RU2395575C1 - STRAIN OF HYBRID ANIMAL CELLS Mus musculus L - PRODUCER OF MONOCLONAL ANTIBODIES FOR EXPOSING VP40 PROTEIN OF MARBURG VIRUS, (Popp STRAIN) (VERSIONS), MONOCLONAL ANTIBODY PRODUCED BY SAID STRAIN (VERSIONS) AND SET FOR "SANDWICH" FORMAT IMMUNOENZYMOMETRIC SYSTEM FOR EXPOSING VP40 MATRIX PROTEIN OF MARBURG VIRUS (Popp STRAIN) - Google Patents

Abstract

FIELD: chemistry; biochemistry.

SUBSTANCE: invention discloses a strain of hybrid animal cells Mus musculus L. 7D8, 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 VP40 matrix protein of the Marburg virus (Popp strain) and are used as binding antigens in a "sandwich" format immunoenzymometric system for exposing the VP40 matrix protein of the Marburg virus (Popp strain) and a strain of hybrid animal cells Mus musculus L. 7H10 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 VP40 matrix protein of the Marburg virus (Popp strain) and are used as biotin labelled indicators in the "sandwich" format immunoenzymometric system for exposing the VP40 matrix protein of the Marburg virus (Popp strain). Monoclonal antibodies 7D8 which are produced by the strain of hybrid animal cells Mus musculus L. 7D8 relate to a subclass of immunoglobulins IgGl and have a heavy 55 kDa and a light 25 kDa chain. Monoclonal antibodies 7H10 which are produced by the strain of hybrid animal cells Mus musculus L. 7H10 relate to a subclass of immunoglobulins IgGl and have a heavy 55 kDa and a light 25 kDa chain. Monoclonal antibodies 7D8 and antibodies 7D8 and 7H10 are used together in the "sandwich" format immunoenzymometric system for exposing the VP40 matrix protein of the Marburg virus (Popp strain).

EFFECT: use of the invention increases reliability of enzyme immunoassay.

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Description

The invention relates to biotechnology, in particular to medical virology and immunology, and can be used for immunodiagnostics of Marburg hemorrhagic fever (GLM).

Marburg virus (VM) belongs to the family of filoviruses and causes human hemorrhagic fever with a mortality rate of 40 to 90%, as shown by epidemiological studies during the largest outbreak in 2004-2005 in Angola [1, 2]. The natural reservoir and carriers are not known, effective means of specific prophylaxis for people are absent. Despite the geographically limited range of unexpected outbreaks, the potential for aerosol transmission is high and the risk of spread among the human population in different regions of the world is possible [3, 4, 5]. Attempts to obtain a vaccine based on an inactivated virus have failed [6]. Currently, only one Center (the U.S. Military Medical Research Institute of Infectious Diseases) is searching for approaches to creating a vaccine based on filovirus protein gene delivery vectors. Several candidates for the vaccine have been described: 1) DNA vaccination with plasmids carrying the BM genes, 2) defective virus particles of the Venezuelan encephalomyelitis containing filovirus proteins, 3) an adenovirus vector system, 4) a bivaccine based on adenovirus-like particles carrying proteins of several strains of BM and Ebola virus (RE) [7, 8, 9, 10]. Researchers have decided on the main proteins that cause complete protection of infected animals, these are glycoprotein (GP) and matrix protein VP40. It is possible that the protection of people against deadly filovirus fevers will be achieved over time. Matrix protein VP40 plays an important role in the final stages of virion assembly. It has been shown that for the formation of virus-like particles morphologically similar to natural particles during transfection of 293 cell culture with plasmids containing the gene encoding the VP40 protein, participation of it alone is sufficient [11, 12]. Protein VP40 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, causing the formation of antibodies in the body of people who have had HLM [13, 14 ]. VM appears in the blood and secretions of infected guinea pigs and Masas mulatta monkeys on the 4th day from infection and can be detected by enzyme-linked immunosorbent assay (ELISA) [15, 16]. Guinea pigs were infected with blood, sperm, urine, and a nasopharyngeal flush of an employee infected with VM in case of accidental laboratory infection. All animals died, and VM was isolated from their organs [17]. Currently, in Russia there are no commercially available ELISA test systems for the rapid detection of BM antigen for monitoring imported animals for zoos and research purposes and tourists who came from African countries with symptoms similar to those with HFM. Preparations of purified monoclonal antibodies (MCAs) can serve as the basis for such a system on their own or as a confirmatory test for PCR diagnostics, which has not yet been developed in Russia either.

