RU2491338C2 - Using monoclonal antibodies for identifying yamagata or victorian influenza b viral evolution line, hybridoma 4h7 strain for preparing monoclonal antibodies used to identify influenza b viruses in yamagata branch, hybridoma b/4h1 strain for preparing monoclonal antibodies used to identify influenza b viruses in victorian branch - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention discloses using the monoclonal antibodies (MCAs) produced by the hybridomas B/4H1 and 4H7 and directed to variable determinants as a part of influenza B virus proteins. The hybridoma B/4H1 and 4H7 strains are deposited in the Russian Collection of Vertebral Cell Cultures (RKKK P) under Nos. 719 D and 720 D, respectively. The two current influenza B viral evolution lines are identified with using the MCAs B/4H1 and MCAs 4H7 directed to variable determinants of Victorian-type and Yamagata-type influenza B hemagglutinin, respectively.

EFFECT: using the prepared MCA kit enables identifying the newly recovered influenza B viruses Besides, the MCAs under the invention may be used to study the antigenic structure and variability of this agent, to identify them as belonging to a specific evolution line; the MCAs may be also used to construct the diagnostic test systems.

3 cl, 12 dwg, 8 ex

Description

Type B influenza viruses (Influenza B virus family Orthomyxoviridae) were first isolated in 1940. Until recently, they were significantly inferior to type A influenza viruses in terms of virulence, contagiousness and epidemic significance. This behavior of influenza B viruses was observed until 1983, when as a result of not quite disclosed events, the divergent nature of the evolution of the pathogen was outlined. Since that time, two sharply different antigenic varieties of type B influenza viruses were recorded in circulation: B / Victoria / 2/87 and B / Yamagata / 16/88 (Rota, 1989). At the same time, the activity of influenza B viruses sharply increased, epidemics among the Russian population began to occur much more often (after 1-2 years), and since 1993 - almost every year (Slepushkin, 1998). In addition, a constant antigenic hemagglutinin drift occurs within the lines, the ancestors of the above standards of influenza B viruses. Currently, two variants of the type B influenza virus are of epidemic significance in the world: B / Brisbane / 60/08 of the Victorian line and B / Florida / 04 / O6 of the Yamagata variety (Waicharoen S., 2008., Jian J-W., 2008). This determines the need for a clear and fast identification of influenza B viruses with the determination of their belonging to one or another evolutionary line, which is necessary for timely changes in the strain composition of vaccines and diagnostic drugs.

Currently, virological, serological, electron microscopic methods and PCR analysis are used to diagnose influenza B virus.

The priority areas in the field of creating diagnostic products for identifying pathogens of infectious diseases are:

- Creation of modern highly specific test systems based on the use of monoclonal antibodies and the achievements of immunochemistry

- Creation of molecular biological test systems (real-time PCR, biochips, etc.)

One of the most promising ways to improve immuno-enzyme analysis (ELISA) and immunofluorescence analysis (IFL) is to include in their composition preparations of monoclonal antibodies (MCAs) of a given direction, synthesized by hybridomas, which are obtained by somatic hybridization of myeloma tumor cells and B-lymphocytes producing antibodies after immunization of animals with appropriate antigens. MCAs can significantly increase the specificity and achieve high standardization of analyzes, as well as significantly simplify the technology of production of test systems.

A similar set of proposed strains of hybridomas in the patent and available literature is not described. Work on the creation of hybridomas, including in the patent literature, producing antibodies to type B influenza nucleoprotein was published abroad, however, they do not allow differentiation of isolates belonging to different evolutionary branches, since they synthesize antibodies directed to the conservative determinants present in the composition of viruses of both varieties (Glikmann et al, 1995). Well-known foreign and domestic MCAs for influenza B virus hemagglutinin are directed to antigenic determinants of relict strains (B / Singapore / 222/79) and, therefore, they are not suitable for identification of modern simultaneously circulating strains of influenza B viruses (Busse et al, 1987). The described MCAs do not allow the differentiation of viruses of the Yamagata and Victorian branches, which is of great importance not only for diagnosis, but also for understanding the laws of natural evolution and spread of the pathogen in human populations.

Known used in the diagnosis and study of the evolution of influenza B polyclonal sera to newly isolated influenza B viruses, obtained as a result of immunization of rabbits, rams, rats or ferrets with the corresponding strains of viruses B. The prototype disadvantages are:

- the need to obtain hyperimmune sera, which requires a certain time and does not allow for the immediate identification of isolated viruses, which is extremely important for the operational analysis of the epidemiological situation and surveillance of influenza (Table 1);

- polyclonal immunoglobulins differ in the heterogeneity of the composition of serum antibodies, which is fraught with the danger of cross-nonspecific reactivity with host and heterologous viral antigens when used as part of immunological tests (Malogolovkin, 1998);

- the specificity and repertoire of the immune response when receiving polyclonal sera depends on the type of animal, immunization schedules and varies depending on the series of the drug;

- antibody preparations obtained as a result of immunization of animals are difficult to standardize due to differences in the constituent antibodies in terms of affinity and epitope orientation (Ignatyeva, 1989);

- a characteristic feature of modern influenza viruses is their extremely high sensitivity to non-specific inhibitors of the sera of normal animals, which creates certain difficulties in the initial identification of isolates and distorts the analysis results (Flu and other respiratory viral infections, 2003).

In addition, polyclonal immunoglobulins are not able to differentiate viruses belonging to one of the evolutionary branches from another (Lupulescu E., 1996). The use of high-volume diagnostic sera from WHO kits also does not allow differentiation of the Victorian and Yamagata virus lines from each other, which is extremely important for the correct choice of viruses in the composition of the influenza vaccine for the upcoming epidemic season (Tables 2, 3).

Table 1 Labor costs and time required to obtain polyclonal antisera (PKA) and MCA Stages of receipt Deadlines (PKA) Deadlines (MCA) * Obtaining reference strains 8-10 weeks 0 Virus recovery 1-2 weeks 0 Accumulation of IMPORTANT 1 week 0 Purification and concentration of the virus 1 week 0 Immunization of laboratory animals 4 weeks 0 Getting immune serum 2 days 0 Testing the specificity and activity of the drug 4 days 0 Total: 16-18 weeks 0 * MCA can be prepared in significant quantities in advance. Shelf life at -70 ° is not limited.

table 2 The reactivity spectrum of MCA and sera as part of the WHO kits in rtga with strains of influenza viruses in the Yamagat line Influenza B viruses WHO serum MCA B / 4H1 MKA4N7 B / panama / 45/90 + - + B / quindao / 102/91 + - + B / Beijing / 184/93 + - + B / harbin / 07/94 + - + B / Yamanashi / 166/98 + - + B / Johannesburg / 05/99 + - + B / Florida / 07/04 + - + B / leningrad / 148/91 + - + B / Khabarovsk / 135/93 + - four- B / n. Novgorod / I 10/93 + - + B / Leningrad / 86/93 + - + B / St.-Petersburg / l 41/95 + - + B / St-Petersburg / 210/95 + - + B / Moscow / 91/95 + - + B / Lipetsk / 3/97 + - + B / N. Novgorod / 119/97 + - + B / N. Novgorod / 348/97 + - + B / samara / 240/99 + - + B / samara / 253/99 + - + B / Kalinmgrad / 900/99 + - + B / Kaliningrad / 905/99 + - + B / cheeta / 2/05 + - + B / Kalinmgrad / 4/05 + - +

