascites formation is 10–20 days; it does not lose antibody-producing ability when vaccinated for 15–20 passages in mice (observation period).

 6. Cytogenetic (karyological) characteristic. The karyotype corresponds to the species (mouse). Modal class 86 chromosomes. The modal class for the parental myeloma line PAI-64 chromosome.

 7. Cytomorphological characteristic. The cells are round, of various sizes. Nuclei occupy most of the cell.

 8. Oncogenicity. The strain has oncogenic properties, characterized by the formation of ascites or solid tumors in mice of the line BA L B / s.

 9. Marker characteristics. LDH isoenzymes correspond to murine ones (one isoform with an additional band). Hybrid cells produce Mab to BWA strain UNIIEV 18B.

10. Data on contamination. Microbiological, cytochemical (staining with olivocinin, acridine orange) and electron microscopic methods did not detect contaminants
11. Biotechnological characteristics. The strain produces Mabs belonging to the class JgG and having specific activity in ELISA, pH, RE in mouse suckers. Mabs are found in ascitic fluid in ELISA for 15-20 passages in mice (observation period) at dilutions of 10 ^-3 -10 ^-5 and have specific activity for BWA strain UNIIEV 18B. It was noted in RN-6, Olog ₂ , RZ on mouse suckers -3.2. The culture fluid can be used as a diagnostic antibody preparation, however, to obtain a higher antibody titer of 10 ^5, hybrid cells at a dose of 1-10 million / 0.5 ml are administered intraperitoneally to mice of the BA L B / s line, primed with intraperitoneal Angara oil. 14-20 days after the appearance of ascites or solid tumors in mice, liquid (3-10 ml) is collected, the cells are precipitated by centrifugation at 1000 rpm for 10 minutes. Non-stop liquid contains Mab. The pellet containing hybrid cells is transferred to culture or injected into mice. To increase the activity and specificity of Mabs, they are concentrated and purified, isolating pure immunoglobulins. The immunoglobulin fraction is obtained by double precipitation of 10 PEG with m.m. 6000 (an equal volume of 20 PEG is added to the antibodies and centrifuged at 3500 rpm for 30 minutes).

The precipitate was resuspended in a reduced amount of phosphate buffered saline (PBS). The solution is dialyzed against the FBI for 24 hours at 4 ^o C. The culture fluid contains 10-20 μg / ml, and ascites -20-30 mg / ml of specific immunoglobulins.

12. The method of cryopreservation. Hybridoma cells are preserved in liquid nitrogen in plastic or glass ampoules in a volume of 1 ml with a concentration of 4-10 million / ml. Cryoprotective medium: DMEM-50 fetal serum KRS-43, dimethyl sulfoxide (DMSO) 7 Cell survival after storage 60-90
Once the medium becomes liquid, the cells are transferred to a conical plastic beaker with 50 ml of cold nutrient medium and precipitated by centrifugation at 1000 rpm. The precipitate is resuspended in 5 ml of DMEM medium with 20 fetal serum and GAT and plated in a 50 cm ³ glass mattress. Cells are inhibited at a temperature of 37 ^o C. When the colonies fill 50-70 areas of the mattress, a sample of culture fluid is taken to determine the titer of antibodies to VBA in ELISA. The highest antibody titer was observed with a complete monolayer.

 The proposed strain of hybrid cultured cells was obtained according to a previously developed technique / 22 / as follows.

Mice of the BA L B / c line at the age of 1.5-2 months were injected intraperitoneally with the BBA strain UNIIEV 18B in an amount of 100 μg / mouse in a mixture with Freud's complete adjuvant in a ratio of 1: 1. After 21 days, mice were hyperimmunized intraperitoneally with the same antigen in dose of 100 mcg / mouse without adjuvant. Three days after the repeated administration of the antigen, the blood serum of mice was tested for the presence of specific antibodies in ELISA. Donors with an antibody titer in the blood serum of less than 10 ³ were selected for the experiment.

