RU2478703C1 - STRAIN OF HYBRID ANIMAL CELLS Mus musculus 5G6 - PRODUCER OF MONOCLONAL ANTIBODIES SPECIFIC TO Yersinia pestis V ANTIGEN - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology and can be used to obtain monoclonal antibodies against the Yersinia pestis V antigen. The strain of hybrid animal cells Mus musculus 5G6 is obtained by immunising BALB/c mice. The mice are immunised by four-time administration of a recombinant V antigen in a dose of 100 mcg/mouse. On the third day after the last immunication, splenocytes of immune mice (1×10⁸ cells) are hybridised with mouse myeloma cells RZ-X63 Ag/8-653 (1×10⁷ cells). The fusion agent used is polyethylene glycol (Sigma, USA). Hybridisation is followed by selection, screening, cloning and cryopreservation of the hybridoma. The strain is deposited in the state collection of pathogenic microorganisms and cell cultures (GKPM-Obolensk) under number N-19.

EFFECT: strain of hybrid cultured cells, which produces monoclonal antibodies which are specific to the Y pestis V antigen, is suitable for constructing test systems for detecting plague pathogens.

8 dwg, 2 tbl, 7 ex

Description

The invention relates to biotechnology and can be used to obtain monoclonal antibodies (MCAs) to the V antigen of Yersinia pestis.

The causative agent of plague, Yersinia pestis, is one of the most virulent microorganisms and belongs to the gram-negative bacteria of the genus Yersinia [Hoult J. et al., 1997].

After the events of September 11, 2001, a list of the most likely and dangerous pathogens was compiled in New York. Along with the causative agents of smallpox, anthrax, Ebola and Marburg viruses, a plague microbe is noted. The susceptibility of a person to the plague is extremely high. The pathogen enters the human body through the bite of an infected flea or through damaged skin. In these cases, a bubonic or septic form of the plague develops, which, with timely antibiotic therapy and the absence of complications in the form of secondary pulmonary pneumonia, does not pose a great epidemic danger.

Plague so far remains a serious problem for endemic areas, as well as for global health in general. About 2,000 cases of human infection are recorded annually. An acute natural focal especially dangerous infectious disease, several pandemics of which claimed the lives of millions of people, is recognized as a “resurgent infection" and is one of the potential agents of bioterrorism [Gage et al., 2005, Inglesby et al, 2000]. Large outbreaks of plague recorded in recent years dictate the relevance of scientific research to improve diagnostic and prophylactic drugs aimed at preventing the spread and elimination of these infections.

Increasingly, traditional, and sometimes recently seemingly promising diagnostic methods are insufficient and fundamentally unacceptable, which forces us to search for new and unify existing methods for identifying and identifying infectious disease pathogens with expressivity, reliability, and high specificity.

Early laboratory diagnosis, timely identification of the source of infection occupy a major place in the system of anti-infection measures. In this regard, there is a need for the development and improvement of immunobiological methods, the construction of new rapid diagnostic and indication methods aimed at reducing the analysis time, simplifying it while increasing the reliability and ease of interpretation of the results with high sensitivity and specificity. Diagnostics prepared on the basis of monoclonal antibodies (MCAs) have such advantages, since they are aimed at identifying a single antigen.

The objective of the invention is to obtain a strain of hybrid cultured cells that produce monoclonal antibodies specific for the Y.pestis V antigen and suitable for constructing test systems based on them to identify the plague pathogen.

The problem is solved by the fact that a new strain of hybrid cultured animal cells of animals Mus musculus 5G6 is proposed - the producer of highly specific monoclonal antibodies to the V antigen Y. pestis.

The strain of hybrid animal cells Mus musculus 5G6 was deposited in the state collection of pathogenic microorganisms and cell cultures of the Federal budget institution of science of the State scientific center of applied microbiology and biotechnology (FBUN SSC BCH), collection number - H19.

Mus musculus 5G6 animal hybrid cell strain is obtained by immunizing BALB / c mice. Mice are immunized with 4 times the introduction of recombinant V antigen at a dose of 100 μg / mouse. On the third day after the last immunization, splenocytes of immune mice (1 × 10 ⁸ cells) are hybridized with mouse P3-X63 Ag / 8-653 mouse myeloma cells (1 × 10 ⁷ cells). Polyethylene glycol (Sigma, USA) is used as a fusion agent. After hybridization, selection, screening, cloning, and cryopreservation of the hybridoma are performed.

