RU2395576C1 - STRAIN OF HYBRID ANIMAL CELLS Mus museums L - PRODUCER OF MONOCLONAL ANTIBODIES FOR EXPOSING NUCLEOPROTEIN OF EBOLA VIRUS, ZAIRE SUBTYPE (Mainga STRAIN) (VERSIONS), MONOCLONAL ANTIBODY PRODUCED BY SAID STRAIN (VERSIONS) AND SET FOR "SANDWICH" FORMAT IMMUNOENZYMOMETRIC TEST SYSTEM FOR EXPOSING NUCLEOPROTEIN OF EBOLA VIRUS, ZAIRE SUBTYPE (Mainga STRAIN) - Google Patents

Abstract

FIELD: chemistry; biochemistry.

SUBSTANCE: invention discloses a strain of hybrid animal cells Mus musculus L. 1B2, which is deposited in the Collection of cell cultures of the State Research Center of Virology and Biotechnology VECTOR, which is a producer of monoclonal antibodies which are specific to the nucleoprotein of the Ebola virus, Zaire subtype (Mainga strain) and are used as binding antigens in a "sandwich" format immunoenzymometric system for exposing the neucleoprotein of the Ebola virus, Zaire subtype (Mainga strain), and a strain of hybrid animal cells Rattus Norvegicus 7B11 which is deposited in the Collection of cell cultures of the State Research Center of Virology and Biotechnology VECTOR and which is a producer of monoclonal antibodies which are specific to the nucleoprotein of Ebola virus, Zaire subtype (Mainga strain) and are used as biotin labelled indicators in the "sandwich" format immunoenzymometric system for exposing nucleoprotein of the Ebola virus, Zaire subtype (Mainga strain). The invention describes monoclonal antibodies 1B2 which are produced by the strain of hybrid animal cells Mus musculus L. 1B2, which relate to the subclass of immunoglobulins IgGl which have a heavy 55 kDa and a light 25 kDa chain, and monoclonal antibodies 7B11 which are produced by the strain of hybrid animal cells Rattus Norvegicus 7B 11 related to the subclass of immunoglobulins IgG. The antibodies are used together in the "sandwich" format immunoenzymometric system for exposing nucleoprotein of the Ebola virus, Zaire subtype (Mainga strain).

EFFECT: use of the invention enables to obtain results during "ВЭ" laboratory reseach and when designing a test system for highly reliable exposure of an antigen.

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Description

The invention relates to biotechnology, in particular to medical virology and immunology, can be used to carry out immunodiagnostics of Ebola hemorrhagic fever (GLE).

Ebola virus (VE) belongs to the family of filoviruses, spreads from person to person through direct contact with biological fluids. An aerosol route of transmission is also possible, as studies on experimentally infected animals have shown [1, 2] and high mortality among medical personnel during the 2000–2001 outbreak in Uganda [3]. The natural reservoir and carriers are unknown, effective means of specific prophylaxis for people are absent. In this disease, quick and differential diagnosis is important, since rapidly developing hemorrhagic fever can be fatal in a few days [4, 5, 6, 7, 8, 9, 10] and the symptoms are similar to those caused by diseases caused by other pathogens [11 ] for which vaccines and treatment methods have been developed [12]. Ebola virus is dangerous for the human population due to the development of tourism in African countries, where contact with sick monkeys is possible [13]. Filovirus nucleoprotein (NP) is one of the three major components, accounting for 17% (VP35 protein - 24.5% and VP40 protein - 37.7%, respectively) by weight of the virion [14]. A study of the body's immune response against RE during an outbreak in 1996 in Gabon showed that subsequently surviving patients showed early and quantitatively increased IgG in serum, mainly against nucleoprotein and matrix VP40 [15]. VE appears in the culture fluid upon infection of Vero and L-68 cell culture on 3-4 days, in the blood of infected guinea pigs on 4-7 days, in the blood of Papio Hamadryas monkeys on 7 days from infection (on the day the temperature appears) and may be detected by enzyme immunoassay (ELISA) [16, 17]. In patients suffering from GLE, the virus is present in tissues and blood in high titers of 10 (6) -10 (8) virus particles per milliliter and can be detected on the third day after the onset of symptoms [18]. Currently, in Russia there are no commercially available PCR and ELISA test systems for the rapid detection of antigen VE. Preparations of purified monoclonal antibodies (MCAs) can serve as the basis of an ELISA test system for the detection of viral antigen and as a confirmatory test for PCR diagnostics. In [19], the authors use primers for PCR diagnostic primers for the gene fragment of the nucleoprotein CE and conclude that it is advisable to use an additional reliable ELISA for the detection of viral antigen.

