RU2395577C1 - STRAIN OF HYBRID ANIMAL CELLS Mus musculus L - PRODUCER OF MONOCLONAL ANTIBODIES FOR EXPOSING VP40 PROTEIN 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 VP40 PROTEIN 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.4 A2, 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 matrix protein VP40 of the Ebola virus, Zaire subtype (Mainga strain), and a strain of hybrid animal cells Mus musculus L. 1C1 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 matrix protein VP40 of the Ebola virus, Zaire subtype (Mainga strain). The invention is also aimed at obtaining monoclonal antibodies 4A2 which are produced by the 4A2 hybridome, (subclass of immunoglobulins IgGl which have a heavy 55 kDa and a light 25 kDa chain) and are used as binding antigens in the "sandwich" format immunoenzymometric system for exposing the matrix protein VP40 of the Ebola virus, Zaire subtype (Mainga strain), and monoclonal antibodies 1C1 produced by the 1C1 hybridome (subclass of immunoglobulins IgGl which have a heavy 55 kDa and a light 25 kDa chain), used as biotin labelled indicators in the "sandwich" format immunoenzymometric system for exposing the matrix protein VP40 of the Ebola virus, Zaire subtype (Mainga strain). The disclosed antibodies are used together in a "sandwich" format immunoenzymometric system for exposing the matrix protein VP40 of the Ebola virus, Zaire subtype (Mainga strain).

EFFECT: invention enables to obtain monoclonal antibodies which are specific and do not compete with each other for antigen epitopes and which, when used together in a "sandwich" format immunoenzymometric system, ensure high reliability of results for exposing the matrix protein VP40 of the Ebola virus.

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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, causing severe hemorrhagic fever of a person with a high mortality rate during unexpectedly occurring outbreaks [1, 2, 3, 4, 5, 6]. The natural reservoir and carriers are not known, effective means of specific prophylaxis for people are absent. Filoviruses spread from person to person through direct contact with body fluids. An aerosol route of transmission is also possible, as studies on experimentally infected animals have shown [7, 8] and high mortality among medical workers during an outbreak in Uganda in 2000-2001. [9]. The spread of renewable energy among the human population is possible through the import of monkeys from African countries and the development of tourism in this region [10]. The VE vaccine is still under development. Complete protection of infected guinea pigs [11] and monkeys [12] was shown when they were immunized with a bivaccine in the form of adenovirus-like particles containing glycoprotein (GP) proteins and matrix protein (VP40) of several strains of Ebola and Marburg viruses. The VP40 protein of filoviruses is one of the three major components, accounting for 37.7% (VP35 protein - 24.5% and nucleoprotein (NP) - 17%, respectively) of the virion mass [13]. 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) [14, 15]. 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 [16]. At present, in Russia there are no commercially available ELISA test systems for the rapid detection of Ebola antigen for monitoring imported animals for zoos and research purposes and tourists who came from African countries with symptoms similar to HLE. Preparations of purified monoclonal antibodies (MCAs) can serve as the basis of such a system on their own or as a confirmatory test for PCR diagnostics [17], which has not yet been developed in Russia either.

Murine hybridomas are known that produce MCAs for Ebola virus proteins: 12 lines for recombinant NP [18], 2 lines for glycoprotein [19], and one line for glycoprotein [23]. In work [20], 9 lines are described that produce MAB for the triton inactivated matrix protein VP40 — 100 VE subtype Zaire (Mainga strain), and one laboratory ELISA sandwich-based test system based on two MABs not competing for antigenic matrix protein epitopes VP40. This system successfully detects the virion-associated VP40 from the virus-containing culture medium and the virus inactivated with sodium dodecyl sulfate added to human serum, as well as the recombinant protein VP40, obtained by infection of cells with the recombinant vaccine virus MVA-T7 containing plasmids encoding the VP40 gene VE (subtype Zaire).

The technical result is to obtain such strains of hybrid cells of Mus musculus L. that would produce specific MABs that do not compete with each other for antigenic epitopes, which would make it possible to use them in the sandwich immunoenzyme system for the detection of VP40 BE protein together as "exciting" antigen and "indicator" labeled with biotin, which will increase the reliability of ELISA in laboratory studies of renewable energy and in the design of test systems for the detection of antigen.

The specified technical result is achieved by creating a strain of hybrid cells of the animal Mus musculus 1. 4A2 deposited in the Collection of cell cultures of the SSC VB "Vector", which is a producer of monoclonal antibodies specific for the Ebola virus VP40 matrix protein, subtype Zaire (Mainga strain), and used in as an exciting antigen in the sandwich format enzyme immunoassay system for detecting the VP40 matrix protein Ebola virus, subtype Zaire (Mainga strain).

The specified technical result is also achieved by creating a strain of hybrid cells of the animal Mus musculus l. 1C1, deposited in the Cell Culture Collection of the State Research Center BB "Vector", which is a producer of monoclonal antibodies specific for the Ebola virus VP40 matrix protein, subtype Zaire (Mainga strain), and used as indicator biotin-labeled in the sandwich immunoenzyme system for detection of matrix Ebola virus VP40 protein, subtype of Zaire (Mainga strain).