Known is a strain of hybrid animal cells of Mus musculus L. M1 / N-10G9 animals that produce monoclonal antibodies to the structural protein GP of the Marburg virus for 30 passages in vitro; the Porr strain belonging to the IgG isotype is suitable for preparing diagnostics based on them for specific detection Marburg virus using enzyme immunoassay (RF patent No. 2186107, IPC C12N 5/18, publ. 07.27.2002).

However, the monoclonal antibodies produced by the indicated strain of hybrid cells are specific for a surface protein, which is GP glycoprotein with a molecular weight (MM) of 125 kD.

The closest analogue (prototype) is a murine hybridoma cell line producing MAB to the matrix protein VP40 [7] of Marburg virus (strain Musoke).

However, hybrid animal cell lines producing MABs are not known that do not compete for antigenic epitopes of VP40 matrix protein.

MKAs are known that are used in laboratory ELISA test systems in the “sandwich” format for detecting VM antigen [15, 22, 23, 24].

However, from the indicated laboratory ELISA test systems in the “sandwich” format for detecting the BM antigen, there is no system based on two MCAs that do not compete for antigenic epitopes of the matrix protein VP40.

The technical result of the proposed technical solution is to obtain such strains of hybrid Mus musculus L. cells that produce specific mAbs for VP40 BM protein (Porr strain) that do not compete for antigenic epitopes, which allows their use in the sandwich immunoenzyme system for protein detection VP40 VM with the joint use of MCAs as “capturing” antigens and as “indicators” labeled with biotin, which will increase the reliability of ELISA in laboratory studies of VM and in the design AANII 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 L. 7D8, 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 Marburg virus matrix protein VP40 (Porr strain) and used as " exciting "antigen in the enzyme immunoassay system of the" sandwich "format to detect the matrix protein VP40 of the Marburg virus (strain Porr).

The indicated technical result is also achieved by creating a strain of hybrid cells of the animal Mus musculus L. 7H10 deposited in the Collection of cell cultures of the Federal State Institution of Science and Science of the Scientific Center "Vector" of Rospotrebnadzor, which produces monoclonal antibodies specific for the Marburg virus VP40 matrix protein (Rohrr strain) and used as indicator labeled with biotin in an immunoenzyme system of the "sandwich" format for detection of the matrix protein VP40 of the Marburg virus (strain Porr).

The specified technical result is also achieved by using monoclonal antibodies 7D8 produced by the strain of animal hybrid cells of the animal Mus musculus L. 7D8 belonging to the subclass of IgGI immunoglobulins having a heavy 55 kDa and light 25 kDa chain and monoclonal antibodies 7H10 produced by the strain of hybrid cells of the animal Mus musculus 1 .7H10 belonging to the subclass of IgGI immunoglobulins having a heavy 55 kDa and light 25 kDa chains and used together in the sandwich format enzyme immunoassay for detecting the Marb virus VP40 matrix protein urg (strain Porr).

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

The strains were obtained by fusion of mouse p3-X63 / Ag8.653 (NS / 1) myeloma cells with spleen cells of BALB / c mice immunized with purified, concentrated, inactivated BM preparation obtained from the blood plasma of infected guinea pigs [18] and an inactivated ethyleneimine dimer up to 0.17% as described [19].

The inventive hybrid cell strains of Mus musculus L. SSC VB Vektor - 7D8 and Mus musculus L. SSC VB Vektor - 7H10 were obtained at the State Scientific Center for Virology and Biotechnology Vector of the Russian Federal Service for Supervision of Human Welfare and deposited in the Cell Culture Collection of the SSC VB Vector". The author name of the hybridoma cell lines is 7D8 and 7H10.

The inventive strains of hybrid cells are part of the collection of FGUN SSC VB "Vector", consisting of 21 hybridoma cell lines, of which 11 produce MAB for the Marburg virus VP40 protein (Rohrr strain) [20].