Characterization of the strain of hybrid cells of animals Mus musculus 5G6.

Morphological characteristics. The hybrid cell culture consists of rounded cells loosely attached to the substrate the size of the original myeloma cell.

Cultural properties. The cultivation of the strain of hybrid animal cells of Mus musculus 5G6 animals is carried out at a temperature of 37 ° C in an atmosphere containing 5% carbon dioxide. The cultivation medium is RPMI - 1640 medium containing 20% fetal calf serum, 2 mM / L-glutamine, 100 μg / ml gentamicin. Cells are cultured as a stationary suspension in Costar plastic mattresses. Passage of 5G6 hybridoma cells is carried out 2 times a week with a dilution ratio of 1: 2-1: 3. The inoculum cell concentration is 1 × 10 ⁵ in 1 ml of medium. The maximum concentration of hybrid cells during cultivation is 1 × 10 ⁵ per 1 ml of medium. The hybrid clone does not lose the ability to synthesize antibodies at 10 passages in vitro (observation period).

Cultivation of 5G6 hybridoma in an animal.

BALB / c mice of 20 weeks of age are treated with a pier (Sigma, USA) 0.5 ml intraperitoneally and 1 x 10 ¹⁰ hybrid cells are intraperitoneally injected after 2-3 weeks. The appearance of immunoascites is recorded on the 7-15th day, the selection of the immunoascitic (IAJ) fluid is performed on the 10-21th day. The synthesis of immunoglobulin produced by a hybridoma is determined by indirect enzyme-linked immunosorbent assay (ELISA) [Egorov AM et al., 1997].

The characteristic of a useful product. The mus musculus 5G6 animal hybrid cell strain produces specific monoclonal antibodies to the V antigen Y. pestis, the titer of which is in the culture fluid (QOL) 1: 640000, in the IAI 1: 1000000. Monoclonal antibodies from QOL and IAG were isolated by affinity chromatography on a column of protein G-Sepharose (Protein G Sepharose 4 Fast Flow). The purity of the obtained immunoglobulin fractions was evaluated in SDS-PAGE-electrophoresis under denaturing conditions. The concentration of immunoglobulins was determined spectrophotometrically at a wavelength of 280 nm (Smart Spec Plus spectrophotometer, BIO RAD, USA).

The productivity of the strain of hybrid cells of animals Mus musculus 5G6. MCA production in the cultivation medium is 20-50 μg / ml, in ascitic fluid - 5-10 mg / ml. MCA production in the culture fluid is maintained for 10 passages (observation period) and 5 passages during cultivation in the form of ascites tumors in mice (observation period).

The contamination of the strain of hybrid cells of animals Mus musculus 5G6. Hybrid line contaminants, including bacteria, yeast, fungi, were not detected. Mycoplasmas were not determined.

Cryopreservation. The freezing medium contains 90% fetal calf serum, 10% dimethyl sulfoxide.

Cryopreservation and heating mode. Hybrid cells are introduced into cryovials, placed in a foam container with a wall thickness of at least 1 cm and left for 16-24 hours in a kelvinator at a temperature of -70 ° C and then immersed in liquid nitrogen.

Defrosting is carried out quickly in a water bath at a temperature of 37 ° C.

Ampoules contain 1.0 ml of cryoprotective medium with a concentration of hybrid cells of 1 × 10 ⁶ . The viability of restored hybridomas after cryopreservation is 75-85%.

Properties of a useful product. The hybridoma produces monoclonal antibodies belonging to IgG2 in the mouse immunoglobulin subclass.

The invention is illustrated by the following graphic materials.

Figure 1 Dot blot with MCA 5G6. Determination of nonspecific activity against microbial cells of other microorganisms.

Figure 2 Dot blot with MCA 5G6. Determination of specific activity against representatives of the genus Yersinia.

Figure 3 Dot blot with MCA 5G6. Determination of specific activity for the V antigen of Y. pestis.

4 SDS-PAGE electrophoresis of MCA 5G6.

Figure 5 Determination of subclass affiliation MCA 5G6.

6 Immunoblotting MCA 5G6 with V antigen Y.pestis.

7. Detection of Y. pestis cells in latex agglutination reaction using 5G6 MCA.

Fig. 8 Determination of cross-activity in latex agglutination reaction using 5G6 MCA.

For a better understanding of the invention, the following are examples of its specific implementation.

Example 1. Obtaining a strain of hybrid cells 5G6.

Immunization.