The specified technical result is also achieved by obtaining monoclonal antibodies 4A2 produced by the strain of hybrid cells of the animal Mus musculus l. 4A2 (a subclass of IgG1 immunoglobulins having a heavy 55 kDa and a light 25 kDa chain), and monoclonal antibodies 1C1 produced by a hybrid strain of animal mus musculus 1 animals. 1C1 (a subclass of IgG1 immunoglobulins having a heavy 55 kDa and light 25 kDa chain) used together in the enzyme immunoassay system of the "sandwich" format for the detection of the matrix protein VP40 of the Ebola virus, subtype Zaire (Mainga strain).

It is also possible individual use of MCA in immunofluorescence and enzyme immunoassay methods for the detection of RE in infected cells and tissues.

The strains were obtained by fusion of murine p3-X63 / Ag8.653 (NS / 1) myeloma cells with spleen cells of BALB / c mice immunized with purified, concentrated, inactivated 5% β-mercaptoethanol and 1% sodium dodecyl sulfate with the preparation of VE subtype (strain Mainga) [21]. 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 name of the hybridoma cell lines is 4A2 and 1C1.

Pedigree of strains. The hybrid cell strains were obtained by fusion of mouse p3-X63 / Ag8.653 (NS / 1) myeloma cells with spleen cells of BALB / c mice immunized with a purified, concentrated, inactivated EB preparation. A 45% solution of Sigma polyethylene glycol with a molecular weight of 1300–1600 kD was used as a merging agent according to the method of [22].

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

Marker signs and methods for their assessment. The strains secrete murine monoclonal immunoglobulins of the IgGl subclass (having a heavy 55 kDa and a light 25 kDa chain) that specifically interact with VP40 VE protein. Analysis of murine immunoglobulins is carried out by ELISA, using purified inactivated VE, the subtype Zaire (Mainga strain) or the VP40 recombinant protein (obtained as described [26], as a result of biosynthesis in E. coli cells based on a plasmid construct containing the full gene VP40 VE) and anti-mouse IgG antibodies labeled with horseradish peroxidase.

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. Proliferation index - not less than 5.

The cultivation of hybridomas 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 characteristic of a useful product. Hybridoma immunoglobulins were typed by the method of solid-phase ELISA using monospecific murine antibodies (Sigma, USA). MCAs belong to the IgGl subclass. They specifically interact with native VP40 VE protein (40 kDa) and recombinant VP40 (40 kDa) in the immunoblot reaction. In IFA, the titer of MCA in ascites is 1: 6561000. 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 MCA 4A2 and 1C1 (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 protein VP40, where:

1 - values of the molecular weight of protein markers;

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

3 - purified recombinant protein VP40 (10 μl);

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

5 - designations of viral proteins.

Figure 2c shows an immunoblot of native and recombinant VP40 Ebola virus proteins with MCA, where:

1 - preparation of the Ebola virus, treated with MKA 4A2 at a dilution of 1: 10000;

2 - preparation of the Ebola virus, treated with 1A1 MCA at a dilution of 1: 10000;

3 - recombinant protein VP40, treated with MKA 4A2 at a dilution of 1: 10000;

4 - recombinant protein VP40, processed MKA 1C1 at a dilution of 1: 10000;

5 - Marburg virus preparation, treated with MKA 4A2 at a dilution of 1: 10000 (negative control);

6 - preparation of the Marburg virus, processed 1A1 MCA at a dilution of 1: 10000 (negative control);

7 - lysate of E. coli., Treated with MKA 4A2 at a dilution of 1: 10000 (negative control);

8 - lysate of E. coli., Treated with 1A1 MCA at a dilution of 1: 10000 (negative control).

Figure 3 shows a graph of titration of the Ebola virus antigen and the recombinant protein VP40 with a pair of MCA 4A2 and 1C1 *, where the 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

1C1 * - indicator MCAs labeled with biotin;

a pair of MCA 7D8 and 7H10 * specific for the Marburg virus VP40 protein 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 claimed strains.

Strains of hybrid cells of Mus musculus L. SSC VB "Vector" - 4A2 and Mus musculus L. SSC VB "Vector" 1C1 obtained as follows. Female BALB / c mice (vivarium GNTSVB "Vector") weighing 15-20 g are immunized according to the scheme shown in the table below.

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 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 strains of Mus musculus L. SSC VB "Vector" - 4A2 and Mus musculus L. SSC VB "Vector" 1C1 producing MAB for VP40 VE protein in animals, BALB / c mice.

BALB / c mice (vivarium of the State Scientific and Technical Center for Segment Vector) weighing 20-22 g are injected intraperitoneally with 0.3-0.5 ml pristan not less than 10 days before 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. The selection of purified monoclonal antibodies produced by hybrid cultured cells of strains of Mus musculus L. SSC VB "Vector" - 4A2 and Mus musculus L. SSC VB "Vector" 1C1. 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. Caprylic acid is added dropwise to the diluted sample 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 [25], 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 presented in figure 1.