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 BM preparation. A 45% polyethylene glycol solution of Sigma company with a molecular weight of 1300-1600 kD was used as a merging agent according to the method

[25]. 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 IgG subclass immunoglobulins (having a heavy 55 kDa and a light 25 kDa chain) that specifically interact with the VP40 BM protein. Analysis of murine immunoglobulins is carried out by ELISA using inactivated BM (Porp strain) or VP40 recombinant protein (obtained as a result of biosynthesis in E. coli cells based on a plasmid construct containing the full VP40 BM gene as described [21]) and antibodies against Mouse IgG 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. The proliferation index is not less than 5. Cultivation of hybridomas in the animal organism. 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. Hybridomas are 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 the native VP40 BM protein (32 kDa) and the recombinant VP40 BM protein (36 kDa) in the immunoblot reaction. In ELISA, the titer of MCA in ascites is for 7D8 - 1: 2187000 and for 7H10 - 1: 729000, respectively. Stable production of MCA is maintained for at least 10 passages in vitro with continuous cultivation. 3-5 milligrams of purified monoclonal antibodies 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 1 million / ml cells.

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

Figure 1 presents the electrophoregram of purified monoclonal antibodies, where:

1, 2 - preparations of purified MCAs 7H10 and 7D8 (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 Marburg 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 - drug Marburg virus (10 μl), the arrows indicate viral proteins;

5 - designations of viral proteins;

On figv presents an immunoblot of native and recombinant proteins VP40 with MCA, where:

1 - the Marburg virus preparation was treated with 7: 10 MCA at a dilution of 1: 500;

2 - Marburg virus preparation was processed with 7D8 MCA at a dilution of 1: 500;

3 - recombinant protein VP40 treated with MKA 7H10 at a dilution of 1: 500;

4 - recombinant protein VP40 processed MKA 7D8 at a dilution of 1: 500;

5 - the preparation of the Ebola virus was processed by MCA 7H10 at a dilution of 1: 500 (negative control);

6 - the preparation of the Ebola virus was processed by MCA 7D8 at a dilution of 1: 500 (negative control);

7 - lysate of E. coli. processed by MCA 7H10 at a dilution of 1: 500 (negative control);

8 - E. coli lysate. processed by MCA 7D8 at a dilution of 1: 500 (negative control).

Figure 3 shows a graph of titration of the Marburg virus antigen and VP40 recombinant protein with a pair of MCA 7D8 and 7H10 *, 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;

7Н10 * - indicator MCAs labeled with biotin;

as a negative control, a pair of MKA 4A2 and 1C1 * specific for the Ebola virus VP40 protein was used;

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 Mus musculus L. SSC VB "Vector" - 7D8 and Mus musculus L. SSC VB "Vector" 7N10 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.

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 33 intravenously 12 Saline 45 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 hapoxanthin, 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 VM 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 antispecific conjugate (rabbit immunoglobulins after drinking IgG mice 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" - 7D8 and Mus musculus L. SSC VB "Vector" 7N10 producing MAB for VP40 BM protein 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. The 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. Female BALB / c mice (vivarium of the State Scientific and Technical Center for Segment Vector) weighing 20-22 g each are intraperitoneally injected with 1 ml of a cell suspension containing at least 10 million hybridoma cells. After 7-10 days, the animals are euthanized and 3-5 ml of ascitic fluid are removed from the abdominal cavity under aseptic conditions. Cells from ascitic fluid are separated by centrifugation, and the tiger 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 musculis L. SSC VB "Vector" - 7D8 and Mus musculus L. SSC WB "Vector" 7N10.

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 centrifuged for 30 min at 8000 g and the precipitate was removed, and the supernatant was mixed with ten-fold phosphate-buffered saline (PBS) and adjusted to pH 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 [27] 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% bromophenol 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 3. Determination by ELISA of the specific interaction of MCA produced by hybrid cells of the strains of Mus musculus L. SSC VB "Vector" - 7D8 and Mus musculus L. SSC VB "Vector" 7N10 with BM and recombinant protein VP40.

ELISA was carried out on polystyrene tablets. Working dilution antigen (purified and inactivated Marburg virus 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 FMSF (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. 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 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. Mus musculus L. SSC VB "Vector" - 7D8 and Mus musculus L. SSC VB "Vector" 7H10 with VP40 BM protein (Porr strain) and recombinant protein VP40. Viral proteins and 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) at 37 ° C 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 Ebola virus [26] and E. coli. Lysate transferred after electrophoresis and also treated with MCA. The results are presented in figa and 2B.

