RU2451078C1 - Mus musculus cultivated hybrid cell strain 11d6 producer of monoclonal antibodies specific to lipopolysaccharide francisella tularensis - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: hybridoma strain is produced by immunization of BALB/c mice. The mice are immunised by common technique by double subcutaneous introduction of LPS Francisella tularensis 15/10. On the third day following the last booster injection, splenocytes of the immune mice are hybridised (1×10⁸ cells) and mice myeloma cells RZ-X63 Ag/8-653 (1×10⁷ cells). Polyethylene glycol (Sigma, the USA) is used as a fusion agent. The hybridisation is followed by hybridoma selection, screening, cloning and cryopreservation. The hybridoma 11D6 under the invention produces monoclonal antibodies (MCA) to LPS F. tularensis with titre making in a culture fluid 1:1000, in immunoascitic fluid 1:100000.

EFFECT: hybridoma produced MCAs are high specific to LPS Francisella tularensis and applicable for engineering test systems for rabbit disease agent detection.

7 dwg, 6 tbl, 7 ex

Description

The invention relates to biotechnology and can be used to obtain monoclonal antibodies (MCAs) to lipopolysaccharides (LPS) of Francisella tularensis.

On the territory of the Russian Federation, the presence of natural foci of tularemia is determined, the epizootic activity of which is confirmed by the sporadic incidence of humans and the release of the causative agent of tularemia from rodents, arthropods, from environmental objects or the detection of antigen in bird ridges and litter of predatory mammals. In the last decade (1995-2004), predominantly sporadic and group morbidity are recorded, which varies annually between 50-100 cases [1]. But since 2004, there has been an increase in cases of the disease (an increase of 3 times compared with 2003). In 2005, the number of cases increased by 8.5 times compared with the illogical period in 2004.

A variety of mechanisms and routes of infection: contact (through the skin or mucous membrane of the eye), inoculative (through the skin with a bite of an arthropod or mammal), alimentary (through the digestive tract) and aspiration (through the respiratory tract) determine the polymorphism of the clinical manifestations of tularemia.

The microbe is stable in environmental objects, in the water of surface reservoirs at a temperature of 13-15 ° C, bacteria can survive up to 3 months, at 4 ° C up to 4 months or more, at a temperature of 20-25 ° C they die within a few days. In the soil or at a temperature of 4-7 ° C, bacteria survive up to 3 months. They survive for a long time in milk, cream, stored at low (8-15 ° C) - 8 days, in frozen milk - more than 3 months. On grain and straw at a temperature of -5 ° C - about 7 months, at 8-12 ° C - up to 2 months. The causative agent of tularemia is resistant to drying, especially if it is in the organs and tissues of animals.

In direct sunlight, tularemia bacteria die within 20-30 minutes, in diffuse - can remain for several days.

The microbe is unstable to high temperatures (at 60 ° C it dies after 5-10 minutes, boiling for 1-2 minutes). Low resistance to chemical disinfection: 3% solutions of bleach, chloramine, other disinfectants destroy it after 20-30 minutes.

The Holarctic race exhibits greater stability in the external environment without reducing virulence: in ice for up to 10-11 months, in river water at 1 ° C for up to 9 months, on grain and straw for up to 6 months, at room temperature they are stored in water 30-60 days. The non-Arctic race, respectively, 8 months, up to 5-6 months, 3-4 months, not more than 20 days [2].

In connection with the development of immunochips and multiparameter diagnostic systems, great importance is attached to the production of specific immunoreagents, primarily monoclonal or monoreceptor polyclonal antibodies. The specificity of immunoglobulin preparations, which is the most important determining property of their quality, depends directly on the degree of purification and maximum expression of specific epitopes used to obtain antigen antibodies of a particular microorganism.

Therefore, one of the important approaches in detecting the causative agent of tularemia in environmental objects is the use of MCAs produced by hybridomas.

Hybridomas producing monoclonal bodies specific for LPS F.tularensis are known (3, 4). However, the monoclonal antibodies mentioned in the press are not sensitive enough to construct test systems on their basis for identifying the causative agent of tularemia. Thus, according to M.Pohanka et al. (4), the use of monoclonal antibodies against tularemia pathogen in the enzyme-labeled antibody method allows only 6.9 × 10 ⁶ microbial cells per ml or more to be detected.

The objective of the invention is to obtain a strain of hybrid cultured cells that produce monoclonal antibodies specific for F.tularensis lipopolysaccharides and suitable for constructing test systems based on them to identify the causative agent of tularemia.

The problem is solved by the fact that a new strain of hybrid cultured animal cells of animals Mus musculus 11D6 is proposed - a producer of highly specific monoclonal antibodies to LPS F.tularensis.