The closest analogue is a collection of 12 lines of murine hybridomas that produce MCA for the recombinant nucleoprotein (subtype Zaire) obtained in the baculovirus expression system. A test system based on these MCAs that capture antigen is also known; rabbit polyclonal antibodies were used as indicators [20]. This laboratory test system detected a virion-associated nucleoprotein in clinical samples (blood serum, liver, spleen) obtained from monkeys during the Ebola-Reston outbreak in the Philippines in 1996 and stored at -80 ° C until 2003 [21 ].

However, the reliability of the data using these MCAs is significantly limited.

The technical result of the proposed technical solution is to obtain such strains of hybrid cells of Mus musculus L. and Rattus norvegicus producing specific MABs as “capturing” antigens and as “indicator” labeled with biotin, not competing with each other for antigenic epitopes, which makes it possible to use them in an immunoenzyme system of the "sandwich" format for detecting a nucleoprotein of HE with a higher reliability of results in laboratory studies of HE and in the construction of test systems for detecting antigen but.

The specified technical result is achieved by obtaining a strain of hybrid cells of the animal Mus musculus l. 1B2, deposited in the Cell Culture Collection of the Federal State Institution of Science and Science, VB Vektor, Rospotrebnadzor, which produces monoclonal antibodies specific for the Ebola nucleoprotein, the Zaire subtype (Mainga strain) and is used as a capture antigen in the sandwich immunoenzyme system to detect the Ebenoprotein virus , subtype of Zaire (Mainga strain).

The indicated technical result is also achieved by obtaining a strain of hybrid cells of the animal Rattus Norvegicus 7B11, deposited in the Cell Culture Collection of the Federal State Institution of Science and Science Scientific Center "Vector" Rospotrebnadzor, which produces monoclonal antibodies specific for the Ebola virus nucleoprotein, subtype Zaire (Mainga strain) and used as indicator labeled biotin in an enzyme immunoassay system of the "sandwich" format for the detection of the Ebola virus nucleoprotein, subtype Zaire (Mainga strain).

The specified technical result is also achieved by obtaining monoclonal antibodies 1B2 produced by the strain of hybrid cells of the animal Mus musculus l. 1B2, belonging to the subclass of IgG1 immunoglobulins having a heavy 55 kDa and light 25 kDa chains, and monoclonal antibodies 7B11 produced by the hybrid animal cell strain Rattus Norvegicus 7B11, belonging to the IgG immunoglobulin subclass used together in the sandwich format immunoenzyme system for detecting a sandwich Ebola virus, subtype of Zaire (Mainga strain).

It is also possible to use them individually in immunofluorescence and enzyme immunoassay methods for the detection of EB in infected cells and tissues.

The strains are obtained by fusion of murine myeloma p3-X63 / Ag8.653 (NS / 1) cells with spleen cells of BALB / c mice immunized with purified, concentrated, inactivated EB preparation, subtype Zaire (Mainga strain) [22] and by rat cell fusion myeloma 210 RC.Y3-Ag 1.2.3. (Y-3) with spleen cells of a rat LOU immunized with the same antigen. The inventive strains of hybrid cells obtained at the State scientific center of virology and biotechnology "Vector" Rospotrebnadzor of the Russian Federation and deposited in the Collection of cell cultures SSC WB "Vector". The author's name for hybridoma cell lines is 1B2 and 7B11.

The pedigree of the strain Mus musculus L. SSC VB "Vector" 1B2. The hybrid cells of the strain were obtained by fusion of murine p3-X63 / Ag8.653 (NS / 1) mouse myeloma cells with spleen cells of BALB / c mice immunized with a purified, concentrated, inactivated VE preparation. A 45% solution of Sigma polyethylene glycol with a molecular weight of 1300–1600 kDa was used as a merging agent according to the method [23]. Cells after fusion were grown in selective GAT medium in 96-well culture plates. Peritoneal macrophages of outbred mice were used as feeder cells. Hybridomas stably producing specific MABs were cloned 3 times. The yield of positive clones in the last cloning was 100%.