For immunization, BALB / c mice aged 3-4 months are used. Immunization is carried out according to the following scheme:

0 day - 100 μg of antigen in PBS emulsified in PAP (0.2 ml) subcutaneously;

21 days - 100 μg of antigen in PBS emulsified in NAF (0.2 ml) subcutaneously;

51 days - 100 μg of antigen in PBS intravenously;

58 day - 100 μg of antigen in PBS intravenously;

Blood from immune mice was taken on the third day after the last injection of antigen at a concentration of 100 μg according to Animal protocol No. P02-19 p.7.

The titers of specific antibodies in animal sera are determined using indirect solid-phase ELISA.

Hybridization.

3 days after the last intravenous injection, the mouse spleen is removed and 1 × 10 ⁸ mouse splenocytes are hybridized with 1 × 10 ⁷ cells of the P3-X63 Ag / 8-653 myeloma line in the presence of 1 ml of polyethylene glycol (SIGMA, USA) for 1 minute.

Selection.

After washing the polyethylene glycol, the cells were plated on 96-well plates on a layer of peritoneal macrophages taken from BALB / c mice. Selection of hybrid cells is carried out on a selective medium containing hypoxanthine-aminopterin-thymidine (HAT). After 21 days, aminopterin is removed from the medium. Subsequently, cultivation is carried out on RPMI - 1640 Sigma medium with 20% fetal calf serum, 2 mM / L-glutamine, 100 μg / ml gentamicin.

Screening of hybrid clones.

Indirect solid-state ELISA is used to select positive hybrid clones producing MABs.

To conduct an indirect ELISA, 1 μg / well of V Y. pestis antigen is added to the wells of a 96-well polystyrene plate for ELISA and incubated at 37 ° C for 1 hour or at 4 ° C for 18 hours. Three times washed with phosphate-buffered saline pH 7.4, containing 0.5% tween-20 (FSB-T). Then, 160 μl of a 0.5% solution of bovine serum albumin (BSA), tested for the absence of peroxidase activity, is added to the wells and incubated at 37 ° C for 1 hour. Wash FSB-T three times, add supernatant of culture fluid to the wells in a volume of 100 μl and incubate at 37 ° C for 1 hour. After this, the wells of the tablet are washed three times with a solution of PBS-T and the peroxidase conjugate is added to the wells to the whole mouse IgG molecule (Sigma, USA) in working dilution. The plate is incubated at 37 ° C for 1 hour, then the wells are washed 6 times with FSB-T solution. After that, 100 μl of substrate-indicator solution (10 μl of 30% H ₂ O ₂ and 8 mg of orthophenylenediamine per 10 ml of phosphate-citrate buffer pH 5.0) is added to the wells. A positive reaction is evaluated by the appearance of a tan solution. The reaction is stopped by adding 50 μl of 4N sulfuric acid to the wells. The results are taken into account on a photometer (Picon photometer, RF) for ELISA at a wavelength of 492 nm.

Cloning of 5G6 hybrid cells was carried out by limiting dilution in 96-well plates on a layer of peritoneal macrophages at the rate of 1 × 10 ⁴ cells per well. Clone screening is carried out by indirect solid-phase ELISA, as described above.

Cultivation.

The cultivation of the strain of 5G6 hybrid cells is carried out at a temperature of 37 ° C in an atmosphere containing 5% carbon dioxide. The cultivation medium is RPMI - 1640 medium containing 20% fetal calf serum, 2 MM / L-glutamine, 100 μg / ml gentamicin.

Cryopreservation.

Clones of 5G6 hybridoma are cryopreserved on a freezing medium consisting of fetal calf serum - 90% dimethyl sulfoxide - 10%. Hybridoma clones are stored in Dewar vessels with liquid nitrogen.

Example 2. Determination of nonspecific activity against microbial cells of other microorganisms by dot blot analysis.

To conduct this study, a panel of 31 microorganisms of various species was used. As a positive control, Y. pestis antigen V was used. Inactivated microbial cells were adsorbed at a concentration of 1 × 10 ⁶ cells / ml on a nitrocellulose membrane (GE Water & Process Technologies, USA) using a BIO-DOT device (BIO RAD, USA). Strains of microorganisms were obtained from the collection of microorganisms FBUN SSC BCH. The membranes were then blocked with an inert protein solution and incubated sequentially with 5G6 MCA and peroxidase conjugate to the whole mouse IgG molecule (Sigma, USA). The reaction was visualized with a solution of a substrate mixture based on diaminobenzidine (0.05% diaminobenzidine (Sigma, United States), 0.015% H ₂ O ₂ , 0.01 M phosphate-buffered saline, pH 7.4). The reaction was stopped by washing with distilled water.