The strain is deposited in the collection of microorganisms of the Federal State Institution of Science of the State Scientific Center for Applied Microbiology and Biotechnology (FGUN SSC BCH) collection number - H11.

The hybridoma strain is obtained by immunization of BALB / c mice. Mice are immunized according to the standard method by double subcutaneous administration of LPS F.tularensis 15/10. On the third day after the last booster injection, splenocytes of immune mice (1 × 10 ⁸ cells) are hybridized with mouse cells of myeloma PZ-X63 Ag / 8-653 (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 hybridoma 11D6.

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 hybridoma strain 11D6 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. Passaging of 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).

The cultivation of hybridomas in the body of the animal. 20-week-old BALB / c mice were treated with a pieran (Sigma, USA) 0.5 ml intraperitoneally and after 2–3 weeks 1 × 10 ¹⁰ hybrid cells were intraperitoneally injected. 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 the immunoglobulin produced by the hybridoma is determined by the indirect ELISA [6].

The characteristic of a useful product. Hybridoma 11D6 produces specific monoclonal antibodies to LPS F.tularensis 15/10, the titer of which is 1: 1000 in the culture fluid (QL) and 1: 100000 in the IAH. 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).

Strain productivity. 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 when cultured in the form of ascites tumors in mice (observation period)

Strain contamination. Hybrid line contaminants, including bacteria, yeast, fungi, were not detected. Mycoplasmas were not determined.

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

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 the IgG3 subclass of mouse immunoglobulins; the affinity constant of MAB 11D6 - 1.4 × 10 ^-9 MAB are specific for LPS F.tularensis 15/10.

The invention is illustrated by the following graphic materials.

Figure 1 Dot blot with MKA 11D6. Determination of nonspecific activity against microbial cells of other microorganisms.

Figure 2 Dot blot with MKA 11D6. Determination of specific activity against representatives of the genus Francisella.

Figure 3 SDS-PAGE electrophoresis of MKA 11D6.

Figure 4 The definition of subclass affiliation MCA 11D6.

Figure 5 Immunoblotting MCA 11D6 with LPS F.tularensis 15/10.

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

Example 1. Obtaining hybridoma 11D6.

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 LPS in PBS emulsified in PAF (0.2 ml) subcutaneously;

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

51 days - 100 μg of LPS in PBS intravenously.

Blood from immune mice was taken on the third day after the last injection of LPS at a concentration of 100 μg according to Animal protocol No. P 02-19 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, the 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 F.tularensis 15/10 LPS 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 the supernatant of the culture fluid to the wells in a volume of 100 μl and incubate at 37 ° C for 1 hour. After that, 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 4 N 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.

Cloning of hybrid cells is carried out by the method of limiting dilutions 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.

Hybridoma clones are cultured 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.

Hybridoma clones 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 30 microorganisms of various types was used. Cells of five strains of F.tularensis were used as a positive control. 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). Microorganism strains were obtained from the collection of microorganisms of the Federal State Institution of Science and Science of the Russian Academy of Medical Sciences. The membranes were then blocked with an inert protein solution and incubated sequentially with 11D6 mAb and a 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 11D6 did not show cross activity with microbial cells of other microorganisms (wells 1 through 26 and 28) and interacted only with cells of F.tularensis strains (wells 25-27 and 29-30) (Fig. 1).

ICA specificity verification.

Example 3. Dot blot. Determination of specific activity against representatives of the genus Francisella.

Verification of the specific activity of MCA 11D6 against various strains of F.tularensis was performed by dot blot analysis. Microbial cells of various strains of the genus Francisella 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, 1 × 10 ⁵ cells / ml , 1 × 10 ⁴ cells / ml. Microorganism strains were obtained from the collection of microorganisms of the Federal State Institution of Science and Science of the Russian Academy of Medical Sciences. The membranes were then blocked with an inert protein solution and incubated sequentially with 11D6 mAb and a 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 11D6 interact with microbial cells of F.tularensis at a concentration of 1 × 10 ⁵ cells / ml and do not have cross activity in relation to pc. Francisella novicida. (figure 2).

Example 4. Isolation and purification of MCA 11D6.

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. 3).

Example 5. The definition of subclass affiliation MCA 11D6. 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 determination of the subclass MCA 11D6 showed that the hybridoma produces monoclonal antibodies related to the IgG3 subclass of mouse immunoglobulins (figure 4).

Example 6. Immunoblotting MCA 11D6 with LPS F.tularensis 15/10.