The pedigree of the strain Rattus norvegicus SSC WB "Vector" 7B11. The hybrid cells of the strain were obtained by fusion of rat myeloma 210 RC.Y3-Ag1.2.3 cells. (Y-3) with rat spleen cells from a LOU immunized with a purified, concentrated, inactivated EB preparation. A 45% solution of Sigma polyethylene glycol with a molecular weight of 1300–1600 kDa was used as a merging agent according to the method [23]. Cells after fusion were grown in selective GAT medium in 96-well culture plates. Peritoneal macrophages of rat LOU were used as feeder cells. Hybridomas stably producing specific MABs were cloned 3 times. The yield of positive clones in the last cloning was 100%.

The number of passages at the time of deposit: 7-10 passages.

Marker signs and methods for their assessment.

The strain Mus musculus L. SSC VB "Vector" 1B2 secrets murine monoclonal immunoglobulins of the IgG1 subclass (having heavy 55 kDa and light 25 kDa chains) that specifically interact with the NP protein VE. Analysis of murine immunoglobulins is carried out by ELISA using inactivated SE, the Zaire subtype (Mainga strain), or recombinant nucleoprotein (rec. NP) as an antigen (obtained as described [25] as a result of biosynthesis in E. coli cells based on a plasmid construct including complete NP gene VE) and antibodies against mouse IgG labeled with horseradish peroxidase.

The strain Rattus norvegicus SSC WB "Vector" 7B11 secrets rat monoclonal immunoglobulins of the IgG subclass that specifically interact with the NP protein VE. Rat immunoglobulins are analyzed by ELISA using inactivated SE, the Zaire subtype (Mainga strain) or recombinant NP protein and rat anti-IgG antibodies labeled with horseradish peroxidase as antigen.

Contamination by bacteria and fungi was not detected.

Cultural properties. Medium for cultivation of DMEM / F12 with glutamine, pyridoxine, Hepes (FGUN SSC WB "Vector" Rospotrebnadzor). The content of fetal serum optimized for hybridomas (HyClone, USA) in the growth medium is 10%. 80-160 μg / ml gentamicin sulfate is also added to the medium.

Strains are monolayer-suspension cultures in which up to 20% of the cells are in suspension, without attaching to the surface of the dishes for cultivation. Cells from the surface of culture dishes are removed with a solution of trypsin / versene = 1/1 (volume / volume). Sowing dose of 200 thousand cells / ml. Passaging frequency after 3-4 days. The proliferation index is not less than 5. Cultivation of the strain Mus musculus L. SSC VB "Vector" 1B2 in the body of the animal. Female BALB / c mice (vivarium of the State National Center for Seventeenth Century "Vector") are pre-injected intraperitoneally with 0.3-0.5 ml pristan (Sigma). After 2-4 weeks, animals are inoculated intraperitoneally with 10 million hybrid cells. Ascitic tumor forms on the 7-10th day. Hybridoma is vaccinated in 100% of cases. 3-5 ml of ascitic fluid containing MCA can be obtained from one animal. The cultivation of the strain of Rattus norvegicus SSC WB "Vector" 7B11 in the body of the animal Rattus norvegicus. Female LOU rats (vivarium of the State Scientific and Technical Center for Seeds Vector) are previously injected intraperitoneally with 5 ml of paraffin oil or pristan (Sigma). After 2-4 weeks, animals are inoculated intraperitoneally with 30-50 million hybrid cells. Ascitic tumor forms on 10-12 days. Hybridoma is vaccinated in 100% of cases. From one animal, 15-30 ml of ascitic fluid containing MCA can be obtained.

Characterization of a useful product of strain Mus musculus L. SSC VB "Vector" 1B2. Typing of hybridoma immunoglobulins 1 B2 was carried out by the method of solid-phase ELISA using monospecific murine antibodies (Sigma, USA). MCAs belong to the IgGl subclass. They specifically interact with the native protein NP VE (96 kDa) and rivers. NP (96 kDa) in the immunoblot reaction. In IFA, the titer of MCA in ascites is 1: 2187000. Stable production of MCA is maintained for at least 10 passages in vitro with continuous cultivation. 3-5 milligrams of purified MCA can be obtained from one milliliter of ascitic fluid.