As the analysis showed, MCA 5G6 specifically interacted with the V antigen Y. pestis and did not give cross-reactions with microbial cells of other microorganisms (Fig. 1).

Example 3. Determination of specific activity against representatives of the genus Yersinia.

The specific activity of MCA 5G6 against various strains of Y. pestis was checked by dot blot analysis. Microbial cells of various strains of the genus Yersinia were sorbed onto a nitrocellulose filter using a Bio-Dot device (Bio-Rad, USA) at a concentration of 1 × 10 ⁷ cells / ml, 1 × 10 ⁶ cells / ml. Microorganism strains were obtained from the collection of microorganisms of the Federal State Health Institution Irkutsk Research Anti-Plague Institute of Siberia and the Far East. The membranes were then blocked with an inert protein solution and incubated sequentially with 5G6 MCA and peroxidase conjugate to the whole mouse IgG molecule (Sigma, USA). The reaction was visualized with a solution of a substrate mixture based on diaminobenzidine (0.05% diaminobenzidine (Sigma, United States), 0.015% H ₂ O ₂ 0.01 M phosphate-buffered saline, pH 7.4). The reaction was stopped by washing with distilled water.

As a result of the analysis, it was found that MCA 5G6 interact with microbial cells of Y. pestis at a concentration of 1 × 10 cells / ml and do not have cross activity against other closely related Yersinia (figure 2).

The specificity check of MCA 5G6.

Example 4. Determination of specific activity for the V antigen Y. pestis.

The specific activity of QOL 5G6 hybridomas against the V antigen of Y. pestis was tested by dot blot analysis. A V antigen with an initial concentration of 0.25 μg / ml was sorbed onto a nitrocellulose filter using a Bio-Dot device (Bio-Rad, USA) and titrated in two steps to a final concentration of 0.002 μg / ml. Next, the membrane was blocked with an inert protein solution and sequentially incubated with 5G6 CL hybrid and peroxidase conjugate to the whole mouse IgG molecule (Sigma, USA). The reaction was visualized with a solution of a substrate mixture based on diaminobenzidine (0.05% diaminobenzidine (Sigma, United States), 0.015% H ₂ O ₂ 0.01 M phosphate-buffered saline, pH 7.4). The reaction was stopped by washing with distilled water.

As a result of the analysis, it was found that MCA 5G6 detect up to 7 ng / ml of V antigen (figure 3).

Example 5. Isolation and purification of MCA 5G6.

The allocation of MCA from ascitic fluid is carried out by affinity chromatography on a column of protein G-sepharose (Protein G Sepharose 4 Fast Flow). Cellular components from the culture and ascitic fluids are removed by centrifugation (2000 g × 20 min), the pH of the culture fluid is adjusted to 8.6 using a 1 N NaOH solution, the ascitic fluid is diluted 1: 1 with planting buffer (0.05 M Tris, 0.15 M NaCl, 0.02% NaN ₃ , pH 8.6). The column is balanced with landing buffer and samples are applied at a rate of 10 ml / hour at room temperature. After applying the samples, the column is washed from unbound components and the immunoglobulins are eluted with 0.05 M glycine buffer, 0.15 M NaCl, pH 2.3. The control of the yield of the immunoglobulin fraction is carried out using a UV detector (Pharmacia LKB, Sweden).

The isolated immunoglobulins are concentrated by precipitation with dry ammonium sulfate (up to 50% saturation) and transferred to a phosphate-buffered saline by gel filtration on a Sephadex G-25 column (PD-10, Pharmacia LKB, Sweden) with a capacity of 10 ml. The purity of the obtained immunoglobulin fractions was evaluated in SDS-PAGE-electrophoresis under denaturing conditions.

As a result, purified immunoglobulin preparations were obtained (FIG. 4).

Example 6. The definition of subclass affiliation MCA 5G6.

Subclasses of the obtained MCAs are determined using the isotype kit for murine monoclonal antibodies (Roche Diagnostic Corporation, USA). An immunochromatographic strip is immersed in a culture liquid of 500 μl to determine the isotypes of murine monoclonal antibodies no deeper than 1.5 cm for 5 minutes. After the development of the control band and the band corresponding to the tested Ig, the antibody subclass is determined using the control immunochromatographic strip (attached to the kit for determining isotypes of murine monoclonal antibodies).