Table 3 The reactivity spectrum of MCA and sera as part of the WHO kits in RTGA with strains of influenza viruses In the Victorian line Influenza B viruses WHO serum MCA B / 4H1 MKA4N7 B / Victoria / 2/87 + + - B / shandong / 07/97 + + - B / Beijing / 243/97 + + - B / shiga / 51/98 + + - B / tokyo / 53/99 + + - B / HongKong / 330/01 + + - B / Malaysia / 2506/04 + + - B / Kaliningrad / 6/05 + + - B / Khabarovsk / 13/05 + + - B / Khabarovsk / 14/05 + + - B / Murmansk / 57/06 + + - B / ryazan / 5/06 + + - B / samara / 103/06 + + - B / St.-Petersburg / 12/06 + + - B / Tula / 1/06 + + - B / Chelyabinsk / 5/06 + + - B / Astrakhan / 1/07 + + - B / Voronezh / 19/07 + + - B / Irkutsk / 97/07 + + - B / St.-Petersburg / 15/07 + + - B / St-Petersburg / 18/07 + + - B / St.-Petersburg / 22/07 + + - B / Khabarovsk / 7652/07 + + -

All of the above disadvantages are absent in the alleged invention. Distinctive characteristics and advantages of MCAs compared to polyclonal ones are:

1. MCAs provide a high standardization of the analyzes in which they are used, since they have fixed molecular, biochemical and biological properties:

- a high degree of homogeneity in antibody isotypes: they are characterized by only one class (subclass) and one heavy chain allotype, one type and light chain allotype, identified by the idiotype (Fuchs, 1982).

- MCAs have immunological homogeneity in epitope orientation.

2. MCAs can provide high specificity, sensitivity and reproducibility of immunological analysis techniques.

3. Hybridomas with established characteristics allow to obtain unlimited quantities of antibodies with a given desired affinity - high for immunosorbent methods and low for affinity chromatography and immunochemistry (Spira 1985).

4. MCAs are directed to only one epitope (determinant) of an antigenic molecule and are able to recognize single amino acid substitutions as part of individual viral determinants, thereby they can serve as an effective tool for revealing and studying the mechanisms of evolution and differentiation of protein molecules in viruses (Manko 1990, Drobyshevskaya 1980) .

5. The possibility of freezing the hybridoma culture and replacing it with the receipt of MCA at certain times (if necessary in advance), availability in relatively large quantities at a relatively low cost (Englenberg 1984, Spira 1985).

6. The possibility of creating immunotherapeutic drugs with targeted action using MCA.

7. The methodological method of obtaining escape mutants in the presence of MCAs with virus-neutralizing activity, followed by analysis of the primary structure of the genome and identification of amino acid substitutions allows one to recognize sites responsible for the induction of virus-neutralizing antibodies, which is essential for the directed design of modern genetically engineered vaccines, as well as understanding the structural composition of the virus, the pathways and direction of its natural evolution and its modeling in vitro (Nakagava 2005, 2006).

SUMMARY OF THE INVENTION

A hybrid kit is described, including:

1. hybridoma B / 4H1 producing MAB to the antigenic determinant of hemagglutinin of influenza viruses in the Victorian branch, which do not react with the viruses of the Yamagata branch, but actively detect all studied Victorian strains in RTGA and ELISA (Scheme 1).

2. Hybridoma 4H7, producing MCA to the antigenic determinant of hemagglutinin of influenza viruses in the Yamagata branch, which do not react with the viruses of the Victorian branch, but actively detect all studied strains of the Yamagat line in RTGA and ELISA (Scheme 2).

The method of the present invention allows to increase the sensitivity and specificity of immunological test systems for the diagnosis and differentiation according to the evolutionary affiliation of influenza B viruses due to the inclusion of the proposed MCA in their composition. In addition, the pronounced virus-neutralizing activity of MCA against hemagglutinin of influenza viruses in the Yamagat and Victorian varieties opens up prospects for their use in studying the antigenic structure and evolutionary variability of this glycoprotein.

The present invention relates to a method for carrying out detection of influenza B virus antigens in biological materials from patients depending on their affiliation with various modern evolutionary lines (Yamagata or Victorian), as well as sites specific to each of the branches.

A method for producing a panel of multidirectional MCAs for influenza B viruses is described using hybridoma technology, which provides for the selection and accumulation of viral antigens relevant for the tasks, immunization of splenic cell donor mice, immunization of hybridization of immunocytes with mouse myeloma, selection of positive cultures using ELISA and their subsequent cloning, the accumulation of cell mass of specific hybridomas to obtain ascitic preparations of MCA on the sensitive line of mice.

A brief description of the graphic material

Figure 1. Schemes for producing hybridomas and MCA produced by them.

A murine myeloma culture was fused with splenocytes of immune mice in suspension using 50% PEG. The cells were then transferred to selective HAT medium and distributed into the cells of 96-well plates. The main testing method was solid-phase ELISA. Positive cultures were cloned 2-4 times by limiting dilution method. Homogeneous hybridoma strains were grown in culture, the supernatant was collected to study the properties of MCA, part of the cell population was frozen, and part was inoculated to syngeneic mice to obtain MCA-containing ascitic fluid.

Figure 2. This is a series of graphs illustrating the concentrations of the studied MCAs to which they specifically interact in ELISA with a purified homologous virus concentrate. Viral concentrates were applied to the substrate at a concentration of 2 μg / ml. The result was taken into account by a change in optical density at a wavelength of 450 nm.

Figure 3. The study of the neutralizing activity of MCA in relation to homologous influenza B viruses was carried out under microplate culture. The results of neutralization of MCA virus reproduction of the virus (infection dose - 100 TCD ₅₀ ) were taken into account using an ELISA microplate with staining of the cell monolayer with peroxidase conjugate MKA 1/22 directed to a highly conserved epitope in the composition of influenza B nucleoprotein B.

Figure 4. Specific binding of MCA to viral proteins in Western blot analysis. Lane A illustrates the binding of MAB to a protein of a homologous antigen under reducing conditions, lane B under non-reducing conditions.

Figure 5. The graphs show the limiting dilutions of the peroxidase conjugates of MABs, which detect in the ELISA a homologous antigen adsorbed on the solid phase at a concentration of 2 μg / ml.

DETAILED DESCRIPTION OF THE INVENTION

In the modern world, including Russia, two evolutionarily different lines of the influenza B virus are circulating - Victorian and Yamagatskaya. The present invention relates to a method for identifying influenza B viruses heterogeneous by antigenic properties, followed by determining their belonging to one of the evolutionary branches.

The present invention relates to a new set of hybridomas producing MABs directed to conservative and variable antigenic determinants of influenza B viruses. These MABs are suitable for the preparation of immunodiagnostic preparations.

Hybridoma B / 4H1 produces MCA to the antigenic determinant in the hemagglutinin of the influenza viruses of the Victorian branch, which allows only strains of this evolutionary line to be identified. Hybridoma 4H7 produces MCA to an antigenic determinant in the composition of the hemagglutinin of influenza viruses in the Yamagata branch, suitable for detecting isolates of only this species.