Characterization of a useful product of the strain Rattus norvegicus SSC WB "Vector" 7V11. MCAs specifically interact with the native NP protein VE (96 kDa) and the recombinant protein NP (96 kDa) in the immunoblot reaction. In IFA, the titer of MCA in ascites is 1: 512000. Stable production of MCA is maintained for at least 10 passages in vitro with continuous cultivation. 3-5 milligrams of purified MCA can be obtained from one milliliter of ascitic fluid.

Cryopreservation. The freezing medium is DMEM (M) medium - 50%, fetal serum - 40%, dimethyl sulfoxide - 10%. 1-1.5 ml of the cell suspension is transferred into plastic cryovials and placed in a foam container with a wall thickness of 1 cm. The container is placed in a pair of liquid nitrogen and after 18-24 hours the tubes are transferred into liquid nitrogen. Defrosting is carried out by lowering the tubes into water at a temperature of 37-41 ° C. Cells are diluted with DMEM (M) and centrifuged at 1000 rpm. The sediment is resuspended in a growth medium and the cells are transferred into culture bottles at a concentration of 200-300 thousand per milliliter. Cell viability after thawing is 60-80% (according to the results of staining with 0.25% trypan blue).

Each ampoule contains at least 10 million / ml cells.

The invention is illustrated in the graphic material shown in figures 1-3.

Figure 1 presents the electrophoregram of purified MCA, where:

1, 2 - preparations of purified MCAs 1B2 and 7B11 (1 μl each), arrows indicate immunoglobulin chains:

t - heavy chain (55 kDa); l - light chain (25 kDa);

3 - protein markers of molecular weight (10 μl) (Bio-Rad);

4 - values of the molecular weight of protein markers.

On figa presents an electrophoregram of native proteins of the Ebola virus and recombinant nucleoprotein (NP), where:

1 - purified recombinant nucleoprotein (NP) (10 μl);

2 - the preparation of the Ebola virus (20 μl), the arrows indicate the viral proteins;

3 - designations of viral proteins.

Figure 2c shows an immunoblot of a native and recombinant nucleoprotein, where:

1 - the preparation of the Ebola virus, treated with MKA 1B2 at a dilution of 1: 500;

2 - a preparation of the Ebola virus, processed by MCA 7B11 at a dilution of 1: 500;

3 - recombinant NP, treated with MKA 1B2 at a dilution of 1: 500;

4 - recombinant NP, treated with MCA 7B11 at a dilution of 1: 500;

5 - preparation of the Marburg virus, treated with MKA 1B2 at a dilution of 1: 500 (negative control);

6 - preparation of the Marburg virus, processed by MCA 7B11 at a dilution of 1: 500 (negative control);

7 - E. coli. Lysate, treated with 1: 500 MKA 1: 500 dilution (negative control);

8 - E. coli. Lysate, treated with MKA7 B11 at a dilution of 1: 500 (negative control).

Figure 3 shows a graph of titration of an Ebola virus antigen and a recombinant nucleoprotein with a pair of MCA 1B2 and 7B11 *, where: initial concentration of antigen preparations in SF 1 mg / ml, the first titration point at a dilution of 1: 400 corresponds to a concentration of 2500 ng / ml;

7B11 * - indicator MCAs labeled with biotin;

a pair of MCA 9C7 and 5F11 * specific for the Marburg virus nucleoprotein was used as a negative control [24];

the concentration of MCA for the "capture" of antigens - 10 μg / ml;

the concentration of indicator MCAs labeled with biotin is 1 μg / ml.

The method of obtaining the strain of Mus musculus L. SSC VB "Vector" - 1B2.

The strain of hybrid cells Mus musculus L. SSC WB "Vector" - 1B2 receive as follows. Female BALB / c mice (vivarium GNTSVB "Vector"), weighing 15-20 g, are immunized according to the scheme that is shown below in table 1.