Example 3. Determination by ELISA of the specific interaction of MCA produced by hybrid cells of the strains of Mus musculus L. SSC VB "Vector" - 4A2 and Mus musculus L. SSC VB "Vector" 1E1 with VE or recombinant protein VP40.

ELISA was performed on polystyrene tablets; working dilution antigen (purified and inactivated VE or VP40 recombinant protein) was sorbed in FSB, 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 MCA to the antigen was detected by antispecies peroxidase-labeled antibodies against mouse IgG. Next, a chromogen, 0.1% O-phenylenediamine, was added in nitrate 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 sera were used, respectively.

Example 4. Detection in the immunoblot of the interaction of MCA produced by hybrid cells of the strains of Mus musculus L. SSC VB "Vector" - 4A2 and Mus musculus L. SSC VB "Vector" 1C1 with VP40 VE protein (subtype Zaire) and recombinant protein VP40.

Viral proteins and the recombinant protein VP40 after 12% PAG electrophoresis were transferred to a 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. The specific binding of antibodies interacting with viral proteins was detected using a conjugate of anti-mouse anti-mouse IgG 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 FIGS. 2a and 2c.

Example 5. 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 solution of immunoglobulins 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 6. Competitive ELISA format "sandwich" to detect native protein VP40 VE, subtype Zaire (strain Mainga), and recombinant protein VP40.

In wells of highly sorption polystyrene plates (Testiks or Nunc), purified MCA 4A2 was sorbed 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 (inactivated virus or recombinant protein) was carried out with a 1: 400 dilution in two steps overnight at 4 ° C or at 37 ° C for one hour. Then, the MKA 1C1 conjugate with biotin was added to the wells of the plates in working dilution (1 μg / ml), kept at 37 ° С for an hour, and after 3 times washing the wells, the streptavidin conjugate with peroxidase was added to them. Specific binding of biotin-labeled antibodies to the conjugate was detected by the 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 IN 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 MCAs was used to detect Marburg virus VP40 protein. The antigen titration schedule is shown in FIG. 3.

The above properties of the hybrid strains of Mus musculus L. SSC VB "Vector" - 4A2 and Mus musculus L. SSC VB "Vector" 1C1 (the author's name of the cell lines 4A2 and 1C1) allow us to conclude that for the first time on the basis of mouse myeloma hybridomas Mus musculus L were obtained SSC VB "Vector" - 4A2 and Mus musculus L. SSC VB "Vector" 1C1 - producers of MABs that do not compete for antigenic epitopes of VP40 VE protein. The strains of hybrid cells provide mouse monoclonal immunoglobulins of the IgGl class 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 VP40 recombinant protein (MAB titer was not less than 1: 6561000); the virus-associated VP40 protein and the VP40 recombinant protein obtained as a result of biosynthesis in E. coli cells on the basis of a plasmid construct including the full VP40 VE gene were 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 1ng / ml. The use of these drugs MCA and the recombinant protein VP40 as a positive control for the antigen will effectively detect cases of GLE in Russia in the ELISA format "sandwich".

The above properties of the strains of Mus musculus L. SSC VB Vektor 4A2 and Mus musculus L. SSC VB Vektor 1C1 (the author's name for cell lines 4A2 and 1C1) 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. 4A2, deposited in the Collection of cell cultures of the SSC VB "Vector", which is a producer of monoclonal antibodies specific for the Ebola virus matrix VP40 protein, subtype Zaire (Mainga strain), and used as capture antigen in enzyme immunoassay system of the "sandwich" format for the detection of the matrix protein VP40 of the Ebola virus, subtype Zaire (Mainga strain). 2. Monoclonal antibody 4A2 produced by the strain of animal hybrid cells of Mus musculus L. 4A2 (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 matrix protein VP40, the Zaire subtype (Mainga strain). 3. The strain of hybrid cells of the animal Mus musculus L. 1C1, deposited in the Collection of cell cultures of the SSC VB "Vector", which is a producer of monoclonal antibodies specific for the Ebola virus matrix VP40 protein, subtype Zaire (Mainga strain), and used as indicator labeled with biotin in the enzyme immunoassay system of the "sandwich" format for the detection of the matrix protein VP40 of the Ebola virus, subtype Zaire (Mainga strain). 4. Monoclonal antibody 1C1 produced by the hybrid strain of animal cells of the mus musculus L. 1C1 animal (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 matrix protein VP40, the Zaire subtype (Mainga strain). 5. A kit for an enzyme immunoassay system of the sandwich format for detecting the Ebola virus VP40 matrix protein, the subtype Zaire (Mainga strain), including monoclonal antibodies 4A2, produced by the mus musculus L. 4A2 animal hybrid cell strain and adsorbed on polystyrene plates, biotin conjugates with monoclonal antibodies 1C1 produced by the strain of hybrid cells of the animal Mus musculus L. 1C1, a streptavidin conjugate with peroxidase and tetramethylbenzidine, the quantitative content of which in the kit is sufficient for enzyme immunoassay eaktsii.

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