To date, a similar panel of hybridomas producing MCAs with the above diagnostic capabilities has not been described in the domestic and foreign literature. The MCA of the present invention has proven itself in diagnostic practice. MCAs specific for each of the evolutionary branches of the influenza B virus produced by 4H7 and B / 4H1 hybridomas allow the construction of enzyme-linked immunosorbent and immunofluorescence tests to identify pathogens belonging to different evolutionary lines.

Hybridomas producing MABs for various antigenic determinants of influenza B virus can be obtained by immunizing mice with purified concentrates of various virus strains, followed by fusion of spleen cells taken from an immune mouse with mouse myeloma cells.

Obtaining a hybridoma and produced by it MCA includes, for example, the following stages:

1. purification and concentration of the virus for use as an immunizing antigen;

2. obtaining antibody-producing cells as a result of infection of the mouse with the corresponding antigen under control in the ELISA titer of specific serum antibodies;

3. accumulation of mouse myeloma cells;

4. fusion of antibody-producing cells with myeloma in the presence of PEG;

5. selection in ELISA hybridomas of a given specificity, with good growth, using a wide panel of antigens;

6. cloning of selected hybridomas by the method of limiting dilution in microplates on HAT medium;

7. ELISA selection of well-growing clones from wells that contain specific antibodies in high titers;

8. the reclamation of hybridomas, followed by the accumulation of cell mass;

9. cultivation of hybrid cells in mice in order to obtain ascitic preparations of MCA;

10. testing the resulting MCA for biological activity and specificity, the study of its parameters in various immunological and biochemical reactions;

11. hybridoma cryopreservation

The general procedure for the preparation of 4H7 and B / 4H1 hybridomas is described in more detail below in accordance with each of the above steps. This method for producing hybridomas of the present invention is provided only to illustrate methods for producing hybridomas and is not limiting. Other known procedures or the following modified method may be used.

1. Antigen production

The purified concentrates of influenza B viruses (B / Shandong / 7/97 of the Victorian line, B / Panama / 45/90 of the Yamagat line) used as antigens for immunization of splenic cell donor mice were obtained in several stages by the method of differential ultracentrifugation of virus-containing allantoic fluid. The specificity of the obtained viral concentrates before the start of the immunization stage was confirmed in the reaction of inhibition of hemagglutination with reference serums.

2. Obtaining antibody-producing cells

The mouse was immunized with an immunogen produced in stage 1 and mixed with an adjuvant, such as Freund's complete or incomplete adjuvant or alum. Other suitable experimental animals are, for example, rats, guinea pigs, rabbits, dogs, chickens, horses, pigs, cows and sheep.

Suitable methods for administering an immunogen for immunization of an experimental animal are subcutaneous, intraperitoneal, intravenous and intramuscular injection, with subcutaneous and intraperitoneal immunization being preferred. Immunization can be carried out by introducing a single dose or several multiple doses at appropriate time intervals (preferably 1-3 weeks).

Then, immunized animals are monitored for a titer of specific antibodies in sera and an animal with a sufficiently high antibody titer is selected as a source of antibody-producing cells. Selecting an animal with a high titer of antibodies makes the subsequent process more efficient. Cells for subsequent fusion are harvested from the animal 3-4 days after the last immunization.

Specific antibody titer assays are a well-known technique such as radioimmunoassay, enzyme-linked immunosorbent assay (hereinafter referred to as ELISA), fluorescence analysis, hemagglutination inhibition reaction, with ELISA being preferred because of its sensitivity, speed, accuracy and automation capability.

Determination of antibody titer can be carried out, for example, using ELISA as follows. First, the purified homologous influenza B virus concentrate is adsorbed onto a solid surface, such as a 96-well ELISA plate, and then the rest of the surface to which the virus does not bind, is blocked with a protein that is not related to the antigen, such as 0.5% gelatin. After washing to bind the antiviral MCA to the antigen, the surface of the wells is contacted with serially diluted samples of mouse serum. To bind to a murine antibody, an enzyme-labeled anti-mouse immunoglobulin antibody is added as the “second” antibody. After washing, the substrate for the enzyme is added and the antibody titer is evaluated by determining the change in optical density due to color development caused by the change in substrate.

3. The accumulation of myeloma cells

The source of myeloma cells is 8-azaguanine-resistant myeloma strains, such as P3 / NS1 / 1-Ag4-1, P3X63Ag8U.1, SP / 20-Ag14, P3X63Ag8.653 and P3X63Ag8. The selected cell line was cultured on RPMI-1640 medium supplemented with glutamine, 2-mercaptoethanol, gentamicin and animal serum to provide at least 2 × 10 ⁷ cells / ml on the day of the fusion.

An X63Ag8.653 mouse murine myeloma cell culture adapted to growth on media supplemented with SCRS obtained from the TsNIIRRI collection (Klimovich 1986) was used as a fusion partner for producing the hybridomas of the present invention. This strain is resistant to 8-azaguanine, that is, defective in the hypoxanthine-guanine-phosphoribosyltransferase gene.

Myeloma was cultured in plastic culture plates or in bottles of Sarstedt or Nunc production. Cells were kept at + 37 ° C and 100% humidity in the atmosphere with 6-7% CO ₂ . The basic culture medium contained 90% RPMI-1640 and 10% blood serum of cattle (SCRS) manufactured by Biolot (St. Petersburg).

4. Cell fusion

To obtain hybridomas producing MAB, lymphocytes or plasma cells obtained from any suitable part of the animal's body are used as precursor cells. Such sources of lymphocytes or plasma cells are, for example, the spleen, lymph nodes, peripheral blood, or any suitable combination thereof, with spleen cells being used most often.

After the last booster immunization to obtain antibody-producing cells in a mouse having a pre-high titer of specific antibodies, the tissue in which the antibody-producing cells are present is removed. In the current preferred technique for fusion of spleen cells with myeloma cells obtained as described in step 3, polyethylene glycol is used.

The indicated fusion technique involves washing the spleen and myeloma cells with serum-free medium (such as RPMI-1640) or buffered saline and mixing them so that the ratio of spleen cells to myeloma cells is approximately 5: 1-10: 1, after which the cells are centrifuged. Then the supernatant is discarded and the precipitated cells are sufficiently loosened, after which 1 ml of serum-free medium containing 50% (w / v) polyethylene glycol (Mw 1000-4000) is added dropwise with stirring. Then 10 ml of serum-free medium are slowly added, after which the mixture is centrifuged. The supernatant is discarded again, and the precipitated cells are suspended in an appropriate amount of selective HAT medium containing a solution of hypoxanthine, aminopterin and thymidine. Then the suspension is distributed into the wells of the tablet for cultivation and incubated in the presence of 5-8% CO ₂ at 37 ° C, for about 2-3 weeks with the addition of selective medium.

5. Hybrid selection

If the myeloma strain used is resistant to 8-azaguanine, that is, if it is deficient in the hypoxanthine-guanine-phosphoribosyltransferase enzyme, then any non-fused myeloma cells and any myeloma-myeloma hybrids are not able to survive in HAT medium. On the other hand, hybrids of antibody-forming cells formed with each other, as well as hybridomas formed by anti-body-producing and myeloma cells, can survive in this environment, and the former have only a limited lifetime. Accordingly, prolonged incubation in HAT leads to the selection of only the desired hybridomas. Aliquots of the supernatant are then taken to determine the titer of specific antibodies in an ELISA (the procedure is described in detail in paragraph 2).