To verify the specificity of MCA 11D6 with LPS F.tularensis 15/10, SDS-PAGE electrophoresis was performed on 10% polyacrylamide gel (PAGE) for 1 hour at 220 V, 20 mA. LPS from PAG is transferred to 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 solution of MCA hybridoma 11D6 for one hour at a temperature of 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 11D6 interact with LPS F.tularensis 15/10 (figure 5).

Example 7. Detection of F.tularensis 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 MKA 11D6. The immobilization of MCA on latex particles is carried out according to the manufacturer's protocol: 100 μl of MCA 11D6 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 round-bottom 96-well plates with a U-shaped profile of the wells (company Pan Eco, Russia). As control strains, the cells of the strains are used: F.tularensis 15/10, F.tularensis Schu, F.tularensis 503, F.tularensis miura, F.tularensis 120. To control specificity, strains F.novicida 112, Y.pestis EV NIIEG are used , Y.enterocolitica 287, Y. pseudotuberculosis III, S.typhi 65, S.typhimurium 79, E.coli ATCC 25922, E.coli O157: H7 T 3691 and B. abortus LPS. 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 round-bottom 96-well plates in borate buffer. After adding 25 μl of latex particles with immobilized MCA 11D6 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 11D6 detect cells of tularemia strains at a concentration of 9 × 10 ⁴ -7.5 × 10 ⁵ cells / ml and do not reveal heterologous cultures of microorganisms at a concentration of 2 × 10 ⁸ cells / ml and below (Fig.6, Fig.7, table 1, table 2).

Example 8. Detection of F.tularensis cells by ELISA using magnetic particles with immobilized MAB 11D6.

Magnetic immunosorbents (MIS) developed using the method described in the invention (Pat. RF. No. 2138813, G-01, No. 33/543, dated September 27, 1999. Bull. No. 27) are used for ELISA using magnetic particles (7) . The activation of magnetic particles is carried out by oxidation using sodium perchlorate (ChemMed, RF) as follows: 3 ml of distilled water containing 0.20 g of sodium perchlorate is poured into 0.5 g of magnetic particles. Incubated at a temperature of (22 ± 4) ° C for 1 hour in the dark. Then the magnetic particles are washed with buffered saline solution (PBS) until the extinction value is absent on a spectrophotometer at a wavelength of 280 nm. The immobilization of MCA 11D6 on magnetic particles is carried out as follows: 2 ml of MCA 11D6 with a concentration of 2 mg / ml is added to 0.4 g of activated magnetic particles. Incubated for 2 hours at a temperature of (22 ± 4) ° C. For ELISA, immunoperoxidase conjugates are prepared by the method of periodic oxidation according to PKNakane, A. Kawaoi [8]: 1 ml of 0.3 M sodium hydrogencarbonate solution and 0.025 ml of 32% formalin are added to 5 mg of horseradish peroxidase, and the mixture is incubated on a shaker for 30 minutes. Next, 1 ml of sodium periodate was added to the reaction mixture and incubated on a shaker for 30 minutes. Then, 1 ml of 0.16 M ethylene glycol was added to the reaction mixture and incubated for 1 hour on a shaker. Next, the reaction mixture was dialyzed overnight at + 4 ° C against 0.01 M carbonate-bicarbonate buffer, pH 9.6. After dialysis, 1 ml of MCA 11D6 at a concentration of 5 mg / ml is added to activated horseradish peroxidase and incubated on a shaker for 2 hours, 5 mg of sodium borohydride are added and incubated in the dark for 2 hours at + 4 ° C. Then dialyzed night against 0.1 M PBS. After dialysis, BSA (10 mg BSA per 1 ml conjugate) is added.

ELISA using magnetic particles with immobilized MCA 11D6 (MIS). 50 μl of 10% suspension of MIS is added to eppendorfs. Then, 200 μl of FSB with albumin-tween (USB, USA) (FSB-AT) is added to eppendorf with a negative control, 200 μl of suspensions of killed cultures of tularemia microbe and strains of heterologous microorganisms are added to other eppendorfs. All suspension eppendorfs are incubated at a temperature of (37 + 1) ° C for 40 minutes. After incubation, the liquid is removed by holding the eppendorfs with a permanent magnet (Invitrogen, Norway), and the MIS is washed twice with phosphate-buffered saline with tween (FSB-T). Then, 200 μl of working dilution of the conjugate are added to each eppendorf. The eppendorfs with the conjugate are incubated at a temperature of (37 + 1) ° C for 15 min and washed with FSB-T 6 times. After that, 200 μl of substrate-indicator solution is added to the eppendorfs. Take into account the color change of the solutions in eppendorfs for 1-3 minutes. The supernatant is transferred 100 μl into the wells of a polystyrene plate for enzyme immunoassay (Table 1), holding eppendorf with a permanent magnet, and the reaction is stopped by adding 50 μl of a 4 N sulfuric acid solution (ChemMed, RF). The results are taken into account on a photometer (Picon photometer, RF) for ELISA at a wavelength of 492 nm. Positive are considered the results if the optical density of the samples was two or more times higher than the optical density of the negative control.