Table 1 Immunization schedule days antigen dilution solution antigen dose (mcg / mouse) injection area 0 Freund's complete adjuvant 10 intraperitoneally 7 Freund's incomplete adjuvant 10 intraperitoneally eleven saline 40 intravenously 12 saline 60 intravenously fourteen Hybridization (spleen fence)

For fusion, a 3/1 ratio of mouse splenic cells and NS / 1 murine myeloma cells is used. The mixture of cells is centrifuged, the supernatant is thoroughly removed and 0.4 ml of a 45% solution of polyethylene glycol (PEG) with a molecular weight of 1300-1600 is added to the cell pellet. The mixture is centrifuged for 15 minutes at 1000 rpm. After a 3 min pause, the PEG layer was slowly diluted with 5 ml of versene solution, after which the precipitate was resuspended. Then the cells are pelleted again (10 min at 1000 rpm) and the pellet is dissolved in the growth medium. Cells are dispensed into five 96-well microplates (Costar) at 100 μl per well. Selection of hybrid cells is carried out in GAT medium consisting of DMEM (M) nutrient medium, to which 10% fetal bovine serum (Gipco), 0.1 mM hypoxanthine, 0.04 mM thymidine and 0.01 mM aminopterin are added. Specific hybrids are selected by enzyme-linked immunosorbent assay (ELISA). In the wells of microplates (VNIIMedPolymer), 100-200 ng of purified VE are adsorbed as antigen. Non-specific binding sites are saturated with a 0.5% casein solution (ICN). Then, 100 μl of the culture medium of the studied hybridomas are transferred to the wells and incubated for 45 min at 37 ° C. After incubation, the wells are washed 3-5 times with saline containing 0.05% tween-20 (Sigma). Then, 100 μl of an antispecific conjugate (rabbit immunoglobulins against mouse IgG labeled with horseradish peroxidase) were added to the tablets and incubated for 45 min at 37 ° C. The tablets are washed and carry out an enzymatic reaction. The results of the analysis are determined on a Multiscan spectrophotometer (Finland).

The method of obtaining the strain of Rattus norvegicus SSC WB "Vector" 7B11.

The strain of hybrid cells Rattus norvegicus SSC WB "Vector" 7B11 receive as follows. Female rats Lou (vivarium GNTSVB "Vector"), weighing 180-200 g, are immunized according to the scheme that is shown below in table 2.

table 2 Immunization schedule days antigen dilution solution antigen dose (mcg / rat) injection area 0 Freund's complete adjuvant 10 intraperitoneally 7 Freund's incomplete adjuvant 10 intraperitoneally eleven saline fifty intravenously 12 saline one hundred intravenously fourteen Hybridization (spleen fence)

For fusion, a ratio of 3/1 of rat splenic cells and rat myeloma 210 RC.Y3-Agl.2.3 cells is used. (Y-3). The mixture of cells is centrifuged, the supernatant is thoroughly removed and 0.4 ml of a 45% solution of polyethylene glycol (PEG) with a molecular weight of 1300-1600 is added to the cell pellet. The mixture is centrifuged for 15 minutes at 1000 rpm. After a 3 min pause, the PEG layer was slowly diluted with 5 ml of versene solution, after which the precipitate was resuspended. Then the cells are pelleted again (10 min at 1000 rpm) and the pellet is dissolved in the growth medium. Cells are dispensed into five 96-well microplates (Costar) at 100 μl per well. Selection of hybrid cells is carried out in GAT medium consisting of DMEM (M) nutrient medium, to which 10% fetal bovine serum (Gipco), 0.1 mM hypoxanthine, 0.04 mM thymidine and 0.01 mM aminopterin are added. Specific hybrids are selected by enzyme-linked immunosorbent assay (ELISA). 100-200 ng of purified Ebola virus is adsorbed into the wells of microplates (VNIIMedPolymer) as antigen. Non-specific binding sites are saturated with a 0.5% casein solution (ICN). Then, 100 μl of the culture medium of the studied hybridomas are transferred to the wells and incubated for 45 min at 37 ° C. After incubation, the wells are washed 3-5 times with saline containing 0.05% tween-20 (Sigma). Then, 100 μl of antispecific conjugate (rabbit immunoglobulins against rat IgG labeled with horseradish peroxidase) were added to the tablets and incubated for 45 min at 37 ° C. The tablets are washed and carry out an enzymatic reaction. The results of the analysis are determined on a Multiscan spectrophotometer.

The obtained hybrid strains were cloned twice by the method of limiting dilutions, transferred to mass culture and frozen in liquid nitrogen. The following examples detail the useful properties of the objects of the invention.

Example 1. The cultivation of hybrid cells of the strain Mus musculus L. SSC VB "Vector" - 1B2, producing MAB for nucleoprotein VE in animals, BALB / c mice.