6. Cloning, testing and re-cloning of hybridomas.

Suitable methods for cloning are: the limiting dilution method, in which hybridomas are diluted so that one cell per well of the plate is grown and then cultured; a method in which colonies are isolated by cultivation in a medium with soft agar; micromanipulator method for isolating a single cell; and “clone sorting,” in which single cells are separated on a cell sorter.

The cloning procedure, carried out, for example, by the method of limiting dilution, is repeated 2-4 times for each clone producing specific antibodies. Clones, stable producers of antibodies, are selected to obtain MCA.

The hybridoma obtained by cloning is then cultured in a growth medium in order to accumulate a sufficient number of cells for passage on syngeneic mice. Large-scale cultivation is carried out in culture roller bottles or large culture bottles.

The mouse-mouse hybridomas of the present invention, on the basis of which MCA B / 4H1, 4H7 are obtained, were deposited in the Specialized Collection of Vertebrate Cell Cultures of the Russian Cell Culture Collection 01/13/2009 under registration numbers RKKK (P) 719D, RKKK (P) 720D, respectively. Accordingly, the production of MCAs using B / 4H1 and 4H7 hybrids can be achieved in accordance with the procedure described below in step 9 without carrying out steps 1-6.

9. Cultivation of hybridoma in mice with the aim of obtaining MCA

Ascitic fluids containing MAB secreted by the presented hybridomas were obtained according to the methods described by Novokhatsky A.S. with colleagues (Novokhatsky 1985), as well as by a number of other authors (Novikov, 1988, Hoogenraad N., 1983). To pass the hybridomas, recipient mice were injected intraperitoneally with 0.5 ml pristan (Sigma) 7-14 days before vaccination. Cells in the amount of 5-10 million were inoculated into the abdominal cavity. After ascites formation, the abdominal wall was punctured and leaking fluid was collected.

A saturated solution of ammonium sulfate is used to clean the collected ascites of MCA. The first salting out was carried out using a saturated solution of (NH ₄ ) ₂ SO ₄ in the ratio with ascitic liquid 1: 1 (50% solution). A saturated solution of (NH ₄ ) ₂ SO was prepared at the rate of 70.7 g per 100 ml of distilled water. For the second and third (if necessary) salting out, a 40% solution of ammonium sulfate was used.

10. Analysis of MCA

MCAs produced by the hybridomas of the present invention have various functional properties described below.

10.1 MCA 4H7 for influenza B virus, strain B / Panama / 45/90, was characterized by enzyme-linked immunosorbent assay and hemagglutination inhibition reaction. It was established that the titer of MCA with purified influenza viruses in the Yamagata evolutionary line adsorbed on the solid phase was 10 ^-5 -10 ^-6 , and the level of nonspecific reaction with various subtypes of influenza A virus, influenza B strains of early isolation and influenza B virus strains The Victorian line did not exceed background values.

In RTGA, MCA 4H7 had a pronounced inhibitory activity. The study of the response spectrum of MCA 4H7 with a large set of strains of influenza virus type In 1940-2006, isolation significantly differing in the antigenic characteristics of HA, showed that the hemagglutinating activity of the 1988-2006 strains, originating from the reference B / Yamagata strain / 16/88, as a rule, was suppressed in high dilutions of MCA, up to a titer of 1/5120. Strains originating from the B / Victoria / 2/87 virus and strains isolated before 1988 did not interact with 4A7 MCA in either ELISA or rtga.

The ability of antibodies to neutralize the virus, thereby inhibiting the infectious process, is an essential characteristic of their functional activity. The use of MCAs allows one to determine whether the antigenic determinant of the viral protein with which they interact is an inducer of the synthesis of virus neutralizing ATs. To assess the virus-neutralizing properties of AT, the microcultural method was used, based on taking into account the residual reproduction of the virus in monolayer cultures after incubation with MCA using ELISA. T.O. it was found that MCA 4H7 suppressed the reproduction of homologous virus B / Panama / 45/90 to a concentration of 2 μg / ml

Assessment of the epitope orientation of MCA was carried out using immunoblotting. After electrophoretic separation of viral proteins under non-reducing conditions, HA was detected by 4A7 MCA in the form of several oligomeric polypeptides with molecular masses (MM) of 82 kD, 99 kD and 205 kD. To make sure that these polypeptides are indeed oligomeric forms of HA, these proteins were analyzed in two-dimensional electrophoresis. Under reducing conditions, HA 82 and HA 205 polypeptides were divided into two subunits corresponding in relative molecular weight to HA1 (52 kD) and HA2 (29 kD). HA 99 under the same conditions passed into the HA 82 form, but was not divided into HA1 and HA2, i.e., probably, this polypeptide is part of HA molecules that did not undergo post-translational proteolysis by host heavy and light chain proteases. These data once again confirm that the 4H7 MCA is directed to the HA molecule. Under reducing conditions, MCA 4H7 interacted with two proteins with MM 52kD and 82 kD, which can be reliably identified as HA1 and HA0. Thus, immunoblotting showed that 4H7 MABs interact with epitopes of the HA molecule localized in the HA1 domain.

The preparation and characterization of escape mutants (EMs) that are resistant to the effect of neutralizing MABs is a valuable tool in studying the antigenic structure of viral proteins. The standard method for obtaining EM is based on a single cloning of a strain of DT in the presence of virus-neutralizing MCAs (Berton M, Webster R., 1985). EMs thus obtained have, as a rule, 1 or 2 nucleotide and corresponding amino acid substitutions, while, as a rule, an 8-32-fold decrease in antibody titers in rtga is observed (with MCAs used for selection). Sequencing of the HA EM gene cloned in the presence of 4A7 MAB showed that these MABs interact with an epitope that includes amino acid residues at positions 75 and 150 (Fig. 6).

10.2 Analysis of B / 4H1 MCAs obtained for influenza virus B / Shandong / 7/97 in ELISA and RTGA showed that these antibodies reacted in ELISA only with influenza B strains of the Victorian evolutionary line and did not interact with different subtypes of influenza A virus , influenza strains In the early years of isolation and influenza virus strains in the Yamagata branch. In the RTGA, MCA B / 4H1 had a pronounced hemagglutinating activity with B / Victoria / 2/87-like strains from 1987-2007. excretion up to titer 1/20480 in the absence of binding to influenza strains In the early years of excretion and influenza viruses In the Yamagata branch.

The study of the virus-neutralizing activity of MAB B / 4H1 with a homologous strain B / Shandong / 7/97 showed that these MAB inhibit the reproduction of the virus by 100% at a concentration of 1 μg / ml or more.

Using immunoblotting, it was found that MCA B / 4H1 under non-reducing conditions detected proteins with molecular weights of 82 kD, 99 kD and 205 kD, which corresponds to oligomeric forms of hemagglutinin. Under reducing conditions, B / 4H1 mAbs interacted with two proteins with 52 kD and 82 kD MM, which are HA1 and HA0 polypeptides. Thus, immunoblotting showed that B / 4H1 MABs interact with epitopes of the HA molecule localized in the heavy HAL subunit

Sequencing of the HA EM gene cloned in the presence of B / 4H1 MAB allowed us to establish that these MABs are directed to the epitope with a significant amino acid residue at position 218 (Fig. 7).