The subclass of the obtained MCAs showed that the strain of 5G6 hybrid cells produces monoclonal antibodies belonging to the IgG2b subclass of mouse immunoglobulins (Fig. 5).

Example 6. Immunoblotting MCA 5G6 with V antigen Y. pestis.

To verify the specificity of MCA 5G6 with the Y.pestis V antigen, SDS-PAGE electrophoresis was performed on 10% polyacrylamide gel (PAGE) for 1 hour at 220 V, 20 mA. V antigen from PAG is transferred onto a nitrocellulose membrane (GE Water & Process Technologies, USA) for 1 hour at a current of 380 mA. After the transfer is complete, the membrane is blocked by immersing in a 1% BSA solution in FSB pH 7.4 for 1 hour at room temperature. For washing the membranes using FSB-T pH 7.4. The blocked and washed membrane is incubated in a 5G6 hybridoma MCA solution for one hour at 37 ° C. Further, after washing with a buffer, the membrane is incubated with a peroxidase conjugate to the total mouse Ig fraction (Sigma, USA) in working dilution under the same conditions, washed with PBS-T. The reaction was visualized with a solution of a substrate mixture based on diaminobenzidine (0.05% diaminobenzidine (Sigma, United States), 0.015% H ₂ O ₂ , 0.01 M phosphate-saline buffer - FSB, pH 7.4). The reaction is stopped by washing with distilled water.

As a result of the analysis, it was found that MCA 5G6 interact with the V antigen Y. pestis (Fig 6).

Example 7. Detection of Y. pestis cells in latex agglutination reaction.

For the formulation of the latex-agglutination reaction (RLA), polyacrolein latex particles with a diameter of 1 and 1.2 μm are used (Institute of Bioorganic Chemistry named after Academician M.M.Shemyakin and Yu.A. Ovchinnikov RAS, Moscow) and MCA 5G6. The immobilization of MCA on latex particles is carried out according to the manufacturer's protocol: 100 μl of MCA 5G6 with a concentration of 1 mg / ml is added to 50 μl of a 10% latex suspension, the volume is adjusted to 0.5 ml and incubated for 2 hours at room temperature with stirring. To block unreacted groups add 4 ml of a 1% solution of ovalbumin (Sigma, USA) in 0.1 M borate buffer (BB), pH 8.2. Latex particles are washed from unbound MCA in the BB by three-fold 10-minute centrifugation at 1200 g. The pellet was resuspended in 5 ml BB with 1% ovalbumin.

The latex agglutination reaction is carried out in 96-well plates with a V-shaped profile of the wells (company Greiner Bio one, Germany). As control strains use: Yersinia pestis I-2377, Yersinia pestis I-2422, Yersinia pestis I-2638. For specificity control, the following strains were used: Y. pseudotuberculosis I-722, Y. pseudotuberculosis I-716, Y.enterocolitica 287, Y.enterocolitica I-131, E. coli 3912/41, E. coli ATCC 25922, P.aeruginosa ATCC 27853 , S.typhimurium 21, S.enteritidis 1127, S.typhi H-901, S.paratyphi A-225, Sh.disenteriae 1362.

As a negative control using 0.1 M borate buffer, pH 8.2.

Dilutions of cells of microorganism strains with step 2 are prepared in a volume of 25 μl in 96-well plates in borate buffer. After adding 25 μl of latex particles with immobilized MCA 5G6 to all wells, the plate is shaken and incubated for 1.5-2.0 hours at room temperature.

The results are taken into account visually using a four-cross system on a light background in good light.

4+ - all latex particles are agglutinated, uniformly cover the bottom of the hole, forming an "umbrella".

3+ - almost all latex particles are agglutinated, which is reflected in a decrease in the diameter of the "umbrella".

2+ - half of the latex particles are agglutinated.

1+ - most particles are not agglutinated and settled on the bottom of the hole, but the diameter of the “button” is still different from the “point”.

“-” - there are no signs of agglutination, latex particles settled on the bottom of the hole in the form of a “dot” of bright blue in the center of the hole.

A positive reaction is considered to be rated at least 3+.

As a result of the latex agglutination reaction, it was found that latex particles with immobilized MCA 5G6 cells of the strain Y. pestis at a concentration of 1.5 × 10 ⁶ cells / ml and do not reveal heterologous cultures of microorganisms at a concentration of 5 × 10 cells / ml and lower ( Fig.7, Fig.8, table 1, table 2).