BALB / c mice (vivarium of the State Scientific and Technical Center for Segment Vector), weighing 20-22 g, were injected intraperitoneally with 0.3-0.5 ml pristan not less than 10 days before the vaccination of hybridoma cells. Cultured cells located in the logarithmic phase of growth are sterilized by centrifugation for 5-10 minutes at 1000 rpm in an OPN-3 centrifuge. The supernatant is removed and the cell pellet is suspended in a sterile Earl or Hanks solution. After 2-4 weeks, animals are inoculated intraperitoneally with 10 million hybrid cells in a volume of 1 ml of cell suspension. After 7-10 days, the animals are euthanized and 3-5 ml of ascitic fluid is removed from the abdominal cavity. Cells from ascitic fluid are separated by centrifugation and the titer of the MCA is determined in the supernatant by ELISA, as described above.

Example 2. Cultivation of hybrid cells of the strain Rattus norvegicus SSC VB "Vector" 7B11, producing MCA for nucleoprotein VE in animals, rats Lou.

To rats Lou (vivarium of the State Scientific and Technical Center for Severe Vector), weighing 180-200 g, not less than 10 days before vaccination of hybridoma cells, 5 ml of petroleum jelly or pristan are injected intraperitoneally. Cultured cells located in the logarithmic phase of growth are sterilized by centrifugation for 5-10 minutes at 1000 rpm in an OPN-3 centrifuge. The supernatant is removed and the cell pellet is suspended in a sterile Earl or Hanks solution. After 2-4 weeks, animals are inoculated intraperitoneally with 30-50 million hybrid cells in a volume of 3 ml of cell suspension. After 10-12 days, the animals are euthanized and 15-30 ml of ascitic fluid are removed from the abdominal cavity. Cells from ascitic fluid are separated by centrifugation and antibody titer is determined in the supernatant by ELISA as described above.

Example 3. Isolation of purified monoclonal antibodies produced by hybrid cultured cells of strains of Mus musculus L. SSC VB "Vector" - 1B2 and Rattus norvegicus SSC VB "Vector" 7V11

One volume of ascitic fluid containing MCA is diluted with 4 volumes of 0.6 M acetate buffer (0.04 M citric acid, 0.2 M sodium acetate), pH 4.0, and adjusted to pH 4.5 with 0.1 N caustic soda solution. To the diluted sample, caprylic acid is added dropwise, with constant stirring, at the rate of 25 μl per 1 ml of solution and incubated overnight at + 4 ° C. Then it is centrifuged for 30 min at 8000 g and the precipitate is removed, and the supernatant is mixed with 10-fold phosphate-buffered saline (PBS) and the pH is adjusted to 7.4 with a solution of 1.0 N sodium hydroxide. An equal volume of a saturated solution of ammonium sulfate is added to this solution, shaken and incubated overnight at + 4 ° C or 30 min at 20-25 ° C. Centrifuge for 15 min at 5000 g. The supernatant is drained, and the precipitate is dissolved in PBS, pH 7.4. Residues of ammonium sulfate are removed by dialysis against 50-100 volumes of FSB, pH 7.4. Electrophoresis of purified MCA preparations was carried out according to the method described in [26] in a discontinuous buffer system using 12-15% polyacrylamide gel with 0.1% sodium dodecyl sulfate in Tris-glycine buffer. The separating gel contained 0.0625 M Tris-HCl (pH 8.8), 0.1% SDS, 10 (15)% acrylamide, 1% N, N-methylene bisacrylamide. MCA preparations (1 μl each) were applied to the lane in a volume of 30 μl of buffer containing 0.0625 M Tris-HCl (pH 6.8), 2% SDS, 5% 2-mercaptoethanol, 10% glycerol, 0.01% bromphenol blue . Before application, the buffer containing the MCA was heated for 3 min at 95 ° C. Electrophoresis was carried out at 10 V / cm. The gel was stained using Coomassie G-250. Purified preparations in the form of an electrophoregram are presented in figure 1.

Example 4. Determination by ELISA of the specific interaction of MCA produced by hybrid cells of Mus musculus L. strains of the SSC VB "Vector" - 1B2 and Rattus norvegicus SSC VB "Vector" 7V11 with Ebola virus or recombinant protein NP.