10.3 The study of MAB 4H7 to the virus B / Panama / 45/90 in immunological reactions revealed that these MABs work in ELISA, RTGA and neutralization reactions. Testing MKA 4H7 in ELISA with a wide range of influenza strains of type B at different years of isolation (1954-2007) allowed us to establish that MKA 4H7 interact with high titers (10 ^-5 -10 ^-6 ) with all viruses of the Yamagat line, which suggests that the studied MCAs are directed to a variable epitope, which is part of the hemagglutinin of the Yamagat line viruses.

Indeed, according to immunoblotting under reducing conditions, 4A7 MABs interacted with the undigested hemagglutinin (HA0) molecule and the HAL subunit. Under non-reducing conditions, these MABs interacted only with the undigested HA0 molecule (in monomeric and dimeric form).

Commercial kits are preferably used to identify the isotype and subclass of MCA. To determine the MCA isotype secreted by the hybridomas of the present invention, commercial kits were used: Mouse Monoclonal Antibody Isotyping Reagents (SIGMA, USA, Lot 18H4870) and Mouse Monoclonal Antibody Isotyping Kit (SIGMA ImmunoType, USA, Lot 108H4869) according to the manufacturer's recommendations. It was determined that MCA 4H7 belongs to the IgG1 isotype, and the second MCA (B / 4H1) refers to the IgG2a isotype.

11. For cryopreservation of cells used a medium consisting of 90% SCRS and 10% DMSO (Sigma). Cryovials with cells pre-cooled to -70 ° C in nitrogen vapors were placed in liquid nitrogen (for long-term storage). Hybrid ohmic cells in the logarithmic growth phase, forming a continuous monolayer, or cells grown in the peritoneal cavity of syngeneic mice upon receiving ascites fluids were cryopreserved. Before freezing, the cells were subjected to microscopic examination for the absence of microflora contamination. The number of living cells was evaluated by exclusion of trypan blue. The restoration of cell cultures after storage at low temperatures (-70 ° C, -196 ° C) was carried out according to the method described by Oi V.T. & Herzenberg L.A. (25) taking into account the recommendations of Malakhova I.V. et al. (6).

The following examples illustrate the methods and results of the present invention.

Example 1. Obtaining antigens of influenza B viruses

1.1 Cultivation of viruses in developing chicken embryos (RCE).

Influenza B viruses were cultured in 10-11-day RCE. When laboratory strains of influenza B viruses were passivated, the allantoic fluid of infected TBEs was used as the viral inoculum and infected with the virus at a dose of 10 ² -10 ³ EID ₅₀ , introducing 0.2 ml of virus-containing material into the allantoic cavity in physiological solution with the addition of 50 μg / ml gentomycin . Viruses were passaged three to five times to obtain an inoculum with an infectious titer of 7-9 lg EID ₅₀ / 0.2 ml. Influenza B viruses were cultured for 72 hours at + 32 ° C. The concentration of hemagglutinins in the virus-containing allantoic fluid was determined by titration using a standard method using a 1% chicken erythrocyte suspension (Virology 1988).

1.2 Determination of the infectious activity of influenza viruses by titration on chicken embryos.

Infectious activity was determined by TBE titration, infecting 4 TBEs with each of the 10-fold successive dilutions of viral material in FSB. The IA index calculated by the method (Thompson 1947) was expressed as the reciprocal of the decimal logarithm of the EID ₅₀ .

1.3. Preparation of a concentrated purified virus preparation was carried out in several stages according to the method of differential ultracentrifugation (Virology 1988). At the first stage, the virus-containing allantoic fluid (IMA) was clarified by low-speed centrifugation at 4000 rpm. In one hour. Each subsequent stage of purification and concentration of influenza viruses was carried out at 100000g (centrifuge "Beckman") for 2.5 hours. First, the IMPORTANT was precipitated to the bottom by high-speed centrifugation with cold supply. The pellet was resuspended in 10 mM STE buffer (100 mM NaCl, 10 mM Tris-HCl and 1 mM EDTA), pH 7.2-7.4. Purification of the resulting viral concentrate was carried out by centrifugation in a stepwise gradient of sucrose density (20% -60% sucrose solutions in STE) at + 4 ° C. The viral zone was collected in the region of 40% -42% sucrose, diluted with STE 4 times and re-besieged on the bottom of the tube. The pellet was resuspended in 10 mM STE buffer, pH 7.2-7.4, stored until use at -20 ° C. Protein was measured by the Lowry method (Lowry 1951). The concentration of viral protein in the preparation was usually 1-5 mg / ml. Quality control of the preparation was carried out using electron microscopic analysis of the virion morphology by negative contrasting with a 1.5% solution of phosphoric tungsten acid Na-salt on carbon substrates.

Example 2. The development of MCA B / 4H1 to influenza virus B / Shandong / 7/97.

2.1 Obtaining antibody-producing cells.

In order to obtain a hybrid oma B / 4H1, a purified concentrate of influenza virus B / Shandong / 7/97 was used as an immunogen. Immunization of female mice of interline hybrids F1 (SGL / J × Balb / c) 7-8 weeks old with purified influenza virus concentrates dissolved in FSB was carried out intraperitoneally at a dose of 50-100 μg / mouse. Injection of antigens was done 3 times with an interval of 3-8 weeks. Three days before the confluence, mice that had the highest titers of specific antibodies were given an intraperitoneal booster immunization with antigen at a dose of 70 μg / mouse.

2.2 To control the quality of immunization of splenic cell donor mice, as well as the nature of the immune response to the antigen, animal blood serum obtained from retrobulbar sinus was investigated in the reaction of the indirect ELISA. Pre-plates for enzyme immunoassay reactions ("Medpolymer") were sensitized with a purified concentrate of influenza virus B / Shandong / 7/97 at a concentration of 2-3 kg / ml in 0.05 M KBB, 18 hours incubation, + 4 ° C. Then, the wells of the plate were washed 3 times from unbound antigen using FSB containing 0.05% Tween-20 (FSB-T). Mouse sera were added in appropriate dilutions on FSB-T containing 0.5% gelatin (FSB-Zh-T), incubation for 1 hour at + 37 ° C or 18 hours at + 4 ° C. Next, the plate wells were washed 3 times with FSB-T and peroxidase conjugate against mouse IgG (Sigma) was added to FSB-Zh-T, incubation for 1 hour at + 37 ° C. After washing FSB-T four times, a substrate-chromogenic mixture consisting of 3.3'5.5 'tetramethylbenzidine (0.1 mg / ml) and 0.003% hydrogen peroxide in 0.1 M acetate-citrate buffer, pH 5 was added to the wells of the plate. , 0. The reaction was stopped with 2N sulfuric acid. The result was taken into account on an Anthos immunoassay analyzer at a wavelength of 450 nm.

2.3 Carrying out the hybridization procedure upon receipt of the hybridoma B / 4H1.

The B / 4H1 hybridoma of the present invention was obtained according to the method described by Galfre and Milstein (Galfre, Milstem, 1981). On the third day after booster immunization, the spleen was removed from the mouse and placed in 10 ml of serum-free RPMI-1640 medium containing 50 mg / l gentamicin, and then splenocytes were isolated by mechanical extrusion in a homogenizer. The resulting suspension was centrifuged to precipitate cells, which were then washed twice with serum-free medium RPMI-1640, after which the cells were resuspended in serum-free medium and counted.