 For hybridization, spleen was sterilized from immunized mice and resuspended in 50 ml of serum-free DMEM culture medium. PAI line myeloma cells in the logarithmic growth phase were washed in serum from DMEM medium, mixed with spleen immune cells in a ratio of 1: 1 to 1: 5, and precipitated by centrifugation at 1000 rpm for 10 min. A 50% PEG solution of m.m was added dropwise to the precipitate over a minute. 4000 containing 5 DMSO. Cells were left for 5 min with PEG. Then 5 ml of serum-free DMEM was added and left to contact for another 5 minutes. The resulting cell suspension was diluted with DMEM medium containing 20 fetal serum and GAT in an amount of 13.6 mg / l; 0.176 mg / L and 3.78 mg / L, respectively, at working concentrations. Cells were introduced into 96-24-well plates of Flow Laboratories (UK) 0.2-1.0 ml at a concentration of 100-20 thousand / well, respectively. After 10-14 days, supernatants of hybrid clones were tested for the presence of antibodies in ELISA. Positive clones comprised 5-20 of the total number of clones. These clones were subcloned twice by limiting dilution. The stable clone with the highest titer of antibodies (MAb-1B4) was stored in the VNIIZH cell bank under the author's name N 1 GVBAU-94 (GV VAI-94/1) and deposited in the All-Russian Collection of Cell Cultures of the Institute of Cytology RAS (St. Petersburg ) under the registration number VSCK (P) N 642 D.

Example. Antibody WBA strain UNIIEV 18B identified in ELISA using mAbs. For this, Mabs diluted in 0, -2-molar carbonate-bicarbonate buffer pH 9.5 in an amount of 200 μl / well at a concentration of 1-10 ng / ml are added to the wells of polystyrene plates for serological reactions and incubated for 2- 14 hours at a temperature of 20-4 ^o C. The antibody solution is removed and the wells are washed 3-5 times with PBS. Then, 200 μl of the test antigen in the FBI with 10 fetal cattle serum is introduced into the wells. The antigen is prepared by five- or ten-fold dilutions in the same solution. The antigen is incubated in the wells of the tablet for 2 hours at 37 ^o C and washed 3-5 times in the FBI. Then a conjugate (MAb labeled with horseradish peroxidase) was added to the wells in working dilution. The conjugate was diluted on PBS with 10 fetal bovine serum to a concentration of 1-5 μg / ml per JgG and incubated for 1-2 hours at room temperature. Then the wells are washed 3-5 times in the PBS and a substrate containing orthophenylenediamine 0.4 mg / ml in 0.1 m citrate-phosphate buffer pH 5.0 and 0.01 H ₂ O _{2 is} introduced into them. After incubation at room temperature for 1-5 minutes, the reaction is counted by determining the optical density of the solution on a Dyna automatic 1-channel photometer. The reaction can be taken into account visually according to the color intensity of the orange color. It was found that the strain of hybrid cells is stable both in cultural and morphological characters, and in the titer of specific antibodies in ELISA, pH and RE.

 The activity and specificity of Mabs for IBA was determined in ELISA, PH, and RE with both heterologous antigens (foot and mouth disease, CHD, CHP, diarrhea, cattle), and homologous BBA antigens.

Tests have shown that Mabs for BWA do not react with foot-and-mouth disease pathogens (A ₂₂ and Asia-1), VBS, VES, and cattle diarrhea.

 The data on the determination of the specific activity of ascitic fluid containing Mab obtained from strain N 1 of HVBAI-94 (GVVAI-94/1) registration number VSCK (P) 642D, with homologous antibodies of VBA are presented in the table.

 The WBA strains Arsky, Bran, BUK, Geneva, Marashtansky were kindly provided by Dr. Sc. Kamalov G.Kh. (KazNIVI, Kazan).

 The Camel strain is highlighted by prof. Ph.D. Mishchenko V.A. during an outbreak of illness in camels in Turkmenistan in 1992.

 Strain MK was obtained from Dr. Tereklekov P. (Bulgaria).

 According to the table, the highest specific activity of MAb was noted with strains of UNIIEV 18B and BRAN, which probably indicates a high degree of kinship between the above strains. With other homologous antigens, lower activity is established.

 Obtained in accordance with the invention, Mabs as chemically pure reagents can be used in ELISA, PH and RE for the detection of VVA, antiviral antibodies in the blood serum of susceptible farm animals, as well as for genetic engineering and molecular biological characterization of the pathogen.

Mabs against BW can be very useful in veterinary virology in practical and purely scientific fields with:
identification of antigenic sites at the level of amino acid sequences;
characterization of antigenic sites using various modifications of ELISA;
identification of immunologically important antigenic sites;
the use of mAbs to accelerate the identification of viral isolates;
control of vaccine manufacturing technology;
virus isolation and purification;
immunization and therapy of animals.

 The use of Mabs in veterinary practice in the diagnosis, prevention and treatment of farm animals will optimize the epizootic situation in the country for Aujeszky's disease.

Information sources:
1. Maleev V.V. Syurin V.N. Aujeszky's disease. BME, 1975, ed. 3, v. 2, p. 362.

 2. Guadagnini P.F. Gelmett D. Malatia di Aujeski, ove strategie di profalassi Selez Veter. 1985, 26, 4, 534-541.

 3. Oirschot J.T. Van Ctielkens A.L.J. Some characteristies of four attenuated Vaccine Virus strains and a virulent strain of Anijeszky's disease virus Veter. Q, 1984 6, 4, 225-229.