ELISA was performed on polystyrene tablets; working dilution antigen (purified and inactivated Ebola virus or recombinant NP protein) was sorbed in PBS, pH 7.4 in a volume of 100 μl / well per plate. Non-specific binding sites were saturated at 37 ° C for 45 minutes with a 0.5% casein solution in TSB-Tween buffer (0.145 M sodium chloride, 20 mM Tris-HCl, 5 mM PMSF (Sigma), 0.1% Tween-20 (Serva) , pH-7.4) and then incubated with purified MCA for 45 minutes at 37 ° C. Specific binding of the MCA to the antigen was detected by anti-peroxidase-labeled antibodies against mouse IgG for 1B2 or against rat IgG for 7B11. Then added a chromogen, 0.1% O-phenylenediamine, in citrate-phosphate buffer (0.2 M citric acid, 0.5 M Na ₂ HPO ₃ , pH 5.0) with 0.03% hydrogen peroxide). The reaction was stopped by adding 100 μl per well of 1 N HCl and the optical density of the samples was measured on a Multiscan spectrophotometer using a light filter with a maximum transmission of 492 nm. As a negative and positive control, homologous normal (non-immune) and hyperimmune mouse and rat sera were used, respectively.

Example 5. Detection in the immunoblot of the interaction of MCA produced by hybrid cells of Mus musculus L. strains of the SSC VB "Vector" - 1B2 and Rattus norvegicus SSC VB "Vector" 7V11 with the Ebola virus NP protein (Zaire subtype) and recombinant NP protein.

Viral proteins and recombinant protein NP after 12% PAG electrophoresis were transferred to the nitrocellulose membrane (Millipore, USA). Non-specific binding sites were saturated with a 0.5% casein solution in TSB-Tween buffer (0.145 M sodium chloride, 20 mM Tris-HCl, 5 mM PMSF (Sigma), 0.1% Tween-20 (Serva), pH-7.4) 37 ° C for 2 hours. Then, individual strips of the membrane were incubated with purified MAB for 4 hours at 20-22 ° C. Specific binding of antibodies interacting with viral proteins was detected using a conjugate of anti-mouse anti-mouse IgG for 1B2 and anti-rat IgG for 7B11 labeled with horseradish peroxidase. As a negative control, we used membrane strips with the inactivated Marburg virus [24] and E. coli. Lysate, also treated with MCA, transferred after electrophoresis. The results are presented in figa and 2B.

Example 6. Preparation of conjugates of MCA with biotin. The following methodology was used for biotinylation of monoclonal immunoglobulins: a fresh solution of NSB (N-hydroxysuccinimidobiotin) was prepared, for this 2 mg of biotin was dissolved in 0.5 ml of DMSO and the volume was adjusted to 2 ml. Then, a solution of purified MCAs (in 0.1 M NaHCO ₃ pH 9.0) was prepared at a concentration of 1 mg / ml. The NSB solution was added dropwise to the MCA solution in a volume ratio of 1/1 and incubated at room temperature for 4 hours. Then the solution was adjusted to 1 ml with 0.05 M phosphate buffer (PBS) with a pH of 7.0, containing 0.15 M sodium chloride and 0.1% sodium azide. Dialyzed against phosphate buffer. The process of incorporation of biotin into immunoglobulins was controlled by titration of labeled MABs on an antigen immobilized onto plastic with a peroxidase conjugate with streptovidin.

Example 7. Competitive ELISA of the "sandwich" format for the detection of native protein NP VE, subtype Zaire (Mainga strain) and recombinant protein NP.

In wells of highly sorption polystyrene plates (Testiks or Nunc), purified MKA 1B2 was adsorbed in 0.5 M carbonate buffer (pH 9.0) in a volume of 100 μl at a working concentration (10 μg / ml) at 22 ° C for 12 h. Places for nonspecific antibody binding on the plates were saturated with 0.5% casein solution and kept at 37 ° C for 30 minutes. Antigen titration was carried out with a 1: 400 dilution in two steps overnight at 4 ° C or at 37 ° C for one hour. Then, conjugates of MCA 7B11 with biotin were added to the wells of the plates in working dilutions (1 μg / ml), kept at 37 ° C for an hour, and after 3 times washing the wells, streptavidin conjugate with peroxidase was added to them. Specific binding of biotin-labeled antibodies to the conjugate was detected with a TMB liquid substrate system (3.3 ', 5.5' - Tetramethylbenzidine, Sigma) at 100 μl per well. The plates were held for 30 minutes in the dark, the reaction was stopped by adding 100 μl per well of 1N hydrochloric acid, and the optical density of the samples was measured on a Uniscan spectrophotometer at a wavelength of 450 nm. Positive was considered the result of measuring the OD in the well, 2 times greater than that for a well with a negative control. To negatively control the binding of antibodies to the antigen, a pair of MABs detecting the Marburg virus nucleoprotein was used. The antigen titration schedule is shown in FIG. 3.