The culture of mouse myeloma X63Ag8.653 defective in the GGFRT gene was maintained in the logarithmic growth phase by daily screening in RPMI-1640 medium with 10% SCRS at + 37 ° C and 100% humidity in the atmosphere with 6-7% CO ₂ . At the time of fusion, myeloma cells were cultured to a cell density not exceeding 1 × 10 ⁶ cells / ml. Hybridization of splenocytes from hyperimmunized mice with murine myeloma cells was carried out in suspension in a ratio of 10: 1 in the presence of 50% PEG-1000. (Sigma). Then the cells were transferred to a selective NAT medium prepared on the basis of RPMI-1640 and 20% SCRS supplemented with hypoxanthine (10 ^-4 M), aminopterin (4 × 10 ^-7 M) and thymidine (1.6 × 10 ^-5 M) Sigma, as well as 2 mM glutamine, 1 mM sodium pyruvate (Sigma), 1 mg / L amphotericin B, and 50 mg / L gentamicin. The suspension was distributed into the cells of 96-well plates containing a three-day culture of mouse macrophages.

2.4 Cultivation and testing of hybridomas.

After 10 days of cultivation, the NAT medium was replaced with a more gentle NT, and after 20 days the cultures were maintained on RPMI-1640 medium with SCRS. To remove Ig secreted by not merged splenocytes of mice, the culture fluid from cells with growing hybrids was taken at 5-6, then 10-12 and 16-17 days after fusion.

Hybridomas were screened using the solid-phase ELISA described above. To control the nonspecific reactivity of the culture fluids, preparations of purified concentrates of heterologous influenza viruses A and B and the host component (allantoic fluid) were used. At the same time, culture liquids containing immunoglobulins were diluted 5-10 times.

2.5 Cloning.

In total, 7 primary hybridomas were obtained as a result of hybridization, which actively react in ELISA with purified concentrate of influenza virus B / Shandong / 7/97 and do not interact with heterologous viral and host antigens. Positive specifically reactive hybrids were cloned three times by McKearn T.J. under the control of their activity and specificity in ELISA. As a result, 2 hybrid clones of a given specificity were obtained, one of which (B / 4H1) represents the B / 4H1 hybridoma of the present invention. This hybridoma was deposited in the Specialized Collection of Vertebrate Cell Cultures of the Russian Cell Culture Collection on January 13, 2009 under the number RKKK (P) 719D. Cloned hybridomas were cryopreserved in liquid nitrogen (-196 ° C).

2.6 Obtaining ascitic preparations of MCA B / 4H1.

The B / 4H1 hybridoma was grown in vitro to a concentration of 1.5 × 10 ⁶ cells / ml, the cells were besieged by low-speed centrifugation, resuspended in 0.01 M FSB and intraperitoneally administered to mice, F1 interline hybrids (SGL / J × Balb / c), previously primed we stick, at 5 × 10 ⁶ cells / mouse in a volume of 1 ml. Ascitic tumors formed 10-16 days after the introduction of the hybridoma. After centrifugation of ascites, the supernatant containing specific MAB in high titers was used as a semi-finished product for the isolation of immunoglobulin, which was used to obtain immunological preparations. Passed cells on mice were cryopreserved and subsequently used for mass production of ascitic preparations MKA B / 4H1.

Example 3. The use of a set of MCA produced by the proposed hybridomas, for identification in ELISA of various strains of influenza B.

An analysis of the interaction of ascitic preparations containing MAB with reference strains of influenza virus type B in different years of the isolation of various evolutionary lines was carried out. ELISA was carried out according to the method described in example 2. Purified concentrates of the studied strains of influenza B were sorbed on a substrate at a concentration of 2 μg / ml. MCAs were introduced in dilutions of 10 ^-2 -10 ^-6 . ELISA results showed that MCA B / 4H1 allows only strains of the Victorian evolutionary line to be identified, and MCA 4H7 are suitable for detecting isolates of only the Yamagata variety.

Evolution line Influenza B virus strains Monoclonal antibodies 4H7 B / 4h1 Relic strains B / great lake / 54 <2 lg <21g B / Singapore / 222/79 <2 lg <21g B / USSR / 100/83 <2 lg <21g Victorian strains B / Victoria / 2/87 <2 lg 61g B / shandong / 7/97 <2 lg 61g B / Beijing / 243/97 <2 lg 61g B / shiga / 51/98 <2 lg 61g B / tokyo / 53/99 <2 lg 51g B / HongKong / 330/01 <2 lg 61g B / Malaysia / 2506/04 <2 lg 61g Yamagata strains B / panama / 45/90 6 lg <21g B / quindao / 102/91 5 lg <21g B / Beijing / 184/93 5 lg <21g B / harbin / 07/94 6 lg <21g B / Yamanashi / 166/98 5 lg <21g B / Johannesburg / 05/99 6 lg <21g B / Florida / 07/04 6 lg <21g B / Florida / 04/06 6 lg <21g

Example 4. Formulation of the reaction of inhibition of hemagglutination (RTGA).

RTGA was used to determine the reactivity spectrum of MCAs produced by 4H7 and B / 4H1 hybridomas with various strains of influenza B virus belonging to two evolutionary lines. RTHA was set according to the method recommended by WHO. MCA was titrated in steps of 2 in a volume of 50 μl per 0.1 M FSB, after which 25 μl of a viral suspension containing 8 hemagglutinating units was added. After the mixture was incubated at room temperature for an hour, an equal amount (75 μl) of a 1% chicken erythrocyte suspension was added. The highest dilution, completely suppressing hemagglutination, was taken as the MCA titer.

Interaction of MCA 4H7 and B / 4H1 in rtga with strains of different evolutionary lines of virus B.

Evolution line Influenza B virus strains Titer (10- ¹⁾ ICA HAI B / 4H1 4H7 Strains of early release B / great lake / 54 <20 <20 B / Singapore / 222/79 <20 <20 B / USSR / 100/83 <20 <20 B / Victoria / 2/87 > 640 <20 B / shandong / 07/97 20480 <20 B / Beijing / 243/97 > b40 <20 B / shiga / 51/98 > 640 <20 B / tokyo / 53/99 > 640 <20 B / HongKong / 330/01 > 640 <20 B / Malaysia / 2506/04 > 640 <20 Victorian line strains B / Kalinmgrad / 6/05 > 640 <20 B / Khabarovsk / 13/05 > 640 <20 B / Khabarovsk / 14/05 > 640 <20 B / Murmansk / 57/06 > 640 <20 B / ryazan / 5/06 > 640 <20 B / samara / 103/06 > 640 <20 B / St.-Petersburg / 12/06 160 <20 B / Tula / 1/06 > 640 <20 B / Chelyabinsk / 5/06 > 640 <20 B / Astrakhan / 1/07 > 640 <20 B / Voronezh / 19/07 > 640 <20 Bnrkutsk / 97/07 > 640 <20 B / St-Petersburg / 15/07 > 640 <20 B / St.-Petersburg / 18/07 > 640 <20 B / St.-Petersburg / 22/07 > 640 <20 B / Khabarovsk / 7652/07 > 640 <20 B / panama / 45/90 <20 5120 B / quindao / 102/91 <20 1280 B / Beijing / 184/93 <20 1280 B / harbin / 07/94 <20 5120 B / Yamanashi / 166/98 <20 1280