Example 7. Detection of V antigen Y. pestis by ELISA using MCA 5G6.

MCA 5G6 in phosphate-buffered saline, pH 7.4 with an initial concentration of 5 μg / well, was titrated twice in vertical steps to a final concentration of 0.035 μg / well (100 μl / well in PBS). For titration, 96-well stripped ELISA plates were used. Positive (K +) and negative controls (K-) controls were added to the first vertical row of the tablet. Well A1 with negative control was used as blank. The antibody plate was incubated for 1 h on a shaker at a temperature of 37 ° C, then washed with FSB-T 4 times. Non-specific binding sites were blocked by adding 160 μl of milk with 0.5% fat to the wells, incubated for 1 h on a shaker at 37 ° C, and washed 3 times with PBS-T. V antigen was titrated twice in a horizontal direction with an initial concentration of 0.1 μg / well in FSB to a final concentration of 0.000097 μg / well in FSB (100 μl / well in FSB), incubated under the same conditions, and then washed plate 4 times in the FSB-T. Biotinylated MAB 2 B8 (fraction No. 3-1.52 mg / ml) was obviously added in excess (1: 500 dilution) of 100 μl / well into the FSB, the plate was incubated and washed four times, as described above. Then, 100 μl horseradish streptavidin-peroxidase conjugate (Imtek, Russia) was added to the plate wells at a dilution of 1: 5000 (according to the instructions for use) in the FSB. After incubation and four times washing the plate, 100 μl of the substrate-indicator mixture were added to the wells (0.008 mg of orthophenylenediamine in 10 ml of phosphate-citrate buffer, pH 5.0 with 1 μl / ml 33% H ₂ O ₂ ). The plate was placed for 3-5 min in a dark place at room temperature, then the reaction was stopped with 4N sulfuric acid (50 μl / well). The results were taken into account using an xMark plate spectrophotometer (Bio-Rad, USA) at a wavelength of 492 nm.

As a result of the analysis, it was found that for immobilized MCA 5G6, the most optimal concentration is 5 μg / well, which allows you to confidently determine the minimum amount of V antigen (4 ng / ml) (table 3).

Table 1 Latex-agglutination reaction results one 2 3 four 5 6 7 8 9 10 eleven 12 BUT 4+ 4+ 4+ 4+ 3+ 2+ 1+ - - - - - AT 4+ 4+ 4+ 4+ 3+ 2+ 1+ - - - - - FROM 4+ 4+ 4+ 4+ 4+ 3+ 2+ 1+ - - - - D - - - - - - - - - - - -
table 2 Latex-agglutination reaction results one 2 3 four 5 6 7 8 9 10 eleven 12 BUT - - - - - - - - - - - - AT - - - - - - - - - - - - FROM - - - - - - - - - - - -

Table 3 Detection of V antigen of Yersinia pestis by ELISA The concentration of MCA 5G6 (μg / well) The concentration of V antigen (μg / well) 0.5 0.25 0.125 0,062 0,03 0.015 0.0075 0.0037 0.0018 0.001 0,0005 Optical density at a wavelength of 492 nm (p.u.) 5.00 2,332 1,687 1,376 1,190 0.656 0.403 0.242 0.174 0,075 0,048 0,028 2,50 1,942 1,570 1,227 0.701 0.335 0.173 0,063 0.008 -0.008 -0.005 -0.008 1.25 1,080 0.662 0.481 0.246 0,080 0,022 -0.027 -0.016 -0.016 -0.013 -0.009 0.63 0.372 0.238 0.199 0.119 0,037 -0.031 -0.028 -0.025 -0.024 -0.021 -0.003 0.30 0,095 0,046 0,030 0.003 -0.049 -0.047 -0.047 -0.038 -0.035 0,030 -0.009 0.15 0.014 0.014 0.005 0.001 -0.049 -0.045 -0.042 -0.040 -0.038 -0.038 -0.025 0,07 -0,053 -0.048 -0.044 -0.044 -0.044 -0.044 -0.043 -0.035 -0.032 -0.025 -0.019 0,03 -0.062 -0,053 -0.046 -0.045 -0.045 -0.042 -0.042 -0.039 -0.038 -0.035 -0.032

Claims (1)

The strain of hybrid cultured animal cells Mus musculus 5G6, a producer of monoclonal antibodies specific for the Yersinia pestis V antigen, was deposited in the state collection of pathogenic microorganisms and cell cultures "GKPM-Obolensk", collection number H-19.

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