The above properties of the hybrid strains of Mus musculus L. SSC VB Vektor - 1B2 and Rattus norvegicus SSC VB Vektor 7V11 (the author's name for the cell lines 1B2 and 7B11) allow us to conclude that for the first time on the basis of mouse and rat myeloma, hybridomas Mus musculus L SSC VB "Vector" - 1B2 and Rattus norvegicus SSC VB "Vector" 7B11 - producers of MCAs that do not compete for antigenic epitopes of nucleoprotein VE.

The strain of hybrid cells of Mus musculus L. SSC VB "Vector" - 1B2 provides the production of murine monoclonal immunoglobulins of the IgG1 subclass in the amount of 3-5 mg of purified antibodies from a milliliter of ascitic fluid. The strain of hybrid cells Rattus norvegicus SSC WB "Vector" 7B11 provides obtaining rat monoclonal immunoglobulins of the IgG subclass in the amount of 3-5 mg of purified antibodies from a milliliter of ascitic fluid. Purified MABs specifically reacted in ELISA with VE and recombinant nucleoprotein (MAB titer was 1: 2187000 for 1B2 and 1: 512000 for 7B11, respectively); the virus-associated nucleoprotein and recombinant protein obtained as a result of biosynthesis in E. coli cells on the basis of a plasmid construct containing the complete NP gene of vee was detected in the immunoblot. The joint use of MCA in ELISA in the sandwich format allows the detection of native and recombinant proteins with a sensitivity of less than 1 ng / ml. The use of these drugs MCA and recombinant nucleoprotein as a positive control for the antigen will effectively detect cases of HLE in Russia in the ELISA format "sandwich".

The above properties of the strains of Mus musculus L. SSC VB Vektor 1B2 and Rattus norvegicus SSC VB Vektor 7B11 (the author's name for the cell lines 1B2 and 7B11) distinguish them from all previously described hybridomas producing MABs for Ebola virus proteins.

Information sources

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Claims (5)

1. The strain of hybrid cells of the animal Mus musculus L. 1B2, deposited in the Cell Culture Collection of the Federal State Institution of Science and Science, VB Vektor Rospotrebnadzor, which produces monoclonal antibodies specific for the Ebola virus nucleoprotein, subtype Zaire (Mainga strain) and used as capture antigen in the enzyme immunoassay a sandwich format system for detecting the Ebola virus nucleoprotein, subtype Zaire (Mainga strain). 2. Monoclonal antibody 1B2 produced by the strain of hybrid cells of the animal Mus musculus L. 1B2 (a subclass of IgG1 immunoglobulins having a heavy 55 kDa and light 25 kDa chains) used in the sandwich immunoenzyme system to detect the Ebola virus nucleoprotein, subtype Zaire (strain Mainga). 3. The strain of hybrid cells of the animal Rattus Norvegicus 7B11, deposited in the Collection of cell cultures of the Federal State Institution of Science and Science VB Vektor Rospotrebnadzor, which produces monoclonal antibodies specific for the Ebola virus nucleoprotein, subtype Zaire (Mainga strain) and used as indicator biotin-labeled immunofluores sandwich format for the detection of the Ebola virus nucleoprotein, subtype Zaire (Mainga strain). 4. Monoclonal antibody 7B11 produced by the hybrid animal cell strain Rattus Norvegicus 7B11 (IgG immunoglobulin subclass), used together in the sandwich immunoenzyme system to detect the Ebola virus nucleoprotein, subtype Zaire (Mainga strain). 5. A kit for an enzyme immunoassay system of the sandwich format for the detection of an Ebola virus nucleoprotein, a subtype of Zaire (Mainga strain), including monoclonal antibodies 1B2 produced by a hybrid strain of animal cells of mus musculus L. 1B2 and adsorbed on polystyrene plates, biotin conjugates with monoclonal antibodies 7B11 produced by the strain of hybrid cells of the animal Rattus Norvegicus 7B11, conjugate streptavidin with peroxidase and tetramethylbenzidine, the quantitative content of which in the kit is sufficient to carry out enzyme immunoassay ktsii.

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