Example 5. Preparation of conjugates of ICA 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 MAB (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 conjugate of peroxidase with streptovidin.

Example 6. Competitive ELISA of the "sandwich" format for the detection of native VP40 protein of the BM virus (Porr strain) and the recombinant protein VP40.

In wells of highly sorption polystyrene plates (Testiks or Nunc), purified 7D8 MCA 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 was carried out with a dilution of 1: 400 in two steps overnight at 4 ° C or at 37 ° C for an hour. Then conjugates of MCA 7H10 with biotin in working dilutions (1 μg / ml) were added to the wells, kept at 37 ° C within an hour and, after washing the wells three times, a streptavidin-peroxidase conjugate was added to them. Specific binding of biotin-labeled antibodies to the conjugate was detected with 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 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 non-competing MCAs was used to detect the Ebola virus VP40 protein. The antigen titration schedule is shown in FIG. 3.

The above properties of hybrid strains of Mus musculus L. SSC VB "Vector" - 7D8 and Mus musculus L. SSC VB "Vector" 7H10 (the author's name for the cell lines 7D8 and 7H10) allow us to conclude that for the first time on the basis of mouse myeloma, hybridomas Mus musculus L were obtained SSC VB "Vector" - 7D8 and Mus musculus L. SSC VB "Vector" 7H10 - producers of MCAs that do not compete for antigenic epitopes of VP40 BM protein. Strains of hybrid cells provide mouse monoclonal immunoglobulins of the IgGI class in the amount of 3-5 mg of purified antibodies from a milliliter of ascitic fluid. The purified MABs specifically reacted in ELISA with BM (MAB titer 7D8 and 7H10 was 1: 2187000 and 1: 729000, respectively) and recombinant protein VP40 (MAB 7D8 and 7H10 titer was 1: 243000 and 1: 729000, respectively); virus-associated protein VP40 and recombinant protein VP40 (obtained as a result of biosynthesis in R. coli cells based on a plasmid construct containing the full VP40 VM 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 1 ng / ml. The use of these MCA preparations and the recombinant protein VP40 as a positive control for the antigen will allow us to efficiently detect cases of HLM in Russia in the ELISA format “sandwich”.

The above properties of strains of Mus musculus L. SSC VB Vektor - 7D8 and Mus musculus L. SSC VB Vektor 7H10 (the author's name for cell lines 7D8 and 7H10) distinguish them from all previously described hybridomas producing MABs for Marburg virus proteins.

Claims (5)

1. The strain of hybrid cells of the animal Mus musculus L. 7D8, 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 Marburg virus matrix protein VP40 (Porr strain) and used as antigen tracers in the enzyme immunoassay system sandwich format for detecting the Marburg virus matrix protein VP40 (strain Porr). 2. Monoclonal antibody 7D8 produced by the hybrid strain of animal cells of mus musculus L. 7D8 (immunoglobulin IgG1 subclass) having a heavy 55 kDa and a light 25 kDa chain and used in the sandwich immunoenzyme system to detect the Marburg virus VP40 matrix protein (Rohrrr strain ) 3. The strain of hybrid cells of the animal Mus musculus L. 7H10, 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 Marburg matrix VP40 protein (Porr strain) and used as indicator proteins labeled with biotin, in the enzyme immunoassay system of the "sandwich" format for the detection of the matrix protein VP40 of the Marburg virus (strain Porr). 4. Monoclonal antibody 7H10 produced by the strain of animal hybrid cells of the mus musculus L. 7H10 animal (IgG1 immunoglobulin subclass), having a heavy 55 kDa and light 25 kDa chains and used in the sandwich immunoenzyme system to detect the Marburg virus VP40 matrix protein (Rohrrr strain ) 5. A kit for the sandwich immunoenzyme test system for detecting the Marburg virus VP40 matrix protein (Porr strain), including monoclonal antibodies 7D8 produced by the hybrid strain of animal cells of Mus musculus L. 7D8 and adsorbed on polystyrene plates, biotin conjugates with monoclonal antibodies 7H10 produced by the strain of hybrid cells of the animal Mus musculus L. 7H10 conjugate streptavidin with peroxidase and tetramethylbenzidine, the quantitative content of which in the kit is sufficient for carrying out an enzyme-linked immunosorbent reaction.

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