Strains of the Yamagat line B / Johannesburg / 05/99 <20 5120 B / Florida / 07/04 <20 5120 B / leningrad / 148/91 <20 1280 B / Khabarovsk / 135/93 <20 320 B / n. Novgorod / I 10/93 <20 640 B / Leningrad / 86/93 <20 640 B / St.-Petersburg / 141/95 <20 320 B / St-Petersburg / 210/95 <20 1280 B / Moscow / 91/95 <20 640 B / Lipetsk / 3/97 <20 640 B / N. Novgorod / 119/97 <20 5120 B / N. Novgorod / 348/97 <20 2560 B / samara / 240/99 <20 5120 B / samara / 253/99 <20 5120 B / Kaliningrad / 900/99 <20 2560 B / Kaliningrad / 905/99 <20 2560 B / cheeta / 2/05 <20 1280 B / Kaliningrad / 4/05 <20 5120

Example 5. The study of the neutralizing activity of MCA 4H7 and B / 4H1 to HA of influenza viruses B.

The neutralization reaction was carried out by the method of microcultural ELISA. The cell culture of MDSK was grown in 96-tray tablets from Sarstadt (Germany) at + 37 ° C in a CO ₂ thermostat in Igla MEM medium supplemented with 10% cattle serum (SCRS), 0.3 mg / ml glutamine and 100 μg / ml gentamicin. MCA was precipitated from a suspension of semi-saturated ammonium sulfate by centrifugation at 6000 rpm for 20 minutes. The precipitate was dissolved in a minimum volume of distilled water. Next, the MCA was dialyzed against FSB for 12-18 hours on a magnetic stirrer at + 4 ° C. 2-fold dilutions of MCA preparations were combined with equal volumes of 100 TCD of ₅₀ influenza B viruses diluted in Igla MEM medium without SCRS with the addition of 0.126 mg / ml arginine and 2 mgk / ml TRSC trypsin (Sigma), and incubated for 1 hour at +37 ° C. 100 μl of the virus - MCA mixture was applied onto the washed FSB wells with a monolayer of MDS cells. The dose of the virus was controlled by infecting the cells with 10-fold dilutions. The plates were kept for 3 days in a CO ₂ thermostat until the development of the CPD in the control wells. After removing the medium, the cells were fixed for 20 min with 80% cold acetone. 100 μl of conjugate MCA A / 4H1 specific for HPV type A virus with horseradish peroxidase were added to the wells and incubated for 1 hour at + 37 ° C. Working dilution of the conjugate in 0.01 M FSB with 5% skim milk was 1: 5000. After washing the FSB, 100 μl of a substrate-chromogenic mixture containing 0.05% H ₂ O ₂ and 0.1 mg / ml 3.3 ', 5.5' tetramethylbenzidine (TMB) per 0.1 M acetate was added to the wells. citrate buffer. pH 5.0. The reaction was stopped 2N H ₂ SO ₄ . The optical density at a wavelength of 450 nm (OD ₄₅₀ ) was determined using an Anthos photometer (Austria). The neutralization titer in the pH was considered the last dilution of serum, in which there was a 2-fold decrease in OD ₄₅₀ compared to the control of the virus.

Example 6. Determination of the epitope orientation of the MCA.

6.1. Electrophoresis

Electrophoresis was carried out according to the method of Laemmli U.K. (Laemmli, 1970) using an EU 135 apparatus (USA). Polyacrylamide gel (PAGE) with a linear concentration of 5-20% was used as a separating one. Separating gel was prepared on a buffer containing 1.5 M Tris (pH 8.8), a concentrating gel was prepared using 0.5 M Tris (pH 6.8). They also included sodium dodecyl sulfate (SDS) at a concentration of 1%. Under reducing conditions, electrophoresis was performed with 2-mercaptoethanol). The viral concentrate purified in a sucrose density gradient was diluted in lysis buffer (0.062M Tris, 2% SDS, 5% mercaptoethanol, 10% glycerol, 2% bromphenol blue pH 6.8) to a concentration of 5 μg / ml and warmed up for 2-3 minutes in a water bath. The resulting mixture of viral polypeptides was injected into the gel pockets under an electrode buffer (0.025 M Tris, 0.192 M glycerol, 0.1% SDS pH 8.4) using a syringe. Electrophoresis was carried out at a constant voltage of 100 mV, after which the separating gel was removed and used for immunoblotting. Viral proteins were stained with Coomassie R-250 stain (Sigma).

6.2 Immunoblotting.

Viral proteins separated under reducing and non-reducing conditions in PAAG were electrophoretically transferred onto nitrocellulose membranes (SCs) (Millipore Co.) as described (Towbin H., 1979) in transfer buffer (20 tM Tris, pH 8.3, 192 mM glycine, 20% ethanol) for 1 hour at a constant voltage of 26 mV. Free NC binding centers were blocked with 5% skim milk on FSB (FSB-M) (18 hours incubation, + 4 ° С). Then, NTs strips were incubated in an MCA solution (1/1000 dilution) on FSB-M, 1 hour incubation at + 37 ° С. Next, the NC was washed in PBS and incubated in a solution of peroxidase conjugate against mouse IgG (Sigma), incubation for 1 hour at + 37 ° C. After washing with SC FSB, the strips were treated with 1% dextran sulfate in 10 mM EDTA citrate buffer, pH 5.0. The substrate-chromogenic mixture consisted of 3.3'5.5 'tetramethylbenzidine (0.25 mg / ml) and 0.125% hydrogen peroxide in 10 mM EDTA citrate buffer. The reaction was stopped by washing the SC with distilled water.

Example 7. Determination of the activity of MCA 4H7 in the direct and indirect method of fluorescent antibodies (MFA).

7.1 Obtaining test preparations of various strains of influenza viruses In MFA.

The culture of transplantable dog kidney cells (MDSK) was grown in test tubes with 6 × 18 coverslips in growth medium Eagle with 10% fetal serum during the day. After the monolayer was formed, at least 75% of the medium was decanted and the monolayer was washed twice with PBS or Eagle's medium. Cells were infected with a virus-containing liquid in a dose of 4.0-5.0 lg EID ₅₀ /0.1 ml, inoculum dilutions were prepared on Eagle's medium (pH 7.2-7.3). Influenza B viruses were adsorbed onto the monolayer for 60 minutes at 32 ° C, after which the inoculum was removed, and the culture was washed three times with Eagle's medium. After that, a support medium Eagle was added to the cell culture with the addition of arginine and glutamine, 50 mg / l gentamicin. Virus-infected cells were incubated for 48-120 hours at 32 ° C. Then the medium was drained, the infected monolayer was washed twice with FSB, dried, and then fixed in acetone for 10 minutes at + 4 ° C.

7.2 Determination of the activity of MCA 4H7 in indirect MPA.

In indirect MFA, fixed test preparations were incubated with 4H7 MAB in various concentrations for 30 minutes at room temperature. After this, the preparations were washed with FSB for 20 minutes (double change of buffer), dried and incubated with FITC conjugate of anti-mouse IgG in working dilution for 20 minutes, followed by two times washing.