As control strains, cells of the strains are used: F.tularensis 15/10, F.tularensis Schu, F.tularensis 503, F.tularensis miura, F.tularensis 120, F.tularensis 250, F.tularensis 385, F.tularensis 356, F.tularensis 358, F.tularensis 355. To control specificity, strains F.novicida 112, F.novicida 383, Y. pestis EV NIIEG, Y. pestis I 2638, Y.enterocolitica 287, Y.enterocolitica 8, Y.pseudotuberculosis are used III, Y. pseudotuberculosis And 748, S.typhi 65, S.typhi 33221, S.typhimurium 79, S.typhimurium 441, E.coli ATCC 25922, E.coli 0157: H7 T 3691, E.coli 157.

As a result of the tests, it was found that magnetic particles with immobilized MCA 11D6 detect cells of tularemia strains at concentrations of 1 × 10 ² -1 × 10 ³ spores / ml and do not reveal control heterologous cultures of microorganisms at a concentration of 1.0 × 10 ⁵ m.k. ./ml and below when using the conjugate in a working dilution of 1: 600 (Table 3).

Information sources

1. MU 3.1.2007-05 “Epidemiological surveillance of tularemia”.

2. Olsufiev N.G., Dunaeva T.N. Natural foci, epidemiology and prevention of tularemia. - M .: Medicine, 1970 - 261 p.

3. A.P. Suslov, V.G. Lunin et al. Immunodetection systems for especially dangerous infections, including the use of phage display technologies.

4. M. Pohanka, O. Pavlis, M. Croca ELISA detection of Francisela tularensis using polyclonal and monoclonal antibodies. Defense science J., vol. 58, No. 5, p. 608-702. 2008

5. Zharikova T.V. Improving the methods of indication of Francisella tularensis and Leptospira interrogans using immobilized systems, abstract, Saratov, 2007.

6. Egorov A.M., Osipov AP, Dzantiev BB, Gavrilova EM Theory and practice of enzyme immunoassay. / T 33 - M .: Higher. Shk., 1991 .-- S.174-175.

7. Zharnikova I.V., Tyumentseva I.S., Efremenko V.I., Afanasyev E.N., Binatova V.V. A method of producing an immunosorbent (options) / Patent for the invention No. 2138813 ⁶ G01N 33/543, A61K 39/385, C12N 11/14. Zaregistr. in the state. the register of inventions of the Russian Federation 09/27/99, bull. Number 27.

8. Nakane P.K., Kawaoi A. Peroxidase-labelled antibody-a new method of conjugation // J. Histochem. Cytochem. - 1974. - V. 22, N 4. - P.506-508; 1084-1091.

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

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

Table 4. ELISA spectrometric results one 2 3 four 5 6 7 8 9 10 BUT 0,000 - - - - - - - - - AT 0,607 0.458 0.509 0.413 0.510 0.578 0.389 0.618 0.483 0.571 FROM 0.702 0.509 0.589 0.489 0.621 0.642 0.453 0.689 0.538 0.649 D 0.771 0.545 0.648 0.587 0.687 0.749 0.581 0.741 0.597 0.734 E 0.798 0.674 0.699 0.658 0.734 0.823 0.653 0.812 0.648 0.801 F 0.815 0.725 0.748 0.729 0.867 0.937 0.789 0.889 0.793 0.876 G 1.014 0.967 0.984 0.899 1.018 1,026 0.879 0.981 0.873 0.982 N 0.150 0.132 - - - - - - - -

Table 6. ELISA spectrometric results one 2 3 four 5 BUT 0,000 - - - - AT 0.211 0.158 0.201 0.189 0.191 FROM 0.187 0.187 0.168 0.197 0.124 D 0.156 0.143 0,111 0.165 0.136 E 0.147 0.147 0.141 0.142 0.145 F 0.201 0.136 0,132 0.182 0.174 G 0.178 0.154 0.123 0.163 0.179 N 0.161 0.147 - - -

Claims (1)

The strain of hybrid cultured animal cells Mus musculus 11D6, the producer of monoclonal antibodies specific for LPS Francisella tularensis, was deposited in the collection of microorganisms of the Federal State Institution of Science of the State Scientific Center for Applied Microbiology and Biotechnology (FGUN SSC PMB), collection number H-11.

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