 4. Mishchenko V.A. Dudnikov A.I. Bondarenko A.F. Evaluation of the immunogenic activity of an inactivated vaccine against Aujeszky's disease in rabbits. Mater. scientific conf. VNIIVViM, Pokrov, 1992 1, 194.

 5. Prokhorova E.M. Posabtseva L.A. The activity of three virulent strains of Aujeszky's disease. Specific prevention and diagnosis of animal diseases, 1986 16-18.

 6. Ben-Porat T. De Marchi J.M. and all Role of glycoproteins of psendorabis virus in eliciting neutralizing antibodies Virologi, 1986, 154, 325-324.

 7. Chang-Hee Kweon, Dong-Sup Lul, Moo Huyng Jun. Serological diagnosis of pseudorabies uting monoclonal antibody Veterinary Viral diseases, 1985, 478-479.

 8. Eliot M. Bourghaia H. and all. Identification of antigenic sites on pseudorabies virus glycoprotein gp 50 implicated in virus penetration of the host cell J. Gen Virol, 1990, 71.9, 2179-2183.

 9. Eliot M. Fageaud D. Vannier P. Toma B. Development of an ELJSA to differentiable between animals either vacianated with or infected by Anjeszky's disease virus veter. Res, 1989, 4, 91-94.

 10. Jacobs L. Meloen R.N. Gielkeus A.L. Van Oirschot J.T. Epitope analisi's of glycoprotein 1 psendorafies virus J Gen Virol, 1990, 71, 4, 381-387.

 11. Mastuda Asami, Okada Nobutaka, Katayama Shigeyi e.a. Characterizaton of protective viral glycoproteins for pseudorabies virus infection. J Vet. Med. Sci. 1991, 53, 4, 734-741.

 12. Osorio F.A. Doster A.R. Rapid diagnosis on pseudorabies EC (extension curcular) Nebraska cooperative extension service, Nebraska agr. -res. div 1987, 87-219, 14-15.

 13. Wather M.W. Wather L.M.K. Isolation, characterization and physical mapping of a psendorabie virrus mutant containing antigenically altered gp 50. J. Virol. 1984, 51, 1, 57-62.

 14. Quist R. Sorensen K.J. Meiling A. Monoclonal blocking ELJSA detecting serum antibodies to the glycoprotein g. 2 of Anjeszky's disease virus. J. Virol. Meth. 1989, 24, 169-180.

 15. Morenkov O.S. Mantsygin Yu.A. Sergeev V.A. and others. Obtaining and characterization of monoclonal antibodies to glycoprotein G2 of the Aujeszky's disease virus and their use for epitope mapping of G2. Questions virologist. 1994, 4, 174-177.

16. EPO N 0231641, IPC ³ A 61 K 39/42, C 12 P 21/00, C 07 K 15/00, C 12 5/00, C 12 R 1:91, 12.19.86, USA 09.01 .86, authors: Marchidi CC Jancey RJ Aujeszky's disease vaccine.

 17. Tsymbal A.M. and others. Inactivated culture vaccine UNIIEV against Aujeszky's disease of pigs, sheep and fur animals. Thes. doc. 3 All-Union. conf. "Scientific basis of technology for the industrial production of veterinary biological products." M. October 21-23. 1987 49-50.

 18. Tsymbal A.M. Lysenko I.P. Konarzhevsky K.E. et al. Laboratory and production testing of inactivated culture vaccine UNIIEV against Aujeszky's disease. Scientific the basics of the profile. and fight against the disease. village live, 1987 17-23.

 19. Tsymbal A.M. Konarzhevsky K.E. Belokon V.S. et al. Virulent and immunogenic properties of Aujeszky's disease virus reproduced on transplantable cells of the SPEV line cultivated on various nutrient media. Veterinary medicine (Kiev), 1989 64, 3-4.

 20. Guidance on the use of emulsion inactivated concentrate vaccines against Aujeszky's disease from a culture virus. Approved 10/05/92 g.

 21. Emulsion inactivated vaccine VNIIII-UNIIEV against Aujeszky's disease from the culture virus. TU, approved. GUV of Ukraine 01/25/92

 22. The method of obtaining and maintaining hybrid cells secreting monoclonal antibodies to foot and mouth disease Asia-1. Approved by the Director of ARRIAH 06/22/94

Claims (1)

 The strain of hybrid cultured animal cells Mus musculus L VSCK (P) N 642D producer of monoclonal antibodies to the virus of Aujeszky's disease strain UNIIEV 18B.

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