The fluorescence brightness of the affected cells was evaluated on a conventional four-cross scale. For control, it was compared with fluorescence of uninfected cells. The presence in the preparation of cells with typical cytoplasmic or nuclear fluorescence, with a brightness of at least ++ (two crosses), was sufficient to regard the result as positive (provided that there was no background fluorescence in the control preparations). It was found that 4A7 MAB gave a specific cytoplasmic luminescence of cells infected with influenza B viruses belonging only to the Yamagata evolutionary line with a brightness of +++ / ++++ in a working dilution of 5-10 μg / ml.

7.3 Preparation of MCA 4H7 labeled FITC.

MCA was precipitated from a suspension of semi-saturated ammonium sulfate by centrifugation at 6000 for 20 minutes. The precipitate was dissolved in a minimum volume of distilled water. Next, MCA was dialyzed against 0.01 M carbonate-bicarbonate buffer, pH 9.5 for 10-18 hours on a magnetic stirrer at + 4 ° C. For the most complete removal of ammonium sulfate, several (depending on the ratio of drug volumes to buffer) buffer changes were performed.

Then, 1 μg of FITC was dissolved in 1 ml of 0.2 M sodium carbonate (Na ₂ CO ₃ ), pH 9.5 in the ratio of 12.5 μg of FITC per 1 mg of MKA 4H7 protein. The required volume of FITC was added dropwise, constantly stirring, to the protein solution, the procedure was carried out in a dark glass dish on ice. During the first 30 minutes, the pH was periodically monitored and, if necessary, adjusted to 9.0 with a 0.15 M solution of disubstituted potassium phosphate.

Incubation was continued in an ice bath in a refrigerator on a magnetic stirrer for 18 hours.

The conjugate was released from unbound FITC on a Sephadex G-25 column equilibrated with phosphate-buffered saline (without sodium chloride). To determine the protein concentration and the FITC / protein molar ratio, the sample was diluted 50 times with 0.1 M sodium hydroxide and absorbance was measured at 280 and 495 wavelengths.

$$F\mathrm{AND}T\mathrm{Ts}/B=\frac{2.87\times \mathrm{ABOUT}P495}{\mathrm{ABOUT}P280-(0.35\times \mathrm{ABOUT}P495)}$$

7.4 Determination of the activity of FITC conjugates MCA 4H7. When stained by the direct method, FITC conjugates of MKA 4H7 were applied to the studied preparations and incubated for 30 minutes at room temperature, after which they were washed for 10 minutes in two shifts of 0.01 M FSB and dried. The results were taken into account by analogy with indirect MPA. FITC conjugates of MKA 4H7 gave predominantly homogeneous luminescence in the perinuclear zone of the cytoplasm of cells infected exclusively with strains of influenza viruses of the Yamagata variety. Coloring titers of the obtained conjugates ranged from 1:10 to 1:80 and correlated with the concentration of MCAs taken in conjugation.

For differential diagnosis of viral infections caused by influenza strains in the Yamagata branch, using FITC conjugates MKA 4H7 when staining clinical materials (epithelial cells of the lower nasal passages), it is recommended to use working dilutions 2-4 times lower than staining titers.

Example 8. Obtaining escape mutants of the influenza virus / Shandong / 7/97 in RCE, their biological and genetic characteristics.

8.1 Escape mutants (EM) were prepared according to standard procedures (Nakagawa et al., 2005, 2006). For this, the allantoic fluid containing the wild-type strain B / Shandong / 7/97 was mixed with an equal volume of previously dialyzed against 0.1 M phosphate-buffered saline (pH 7.2-7.4) MCA B / 4H1. A mixture of virus and MCA was incubated at room temperature for 1 hour. Then the mixture was introduced into six 10-day developing chicken embryos (RCEs) in a volume of 0.4 ml / embryo and incubated for 72 hours at a temperature of + 32 ° C. After this, the RCE was opened and the collected allantoic fluid was analyzed in RGA and RTGA. According to the RGA, as a result of the first cloning, hemagglutinating activity to titers 1 / 128-1 / 256 was possessed by clones (4) and (6). In RTGA, clones (4) and (6) showed a 256-fold decrease in the level of interaction with B / 4H1 MAB compared to the wild-type strain B / Shandong / 7/97 (titers in RTGA 1/80, 1/80 and 1/20480 respectively). Further, clones (4) and (6) were 4 ^x - multiple cloned by limiting dilutions (10 ^-6 -10 ^-8 ) in RCE already in the absence of MCA.

The resulting clones were designated as EM-B / 4H1 (4) and EM-B / 4H1 (6). 8.2 EM test results are shown in the table. Analysis of EM B / 4H1 with MCA in RTGA

Strain Caption of the ICA in RTGA MKA 5V7 MCA B / 4H1 B / shandong / 07/97 320 20480 EM-B / 4H1 (4) 320 <20 EM-B / 4H1 (6) 320 <20

For a more complete characterization of EM, in addition to the MCA B / 4H1 to which they were obtained, MCA 5 B7, also obtained for strain B / Shandong / 7/97, were used. As can be seen from the table, EM-B / 4H1 (4) and EM-B / 4H1 (6) completely lost the ability to interact in RTGA with MCA B / 4H1, which were used for selection. At the same time, both EMs showed the same level of activity in RTGA with 5B7 MCA as the wild-type strain B / Shandong / 7/97.

9.3 Sequencing of the gene EM-B / 4H1 (4) showed that this EM had one amino acid substitution at position 218 — lysine to threonine — compared to the wild type. EM-B / 4H1 (6) also had a single amino acid substitution at the same position 218, but this was a replacement of lysine with isoleucine.

Thus, sequencing of the HA gene of escape mutants cloned in the presence of B / 4H1 MAB allowed us to establish that these MABs were directed to an epitope with a significant amino acid residue at position 218.

The present invention is not limited to the above deposit or its embodiments described in the examples, which illustrate only a few aspects of the present invention. The scope of the present invention includes any options that are functional equivalents of the present invention. In addition to the options described and mentioned here, various modifications can be made to the present invention that are obvious to every person skilled in the art and are not beyond the scope of the attached claims.

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Claims (3)

1. The use of monoclonal antibodies B / 4H1 of the IgG2a isotype produced by B / 4H1 hybridoma and the 4H7 monoclonal antibodies of the IgG1 isotype produced by 4H7 hybridoma to identify the Yamagata and Victorian evolutionary lines of type B influenza virus. 2. The strain of hybridoma B / 4H1 producing monoclonal antibodies B / 4H1, belonging to the IgG2a isotype, designed to identify type B influenza viruses of the Victorian evolutionary line, deposited in the Specialized Collection of Vertebrate Cell Cultures of the Russian Collection of Cell Culture Cells on January 13, 2009 under inventory number RKKK (P) 719D. 3. The 4H7 hybridoma strain producing 4H7 monoclonal antibodies of the IgG1 isotype, designed to identify type B influenza viruses of the Yamagata evolutionary branch, deposited in the Specialized Collection of Vertebrate Cell Cultures of the Russian Cell Culture Collection on 13.01.2009 under RSCC inventory number (P ) 720D.

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